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The role of neutrophils in the response of solid cancers to photodynamic therapy Cecić, Ivana F. K. 1999

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T H E R O L E OF N E U T R O P H I L S I N T H E R E S P O N S E OF S O L I D C A N C E R S T O P H O T O D Y N A M I C T H E R A P Y by Ivana F . K . Cecic B . S c , The University of British Columbia, 1995 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Department of Pathology) We accept this thesis as conforming to the required standard The University of British Columbia A p r i l , 1999 © I v a n a F . K . Cecic, 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbi; Vancouver, Canada Department DE-6 (2/88) A B S T R A C T Photodynamic Therapy (PDT) has been employed as a treatment for the eradication of solid neoplastic lesions over the last decade. With the F D A approval of Photofrin for use in P D T , this therapy has been clinically established for the treatment of several cancers in the Netherlands, Japan, France, the U S A , and Canada. P D T involves the administration of a photosensitive drug, excited by light of appropriate wavelength, causing localized tumor cell death in the presence of oxygen. The mechanism of cell death by P D T is complex, yet is increasingly understood, with accumulating evidence suggesting that the host response to P D T plays a major role in the success of this treatment. A marked feature o f P D T is the induction of strong, acute inflammation characterized by edema formation, and a wave of infiltrating inflammatory cells, first of which is the neutrophil. It has been documented that activated neutrophils sequester in tumors during and following PDT, and their role is indispensable for the effectiveness of this treatment modality. For instance, as shown in this thesis, in the absence of circulating neutrophils (achieved by the administration of 5 mg/kg of the monoclonal a n t i - G R L antibody) long-term tumor control by P D T is diminished for the murine squamous cell carcinoma S C C V I I grown in syngeneic C3Ff/HeN mice. Using this tumor model and also the murine E M T 6 mammary sarcoma, we further examined the systemic response o f neutrophils to P D T . Based on Wright stain analysis, it was determined that P D T induced a rise in relative circulating neutrophil content up to 2.3 times normal levels, from approximately 25% to 60% of nucleated cells in blood. Consequently, total neutrophil numbers in circulation rose from 2.95 ± 1.1 x 10 6 to 18.3 ± 5 x 10 6 per ml of blood at the peak interval 3 hours following P D T light treatment of subcutaneous back tumors. These increases were significantly higher than the effect on neutrophil levels by any stress-related reaction the animals experienced in handling ii during treatment (tail bleeding for blood collection, i.v. injection, immobilization in lead holders, P D T light only). N o changes were observed in the total numbers o f other white blood cell types, such as lymphocytes and monocytes. Identifying neutrophils by their high level o f GR1 expression, flow cytometry analysis was used to compare the neutrophil cell content in tumors, blood, lungs, and bone marrow of tumor-bearing mice over a 24-hour interval following P D T . A temporal rise in the levels of these cells in tumors, blood, and lungs, corresponded with a 50% drop in the granulocyte cell content of bone marrow. It therefore appears that a release of neutrophils from bone marrow occurred in response to P D T . L-selectin (CD62L) expression was analyzed in these neutrophil populations, as an indicator o f age and activation state. F low cytometry analysis detected a significant increase in L-selectin expression in the neutrophil populations of bone marrow and tumors from 2 and 8%, to 33 and 53%, respectively. In circulation, the majority of neutrophils expressed medium concentrations of L-selectin, with a very small fraction expressing high levels. There was an increase in L - s e l e c t i n m e d l u m with a corresponding decrease in L-select in l o w populations 10 hours after P D T treatment, while a marked rise in L-select in h l g h neutrophils was detected 24 hours post P D T . N o significant changes were observed in the lungs. Collectively, these results suggest that localized P D T induced a systemic response characterized by the release of younger, less mature, L-se lec t in h l g h neutrophils from bone marrow. Hence, tumor-infiltrating neutrophil populations may consist o f both mature and young neutrophils (present due to their accelerated release from bone marrow) which together contribute to tumor eradication by PDT. Therefore, PDT-induced inflammation partly characterized by the continuous sequestration o f neutrophils into P D T treated mouse tumors during the first day following P D T treatment, is causing a systemic response dominated by neutrophilia and an increased activation status of these cells. This condition reflects a massive mobilization of neutrophils from their iii storage pools and myeloid precursors, as they are recruited to participate in the destruction of P D T treated tumor tissue. iv Table of Contents Abstract i i Table o f contents v List o f Tables v i i i List o f Figures ix Abbreviations x i Acknowledgments x i i i Introduction 1 Section 1: History and present day clinical use of photodynamic therapy. 1 I. History of Photodynamic Therapy. 1 II. Advantages of P D T 2 III. Cl inical use and studies involving Photofrin® -based P D T 2 IV. Second generation sensitizers in clinical trials 4 Section 2: Mechanism of PDT-induced cell death 5 Part 1: Primary cell death 5 I. Light delivery 6 II. Photosensitizers 6 III. Cellular drug uptake 7 c IV. Drug uptake and localization in plasma membranes, lysosomes, and mitochondria. 7 Part 2: Secondary cell death 10 I. P D T induced inflammatory response 10 Section 3: Act ivi ty of neutrophils in inflammation 12 I. Function of neutrophils in inflammation 12 II. Interplay of inflammatory signals and adhesion molecules promote neutrophil extravasation. 13 III. Mechanism o f tissue destruction by neutrophils 14 IV. Neutrophil clearance from an inflamed site 15 Apoptosis in resolving acute inflammation. 16 v Section 4: Activi ty of neutrophils in cancer 17 I. Neutrophil-mediated damage of cancerous tissue 17 II. Activi ty of neutrophils in ischemia-reperfusion injury 18 III. P D T and apoptosis 19 Section 5: Neutrophil response to P D T of solid tumors 20 I. Accumulation of neutrophils in PDT-treated tumors 20 II. The role of neutrophils in the induction of a tumor-specific immune response to PDT-treated lesions 21 III. Modulation of blood flow in PDT-treated tumors by alteration of neutrophil activity 21 IV. Influence of modifications in neutrophil activity on the response of tumors to P D T 22 Hypothesis 24 Specific aims of this project 24 Materials and Methods 25 I. Tumor models 25 II. P D T 26 1 .Photosensitizers 26 2. Light treatment 27 3. Light dose 27 III. GR1 antibody. 28 IV. B C G vaccine 28 V . Growth delay 28 V I . Relative blood neutrophil levels 29 VII . Obtaining total leukocyte numbers in circulation. 29 VIII. Control groups in blood neutrophil analysis. 30 IX. Systemic response of neutrophils to photodynamic therapy 30 1. Tumor model and PDT. 30 2. Harvest of tumor, lung, blood, and bone marrow. 31 3. Antibody staining and flow cytometry 32 4. GR1 and L-selectin analysis 33 X . Statistical analysis 33 Results 3 4 Section 1 34 v i I. Growth delay following P D T is shortened with administration of GR1 antibody. 34 II. Photofrin and mTHPC-based P D T stimulate an increase in circulating neutrophil levels in S C C V I I tumor-bearing mice 36 III. P D T induces a marked increase in circulating neutrophil levels 38 IV. P D T induces a rise in circulating neutrophil levels in EMT6-tumor bearing mice, but is suppressed in a combined treatment with B C G vaccine 40 Section 2 ; 42 I. Intravenous drug administration and tail bleeding capable o f inducing an increase in circulating neutrophil levels 42 II. Changes in relative circulating neutrophil levels in non-PDT treated mice 45 a.) i.v. injection of D 5 W or 20 minute restraint, or light alone of Balb/c tumor-free footpad 45 Section 3 ; 47 I. Increase in circulating neutrophil levels is PDT-specific 47 Section 4 50 I. Analysis of total circulating leukocyte numbers following P D T treatment of tumors and normal skin 50 II. Control treatments also induced a change in total circulating leukocyte and neutrophil numbers 54 Section 5: Systemic response of GRl-posi t ive cells to P D T 57 I. Tumors 59 II. Lungs 60 III. Blood 62 IV. Bone marrow 63 Section 6: L-selectin expression on neutrophils following P D T 64 Discussion 70 Future directions 79 References 81 v i i List of Tables Table 1: Total circulating leukocyte numbers per ml of blood following Photofrin-based P D T of: i) tumor-free footpad, ii) s.c. E M T 6 back tumor, i i i ) E M T 6 foot tumor, in Balb/c mice 51 Table2: Total circulating neutrophil numbers following Photofrin-based P D T of: i) Balb/c footpad, ii) s.c. E M T 6 back tumor, and ii i) E M T 6 foot tumor 52 Table 3: Total circulating lymphocyte numbers per ml of blood following Photofrin-based P D T of: i) tumor-free footpad, ii) s.c. E M T 6 foot tumor, i i i) E M T 6 foot tumor, in Balb/c mice 53 Table 4: Total leukocyte and neutrophil numbers in mice following either i.v. injection of D5W or 20 minute restraint 54 Table 5: Total circulating leukocyte and neutrophil numbers in mice following a light dose of 60 J/cm 2 , in the absence of photosensitizer 55 viii L i s t of Figures Figure 1: Neutrophil depletion reduces delay for tumor recurrence following P D T 35 Figure 2: Photofrin- and mTHPC-based P D T of s.c. S C C V I I back tumors can induce an increase in circulating neutrophil levels 37 Figure 3: P D T induces an increase in circulating neutrophil levels 39 Figure 4: P D T induces an increase in circulating neutrophil levels in both E M T 6 tumor and tumor-free Balb/c mice, whereas, B C G vaccine has a suppressive effect 41 Figure 5: Increase in circulating neutrophil levels after i.v. administration o f 10 mg/kg Photofrin. 43 Figure 6: Relative changes in circulating neutrophil levels over a 24 hour period o f multiple blood sampling per animal 44 Figure 7: Changes in blood neutrophil levels after: i) i.v. injection o f 0.2 ml/20g mouse 5% dextrose in H 2 0 ( D 5 W ) , ii) 20 minute restraint, or i i i) 60 J/cm 2 , 630 nm of light on tumor-free footpad 45 Figure 8: Changes in circulating neutrophil levels following P D T on the foot 48 Figure 9: Normalized changes in circulating neutrophil levels following P D T on the E M T 6 back tumor 49 Figure 10: Systemic response of GR1 -positive cells to P D T 58 Figure 11: Changes in cell content of GR1-positive cell population in E M T 6 tumors following P D T 59 Figure 12: Change in the levels of GR1 -positive cells in lungs following PDT. 61 Figure 13: Changes in GRl-posi t ive cell content of blood following treatment with P D T 62 Figure 14: Changes in GRl-posi t ive cell content of bone marrow following P D T 63 Figure 15: Changes in the content of granulocytes expressing high levels of L-selectin in bone marrow and lungs 64 Figure 16: Changes in L-selectin expression of GRl-posi t ive cells in tumors following P D T 6 4 ix Figure 17: Changes in L-selectin expression of neutrophils in circulation following P D T 67 Figure 18: F low cytometry analysis dot-plots of PDT-induced changes in L-selectin expression on GR1 cells in peripheral blood 68 Figure 19: F low cytometry analysis dot-plots of PDT-induced changes in L-selectin expression on GR1 cells in tumor and bone marrow. 69 Abbreviations (ADCC ) antibody-dependent cell-mediated cytotoxicity (ALA) aminolevulinic acid (AMD) age-related macular degeneration (ATP) adenosine triphosphate (BCG) Bacillus of Calmette and Guerin (BPD-MA) Benzoporphyrin derivative mono-acid (CO2) carbon dioxide (D5W) 5 % dextrose in H 2 0 (EDTA) ethylenediaminetetraacetic acid (FDA) Food and Drug Association (FITC) fluorescein isothiocyanate (G-CSF) granulocyte colony-stimulating factor (HpD) hematoporphyrin derivative (HBSS) Hank's buffered salt solution (HOC1) hypochlorous acid (H2O2) hydrogen peroxide (ICAM-1) intercellular adhesion molecule-1 (IL-1) interleukin-1 (IL-8) interleukin-8 (LDL) low-density lipoprotein (LFA-1) lymphocyte function-associated antigen (Lu-Tex) Lutetium Texaphrin xi ( M H C ) major histocompatibility complex (mTHPC) meta-tetrahydroxyphenyl chlorin (Npe6) N-aspartyl chlorin e6 (O2") superoxide anion ( O H ) hydroxyl radical (PBS ) phosphate buffered saline (PDT) Photodynamic therapy (PEG-400) polyethylene glycol-400 (PE) phycoerythrin ( P M N ) polymorphonuclear leukocyte (s.c.) subcutaneous (SnET2) T in Etiopurpurin ( T A M ) tumor-associated macrophages (TNF-a) tumor necrosis factor-alpha (TPPS4) tetraphenylporphine Acknowledgments To the staff of Cancer Imaging, Medical Biophysics, and Advanced Therapeutics, for their unending advice and friendship, I am thankful. I would like to acknowledge the receptiveness, support and confidence I received from the members of my supervisory committee. In particular, I thank Dr. Minchinton for unlimited use of laboratory space and helpful advice. I am indebted to Dr. Matisic, for her encouragement and willingness to make time for my questions. To my supervisor, Dr. Korbelik, I express deepest gratitude for the opportunity to be a member of his research team. His constant guidance led me to persevere through this project. M y family has above all given me the foundation o f strength to diligently work through obstacles. True knowledge comes in realizing how little we know." -Josip Cecic xiii INTRODUCTION SECTION 1; HISTORY AND PRESENT DAY CLINICAL USE OF PHOTODYNAMIC THERAPY I. History of Photodynamic Therapy Documentation for the practice of combining light and photosensitive substances for medicinal use dates back several thousand years to ancient cultures of India and Egypt. The condition visiligo was treated in both areas using plants with photosensitive properties combined with sunlight (1). Photochemical sensitization with the intent to induce cell death was also found in the time of ancient Greece when the Greek scholar Herodotus described the healing aspects of light (2). However, it was in 1903 that the first use of Photodynamic Therapy (PDT) was reported for the treatment of neoplastic lesions, when Jesionek et al combined eosin and light to treat skin cancer (3). By 1911, Hausman pioneered the use of the photosensitive drug hematoporphyrin in PDT, which led to a specific interest in porphyrin-based photosensitizers (4). It was in the 1940s that hematoporphyrin was discovered to accumulate with greater specificity in malignant tumors compared to normal tissue (5). As time went on, tumor-localizing properties were improved with the formation of hematoporphyrin derivative (HpD), a mixture of porphyrin monomers, dimers, and oligomers, which was selectively retained in a large percentage of squamous cell carcinomas and adenocarcinomas (6). In the 1970s Dr. T. Dougherty began to study the application of HpD in combination with light for treatment of different animal and human malignancies (7-11). This work led to the development of the most widely used photosensitizer to date, Photofrin, and, in 1993, the approval of Photofrin-based PDT for clinical use. Following the usual practice with new modalities, PDT was introduced into clinics 1 primarily for palliative use where other more conventional treatments had failed (12). In recent years, clinical use of P D T for the treatment of bladder, skin, lung, and other cancers has been regulatory approved in many countries including the Netherlands, U K , Japan, U S A , and Canada. Cl inical trials at present include those for Photofrin-based P D T o f early stage head and neck cancers, certain types of skin cancer, and also in conjunction with surgery for brain and intrathoracic tumors (pleural mesothelioma) (12). II. Advantages of PDT P D T has certain advantages over standard cancer treatments such as chemotherapy and radiotherapy. P D T does not induce resistance and can be used repeatedly (13). This therapy does not wear-out the patient, as it is a localized form of treatment not systemic (12). Few unwanted side effects are associated with this treatment modality, with the exception of skin photosensitivity (14). Quick recovery and cosmetic healing, thereby healing o f normal tissue is favorable, and this therapy can be used in cases where other more conventional therapies have failed (15). III. Clinical use and studies involving Photofrin®-based PDT Although initial studies focused on treating patients with cutaneous or subcutaneous malignancies, many different tumors at various sites have now been treated with P D T . P D T treated malignancies include basal and squamous cell carcinomas o f skin, malignant melanomas, mycosis fungoides, recurrent metastatic breast carcinoma, and A I D S associated Kaposi 's sarcomas. Complete responses lasting up to 4 years have been achieved (16), and the response o f managing basal cell carcinoma is equal or superior to other forms of therapy (17,18). Although problems can arise with pigmented melanomas for example that virtually do 2 not respond to Photofrin-based PDT, since melanin absorbs light efficiently, clinical use of P D T shows exciting progress. P D T may become a treatment of choice for head and neck cancers. Long-term control o f ear, nose, and throat carcinoma in situ has been reported with a 70% response rate (19). A s P D T was found to be effective in eradicating papillomas in an animal model (20), the eradication of laryngeal papillomatosis by P D T is currently being evaluated clinically. In 1995, F D A approval was granted for P D T use in the treatment of advanced stage esophageal tumors (21). Photofrin-based P D T for lung cancers was originally used as palliative care in patients with endobronchial obstruction (22). In 1998, F D A approval was obtained for early stage lung cancer (12). Tumor localized fluorescence of photosensitizers is also being studied as potential means for detecting and/or delineating small carcinomas (in situ lesions) or superficial lung tumors covering a large endobronchial area (23). The treatment of advanced non-small cell lung cancer with P D T has been compared with ablation using N d - Y A G laser therapy alone. Tumor response to P D T was superior as shown in studies conducted in both Europe and Canada/USA (13). In addition, encouraging results comparing the use o f P D T plus radiotherapy and radiotherapy alone for the opening of obstructed airways has been reported by Lam (24). P D T is also investigated in clinical studies for gastrointestinal malignancies (25), colon cancer (26), and in gynecologic (27) and intra-abdominal malignancies (28), results for which show promise in the future use o f P D T in palliation, an alternative to surgery, or as an intraoperative procedure, respectively. In murine and human cases sensitizer is selectively retained in bladder tumors compared to normal bladder mucosa (29). Wi th specific combinations of photosensitizer and light delivery, superficial transitional cell cancers not involving the muscularis of the bladder, can be destroyed successfully (30). 3 IV. Second generation sensitizers in clinical trials A l l o f these studies mentioned above have been conducted using the F D A approved Photofrin-based P D T . New sensitizers have also entered clinical trials for various types of malignancies. T in Etiopurpurin (AnET2) is in phase II trials for the treatment of cutaneous metastatic breast cancer and Kaposi 's sarcoma (31). Lutetium Texaphrin (Lu-tex) is into a phasell/III trial for certain skin lesions (12). Benzoporphyrin Derivative M o n o - A c i d ( B P D -M A ) has undergone phase I/II trials for the treatment of skin cancers, psoriasis, and age-related macular degeneration ( A M D ) (32). The latter is a common cause of blindness whose current treatments using thermal lasers damages overlying retina. Not only does BPD-MA-based P D T manage to avoid damage to overlying retina, close to 50% o f patients treated experienced improved vision (33). The most potent sensitizer to date, tetrametahydroxyphenyl chlorin (mTHPC) is undergoing clinical trials for head and neck cancers in Europe and the U . S . Results indicate that all o f the patients in this study treated for early stage squamous cell carcinoma o f the upper aerodigestive tract, and Barrett's esophagus with superficial adenocarcinoma, were tumor-free after a follow-up of 3-35 months (34). P D T efficacy depends on the depth of light penetration, drug and light doses, as well as the mode of light delivery, combinations of which often differ depending on the type and site of malignancy. It is important therefore, that all o f these parameters are taken into consideration when designing specific protocols. 4 S E C T I O N 2: M E C H A N I S M OF PDT-INDUCED C E L L D E A T H P A R T 1: P R I M A R Y C E L L D E A T H P D T involves the use of three main components to induce cell death: the administration o f a photosensitive drug, light o f appropriate wavelength, and molecular oxygen. Fol lowing selective accumulation of drug in malignant tissue, the tumor is illuminated with light of appropriate wavelength for absorption by the respective sensitizer used. Upon absorption of light energy the photosensitizer molecule, in an excited, triplet state, can undergo two types o f reactions designated as Type I and Type II (35). The Type I reaction occurs when the drug reacts directly with a substrate by a mechanism involving hydrogen or electron transfer to form radicals which, in the presence of oxygen, can form oxygenated products. The Type II reaction, which predominates in P D T , results in the formation of singlet oxygen by the transfer o f energy from the excited triplet state o f the drug to molecular oxygen. Singlet oxygen can then react with substrates causing oxidative damage to cell components such as plasma membranes, liposomes, mitochondria, and D N A (35, 36). Singlet oxygen is accepted as the major free radical formed by P D T , inducing tumor cell death by oxidative damage (14,15). Due to the extremely short radius of action (<0.02 ujn) o f singlet oxygen, localized damage inflicted on a cell by the photodynamic effect depends on the relative accumulation of photosensitizer in a particular organelle at the time of light treatment. Damage inflicted by P D T is highly localized at the cellular level. 5 I. Light delivery Most photosensitizers are excited by tissue-penetrating wavelengths o f the red light spectrum. Photofrin for example absorbs light of 630nm, whereas m T H P C absorbs at 652nm. Light can be delivered by a number o f different light sources including a 250W metal halide lamp coupled with a light guide at its distal end, a 300W short arc plasma discharge or xenon arc lamp, diode lasers, and N d - Y A G lasers (12). Reasons for choosing a particular mode o f photoillumination may depend on the wavelength of light sought, and the fluence requirements of the sensitizer used (37,38). Other issues would encompass power, practicality, cost, and minimal heat emittance (39,40). II. Photosensitizers The first-generation photosensitizer hematoporphyrin derivative (HpD) is a mixture of porphyrin monomers, dimers and oligomers, formed from the treatment o f hematoporphyrin with acetic and sulphuric acids (41). The heterogeneity of H p D however led to unpredictable localization in different parts o f a cell. The need for a more purified form o f sensitizer with less heterogeneity, led to the production o f the most widely used photosensitizer to date, Photofrin® (42). Although much progress has been made in Photofrin-based P D T , there do exist some limitations such as prolonged skin photosensitivity and that it is excited by light of relatively short wavelength limiting depth of treatment. Therefore, intense research in developing new and improved second- generation sensitizers is well underway. Criteria for developing new photosensitive drugs include: (1) selective uptake by neoplastic cells/tissue, (2) low skin photosensitivity, (3) to be excited by light of longer wavelengths to increase treatment depth, and (4) reduced photobleaching and destruction (41). A number of compounds have arose including tin etiopurpurin (SnET2), lutetium texaphrin (Lu-6 tex), benzoporphyrin derivative monoacid ring A (BPD, verteporfin), tetrametahydroxyphenylchlorin (mTHPC), N-aspartyl chlorin e6 (Npe6), and aminolevulinic acid ( A L A ) protoporphyrin I X pro-drug (12). These new drugs are pure forms, not mixtures as is H p D . Compared to H p D , these newly formed sensitizers are more easily targeted towards specific organelles or cell components such as mitochondria, liposomes, and plasma membranes (12). Tumor vasculature has also been targeted (43). III. Cellular drug uptake Uptake o f the drug, prior to light treatment, into cells is crucial for effective P D T (44). W h y porphyrin-based sensitizers are more selectively taken up by neoplastic cells/tissues compared to normal cells has long been debated. Cancer cells seem to have upregulated expression of the low-density lipoprotein ( L D L ) receptor (45). This may enhance binding and entry o f circulating lipoprotiens carrying lipophilic porphyrins, such as B P D - M A (46). Poor lymphatic drainage may result in the build-up of porphyrins in the interstitial space (35). Rapidly dividing tumor cells may have an increased ability to phagocytose porphyrin aggregates or via pinocytosis (36). Recent studies have shown that it is not necessarily malignant cells involved in selective drug uptake, instead it may be stromal elements of the tumor. Korbelik et al have shown that a population of tumor-associated macrophages ( T A M ) collect the large concentration of sensitizer in malignant tissue (47,48,49). Interestingly, experimental tumors can have up to 80% T A M of cellular content and human cancers generally comprise between 20 and 50% of these cells (50). IV. Drug uptake and localization in plasma membranes, lysosomes. and mitochondria Localization into specific cellular components varies among photosensitizers, depending 7 on their lipophilicity, hydrophobicity, and other properties. To date it is well accepted that most sensitizers do not accumulate in nuclei, resulting in little potential for D N A damage or for mutations to occur. Some damage to D N A has been reported including strand breaks and chromosome aberrations. Recovery from the latter however can occur, suggesting that this effect may not necessarily be lethal (51,52). Direct cell damage does nonetheless vary from plasma membrane, to lysosomes, and to mitochondria, initiating different forms of cell injury and death. Damage by HpD-based P D T seems to concentrate to the plasma membrane (53). Porphyrin uptake begins with binding at the level of plasma membrane then migrating with time to other cellular components (54). Many reports have described the damage induced by P D T on plasma membranes. Observations described include swelling, bleb formation, shedding of vesicles containing plasma membrane marker enzymes, cytosol and lysosomal enzymes (55,56). Membrane transport is also affected. There is a reduction of active transport, depolarization, increased uptake o f a photosensitizer, and increased permeability to lactate dehydrogenase (57-60). The activity of numerous plasma membrane enzymes is inhibited including that of N a + K + - A T P a s e and M g 2 + - A T P a s e , and damage to multidrug transporters occurs (61, 62). Furthermore, P D T induces a rise in C a 2 + concentrations, up- and down-regulation of surface antigens, and l ipid peroxidation (63-65). The cumulative effect is a halt in cell division, followed by cell lysis (14). Other membranes also affected by P D T include that of mitochondria, Golg i apparatus, lysosomes, and the endoplasmic reticulum. Aggregated and also hydrophilic sensitizers, such as T P P S 4 (tetraphenylporphines), are likely to be taken up by pinocytosis and endocytosis, thereby accumulating in lysosomes and endosomes (12). The membranes of these vesicles become permeabilized upon light exposure, resulting in the release o f hydrolytic enzymes into the cytosol (66). 8 Photofrin and A L A , a naturally occurring substance converted into the photosensitive protoporphyrin I X by the heme biosynthetic pathway, have been shown to localize in mitochondria (12). P D T damage to this organelle has been described as causing aberrations in functional oxidative phosphorylation and electron transport chain activity, in addition to a decrease in cellular adenosine triphosphate (ATP) levels (67,68). O f particular interest is the induction o f cell death by apoptosis induced upon photoillumination, with a rapid release o f cytochrome C from mitochondria into the cytoplasm. Cytochrome C binds with A P A F - 1 activating apoptotic caspases, inducing cell death by apoptosis (69). Therefore, depending on the site of PDT-induced photo-damage, cells can undergo cell death by necrosis as a result of membrane damage, or apoptosis believed to be associated primarily with mitochondrial damage (70-72). This is an important consideration, for malignant cells often lack the ability to undergo apoptosis, and therefore escape the effects of chemotherapy (73). In this respect, P D T may prove to be an effective treatment against otherwise drug-resistant cell types. 9 P A R T 2 : S E C O N D A R Y C E L L D E A T H It is generally accepted that the anti-tumor effect of P D T combines both direct and indirect cell death processes (12,74). The direct lethal effect is a result o f irreparable photooxidative injury of vital cellular structures. The indirect ki l l ing of cancer cells results from a series o f events triggered by the formation of phototoxic lesions (not necessarily lethal) in cellular and acellular tumor constituents, including parenchymal and host immune cells, tumor vasculature, and extracellular matrix. Secondary tumor cell death is associated with the induction o f a strong, acute inflammatory response mediated by the combined release o f histamine and serotonin, as well as l ipid degradation products, and metabolites of arachidonic acid from photooxidative lesions o f lipids (75). The complex interplay o f secondary anti-tumor effects induced by P D T is dominated by three major responses: the breakdown o f tumor vasculature, an inflammatory reaction, and an immune response. A series o f events that lead up to secondary cell k i l l include ischemic death from vascular damage and blood flow stasis, ischemia-reperfusion injury, cell death mediated by resident and infiltrating inflammatory cells, and a tumor specific immune reaction (76). I. P D T induced inflammatory response The inflammatory response induced by P D T includes the massive infiltration of activated neutrophils into the treated site and emerging evidence suggests that these cells are major contributors to the rapid ablation of PDT-treated cancers (12,77). In addition to inflicting substantial direct damage to the tumor vasculature and malignant parenchyma, neutrophils are the initiators of inflammatory/immune processes involving the mobilization o f various types of non-specific and specific immune effector cells (78). These developments encompass an immune recognition of PDT-treated cancer and the induction of antitumor immunity mediated 10 by T-lymphocyte populations (74,79,80). Interestingly, P D T represents a rare example of an anti-cancer modality whose therapeutic outcome relies on the exploitation of the destructive capabilities o f neutrophils. This particular approach to the treatment of cancer, thus involves the mobilization of host neutrophils, the most powerful immune cell regarding their tissue destructive potential. This aspect of P D T wi l l be examined in detail in this thesis. 11 S E C T I O N 3: A C T I V I T Y OF N E U T R O P H I L S IN I N F L A M M A T I O N I. Function of neutrophils in inflammation In order to show how it is that neutrophils can have anti-tumor activity, it is helpful to understand their origin and function in inflammatory responses. A l l circulating blood cells are derived from a common progenitor stem cell in the bone marrow. The hematopoietic pathway of white blood cell differentiation can be separated into two lineages: lymphoid and myeloid. Neutrophils are phagocytic myeloid cells that serve as the first line of (innate) cellular defense against invading microorganisms and are the principal mediators of acute inflammation (81). Hereditary deficiencies in neutrophil function for example, can lead to bacterial infections causing death i f left untreated (82). Inflammation can be described as the response to infection or physical injury that has evolved to eliminate invading microorganisms and promote repair of damaging tissues. Four classical signs of inflammation include redness and heat which occur as a response to vascular dilatation, swelling as a result o f increased vascular permeability to plasma and leukocytes, and pain (81). In the first phase, inflammatory activity concentrates on preventing the spread of damage from an affected site, inactivation of microbes ( if present) and dissolution o f irreparably injured tissue. The induction of pro-inflammatory damage triggers an almost instantaneous release o f various chemotactic factors that promote, along a path o f their increasing concentration, the migration of inflammatory cells from the bloodstream to an inflamed site. Neutrophils are the first cells engaged in the inflammatory response (81,83). These cells normally remain contained to the bloodstream and do not migrate into healthy tissue. However, when elicited by inflammatory signals, neutrophils arrive rapidly in large numbers at an affected site, and become activated as powerful mediators in the destructive phase of an inflammatory 12 response. Their activity is associated with the release of additional stimuli that propagate the inflammatory process with continuing waves of an influx of neutrophils and other types of inflammatory cells, including mast cells and monocytes/macrophages. There is also evidence that signals released from extravasated neutrophils can directly attract lymphocytes to the affected site (84). Once extravasated from the blood vasculature, neutrophils, characterized by a short constitutive half-life (6 h in human blood), wi l l never emigrate back into circulation but w i l l shortly after invading inevitably meet their fate (85). II. Interplay of inflammatory signals and adhesion molecules promote neutrophil extravasation Intravascular stimulus generated from the complement, coagulation or kinin-generating system of the plasma, phospholipid metabolites, cytokines (IL-1, IL-8, T N F - a ) or other substances, attract the migration of neutrophils to an inflammatory site (78). These stimuli also activate or up-regulate adhesion molecules on both neutrophils and endothelial cells in the vascular l ining of blood vessels in the inflamed area (86). Upon arrival, neutrophils w i l l marginate and roll along the surface of the endothelium, a process mediated by a class o f adhesion molecules called selectins. L-selectin is expressed on the surface o f leukocytes, E -selectin and P-selectin on endothelial cells (87). Transendothelial migration is mediated by the (32-integrin family o f adhesion molecules, which includes L F A - 1 and Mac-1 expressed on the surface o f neutrophils, and I C A M - 1 on the endothelium. These adhesion molecules help to anchor the neutrophil to endothelial cells initiating diapedesis across the vessel wall toward a site of injury (88). Therefore, three major interactions between neutrophils and endothelial cells must unfold in order for extravasation to occur: cell rolling, anchoring, and diapedesis. 13 III. Mechanism of tissue destruction by neutrophils The neutrophil has been identified as the primary mediator o f tissue destruction in a variety of inflammatory diseases, including rheumatoid arthritis, myocardial reperfusion injury, blistering skin disorders, and ulcerative colitis (89). In these diseases and various acute inflammatory disorders, neutrophils are triggered to release a complex mixture o f destructive agents that normally defend the host against invading microbes. Because these cells have no inherent ability to differentiate between foreign and host antigens (this is left to other arms of the immune system), in the above mentioned disorders its destructive agents destroy normal cells and dissolve connective tissues (78). The neutrophil's inventory contains over 50 different toxic agents for mediating inflammatory tissue damage (89,90). These agents are usually grouped into those localized in the plasma membrane and others that are found in the intracellular granules. The enzyme N A D P H oxidase located in the plasma membrane enables the activated neutrophils to generate a family o f reactive oxidative species. The granules contain microbicidal peptides, proteins and enzymes. When specifically triggered by proinflammatory stimuli, the N A D P H oxidase (otherwise dormant in unstimulated neutrophils) starts to generate and release oxygen metabolites, while almost simultaneously the granules fuse with the plasma membrane and discharge their contents into the phagocytic vacuole and/or extracellular medium (89,91). The membrane-associated N A D P H oxidase system is capable o f producing at least three oxygen metabolites: superoxide anion (02~)> hydrogen peroxide (H2O2), and the hydroxyl radical ( O H ) . The bulk of generated oxygen metabolites appears to be processed as hydrogen peroxide by myeloperoxidase into HOC1 (hypochlorous acid, known as household bleach), an extremely powerful oxidant that rapidly inactivates a wide range of biologically relevant molecules (91,92). It is noteworthy to point out that neutrophils contain large quantities of 14 myeloperoxidase in their granules (up to 5% of the cell 's dry weight), so that substantial amounts o f this enzyme are released by activated cells into extracellular fluids (93). This enables neutrophils to generate impressive high quantities of HOC1: 2x l0" 7 mol per mi l l ion cells in two hours, an amount high enough to destroy 150 mil l ion microbes in milliseconds (93). Among over 20 enzymes contained in neutrophil granules, three proteinases - elastase, collagenase and gelatinase - appear to have the greatest potential for mediating tissue destruction (91). These particular enzymes are specialized in selective degradation o f key components of the extracellular matrix, the structure with an indispensable role in orderly function and repair of tissues. Normally, the destructive potential of such enzymes would be very limited because o f the abundance of powerful antiproteinases (proteinase inhibitors) both in the plasma and interstitial fluid. However, HOC1 and other oxidants produced by neutrophils destroy this antiproteinase shield (91,92). Hence, in order to utilize its ultimate destructive potential, the neutrophil uses both the N A D P H oxidase system and proteolytic granule contents in a cooperative and synergistic fashion (91). IV. Neutrophil clearance from an inflamed site Inflammation has long thought to be a beneficial process in which the host can defend itself against foreign invaders (81). However, it is also understood that the functions of activated neutrophils in chronic inflammation can unfortunately be involved in the pathogenesis of certain diseases such as myocardial infarction/reperfusion injury, atherosclerosis, and chronic bronchitis (85). Chronic inflammation is difficult to resolve, posing a major hindrance to the normal healing process of surrounding healthy tissue. Acute inflammation, as is induced by P D T , can however, under normal conditions, clear out completely, with cell death by apoptosis playing a very important role (94). 15 Apoptosis in resolving acute inflammation Apoptosis is a form of cell death in which a cell receives a signal to die from within, and is a natural process in development. Apoptosis contrasts with necrosis by the well described characteristics of internucleosomal D N A cleavage, nuclear degeneration and condensation, the formation o f apoptotic bodies, and phagocytosis of cell residua (81). Cells dying by apoptosis do not release their cytosolic contents into circulation and their immediate surroundings (94). Apoptosis therefore diminishes the induction or propagation o f an inflammatory process and its associated neutrophilic damage. A s discussed, neutrophils are the first inflammatory cell to arrive at a site o f perturbation creating chemotactic fragments from proteolytic cleavage o f matrix proteins, amplifying the inflammatory response. Therefore, in order to resolve an inflammatory state, the first step would be to remove activated neutrophils. In acute inflammation activated neutrophils undergo cell death with morphological changes characteristic of apoptosis (85). Apoptosis in neutrophils appears to occur constitutively most likely due to a short half-life of six hours (95). Apoptotic neutrophils become senescent, with a shutdown o f secretory processes, and are recognized and ingested intact by macrophages through the recognition o f cell surface changes (96). In this process, pro-inflammatory neutrophil granular contents are not released nor are those of macrophages, such as thromboxane, proteolytic enzymes, and cytokines. These inflammatory macrophages which have ingested apoptotic neutrophils in a site o f injury, are cleared via draining lymph nodes, which could lead to antigen presentation to lymphocytes (97). 16 S E C T I O N 4; A C T I V I T Y O F N E U T R O P H I L S IN C A N C E R I. Neutrophil-mediated damage of cancerous tissue The importance o f neutrophils in host rejection of malignant tumors has only recently received quality attention. It is increasingly clear that these cells can become activated as potent tumoricidal effectors, while, on the other hand, they may have a critical role in the initiation of an immune response to cancer cells (98-100). Neutrophils have been shown to destroy cancer cells in vitro by releasing oxygen metabolites or via antibody-dependent cell-mediated cytotoxicity ( A D C C ) (101,102). Direct involvement o f neutrophils was suggested to be responsible for endotoxin-mediated tumor necrosis (103). Using transduction o f the granulocyte colony-stimulating factor (G-CSF) gene into mouse adenocarcinoma cells, Colombo and co-workers presented direct evidence o f neutrophil-mediated tumor inhibition in vivo (104). G-CSF-releasing cancer cells in tumors, attract neutrophils that became engaged in direct contact with the cytokine-releasing cells. Anti-tumor effects o f neutrophils have also been described in a clinical study, in which peritoneum-infiltrating neutrophils, activated by the streptococcal agent OK-432, mediated the destruction of cancer ascites (105). Close contact between neutrophils and macrophages was shown to be essential for cancer cell cytostasis exhibited by cells present in inflammatory granulomas (98). The interaction of neutrophils with other host immune cells may be of critical importance in the development o f a host immune response against malignant tumors. When infiltrating into tumor tissue, neutrophils produce chemotactic factors for other immune cells, including macrophages and lymphocytes, as well as releasing cytokines such as interleukin-1 and tumor necrosis factor alpha (TNF-oc) through which they can exert a variety o f immunoregulatory functions (100,106). Thus, the depletion of neutrophils was demonstrated to abrogate the 17 immune rejection of transplanted tumors (100). However, in some cancer variants, malignant cells subvert neutrophils to produce factors that are stimulatory for malignant growth and their elimination results in the inhibition of tumor growth (107). II. Activity of neutrophils in ischemia-reperfusion injury Ischemia-reperfusion injury is a well-known physiological insult that can cause considerable damage to the affected tissue. The sudden re-introduction o f oxygen at the time o f reperfusion results in the induction of oxidative stress at the level of vascular endothelium and is associated with a massive recruitment and activation of neutrophils. These cells are largely responsible for the damage inflicted by ischemia/reperfusion injury (108). Although such events lead to irreversible neutrophil-induced damage in myocardial infarction, these occurrences can be beneficial i f incurred in tumor tissue (109,110). It was shown that the induction of ischemia-reperfusion injury by transiently clamping the feeding blood vessels to subcutaneous tumors could cause their ablation (111). 18 III. PDT and apoptosis The ability of a host to resolve an acute inflammatory response in a process inhibiting long-term inflammatory damage, can also be advantageous in the response to P D T . It is now known that neutrophils sequestered in tumors treated with mTHPC-based P D T undergo apoptosis in vivo (Korbelik, unpublished results). Furthermore, the induction o f an acute, not chronic, inflammatory host response may explain why the P D T effect tends to be so passive towards normal tissue. It could perhaps be advantageous to enhance apoptosis in the later stages o f inflammation following P D T to reduce any long-term, i l l effects o f neutrophil function on normal tissue, and augment the ki l l ing of remaining cancerous cells. 19 S E C T I O N 5: N E U T R O P H I L RESPONSE T O PDT OF SOLID T U M O R S I. Accumulation of neutrophils in PDT-treated tumors Insult to tumor vasculature by P D T contributes to the induction o f an inflammatory response (12,60,75). Vascular effects of P D T are manifested as the breakdown o f vessel basement membrane, contraction of endothelial cells, platelet aggregation, and vasoconstriction (8,42,44). Inflammation induced by P D T is further propagated by the release o f potent inflammatory mediators such as arachidonic acid metabolites, extracellular matrix components, and the cytokine interleukin (IL)-8 into the immediate surroundings and circulation, and promotes the adherence o f polymorphonuclear leukocytes to vessel walls (60,75,112,113). In response to inflammatory chemotactic signals, neutrophils rapidly and in large numbers accumulate in PDT-treated tumors. For instance, the neutrophil content of S C C V I I squamous cell carcinoma murine tumors increases 200-fold within five minutes of the start o f P D T light treatment (77). In an activated state, sequestered neutrophils presumably release the chemoattractant leukotriene B4 maintaining an influx of neutrophils, and other inflammatory cells including mast cells and monocytes/macrophages in the PDT-treated tumor (77). Similarly, Gollnick et al observed a change in the relative content of various cellular components o f the PDT-treated E M T 6 mammary sarcoma murine tumor. They reported that tumor cell numbers were diminished 24 hours after treatment as they succumbed to treatment, whereas the macrophage and granulocyte content increased. The granulocyte population increased to 50% of all cells retrieved compared to 3% in the untreated controls (114). Similar results were obtained with the E M T 6 tumor treated with P D T mediated by another photosensitizer, m T H P C (76). Activated neutrophils release from their cytosolic granules the enzyme myeloperoxidase that can be utilized as a qualitative indicator of neutrophil presence and activation (93). 20 Myeloperoxidase activity in S C C V I I and E M T tumors has been observed to increase with time following mTHPC-based P D T and was PDT-dose dependent (Cecic, Parkins, and Korbelik, unpublished results). II. The role of neutrophils in the induction of a tumor-specific immune response to PDT-treated lesions A s described earlier, P D T inflicts direct cell damage to cells. Thus, P D T results in inflammation, characterized in part by the sequestration of inflammatory cells to an affected site in response to chemotactic signals (12). Neutrophils are the first cells to arrive to the site o f perturbation. These cells play a dual role of inflicting cell damage and attracting other inflammatory/immune cells, such as phagocytic macrophages, as well as T-lymphocytes with the capability to become tumor-specific immune memory cells, to the P D T treated lesion (12,74). P D T generates a large amount of cellular debris at a tumor site, which can be taken up by macrophages and dendritic cells recruited to the site of P D T damage. This material is then processed and presented on the surface of these professional antigen-presenting cells in the context of M H C (major histocompatibility complex) molecules, to T-lymphocytes (12). These tumor-specific T lymphocytes may then contribute to both, the eradication o f PDT-treated lesions, and long-term tumor control, since it has been shown that functional T-lymphocytes are crucial for the curative outcome o f P D T (80). III. Modulation of blood flow in PDT-treated tumors by alteration of neutrophil activity The activity of sequestered neutrophils has an impact on the blood flow in PDT-treated 2 1 tumors, observed to decrease in a P D T dose-dependent manner (13,115). Sluiter and co-workers demonstrated in vitro that neutrophils adhere to PDT-treated endothelial cells, and that the pVintegrin molecule C D 18 plays a crucial role in this event (116). Blocking leukocyte adhesion to endothelial cells in vivo with the administration of monoclonal antibodies to the C D 18 molecule before P D T light treatment, completely abolished the induction of blood flow decrease in S C C V I I tumors (78). However, this effect of anti-CD 18 was not seen in the E M T 6 tumor model implicating the involvement of other factors in addition to the obstruction of blood flow by adherent and aggregated blood cells, for the modulation of blood flow in PDT-treated rumors. Interestingly, the PDT-induced decrease in blood flow o f both S C C V I I and E M T 6 tumors was markedly enhanced in mice whose circulating pool of neutrophils was depleted by the administration o f monoclonal antibody to the myeloid differentiation marker G R - 1 . A possible explanation for this finding is that invading neutrophils release nitric oxide that could act as a vasodilatory mediator in the vasculature of PDT-treated tumors (78,117). Reduction in tumor blood flow during photodynamic light delivery results in decreased tumor oxygenation, having a negative impact on the therapeutic effect due to a decreased production of cytotoxic oxygen species. On the other hand, impaired perfusion o f tumor tissue after P D T contributes to the antitumor effect based on ischemic necrosis (78). IV. Influence of modifications in neutrophil activity on the response of tumors to PDT Neutrophils accumulate in PDT-treated tumor tissue during and following treatment, and their contribution to the effectiveness of this treatment has been demonstrated by a number o f researchers. DeVree et al demonstrated that the administration of anti-granulocyte anti-serum into tumor-bearing rats before and at least 5 days after P D T , did not affect tumor volume, and the tumors continue to grow at a normal rate. However, when the administration of antiserum 22 was stopped, allowing neutrophil levels to increase, a delay in tumor growth was recorded (118). Conversely, using granulocyte-colony stimulating factor (G-CSF) prior to and after P D T to increase circulating neutrophil numbers, P D T effectiveness in delaying tumor regrowth was enhanced compared to saline controls (119). Similar results have also been observed in murine tumor models with a single intravenous dose of a monoclonal antibody for the myeloid differentiation marker G R 1 , administered one hour prior to P D T light treatment. In these studies, the cure rate o f PDT-treated E M T 6 tumors was markedly decreased, while the recurrence o f treated S C C V I I tumors was accelerated (78). Based on this evidence it may be concluded that neutrophil-associated events in P D T are indispensable for the efficacy of this treatment modality. Other evidence supporting the above conclusion comes from studying the adjuvant effect o f mycobacterium cell wall extract on the curative effect of P D T (76). The beneficial effect o f this agent was attributed to its enhancement of neutrophil infiltration into PDT-treated E M T 6 tumors. However, depending on circumstances, the engagement of activated neutrophils may both negatively and positively affect the tumor response to P D T . The cure rate of PDT-treated S C C V I I murine tumors was augmented by the administration o f the anti-CD 18 monoclonal antibody before light treatment. B y inhibiting leukocyte-endothelium interactions, the occlusion of blood vessels is presumably diminished allowing for an improved supply of oxygen during light treatment. 23 H Y P O T H E S I S The massive and rapid accumulation of neutrophils from circulation into PDT-treated tumors is secured by a strong systemic response o f these cells, which play an indispensable role in the curative outcome of P D T . SPECIFIC A I M S O F THIS P R O J E C T 1. To examine changes in peripheral blood leukocyte counts of tumor-bearing mice following treatment with P D T . 2. To determine whether in mice P D T induces the enhanced production of neutrophils from immature precursors in the bone marrow, as well as the mobilization o f these cells from their storage and marginated pools. 3. To analyze L-selectin expression in neutrophils localized in the bone marrow, peripheral blood, and PDT-treated tumor, to compare the activation status of these cells before and after P D T . 24 MATERIALS AND METHODS A l l plastics and glassware were obtained from V W R Canlab, Missisauga, Canada or G I B C O , Gaithersburg, U S A , unless otherwise specified. A l l chemicals and media were purchased from S I G M A Chemical Co. , St. Louis, U S A , unless otherwise specified. I. T u m o r m o d e l s : 1. S C C V I I squamous cell carcinoma (120) and E M T 6 mammary sarcoma (121) tumors were grown in syngeneic, immunocompetent C 3 H / H e N and Balb/c mice, respectively. These cell lines were routinely maintained in vivo by biweekly intramuscular tumor brei inoculation. M i c e were sacrificed by CO2 inhalation and the tumors removed and minced using two #22 scalpel blades. Subsequently the tumor tissue was repeatedly passed through two 18 gauge and 20 gauge needles, respectively, and diluted 5 times in phosphate buffered saline (PBS). 0.1 m l of tumor brei was inoculated into the thigh muscles of anesthetized mice (122). For experiments, the tumor was removed using aseptic technique, chopped using two #22 scalpel blades, suspended in 5 m l o f P B S and enzymatically digested with gentle rotation at 37°C for 30minutes (SCCVII) or 15 minutes (EMT6) . The three enzyme cocktail used for disaggregation contained: DNase (type I) 0.6 mg/ml, collagenase (type IV) 0.24 mg/ml and Dispase (Boerhinger, Mannheim, Germany) 0.18mg/ml, diluted in 5 mis of cold P B S (122,123). The enzymes were added to the tumor just prior to incubation. The tumor cell suspension was then filtered through a 100pm nylon mesh filter using a 6cc syringe, and pelleted by centrifugation at 600 rpm, and suspended in P B S . Cel l concentration was determined by hemacytometer count. For experiments, 1-2 x 10 6 S C C V I I and E M T 6 cells were inoculated subcutaneously on the sacral region on the dorsal side of animals, or 4 x 10 6 E M T 6 cells inoculated on the dorsal side of the 2 5 hind leg footpad. During inoculation, the animals were anesthetized by inhalation of Metofane® (Associated Veterinary Purchasing Ltd., Abbotsford, Canada). 2. In vitro culture: E M T 6 tumor cells were cultured at 37°C, 5% CO2, and 95% humidity, in alpha-minimal essential medium supplemented with 10% fetal calf serum (HyClone Laboratories Inc., Logan, U S A ) , 100 u,g/ml streptomycin, and 100 Units/ml penicillin, growing adherent to the bottom of T75cm tissue culture flasks. To harvest, a confluent monolayer of cells was treated with Tryps in-EDTA (ethylenediaminetetraacetic acid) solution ( G I B C O ) containing 0.25% trypsin and I m M E D T A 4 N a in H B S S (Hank's buffered salt solution), suspended in P B S , washed once by centrifugation at 600 rpm, and resuspended in P B S . A count of cell viability was obtained using trypan blue dye exclusion. 2 x 10 6 cells were inoculated into the footpad of anesthetized animals. A l l subcutaneous tumors were treated 7-8 days after inoculation and foot tumors 10-11 days after inoculation when the tumors reached an optimal P D T size o f 6-8 mm in largest diameter. The mice treated were 7-9 week old females, and kept in the Joint Animal Facili ty at the B . C . Cancer Research Centre where they were supplied with food and water ad libitum. The Animal Ethics Committee of the University of British Columbia approved all experimental protocols. II. PDT: 1 .Photosensitizers: 1.1 Photofrin®, kindly provided by Q L T (Quadralogics Technologies Inc., Vancouver, 26 B.C.,Canada), was reconstituted in 5% dextrose in H2O and used at a concentration of 10 mg/kg. A volume o f 0.2 ml/20gram mouse was administered intravenously (i.v.) 24 hours prior to light treatment. 1.2 m T H P C (metatetrahydroxyphenylchlorin, Scotia pharmaceuticals, Great Britain) was reconstituted in PEG-400 solution containing ethanol, polyethylene glycol, and H2O in a 2:3:5 v/v/v ratio, respectively. A volume of 0.1ml/20gram mouse was administered i.v. 24 hours prior to light treatment. 2. Light Treatment: Light was administered by a tunable light source (Photon Technology International Inc., Mode l A5000) equipped with a l k W Xenon bulb. This source was used to generate 630±10 nm light for Photofrin-based P D T and 652±10 nm light for mTHPC-based P D T delivered through a 5mm-core diameter liquid light guide (2000A, Luminex, Munich, Germany). The power density at the illuminated spot encompassing the tumor and ~-lmm of surrounding normal tissue was 100-120 m W / c m 2 . During light treatment each animal was restrained unanesthetized in specially designed lead holders exposing either the sacral region o f the back or the dorsal side of the footpad. 3. Light dose: E M T 6 tumor was treated with a dose of 60 J/cm 2 , unless otherwise indicated, and the average time per treatment was 8 minutes. The S C C V I I tumor was treated with 150 J/cm , unless otherwise indicated with an average time o f 20-30 minutes. Time duration was dependent on three parameters: (1) diameter of the tumor, (2) power output of the light source, and (3) light 27 dose. I I I . G R 1 a n t i b o d y The hybridoma cell line, clone RB6-8C5 producing rat IgG2b, was grown with the permission o f Dr. Gerald J. Spangrude (Department of Medicine, University of Utah) for the production of the monoclonal antibody reacting with mouse GR-1 antigen, L y - 6 G , expressed on mouse granulocytes. These cells were grown in Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum (HyClone Laboratories, Inc., Logan, U S A ) at 37°C, 5% CO2, and 95% humidity. The cells were transferred into serum- and protein-free hybridoma medium and incubated for two days at which time the supernatant was collected and the antibody concentrated to 1-1.5 mg/ml using 50,000 M W concentrating filters (Mill ipore Corporation, Bedford, U S A ) (124). 5 mg/kg of the antibody solution was administered i.v. between 30 minutes and 1 hour prior to P D T light treatment. I V . B C G v a c c i n e : The Bacillus of Calmette and Guerin ( B C G ) vaccine ( P A C I S M D , I A F Bio V a c Inc., Laval Quebec, Canada), was administered by lifting the subcutaneous tumor and injected the solution slowly under the tumor using a 26-gauge needle. The dose used was 1 x 10 7 colony forming units (CFU) per mouse. V . G r o w t h d e l a y : S C C V I I subcutaneous (s.c.) tumors were grown on the sacral region o f the back on female 28 C 3 H / H e N mice, 6-8 weeks of age. When the tumors reached optimal P D T size the mice were separated into two groups o f 8. One group was treated with Photofrin-based P D T alone and the second group was depleted of circulating neutrophils with the i.v. adminisration o f 5 mg/kg monoclonal GR1 antibody prior to P D T light treatment. Thereafter, the animals were monitored over a period o f 90 days, the time reference for tumor cures. Additional control groups consisted of tumor-bearing mice treated with ant i -GRl antibody only or isotype control antibody, Rat IgG2b. Rat IgG2b raised against the murine macrophage scavenger receptor, produced by hybridoma clone 2F8, was obtained purified in azide-free form from Cedarlane Laboratories Ltd. (Hornby, Canada) (124). V I . R e l a t i v e b l o o d n e u t r o p h i l l e v e l s : To obtain a white blood cell count, the tail vein of an animal was dilated in a warm water bath. Subsequently, using a sterile scalpel blade a small nick was made in the tail and a sample of blood collected by heparinized micropipet, smeared onto a clean glass slide, and left to air-dry. Samples were made in duplicate. The air-dried specimens were stained using the Wright 's stain (prepared according to manufacturer's instructions, Accustain, Sigma Chemical Co. , St. Louis, U S A ) for 30 seconds, and rinsed with water. Identifying morphological features, a differential white blood cell count was obtained using a Zeiss microscope at 2 0 X magnification. Cells were grouped as neutrophils, lymphocytes, monocytes, neutrophil band, and other, and counted until a total of at least 200 cells were counted, in a non-specific number of fields, on each slide. From that total, percent of each cell type was calculated. The percentages were then converted into relative numbers, with 1 designating normal baseline levels. 29 VII. Obtaining total leukocyte numbers in circulation: To determine total blood leukocyte levels at various times after relative treatments, the tail vein of the animal was dilated in a warm water bath, and a small nick in the tail was made using a sterile #22 scalpel blade. lOpl of blood collected using a 20ul pipet-aid was placed into 90pl o f ice-cold lysing solution containing ammonium chloride 8.3 g/L, sodium bicarbonate 1 g/L, and 0. 1 m M ethylenediaminetetraacetic acid ( E D T A ) , to lyse erythrocytes, and kept on ice for twenty minutes. 900pJ o f lysing solution was added to each sample and a 10pJ aliquot placed on a hemacytometer to determine total leukocyte count per ml of blood. The total number o f white blood cells was multiplied by the percent of neutrophils (described in part IV) to determine the total number of neutrophils in circulation at specific time points after treatment. VIII. Control groups in blood neutrophil analysis Control groups of tumor-free mice were either: (1) restrained for twenty minutes and released into their cages, (2) administered i.v. 0.2ml/20gram weight 5% dextrose in H2O, (3) administered 10 mg/kg o f Photofrin i.v., (4) light dose o f 60 J/cm2 at 630 nm on the footpad, or (5) Photofrin-based P D T on the footpad. A single blood sample was collected from each mouse to determine total neutrophil numbers in circulation, and relative neutrophil levels. IX. Systemic response of neutrophils to photodynamic therapy 1. T u m o r model and P D T : 2 x 10 6 E M T 6 tumor cells were inoculated onto the footpad of anesthetized Balb/c mice. When the tumors reached a diameter of 7 mm, 10 mg/kg of Photofrin was administered i.v. to each mouse 24 hours prior to light treatment. The light dose given to each tumor was 60 J/cm . 30 2. Harvest of tumor, lung, blood, and bone marrow: 1. Tumor: Animals were sacrificed by CO2 inhalation and tumors excised using a #22 scalpel blade and placed in a petri dish on ice. The tumors were chopped and made into a single cell suspension, as described in section I, and suspended in ice-cold Hank's buffered salt solution (HBSS) . 2. Lung: Lungs were removed using small scissors and placed in a petri dish set on ice. The lungs were chopped with a #22 scalpel blade, then suspended in 5 m l o f ice-cold P B S . The suspension was placed in a 6cc syringe. With the plunger, the lung tissue was pushed through a 100pm nylon mesh filter, attached on the end of the syringe. The filtrate was pelleted in a centrifuge at 600 rpm and resuspended in erythrocyte lysing solution for 20 minutes on ice. The suspension was pelleted in a centrifuge at 600 rpm and resuspended in H B S S . 3 .Blood: 200-300pJ of blood was collected by cardiac puncture into an insulin syringe with attached 26 gauge (G) needle and immediately placed in a prepared 5cc polystyrene tube containing 3ml o f lysing solution, on ice for 20 minutes. White blood cells were pelleted by centrifugation at 600 rpm and suspended in H B S S . 4. Bone marrow: The femur of the right hind leg was removed using sharp-tipped forceps and small surgical scissors. Using a 26G needle attached to a l c c syringe filled with P B S , marrow cells were flushed through the end of the femur into a petri dish, and placed on ice. 3ml of P B S were added to the petri dish and the cells transferred into a 6cc syringe. Using a plunger the cell suspension was pushed through a mesh filter, and the filtrate collected into a 12cc polystyrene tube. The cells were pelleted in a centrifuge at 600 rpm for 10 minutes, and resuspended in H B S S . A 10pJ aliquot o f each suspension (tumor, lung, blood, and bone marrow) was placed on a 31 hemacytometer for the determination of total cell count. Each sample was concentrated or diluted, as required, to a total cell count o f 0.5 - 1 x 10 6 in H B S S prior to antibody staining for flow cytometry. 3. Antibody staining and flow cytometry: A l l antibodies were obtained from Pharmingen (San Diego, U S A ) . Monoclonal antibodies used for the detection of cell type specific murine membrane antigens were anti-CD45 (pan-leukocyte marker), an t i -GRl (myeloid differentiation surface marker), and anti-L-selectin. The antibodies G R 1 , L-selectin, and CD45 , were directly conjugated to the fluorescent dyes phycoerythrin, fluorescein isothiocyanate, and Cy-chrome, respectively. A l l samples were centrifuged at 1000 rpm, and 100 p i o f supernatant prepared from hybridoma cells producing anti-mouse Fey receptor antibodies (to block the Fc receptor mediated non-specific binding o f monoclonal antibodies), was added to each pellet and set on ice shielded from light for 10 minutes. Cells were washed once in H B S S . lOOpi o f the antibody solution in H B S S , diluted according to manufacturer's guidelines, was added to each pellet. H B S S used in this and following steps contained 0.02% sodium azide and O.lmg/ml bovine serum albumin. The cells were kept on ice for 25 minutes at which time they were washed once in H B S S and suspended in 0.5ml ice-cold H B S S . The samples were kept on ice and shielded from direct light prior to analysis by flow cytometry using the Coulter Epics Elite ESP apparatus from Coulter Electronics. A 488nm laser was used to excite FITC, P E and C Y - C H R O M , whose emissions were recorded through 530 ± 15, 580 ± 10, and 675 nm bandpass filters, respectively. 20 000 cells were analyzed for each sample. 32 4. GR1 and L-selectin analysis: Dead cells and debris were gated out using forward and side scatter light signals. In tumor and lung samples, white blood cells were separated from other cells, by their expression o f the CD45 pan leukocyte surface marker. Myelo id cells were separated according to their expression o f G R 1 : neutrophils/granulocytes as G R 1 + + + , macrophages as G R + (125). The population of cells expressing high levels of GR1 were then analyzed against their level of L-selectin expression, designated as high (++), and low (+). X . S t a t i s t i c a l a n a l y s i s : In all experiments, data is presented as a mean ± S E M compared to non-treated controls. Analysis o f variance between the means of control and treated sample groups was by one-way A N O V A , unless otherwise indicated. The log-rank test was used when comparing tumor growth delay following P D T (Figure 1). P-values less than 0.05 were considered statistically significant. 33 R E S U L T S S E C T I O N 1 I. Growth delay following PDT is shortened with administration of G R 1 antibody: Two groups of eight S C C V I I tumor-bearing mice were treated with Photofrin-based P D T . One o f the two groups was injected intravenously with 5 mg/kg o f the monoclonal antibody G R 1 . These mice were then observed every 2-3 days for signs o f tumor regrowth. Results of the experiment are shown in Figure 1. A l l tumors responded to P D T , assessed as complete tumor ablation 24 hours after treatment. In the first group treated with P D T alone, all animals were tumor-free at day 16 after light treatment, and by day 23, 12.5 % of the group was without tumor. The second group whose circulating neutrophils were depleted by the administration o f the ant i -GRl antibody prior to light treatment, all animals were tumor-free at day 4 after P D T . Tumors recurred in 50% of the animals by day 10, with no cures by day 14 after treatment. Treatment with GR1 antibody alone showed no obvious effect on tumor growth, and the isotype control treatment with monoclonal antibody against mouse macrophage scavenger receptor (which belongs to the same class of rat immunoglobulin, IgG2b, as GR1) had no effect on the P D T response (124). 34 Time after PDT (days) Figure 1: Neutrophil depletion reduces delay for tumor recurrence following PDT. Two groups of 8 s.c. S C C V I I back tumor bearing C 3 H / H e N mice were treated with P D T and monitored for tumor recurrence. The first group of mice was treated with P D T alone as the second group received a combined treatment of 5 mg/kg of the an t i -GRl antibody lhour prior to P D T light treatment. P D T : Photofrin 10 mg/kg injected i.v. 24 hours before 220 J / cm 2 light delivery. Statistical significance was determined using the log-rank test. The delay for 50% tumor recurrence is significantly different (p<0.002). 35 II. Photofrin and mTHPC-based PDT stimulate an increase in circulating neutrophil levels in SCCVII-tumor-bearing mice In order to investigate PDT-induced changes in systemic levels o f neutrophils, a series of experiments were undertaken in which blood samples were collected from the tail vein of mice at different time intervals before and/or after P D T or related treatments. In these experiments, one of two protocols for blood sampling was followed: consecutive sampling or individual sampling. Consecutive sampling involved cutting nicks using a scalpel blade in the tail o f a mouse to collect multiple blood samples over an observation period. Individual sampling meant that only one nick for a single sample o f blood was taken per mouse, thereby one group of mice would have been used to collect blood at a particular time point. Both m T H P C - and Photofrin-based P D T can cause a similar increase in the level o f circulating neutrophils in S C C V I I tumor-bearing mice, as shown in Figure 2. Neutrophil levels in treated animals rose 2.2 and 2.4 times above normal between 3 and 6 hours for Photofrin- and two different doses of m T H P C - P D T , respectively, after the onset o f P D T light treatment. A l l three doses of P D T induced complete initial ablation of treated tumors, however, permanent cure rates for 0.6 mg/kg m T H P C . 0.3 mg/kg m T H P C , and Photofrin were 100%, 50%, and 0%, respectively (data not shown). 36 2.8-2.6-2.4-2.2-2.0-1.8-1.6-1.4-1.2-1.0-0.8-0.6-•--10 mg/kg photofrin •#- 0.3 mg/kg mTHPC A- 0.6 mg/kg mTHPC 0 2 4 6 8 10 Time after the onset of light treatment (hours) Figure 2: Photofrin- and mTHPC-based PDT of s.c. S C C V I I back tumors can induce as increase in circulating neutrophil levels, s.c. S C C V I I tumors on the backs o f three groups o f 8 C 3 H / H e N mice, were treated with either 10 mg/kg Photofrin and 150 J/cm , 0.3 mg/kg m T H P C and 110 J/cm 2 , or 0.6 mg/kg m T H P C and 110 J/cm 2 . A differential white blood cell count was determined from Wright stained blood smears, using the protocol o f consecutive sampling. Data is presented as mean ± se, * p<0.05, ** p<0.002, *** p<0.0001, compared to the control. 37 III. PDT induces a marked increase in circulating neutrophil levels: Three groups o f S C C V I I tumor-bearing mice were used for this experiment, results o f which are shown in Figure 3. The first group o f mice was treated with Photofrin-based P D T , the second was administered 5 mg/kg GR1 antibody, and the third group received the antibody prior to light treatment of Photofrin-based P D T . Blood samples were collected for each animal over a period of 10 hours. M i c e treated with only the GR1 antibody, were depleted of neutrophils in circulation over a time course of the observation period. P D T induced a marked increase in circulating neutrophil numbers with a peak 2.4 times that of relative normal levels, at 3 hours after the onset of light treatment. This increase persisted for a period of at least 10 hours. A marked increase in circulating neutrophil levels was also observed in mice treated with the antibody prior to light treatment. In this group relative neutrophil levels rose to a peak of 1.5 times that of normal levels at 2.5 hours after the onset o f light treatment, however, this increase did not last more than 1 hour at which time levels fell down below normal. 38 — • — P D T — # — a n t i - G R 1 + P D T - A- a n t i - G R 1 Figure 3: P D T induces an increase in circulating neutrophil levels. A change in circulating neutrophil levels was compared in three groups of 8 s.c. S C C V I I back tumor-bearing C 3 H / H e N mice. Tumors in the first group were treated with P D T only, the second group received a combined treatment of 5 mg/kg GR1 antibody 1 hour prior to P D T light treatment, and the third group was administered the GR1 antibody alone. P D T : Photofrin 10 mg/kg and 150 J/cm2. The light treatment lasted an average of 20 minutes. Neutrophil levels were determined as described in Figure 2. Data are presented as mean ± se, ** p<0.005 compared to controls. 39 IV. PDT induces a rise in circulating neutrophil levels in EMT6-tumor bearing mice, but is suppressed in a combined treatment with B C G vaccine Figure 4 illustrates the effect of Photofrin-based P D T on neutrophil levels in the blood of Balb/c mice when either E M T 6 back tumors, or normal skin on tumor-free mice were treated. In addition, the effect of a peritumoral administration o f 1 x 10 7 C F U B C G vaccine alone, which produces a different type of inflammatory insult, or a combined treatment of B C G and P D T on E M T 6 back tumors is demonstrated. A s shown earlier, P D T induced a peak rise 2.5 times above normal at 2.5 hours after light treatment. P D T of tumor-free, normal skin also caused a rise in circulating neutrophil levels with a peak o f 2.2 times that of normal at 1 hour after treatment. B C G did not stimulate a rise in the level of neutrophils in blood, but induced a significant fall in blood neutrophil levels at 10 and at 24 hours. The combined treatment o f B C G and P D T produced a modest increase in neutrophil levels in circulation. This peak was reached at 1 hour after treatment, persisted for at least 10 hours, and fell back down to normal levels 24 hours later. The peak rise stimulated by the combined treatment was well below the increase in circulating neutrophil levels following either P D T on the E M T 6 tumor or that of normal skin. 40 3.4 -3.2 -3.0 -2.8 2.6 2.4 -2.2 -2.0 -1.8 -1 .6 -1 .4 -1 .2 1 .0 0.8 - | 0.6 0.4 -4 0.2 — • — P D T — • — P D T , tumor-free — A — P D T and B C G — • — B C G 0 T 2 T 6 T 8 - r -1 0 24 T i m e after the onse t of light t rea tment (hours) Figure 4: PDT induces an increase in circulating neutrophil levels in both E M T 6 tumor and tumor-free Balb/c mice, whereas, B C G vaccine has a suppressive effect. Multiple blood samples were collected from each animal after treatment. Groups consisted of 4-6 mice each. The first group o f s.c. back E M T 6 tumor-bearing female mice were treated with P D T , the second was treated with P D T on their tumor-free depilated backs, a third group received a combined treatment of 10 7 C F U B C G vaccine and P D T , and the fourth group was administered B C G peritumoraly. P D T : 10 mg/kg Photofrin and 75 J/cm 2 . Data is based on Wright stain analysis o f blood smears as explained in Figure 2,and is presented as mean + se. * p<0.02, ** p< 0.0005 ompared to controls. 41 S E C T I O N 2 I. Intravenous drug adminstration and tail bleeding capable of inducing an increase in circulating neutrophil levels: Figure 5 shows the change in blood neutrophil levels following an i.v. injection o f 10 mg/kg of Photofrin. A n increase of 2 times that of the normal level was observed 1 hour after treatment. Levels fell back down to normal 24 hours later. Consecutive sampling from the tail vein of otherwise non-treated animals also increased circulating neutrophil levels to a peak o f 2.1 that of normal at 1 hour after the initial nick (See Figure 6). These levels remained slightly elevated 24 hours later. 42 T ime (hours) Figure 5: Increase in circulating neutrophil levels after i.v. administration of 10 mg/kg Photofrin. Blood was collected before, 1, 3, 6, and 24 hours after injection from 4 tumor-free Balb/c mice. Data was collected as explained in Figure 2, and presented as mean ± se. * p<0.05, ** p< 0.005 compared to controls. 43 Figure 6: Relative changes in circulating neutrophil levels over a 24 hour period of multiple blood sampling per animal. Blood was sampled from tail veins of 3 tumor-free Balb/c mice. Data is based on Wright stain analysis, as described in Figure 2 and presented as mean ± se. * p<0.05, ** p<0.005 compared to controls. 44 II. Changes in relative circulating neutrophil levels in non-PDT-treated mice: a.) i.v. injection of D±W, 20 minute restraint, or light alone of Balb/c tumor-free footpad: Shown in Figure 7, 5% dextrose in H2O administered i.v. into a group o f mice can also induce an increase in circulating neutrophil levels. The peak was 1.4 times above normal CD > CD CL O i _ "5 CD C T3 O O X3 CD as CD DC 1 .8 1 .7 1 .6 1 .5 1 .4 1 .3 1 .2 1 .1 1 .0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 • — restraint — • — D ,W — • — l i g h t alone on footpad (tumor-free) 2 4 Tim e (hours) 6 24 Figure 7: Changes in blood neutrophil levels after: i) i.v. injection of 0.2 ml/20 g mouse 5% dextrose in H 20 (DSW), ii) 20 minute restraint, or iii) 60 J/cm 2, 630 nm of light on tumor-free footpad. Differential white blood cell counts were made as described in Figure 2, using the individual sampling protocol. 8 mice were used per time point. Data is presented as mean + se. *p<0.05 compared to control. 45 levels at 1 hour after injection, and dropped down to normal levels by 6 hours after the injection. Twenty-minutes of restraint, as would be experienced during P D T light treatment, also induced a peak increase in blood neutrophil levels 1.6 times that o f normal at 1 hour after the mice were released from the restraint. Neutrophil levels dropped down to normal by 6 hours after treatment. Also shown in Figure 7 is the observed effect of 630nm light delivered at a dose o f 60 J/cm 2 , in the absence of photosensitizer. When light was delivered to the footpad of Balb/c mice results suggest a suppressive effect of light on circulating neutrophil levels, which persist below normal 24 hours later. 46 S E C T I O N 3 I. Increase in circulating neutrophil levels is PDT-specific: Figure 8 demonstrates the change in relative neutrophil levels in circulation following Photofrin-based P D T . P D T on the footpad of tumor-free mice induces a peak increase at 1 hour after light treatment 2 times that of normal levels, an increase statistically higher than any other factor attributed to stress in handling during treatment (discussed in S E C T I O N 2), capable of inducing a rise in neutrophil levels. These levels drop to normal 24 hours after treatment. P D T on the E M T 6 foot tumor also increased the neutrophil levels in blood to a peak of 2 times that of normal, however, this increase occurred at 10 hours after treatment and remained statistically elevated 24 hours after treatment. Figure 9 indicates that P D T had the strongest effect on the s.c. E M T 6 back tumor, with a peak of 2.3 times that of normal levels persisting for at least 10 hours. Levels drop at 24 hours but remain statistically above normal. Light delivered to s.c. E M T 6 tumors grown on the sacral region on the back o f mice, induced an increase in blood neutrophil levels with several peaks over a 24 hour time course. A t 1 hour after light exposure, levels increased 1.8 times that of normal, dropped to 1.3 at 3 hours, rise at 6 hours to 1.5 times that of normal levels and fall down to normal at 10 hours only to rise again 24 hours later. In Figure 9 a third curve represents the normalized P D T effect, corrected for the oscillations in neutrophil levels following light treatment alone. 47 Figure 8: Changes in circulating neutrophil levels following PDT on the foot. P D T (10 mg/kg Photofrin and 60 J/cm 2) was applied to: i.) footpad, and ii.) E M T 6 foot tumor, of Balb/c mice. Blood was sampled from 6 mice at various times after light treatment. Data was collected as described in Figure 7 using individual sampling, and presented as mean ± se. * p< 0.05, ** p< 0.005 compared to non-treated controls. 48 3.0 n EMT6 s.c. back tumor — • — PDT minus light only --O--PDT --A-- light only 2.8-2.6-2.4-2 .2-2.0-1.8-1.6-1.4-1.2-1.0-0.8-0.6-• ~r o 2 4 6 8 - W / - T -10 24 Time after onset of light treatment (hours) Figure 9: Normalized changes in circulating neutrophil levels following PDT on E M T 6 back tumor. Shown in this figure are the data for P D T (10 mg/kg Photofrin and 60 J/cm 2), light alone, and the corrected P D T effect minus light alone on the s.c. E M T 6 back tumor o f Balb/c mice. Blood was sampled from 6 mice at various times after light treatment. Data was collected as described in Figure 7 using individual sampling, and presented as mean ± se. * p< 0.05, ** p<0.005 compared to non-treated controls. 49 SECTION 4 I. Analysis of total circulating leukocyte numbers following PDT treatment of tumors and normal skin: Changes in total blood leukocyte counts and total neutrophil counts in PDT-treated mice are presented in Tables 1 and 2. Total numbers of white blood cells increased dramatically from 11 ± 2.0 x 106 per ml of blood to 24 ± 2.8 x 106 and 18 ± 1.9 x 106 at 3 and 6 hours post treatment, respectively, for mice treated with PDT on their tumor-free footpad (Table 1). A marked increase also occurred in mice whose back EMT6 tumors were treated with PDT, with the greatest difference observed at 3 hours post light treatment when the total leukocyte numbers per ml of blood rose to 29.9 ± 2.6 x 106. Neutrophil numbers (Table 2) also increased significantly at these corresponding time points, with a peak of 2.3 times and 6 times that of numbers normally found in circulation for the treatment on footpad and back EMT6 tumors, respectively, 3 hours after treatment. Although, there does not seem to be a significant change in total leukocyte numbers following PDT on EMT6 foot tumors, an increase in neutrophil numbers was observed at 10, and 16 hours, followed by a peak increase of 1.9 times that of normal numbers, at 24 hours after treatment. Although oscillations did occur in total numbers of lymphocytes (Table 3), changes observed were not consistent. No obvious changes in other leukocytes, such as monocytes, were detected in the blood of treated mice. 50 Table 1: Total circulating leukocyte numbers per ml of blood following Photofrin based-PDT of: i) tumor-free footpad, ii) s.c. EMT6 back tumor, and iii) EMT6 foot tumor, in Balb/c mice. x 106 leukocytes/ml of blood Time after PDT light Footpad back EMT6 tumor Foot EMT6 tumor (hours) non-treated 11 ±2 .0 13.8 ± 1.1 13 ± 1.37 0.5 15 ± 1.3 18.8 ±3 .3 -/-1 16 + 1.3 * 12 ± 1.4 -/-3 24 ±2.8 ** 29.9 ± 2 . 6 * 11 ±2.47 6 18 ± 1.9 * 17.4 ±2 .3 11 ± 1.64 10 10 ±2.25 25 ± 7.16 * 9.3 ± 1.52 16 16 + 2.1 * -/- 13.3 ±0.88 24 14 ± 1.91 14.2 ± 1.0 15.5 ±2 .0 Blood samples for each time point following light treatment were taken from 6-8 mice, by individual sampling protocol. PDT dose was as described in Figure 9. Mean values based on hemacytometer counts are presented ± se. * p< 0.05, ** p<0.005 compared to non-treated controls. 51 Table 2: Total circulating neutrophil numbers following Photofrin-based PDT of: i) Balb/c footpad, ii) s.c. EMT6 back tumor, and iii) EMT6 foot tumor. Time after PDT light (hours) Footpad x 106 neutrophils/ml of blood back EMT6 tumor Foot EMT6 tumor non-treated 2.9 ± 1.1 2.95 ±0.38 3.4 ±0.58 0.5 4.5 ±0.87 8.1 ±2.3 -/-1 5.2 ±0.64 * 7.0± 1.04 -/-3 6.8 ±1.9 ** 18.3 ±5 * 4.2 ± 1.5 6 4.2 ±0.64 * 9.8 ± 1.4 * 5.2 ±0.51 * 10 2.9 ±0.98 10±2.9 * 4.9 ±0.63 * 16 3.3 ±0.47 -/- 5.4 ±0.87 * 24 2.6 ± 0.46 5.6 ±0.58 6.5 ±0.46 * Blood samples for each measurement following light treatment were collected from 6-8 mice, using the individual sampling protocol. Data is based on hemacytometer counts and Wright stain analysis of blood smears. PDT dose was as described in Figure 9. Data is presented as mean ± se. * p<0.05, ** p<0.005 compared to non-treated controls. 52 Table 3: Total circulating lymphocyte numbers per ml of blood following Photofrin-based PDT of: I) tumor-free footpad, ii) s.c. EMT6 back tumor, iii) EMT6 foot tumor, in Balb/c mice. Time after P D T light Footpad (hours) x 106 Ivmphocvtes/ml of blood back E M T 6 tumor Foot E M T 6 tumor non-treated 9.2 ± 1.9 11 ± 0.73 9 ± 0.96 0.5 10.4± 0.72 11± 1.2 -/-1 10.1± 0.57 5.1± 0.9 * -/-3 17 ± 2 . 1 * 12 ±1.01 7.0+1.0 6 13± 1.5* 7.5 ± 1.1 * 5.6± 0.94 * 10 7.2± 1.6 8.9± 2.6 4.3 ± 0 . 9 * 16 13 ± 2 . 4 -/- 7.9 ± 0.1 24 11± 1.5 8.5± 0.96 8.9± 1.5 Blood samples for each time point following light treatment were taken from 6-8 mice, by individual sampling protocol. P D T dose was as described in Figure 9. Mean values based on hemacytometer counts and Wright stain analysis, are presented ± se. * p< 0.05, ** p< 0.005 compared to non-treated controls. 53 II. Control treatments also induced a change in total circulating leukocyte and neutrophil numbers Table 4 shows a stress-related increase in total white blood cells in Balb/c mice. The increase was from normal levels o f 13.8 ± 1.1 x 10 6/ml to a peak level o f 21.5 ± 3 x 10 6/ml and 16.6 ± 2.2<x 10 6/ml at 3 hours after the i.v. administration of D 5 W , or by restraint alone, respectively. The total number of neutrophils can also be elevated in both cases as summarized in Table 4. The peak in mice that were administered D5W i.v. occurred 1 hour after injection, and rose to 8.54 ± 1.3 x 10 6 per ml of blood from 2.3 ± 0.38 x 10 6. The peak for restrained mice, 5.4 ± 0 . 8 1 x 10 6/ml, occurred 3 hours after release. Total numbers of lymphocyte and other leukocytes, such as monocytes, and band neutrophils did not change significantly. Table 4: Total leukocyte and neutrophil numbers in mice following either i.v. injection of D5W or 20 minute restraint. x 106 leukocytes/ml of blood x 106 neutrophils/ml of blood Time after Treatment (hours) D 5 W Restraint D 5 W Restraint non-treated 13.8 ± 1.1 13.8 ± 1.1 2.3 ± 0 . 3 8 2.3 ± 0 . 3 8 1 16.3 ± 2 . 1 9.5 ± 3 . 9 8.4 ± 1 . 3 * 3.9 ± 0 . 9 8 3 21.5 ± 3 * 16.6 ± 2 . 2 * 6.5 ± 1 . 6 * 5.4 ± 0 . 8 1 * 6 16.2 ± 1 13.4 ± 1.9 3.7 ± 0 . 7 1 4.0 ± 0 . 5 8 24 23.2 ± 4 . 6 12.5 ± 1.2 3.6 ± 0 . 7 5 3.1 ± 0 . 5 4 Blood samples for each timepoint following light treatment were taken from 6-8 mice, by individual sampling. Mean values based on hemacytometer counts and Wright stain analysis of blood smears are presented ± se. * p<0.05 compared to non-treatedcontrols 54 Table 5: Total circulating leukocyte and neutrophil numbers in mice following a light dose of 60 J/cm 2, in the absence of photosensitizer. x 106 leukocytes/ml of blood xlO6 neutrophils/ml of blood Time after Light Footpad back E M T 6 tumor Footpad back E M T 6 tumor (hours) treated 10.7 ± 1.0 13.8 ± 1.1 2.9 ± 0 . 5 6 2.9 ± 0 . 3 8 1 ' 15.9 ± 0 . 7 6 9.8 ± 2 . 0 4.4 ± 0.24 4.8 ± 0 . 8 3 3 22.4 ± 4 . 7 * 29.1 ± 5 . 8 * 5.8 ± 1.8 * 9.5 ± 2 . 0 * 6 17.2 ± 1.8 * 13.4 ± 3.1 2.8 ± 0 . 8 8 5.9 ± 1 . 8 * 10 -/- 16.9 ± 2 . 2 -/- 4.0 ± 0.68 24 16.6 ± 2 . 7 14.3 ± 2 . 2 2.2 ± 0 . 2 1 5.5 + 1.1 * Blood samples for each timepoint were collected from Balb/c mice whose footpad or back E M T 6 tumor was illuminated with 60 J/cm 2 o f 630 nm wavelength of red light. Blood samples for each time point following light treatment were taken from 6-8 mice. Mean values are presented ± se, based on hemacytometer counts and Wright stain analysis. * p<0.05 compared to non-treated controls. Table 5 summarizes the increase in total leukocyte and neutrophil numbers in circulation following light treatment in the absence of photosensitizer, on the footpad o f mice or on the E M T 6 tumor grown on the sacral region of their backs. 60 J/cm of light delivered on the footpad of mice induced a peak increase in total white blood cell numbers of 22.4 ± 4.7 x 10 6 at 3 hours after treatment, 2.1 times greater than normal levels o f 10.7 ± 1.0 x 10 6 per ml o f blood. The same light dose induced a very similar increase in total circulating leukocyte numbers when illuminating back E M T 6 tumors. 55 The neutrophil levels increased two-fold at 3 hours, in mice whose tumor-free footpad was illuminated with 60 J/cm of 630nm light. The levels of these cells increased 3.1 times in mice whose back s.c. E M T 6 tumors were treated with light alone. 56 S E C T I O N 5: Systemic response of GRl-positive cells to PDT Four groups o f 3-6 E M T 6 tumor-bearing Balb/c mice were treated with Photofrin-based P D T , and a fifth group was not treated. Flow cytometry analysis was the method used to decipher the GRl-posi t ive cell content of tumors, lungs, blood and bone marrow in these mice, at 1.5, 6, 10, 24 hours following P D T light treatment (Figure 10). The observed changes are commented in detail in the presentation of figures 11-14. 57 6.0 -5.5 -5.0 — "CD -o 4.5 — o > "•+—• 4.0 -vi -o Q. | 3.5 -rr 3.0 -o -<+— 2.5 — o _ 2.0 — > CD 1 .5 — CD > 1 .0 — CO Re 0.5 -0.0 --0.5 * * I L u n g Turn or B l o o d B o n e mar row -2 0 ~r 2 "T 4 T 6 T 8 /h-T-10 12 14 24 T i m e a f te r P D T l ight t r e a t m e n t ( h o u r s ) Figure 10: Systemic response of GRl -pos i t i ve cells to P D T . Four groups of 3-6 E M T 6 foot tumor-bearing Balb/c mice were treated with P D T and a fifth group was untreated. Groups of mice had their lungs, tumor, blood, and bone marrow cells collected at different time points following P D T (10 mg/kg Photofrin and 60 J/cm 2). Data is based on flow cytometry analysis, and is presented as mean ± se. ** p<0.005, * p<0.05 compared to non-treated controls. 58 L Tumors: The cell composition of E M T 6 rumors prior to P D T treatment generally consisted o f 70% tumor associated macrophages ( T A M ) , 10% neutrophils and the remaining 20% were malignant Figure 11: Changes in cell content of GRl-positive cell population in E M T 6 tumors following PDT. E M T 6 foot tumors from 3-6 Balb/c mice were excised and analyzed by flow cytometry for GR1 expression 1.5, 6, 10, and 24 hours after P D T light treatment. P D T : 10 mg/kg Photofrin and 60 J/cm2. Data is presented as mean ± se. ** p<0.005, and p<0.05 compared to non-treated controls. 59 cells. Following P D T treatment a change in the overall cell content occurs with a decrease in malignant and T A M cells which die during treatment, and an increase in infiltrating neutrophil and macrophage populations at 10 and 24 hours post treatment. These changes were also observed by Wright stain analysis of prepared tumor cell suspensions. Mye lo id cells express various levels of G R 1 , where neutrophils express the highest levels, monocytes and less mature neutrophils express medium levels, and macrophages express the lowest. A s shown in Figure 11, P D T induced a continuous influx of GRl-posi t ive cells in E M T 6 tumors over a 24-hour time interval. The majority of these cells expressed high levels of G R 1 , and forward and side scatter signals indicate that these are most likely neutrophils. A significant increase 1.97 ± 0.58 times over normal levels was detected 1.5 hours post P D T . A peak rise was at 10 hours following treatment, 3.12 + 0.41 times over normal levels. A t 24 hours, levels were still elevated at 2.7 ± 0.38 times above normal. II. Lungs : Figure 12 shows that the accumulation o f neutrophils, designated as GRl-pos i t ive cells, in lungs peaked at 6 hours after P D T treatment of E M T 6 tumors, 4.79 ± 0.83 times that of normal levels in pulmonary vessels. The levels of these cells decreased to normal at 10 hours and then rose again at 24 hours to 1.69 ± 0.18 times above normal. The cell content o f resident macrophage and other cell populations, do not change in the 24 hour interval following P D T treatment. 60 T i m e af ter P D T l ight t r e a t m e n t (hou rs ) Figure 12: Change in the levels of GRl-positive cells in lungs following PDT. Lungs o f 3-6 E M T 6 foot tumor-bearing mice were excised and analyzed by flow cytometry for GR1 expression at 1.5, 6, 10, and 24 hours following P D T treatment, and compared to lungs excised from non-treated mice. P D T : 10 mg/kg Photofrin and 60 J/cm 2 . Data are presented as mean ± se. ** p< 0.005, * p< 0.05 compared to non-treated controls. 61 III. Blood: Figure 13 illustrates the change in levels of neutrophils in blood of treated mice. A rise to 2.35 ± 0.24 times that of normal circulating levels of GRl-pos i t ive cells was observed 10 hours after treatment. Levels returned to normal at 24 hours after P D T . T i m e after P D T light t rea tment (hours) Figure 13: Changes in the GRl-positive cell content of blood following treatment with PDT. Blood collected from 3-6 E M T 6 foot tumor-bearing mice 1.5, 6, 10, and 24 hours following P D T light treatment. P D T : 10 mg/kg Photofrin and 60 J/cm 2 . Data is based on flow cytometry analysis and presented as mean ± se. ** p<0.005 compared to non-treated controls. 62 IV. Bone marrow: The change in GRl-posi t ive cell content in bone marrow is shown in Figure 14. There was a marked drop of 20 % from normal levels in the cell population expressing mid-high levels o f G R 1 , at 6 hours and a 55% drop at 10 hours after treatment. Levels return to normal at 24 hours after P D T . Time after P D T light treatment (hours) Figure 14: Changes in GRl-positive cell content of bone marrow following PDT. Bone marrow cells were harvested from 3-6 E M T 6 foot tumor-bearing mice and analyzed by flow cytometry for GR1 expression, 1.5, 6, 10, and 24 hours following P D T light treatment. P D T : 10 mg/kg Photofrin and 60 J/cm 2 . Data is presented as mean ± se. * p<0.05 compared to non-treated controls. 63 Section 6: L-selectin expression on neutrophils following PDT In an attempt to uncover their activation state, flow cytometry analysis was used to detect changes in L-selectin (CD62L) expression in granulocyte populations of the bone marrow and lungs o f E M T 6 foot tumor-bearing mice following P D T (Photofrin 10 mg/kg and 60 J/cm ), over a 24-hour interval. Lungs Bone marrow F i g u r e 15: Change s in the content o f granulocytes express ing h i gh levels o f L -selectin i n bone m a r r o w a n d lungs. Bone marrow cells and lungs were excised from 3-4 E M T 6 foot tumor-bearing Balb/c mice, 1.5, 6, 10, and 24 hours following P D T . Flow cytometry analysis was used to detect changes in the percent o f granulocytes expressing high levels of L-selectin. P D T : Photofrin 10 mg/kg and 60 J/cm . Data is presented as mean ± se. * p< 0.005 compared to non-treated controls. 64 Cells expressing high levels o f L-selectin increased from 2.5% to 36% as shown in Figure 15, with a subsequent decrease from ~97% to 64% of cells expressing low levels of L-selectin, in the total granulocyte population of bone marrow 24 hours following P D T . N o significant change was detected in the lungs. We also examined a change in L-selectin expression in GRl-pos i t ive cells in tumors. Figure 16 illustrates a change from 8% of all GRl-pos i t ive cells expressing high levels of L-selectin to 53%, with a corresponding decrease from 91% to 46% of all GRl-pos i t ive cells expressing low levels of L-selectin over a 24-hour time interval following P D T treatment. Figure 16: Changes in L-selectin expression of GRl-positive cells in tumors following PDT. E M T 6 foot tumors were excised 1.5, 6 and 24 hours from host Balb/c mice, following treatment with P D T . Using flow cytometry techniques, a change in the GRl-posi t ive cell content expressing high levels of L-selectin was detected. P D T : Photofrin 10 mg/kg and 60 J/cm 2 . Data is presented as mean ± se. * p< 0.005 compared to non-treated controls. 65 Changes in the percent o f circulating neutrophils expressing low, medium, and high levels o f L -selectin in blood are shown in Figure 17. In a non-treated control, the majority o f circulating neutrophils in E M T 6 tumor-bearing mice expressed medium levels o f L-selectin, while high levels were expressed by a very small content of the neutrophil population. Ten hours following Photofrin-based P D T , there was a decrease from 20% to 5% o f all neutrophils expressing low levels of L-selectin, and a corresponding increase from 77% to 92% expressing medium levels. A t 24 hours however, 1/3 of all neutrophils expressed high levels of L-selectin, an increase from 4% (1/25) in control levels, which caused a significant decrease in the cell content expressing medium concentrations of L-selectin compared to normal levels. Dot-plots representing changes in L-selectin expression in blood, tumor, and bone marrow, are shown in figures 18 and 19. 66 C O 100-1 90-80-70-60-Q. o 50-1 •*-> ~i CD c 40-| 30 20-10-0 li^ fel L-selectinlow — L-selectinmedium I I L-selectinhigh T m JL non-treated tin fa 1.5 10 24 Time after PDT light treatment (hours) Figure 17: Changes in L-selectin expression of neutrophils in circulation following PDT. E M T 6 foot tumors on Balb/c mice were treated with P D T (Photofrin 10 mg/kg and 60 J/cm 2) and their circulating neutrophils analysed by flow cytometry for a change in L-selectin expression: low, medium, or high. Data is presented as mean ± se. * p< 0.005 compared to non-treated controls. 67 Blood Control 10 hr post PDT 24 hr post PDT o I DO O U H 1000 H c I_a_ n I I r i r Forward scatter Figure 18: Flow cytometry analysis dot-plots of PDT-induced changes in L-selectin expression on GR1 cells in peripheral blood. Representative examples o f flow cytometry-generated dot-plots, with the ordinate showing fluorescence o f F ITC conjugated to anti-mouse C D 6 2 L (L-selectin) in arbitrary units per cell, and light forward scatter on the abscissa. (PDT: Photofrin 10 mg/kg, 60 J/cm 2). 68 Control 24 hr post PDT "5 t/3 00 JO U H 1000 i i 1 1 r i 1 1 1 1 r n i 1 1 r Tumor .v.. . = * ' . 1 B 1 ..... ..... marrow F o r w a r d scatter Figure 19: Flow cytometry analysis dot-plots of PDT-induced changes in L-selectin expression on GR1 cells in tumor and bone marrow. Representative examples o f flow-cytometry-generated dot-plots, with the ordinate showing fluorescence o f F I T C conjugated to anti-mouse C D 6 2 L (L-selectin) in arbitrary units per cell, and light forward scatter on the abscissa. (PDT: Photofrin 10 mg/kg, 60 J/cm 2). 69 DISCUSSION A s the first wave in the host response to PDT-elicited inflammation, neutrophils are attracted to a site o f P D T damage (76,77,114). Inflammatory chemotactic signals released into immediate surroundings and circulation from treated lesions, attract neutrophils which can contribute to the management of tumor cell death in a number of ways. A s large number o f neutrophils accumulate in PDT-treated tumors, their adherence to the endothelium may promote the formation o f a neutrophil aggregate. In this way, neutrophils may enhance blood flow stasis associated with P D T , contributing to tumor hypoxia, and consequent ischemic tumor cell death. Activated neutrophils promote platelet aggregation through the release o f thromboxane, again amplifying reduction in blood flow, which leads to tumor regression (13,78). In addition, activated neutrophils release proteolytic substances and components produced via the N A D P H oxidase membrane system, which in a synergistic manner inflict localized tumor damage. Subsequently, both tumor vasculature and parenchyma become susceptible to cytotoxic capabilities of neutrophils. In particular, endothelial cells are targeted resulting in the breakdown of basement membrane, creating leaky vasculature, and increased vascular permeability with consequent edema formation and hemorrhage (75). I f neutrophils are capable of migrating from the vessel lumen, they may come into direct contact and inflict death to tumor cells. Whether or not neutrophils are able to extravasate into PDT-treated tissue may depend on a number of factors. Adherence to endothelium is crucial and dependent on the expression of adhesion molecules (81,82). L-selectin on polymorphonuclear leukocytes and P/E-selectin on endothelial cells, are crucial for initial contact, and subsequent neutrophil diapedesis between endothelial cells is mediated by members of the integrin family of adhesion molecules (86). 70 The level of endogenous nitric oxide (NO) production in tumors, which can vary significantly in human tumors and among different experimental tumor models (13), may play an important role in regulating adhesion, since N O is a strong inhibitor o f adhesion molecule expression (126,127). This suggests an indirect effect of N O in the control o f neutrophil extravasation, implying a possible hindrance in the rate of tumor cures by P D T . These issues are currently under active investigation in our laboratory. Chemotactic signals, presumably produced and released by activated neutrophils following P D T , initiate and attract an influx of inflammatory cells to the lesion. Neutrophils are followed by macrophages/monocytes, and mast cells, which arrive in waves following P D T treatment (77). A l l of the above mentioned functions of activated neutrophils support the hypothesis that neutrophils play an indispensable role for effective P D T . The rapid, marked sequestration of neutrophils into P D T treated tissue has been well-documented (77,78,114). However, it was not determined whether such a protracted mobilization of neutrophils is associated with a systemic response of these cells to P D T . Does accumulation o f neutrophils into tumors following P D T result in a change in the level of these cells in circulation, or their storage and/or marginated pools in organs such as bone marrow and lungs? This issue has received little attention thus far, aside from a group in the Netherlands. De Vree and co-workers observed a rapid rise in circulating neutrophil levels in the blood o f rhabdomyosarcoma tumor-bearing rats following Photofrin-based P D T (119). This finding and related revelations in earlier work of our laboratory, prompted the first phase o f this thesis project to investigate a change in the circulating level o f neutrophils following Photofrin-based P D T in murine tumor models. The results presented in this thesis show that P D T did induce a marked rise in blood neutrophil levels. This was demonstrated with two tumor models ( S C C V I I squamous cell 71 carcinoma, and E M T 6 mammary sarcoma) growing in two different strains o f mice ( C 3 H / H e N and Balb/c, respectively). A similar, although perhaps somewhat less pronounced effect was observed with P D T treatment of normal skin. PDT-induced changes in circulating leukocytes were almost exclusively limited to alterations in neutrophil levels. Changes in the lymphocyte content of blood were noticed on several isolated occasions, but this was not consistent and may be attributed to experimental fluctuations. There were no indications o f changes in the levels of circulating monocytes or other types of white blood cells, aside from the occasional appearance of immature neutrophils (band cells). The presence of band cells hints to enhanced neutrophil production and differentiation from cell precursors in the bone marrow as a response to P D T -induced inflammation. The leukocyte content in mouse blood is characterized by a marked predominance of lymphoid populations, comprising over 70% of all nucleated cells in circulation. Neutrophils normally make up 25-30% of the total, whereas monocytes and other white blood cells usually do not exceed 1% (78). A marked rise in the level of circulating neutrophils to about 60% of all leukocytes, was observed in PDT-treated S C C V I I tumor bearing mice lasting for at least 10 hours following light treatment. The magnitude of the PDT-induced neutrophilia, is further illustrated by neutrophil total counts which rose over six times above normal levels in mice at peak time intervals, i.e. from below 3 x 10 6/ml (normal levels in mice) to over 18 x 10 6/ml, respectively. This caused the lymphocyte levels to drop below 40%. The total count of circulating leukocytes in these situations increased more than two-fold which can all be attributed to changes in neutrophil levels. These changes are demonstrating that P D T induces a pronounced leukocytosis caused by neutrophils, i.e. neutrophilia. A n increased level of neutrophils in circulation commonly accompanies a strong acute inflammation caused by bacterial infections, tissue injury or acute traumatic disorders 72 (128,129). Neutrophilia is in some cases caused by neoplasia, a phenomenon not observed with our mouse tumor models. Rather, infarction and hemorrhage that occurs in P D T treated tumor tissue are l ikely responsible for the induction of neutrophilia, generally caused by the release of interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-a) from macrophages and other cells, following bacterial infections or tissue injury (129). These cytokines, known to be induced by P D T (119,130), directly promote the accelerated release of neutrophils from bone marrow, with subsequent generation o f colony-stimulating factors by macrophages and T-lymphocytes, inducing the proliferation of bone marrow hemopoietic precursor cells (129). In addition, a pool of neutrophils temporarily marginated along vascular walls can rapidly be mobilized by specific molecular signals (best known are epinephrine, or corticosteroids) (129), some o f which may also be released from PDT-treated sites. Interestingly, the GR1 antibody-mediated depletion o f the circulating neutrophil pool prior to P D T light treatment, reduces the delay for tumor recurrence following Photofrin-based P D T o f the S C C V I I murine tumor (Figure 1). This suggests the relevance of a systemic response o f neutrophils for successful P D T . Surprisingly, it was also observed that the P D T treatment following anti-GRl-mediated neutrophil depletion, induced a marked rise in circulating neutrophil levels, although this did not persist for more than one hour (Figure 3). Therefore, it seems that tumor control by P D T requires a prolonged mobilization o f neutrophils, reflected by a rise in neutrophil levels in blood following treatment. A pool of neutrophils unscathed by the antibody presumably supplied a surge of neutrophils into circulation observed in Figure 3. In addition to the FDA-approved photosensitizer Photofrin, there are several new photosensitizers showing promising results in clinical testing (12). A t least some o f them can be expected to share with Photofrin the ability to induce systemic neutrophilia when 73 photodynamically activated by light energy, as evidenced by our results with the photosensitizer m T H P C . Interestingly, very similar changes in blood neutrophil counts were incited by two different doses of mTHPC-based P D T and Photofrin-based P D T (Figure 2). Another form of inflammatory insult initiated by the peritumoral application o f the live B C G vaccine (used to induce inflammation in the clinical treatment of bladder cancer (131)), however, was not found to provoke a rise in neutrophil levels in circulation (Figure 4), although it does induce neutrophil accumulation in E M T 6 tumors (Korbelik, unpublished). Instead, B C G alone seems to suppress blood neutrophil levels and also abrogates the rise induced by P D T . Since the mice undergo little stress in handling during a peritumoral application o f B C G , the possibility that neutrophilia associated with P D T may originate in other stress-related factors experienced in handling of the animals during P D T treatment, required examination. Neutrophils are known to be the first inflammatory cell to respond and be on alert in any trauma. A neutrophilic state can, for instance, be induced in sheep by a loud noise (132), and as seen in our mouse models, when these animals were subjected to blood collection by tail bleeding. Indeed, the method of consecutive blood sampling by putting a small nick in the tail vein and, in this way, collecting multiple blood samples from each mouse over a period of 24 hours, stimulated an inflammatory response, characterized by neutrophilia (Figure 6). A neutrophilic state is also observed with a single bolus injection of Photofrin administered intravenously, followed by a 24-hour period of multiple, consecutive blood collections (Figure 5). These results prompted us to repeat experiments involving P D T , and follow through on a number of control studies to determine the possibility o f a masked neutrophilic state. Instead o f multiple blood collections per animal over a time course o f 24 hours after treatment, in subsequent experiments, we chose to collect a single sample of blood from a group o f mice at each time point before and following P D T light treatment. In this manner, a 74 neutrophilic response due to repeated tail bleeding was avoided. In the absence o f P D T , mice were also subjected to stress factors such as, intravenous administration o f 5% dextrose in H2O (the solution in which we reconstitute Photofrin prior to use), or the restraint in which mice are immobilized during light treatment. These stress factors involved in the methodology o f P D T in mice, can individually induce an increase in circulating neutrophil levels (Figure 7). Initial experiments collecting multiple blood specimens from individual mice after P D T , were undertaken using mice bearing E M T 6 and S C C V I I tumors on their depilated backs. This created the possibility for light exposure to proximal organs, such as the liver or spleen. This may be a concern since photosensitizer is taken-up by virtually all cells in the body but especially by phagocytic cells of the reticuloendothelial system (35,133). Two scenarios comparing the effect o f light, in the absence of photosensitizer, on (1) the s.c. E M T 6 back tumor and .(2) the tumor-free footpad of mice, produced very different outcomes. P D T light only treatment on the tumor-free foot, as the rest of the body was shielded from light during treatment, seemed to have a suppressive effect on circulating neutrophil levels, whereas oscillations in neutrophil levels above normal occur when light was directed on a s.c. E M T 6 back tumor (Figure 8). A complementary, or alternate explanation for these findings may originate from the fact that the blood flow and vascular network through the foot differs from that on the skin of the back. These factors are also l ikely responsible for the fact that the level of increase in circulating neutrophils following P D T seems to depend on the position of the tumor during treatment. These considerations prompted us to compare the effect o f Photofrin-based P D T on the tumor-free footpad, E M T 6 foot tumor, and s.c. E M T 6 back tumor. In all three cases, P D T had an independent effect of increasing circulating neutrophil levels significantly higher than any other stress-related factor experienced by the animal during treatment, although time 75 kinetics differ slightly in each case (Figure 9). The delayed peak in neutrophil levels 10 hours following P D T on the E M T 6 tumor grown on the foot, compared to the initial rise and peak in the two alternate groups at 1 hour, could be due to a combination o f reasons. A hallmark trait o f the P D T effect is edema formation (12,75). A t the time of light treatment, in the small area as is a mouse footpad a tumor already has limited capacity to swell. Therefore, the release o f signals from the P D T lesion into circulation may be hampered and delayed by severe vasoconstriction. P D T treatment o f the s.c. E M T 6 back tumor induced the most enhanced rise in circulating neutrophil levels. Reasons for this may be an amplified response to PDT-elicited inflammatory signals released from both the treated tumor site, and also organs in close proximity to the tumor affected during light treatment. From the data described here, it has been deduced that P D T results in a massive sequestration of GRl-posi t ive cells, determined to be neutrophils by Wright stain analysis and due to their high expression of the GR1 surface marker compared to other myeloid cells (125), into circulation. In response to inflammatory signals released by P D T , neutrophils are released into circulation presumably from their storage/marginated pools located at different sites in the body (134,135,136). The bone marrow for instance, has been shown to increase the rate of polymorphonuclear leukocyte (PMNs) production, shorten their maturation time, and release both mature and immature neutrophils into circulation in response to inflammation and stress (128,137). Such a response was shown to be induced by bacterial challenge in rabbit lungs (138,139). This phenomenon had up to date not been described for inflammation associated with P D T . To decipher possible sources of neutrophils released into circulation following P D T , the blood, bone marrow, tumor and lungs (representing tissue containing marginated pools of neutrophils) from PDT-treated E M T 6 foot tumor bearing mice were examined (Figure 10). 76 Flow cytometry analysis revealed a 50% drop in the neutrophil cell content o f bone marrow in response to PDT-elicited inflammation. This release o f neutrophils from bone marrow following P D T presumably occurs in response to inflammatory chemotactic signals released into circulation from the treated site. Neutrophils are released into circulation but seem to undergo a bottleneck effect in the pulmonary vasculature. Perhaps blood pressure is lower, or the vessels in the microvasculature of lungs are smaller in diameter than that encountered in other parts of the body, reducing the speed at which neutrophils can pass. The accumulation of neutrophils in tumors and blood at ten hours after treatment appears to occur due to a cumulative effect of neutrophils released into circulation as they manage to travel through the lungs, and also from bone marrow in a continuous wave of release. Although the level of neutrophils in both blood and bone marrow returns to normal 24 hours after treatment, levels in tumors remain elevated. Perhaps this can be attributed to a second wave of neutrophil release from the bone marrow between 10 and 24 hour intervals following treatment, suggested by a second rise in neutrophil levels in the lungs, observed 24 hours after treatment. Recent reports have suggested that P M N s in bone marrow have different characteristics compared to their older counterparts in circulation. Older neutrophils are less deformable, and have a decreased ability to respond to chemotactic stimuli. Younger neutrophils may have an enhanced ability to attach and extravasate in a site of inflammation, whereas mature neutrophils have a greater ability to degranulate and produce cytotoxic, oxygenated products (140). These traits are not particularly useful when trying to separate the two populations. However, granulocytes from the bone marrow express higher concentrations o f L-selectin than do older, mature cells (141). This adhesion molecule that mediates rolling o f leukocytes along vascular endothelium, may aid the migration of immature neutrophils into circulation (81). B y flow cytometry analysis, L-selectin expression can therefore be indicative of the maturation stage o f a 77 neutrophil and also of its activation state, since the expression o f L-selectin is downregulated once neutrophils reach a site of inflammation (86). In our E M T 6 tumor model, neutrophils normally had low levels o f L-selectin with a small percentage expressing high levels. Fol lowing P D T however, GRl-posi t ive cells infiltrating treated tumors express high and low levels o f L -selectin in a 1:1 ratio. In accordance, the concentration of L-selectin on circulating neutrophils, normally at a medium range, increases, as does that of the granulocyte population harvested from bone marrow (Figures 15,17). The majority o f granulocytes in the lungs expressed low concentrations o f L-selectin, with no significant changes following P D T (Figure 15). From these results one could postulate that PDT-elicited inflammation induced a systemic response of neutrophils in our murine tumor models. The massive sequestration of neutrophils into PDT-treated tumors was accompanied by a marked increase of these cells in circulation. This PDT-induced neutrophilia may be attributed to the release of both mature and immature granulocytes from bone marrow in response to inflammatory signals released from the treated lesion. Compiled, our findings seem to suggest that the presumed importance of neutrophils for effective P D T as an anti-tumor modality, relies on the synergistic actions o f older and less mature neutrophils. Neutrophils invading PDT-treated tumors inevitably die (mostly by apoptosis) at the site within a short time interval after their arrival (Korbelik, unpublished results). These dying cells appear to be replaced constantly by new waves of neutrophils sequestered from circulation, a process which seems to last throughout the first day following P D T treatment. It seems therefore, that huge numbers of neutrophils are required during this period, a process secured by their mobilization from storage pools and accelerated generation of young neutrophils from myeloid precursors. 78 FUTURE DIRECTIONS We have only begun to uncover the knowledge on the activity of neutrophils that contributes to the destruction o f PDT-treated solid cancers. The extent o f its relevance for clinical P D T practice remains to be elucidated by further research. Several avenues of investigation have opened-up based on the findings of this thesis. One of the main questions is whether or not the variable sensitivity o f tumors to P D T stems from the engagement o f neutrophils in treated lesions. In this respect it would be important to identify biological characteristics of tumors critical in determining neutrophil activity following P D T treatment. Factors such as tumor perfusion/vascularization, stromal structure including types of tumor associated host immune cells, profiles o f locally released cytokines, nitric oxide and other mediators, seem to be obvious targets in such an investigation. The evidence of superoxide generation, and of decreased then subsequent increased tumor blood flow, following P D T , strongly suggests the manifestation o f ischemia-reperfusion insult in this treatment. The dominant role o f neutrophils in the infliction of damage mediated by this form of insult, has to be taken into account and investigated. Can the curative effect of P D T be improved by modulating the sequestration and activity of neutrophils in treated tumors? Ongoing studies in our laboratory suggest that a positive answer to this question appears highly likely. For instance, agents modulating the engagement of integrins or selectins, or the levels of nitric oxide can enhance the curative effect o f P D T . Further research for supporting this approach should be very productive. Further studies to supplement the results collected in this project could be aimed at verifying that inflammatory signals specific for neutrophils are indeed being released into circulation from a PDT-treated site. Collecting the serum of treated tumor-bearing mice at 79 various time intervals following PDT, and subsequently introducing it intravenously to naive recipient mice, may confirm the presence of chemotactic signals in circulation, by the induction of neutrophilia in those recipient animals. If this does turn out to be the case, using molecular biology techniques, the next step would be to identify these inflammatory signals. Although IL-1 and TNF-oc are primary candidates in the specific mediation of neutrophilia, other well-established neutrophil attractants such as complement factor 5 a, and the cytokine interleukin-8 (IL-8), or even novel previously undefined molecules cannot be ruled out. The accumulation of activated neutrophils in PDT-treated tumors is well known, however, their localization is not well documented. It would be revealing to find out the extent of extravasation of these sequestered neutrophils from the vasculature in the PDT-treated lesion, and whether this pattern varies between tumors of different sensitivity to PDT. This may clarify which cell type(s) in a tumor are being affected by the cytotoxic capabilities of neutrophils. Such details may possibly be elucidated using fluorescence microscopy methods to study PDT-treated tumor sections. One issue to be taken into consideration when interpreting the results of this thesis is the relative size and location of treated murine tumors. Obviously, the tumor size (and consequently the PDT light-exposed area) related to the body size in the mouse is much larger than that encountered in the human situation. 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