UBC Faculty Research and Publications

Podocalyxin enhances breast tumor growth and metastasis and is a target for monoclonal antibody therapy Snyder, Kimberly A; Hughes, Michael R; Hedberg, Bradley; Brandon, Jill; Hernaez, Diana C; Bergqvist, Peter; Cruz, Frederic; Po, Kelvin; Graves, Marcia L; Turvey, Michelle E; Nielsen, Julie S; Wilkins, John A; McColl, Shaun R; Babcook, John S; Roskelley, Calvin D; McNagny, Kelly M Mar 27, 2015

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RESEARCH ARTICLE Open AccessPodocalyxin enhances breast tumor growth andmetastasis and is a target for monoclonalantibody therapyConclusions: We show that podocalyxin plays a key role in the formation of primary tumors and distant tumorSnyder et al. Breast Cancer Research  (2015) 17:46 DOI 10.1186/s13058-015-0562-7Sciences Mall, Vancouver, BC V6T 1Z3, CanadaFull list of author information is available at the end of the articlemetastasis. In addition, we validate podocalyxin as potential target for monoclonal antibody therapy to inhibitprimary tumor growth and systemic dissemination.* Correspondence: kelly@brc.ubc.ca†Equal contributors1The Biomedical Research Centre, University of British Columbia, 2222 Healththese antibodies, PODOC1, potently blocked tumor groKimberly A Snyder1†, Michael R Hughes1†, Bradley Hedberg2, Jill Brandon2, Diana Canals Hernaez1, Peter Bergqvist2,Frederic Cruz2, Kelvin Po2, Marcia L Graves3, Michelle E Turvey4, Julie S Nielsen1, John A Wilkins5, Shaun R McColl4,John S Babcook2, Calvin D Roskelley3 and Kelly M McNagny1*AbstractIntroduction: Podocalyxin (gene name PODXL) is a CD34-related sialomucin implicated in the regulation of celladhesion, migration and polarity. Upregulated expression of podocalyxin is linked to poor patient survival inepithelial cancers. However, it is not known if podocalyxin has a functional role in tumor progression.Methods: We silenced podocalyxin expression in the aggressive basal-like human (MDA-MB-231) and mouse (4T1)breast cancer cell lines and also overexpressed podocalyxin in the more benign human breast cancer cell line,MCF7. We evaluated how podocalyxin affects tumorsphere formation in vitro and compared the ability ofpodocalyxin-deficient and podocalyxin-replete cell lines to form tumors and metastasize using xenogenic orsyngeneic transplant models in mice. Finally, in an effort to develop therapeutic treatments for systemic cancers,we generated a series of antihuman podocalyxin antibodies and screened these for their abilityto inhibit tumor progression in xenografted mice.Results: Although deletion of podocalyxin does not alter gross cell morphology and growth under standard(adherent) culture conditions, expression of PODXL is required for efficient formation of tumorspheres in vitro.Correspondingly, silencing podocalyxin resulted in attenuated primary tumor growth and invasiveness in miceand severely impaired the formation of distant metastases. Likewise, in competitive tumor engraftment assayswhere we injected a 50:50 mixture of control and shPODXL (short-hairpin RNA targeting PODXL)-expressing cells,we found that podocalyxin-deficient cells exhibited a striking decrease in the ability to form clonal tumors in thelung, liver and bone marrow. Finally, to validate podocalyxin as a viable target for immunotherapy, we screeneda series of novel antihuman podocalyxin antibodies for their ability to inhibit tumor progression in vivo. One ofwth and metastasis.© 2015 Snyder et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly credited. The Creative Commons Public DomainDedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,unless otherwise stated.Snyder et al. Breast Cancer Research  (2015) 17:46 Page 2 of 14IntroductionAlthough most human cancers begin as primary focallesions, metastasis of these primary tumors to distantsites heralds advanced stage disease, poor prognosisand eventual patient death [1]. For this reason, bio-markers that identify tumors likely to metastasize, andthe generation of therapeutics that can inhibit metasta-sis, are key to improving patient survival. Although ad-juvant therapies have been developed for several typesof breast tumors, triple-negative breast cancers (estro-gen receptor (ER)-, progesterone receptor (PgR)- andhuman epidermal growth factor receptor 2 (HER2)-negative) are particularly challenging to treat becauseof their highly aggressive nature and a lack of well-defined therapeutic targets on these cells [2].Podocalyxin (also known as PCLP1, MEP21, gp135,TRA-1-60, TRA-1-81 and GCTM2) is a CD34-relatedsialomucin and a well-known marker of embryonic stemcells, embryonal carcinomas, neoplastic hematopoietic cells[3-6] and a variety of normal cells during embryonic deve-lopment, where it plays a key role in tissue morphogenesis[7-9]. We previously showed that podocalyxin (gene namePODXL) is upregulated on a subset of primary breast tu-mors and is an independent predictor of progression, me-tastasis and poor outcome [10]. Subsequent studies haveconfirmed podocalyxin as a prognostic indicator of pooroutcome in a variety of malignancies, including ovarian,prostate, renal, pancreatic, thyroid, glioblastoma, astrocy-toma, colorectal and bladder cancers [10-18]. Ectopic ex-pression of PODXL enhances tumor aggressiveness in vitro.MCF7 breast tumor cells engineered to express highlevels of murine podocalyxin (MCF7Podxl) exhibitincreased migration in vitro, altered morphogenesisand disrupted cell–cell and cell–matrix contacts[10,19,20]. In addition, podocalyxin has been shown toplay a role in the control of cell migration and the ex-pression of matrix metalloproteinases MMP1 andMMP9 [17,21]. Collectively, these studies establish acorrelation between podocalyxin expression, tumor ag-gressiveness and poor outcome (reviewed by McNagnyet al. [22]). However, the functional significance ofpodocalyxin expression by primary tumors and its in-fluence on metastatic progression in vivo have yet to bethoroughly evaluated. In the present study, we have ad-dressed this issue by silencing podocalyxin expressionin the highly aggressive triple-negative basal-like hu-man breast cancer cell line, MDA-MB-231, or overex-pressing it in a well-differentiated, ER-positive andPgR-positive, luminal-like human breast cancer cellline, MCF7 [23]. We found that podocalyxin is requiredfor efficient tumorsphere formation in both MCF7 andMDA-MB-231 cells. Moreover, suppression of PODXL inMDA-MB-231 cells profoundly impairs formation ofprimary tumors and secondary metastasis in xenograftedmice. We recapitulated this finding in an immunocompe-tent mouse tumor model by silencing podocalyxin expres-sion in 4T1 cells (a mouse mammary tumor line) andengrafting these cells in syngeneic BALB/c mice. Finally,we developed a novel podocalyxin-specific monoclonalantibody (mAb) that delays xenografted tumor formationand metastatic disease in mice. These data validate podo-calyxin as a regulator of tumor progression and a noveltherapeutic target.MethodsCell cultureMDA-MB-231, MCF7 and 4T1 cells (American Type Cul-ture Collection, Manassas, VA, USA) were grown as mono-layers on tissue culture-treated plastic plates. All cell lineswere maintained in low passage (<15). Both MDA-MB-231and MCF7 human breast tumor cell lines were cultured inDulbecco’s modified Eagle’s medium (DMEM) supple-mented with 10% fetal bovine serum (FBS), penicillin andstreptomycin. 4T1 BALB/c mouse-derived mammary tumorcells were cultured in DMEM supplemented with 10% FBS,2 mM glutamine, nonessential amino acids, penicillin andstreptomycin. All cell lines were cultured in a humidified 5%CO2 incubator at 37°C.TransductionMDA-MB-231 cells were labeled with green fluorescentprotein (GFP) or red fluorescent protein (RFP) using retro-viral vectors pLNCX2-GFP or pLNCX2-RFP, respectively(Clontech Laboratories, Mountain View, CA, USA). Hu-man PODXL was silenced in MDA-MB-231 cells by lenti-viral infection using pLKO.1 containing either a scrambledshort-hairpin RNA (shRNA) (shCTRL) or a PODXL-target-ing shRNA (RHS3979-9848792, shPODXL) as recom-mended by the manufacturer (Dharmacon, Lafayette, CO,USA). All cell lines were derived from pooled cultures ofinfected cells. Cells were cultured under continuous drugselection with puromycin (4 μg/ml; Invitrogen, Carlsbad,CA, USA) and G418 (1 mg/ml; Calbiochem, San Diego,CA, USA). PODXL-transfected MCF7 cells were describedpreviously [10,19]. Cells were cultured under continuousselection with gentamicin (50 μg/ml; Calbiochem).Predicted shRNA sequences to target murine 4T1 Podxlwere identified using pSicoOligomaker v1.5 freeware (http://web.mit.edu/jacks-lab/protocols/pSico.html). Three individ-ual shRNA oligomers were each cloned into the HpaI andXhoI sites of the pLL3.7 lentiviral vector. Firefly luciferase-expressing 4T1 (4T1-luc) cells were maintained under selec-tion in G418 (400 μg/ml; Calbiochem). To produce lentiviralparticles, 293T cells were cotransfected with 10 μg of pLL3.7and the appropriate packaging plasmids (3.5 μg of pVSVg,3.5 μg of pRSV-Rev, 6.5 μg of pMDLgag/pol) by calciumphosphate transfection. Lentivirus-containing media werecollected 36 hours post-transfection and transferred to(reverse). Relative expression of PODXL was determinedwas calculated as follows: number of tumorspheres dividedPrimary tumor development was examined followingSnyder et al. Breast Cancer Research  (2015) 17:46 Page 3 of 14subcutaneous (s.c.) injection of MDA-MB-231 cells (1 ×106) prepared in BD Matrigel™ (BD Biosciences, SanJose, CA, USA) into the right hind flank of NSG mice.Tumor growth was measured using manual calipers,and the tumor volume was estimated using the follow-ing formula: length times width2 divided by 2. Finaltumor masses were measured after excision and thetumors were retained for histochemical analyses. Flowby number of cells initially plated times 100.In vivo tumor growth and lung metastasisFor in vivo experiments, we used 6- to 12-week-oldfemale nonobese diabetic severe combined immunodefi-ciency, interleukin 2 receptor gamma chain deficient,NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice or BALB/cJmice (The Jackson Laboratory, Bar Harbor, ME, USA). Ani-mals were maintained in a specific pathogen-free facilityat the University of British Columbia (UBC) BiomedicalResearch Centre. All experiments were conducted withapproval of the UBC Animal Care Committee.relative to GAPDH in each reaction.Tumorsphere assayMDA-MB-231 and MCF7 cells were harvested, andspheres were cultured in MammoCult™ medium (StemCellTechnologies, Vancouver, BC, Canada). After 7 days, tumor-spheres larger than 60 μm in diameter were counted manu-ally using a counting grid. Tumorsphere-forming efficiencysubconfluent 4T1 cells seeded 1 day earlier. The virus-containing medium was replaced with regular growth mediaafter 48 hours and incubated for an additional 48 hours.The cells were then harvested for analysis of expression ofmouse podocalyxin RNA and protein. 4T1 cells with themost efficient knockdown were used for all studies and cul-tured with gentamicin (50 μg/ml; Calbiochem).Quantitative RT-PCRRNA isolation was performed using TRIzol reagent (LifeTechnologies, Carlsbad, CA, USA) according to themanufacturer’s instructions. Total RNA (2 μg) wasreverse-transcribed using a high-capacity cDNA reversetranscription kit (Life Technologies). Real-time quantitativePCR was performed using a SYBR FAST qPCR kit (KapaBiosystems, Wilmington, MA, USA). The PODXL-specificprimers used were 5′-CTCACCGGGGACTACAACC-3′ (forward) and 5′-GCCTCCTCTAGCCACGGTA-3′cytometry was performed on lung digests to enumeratetumor cells based on detection of GFP or RFP fluorescence.Competitive experimental metastasesTo examine experimental metastasis, a 50:50 mixture of 0.5to 2.0 × 105 shCTRLRFP (or shCTRLGFP) and shPODXLGFP(or shPODXLRFP) MDA-MB-231 cells were resuspended in100 μl of Hanks’ balanced salt solution and injected intothe tail vein of NSG mice. At day 3, 7 or 14 postinjection,mice were killed using 2,2,2-tribromoethanol (Avertin;Sigma-Aldrich, St Louis, MO, USA), then perfused throughthe right ventricle with 10 ml of phosphate-buffered saline(PBS) containing 2 mM ethylenediaminetetraacetic acid(EDTA), and the lungs (and, in some experiments, liver, fe-murs and tibias) were removed. Lungs were digested in col-lagenase/dispase solution as described elsewhere [24], andGFP-positive or RFP-positive tumor cells were detected byflow cytometry. At 6 weeks postinjection, NSG mice werekilled and perfused as described above, but tumor noduleson the surface of lungs and livers were manually countedusing a Leica Fluo™ dissecting microscope (Leica Microsys-tems, Buffalo Grove, IL, USA) and QImaging™ software(QImaging, Surrey, BC, Canada). In addition, lung, liverand bone marrow cells were prepared as described previ-ously and analyzed by flow cytometry.Flow cytometryStaining was performed with PBS containing 2% FBS, 2mM EDTA and 0.05% sodium azide. MDA-MB-231 cellswere stained with a primary antibody (Ab) against podo-calyxin (goat antihuman podocalyxin antibody (anti-PODO Ab); R&D Systems, Minneapolis, MN, USA) or agoat immunoglobulin G (IgG) isotype control (Iso) andfollowed with a chicken anti-goat Alexa Fluor (AF) 647–coupled secondary Ab (Molecular Probes, Eugene, OR,USA) for 30 minutes at 4°C and analyzed using a BDLSR II flow cytometer (BD Biosciences). Murine 4T1-luccells were labeled with allophycocyanin-conjugated ratanti-mouse podocalyxin Ab (R&D Systems) and analyzed byflow cytometry. Rat IgG2b was used as an isotype control.Experimental lung metastasisA total of 1 × 105 vector control (VC) or shPODXL 4T1-luc cells were injected intravenously (i.v.) into the lateral tailvein BALB/c mice. Lungs were perfused and excised as de-scribed above. Tumor burden was assessed by countingnodules visible on the surface of the lungs using a dissect-ing microscope and then corroborated by performing a lu-ciferase assay of homogenized lung tissue.Luciferase enzymatic assayTotal luciferase activity was assayed from lungs harvestedfrom BALB/c mice injected i.v. with 4T1-luc cells. Lungswere homogenized in cell lysis buffer (Promega, Madison,WI, USA). Protein concentration was determined using aThermo Scientific Pierce bicinchoninic acid protein assaykit (Pierce Biotechnology, Rockford, IL, USA). The Dual-Luciferase Reporter Assay System (Promega) was used todetect luciferase activity. In these experiments, 20 μl ofsample supernatant was mixed with 50 μl of luciferaseassay reagent, and luciferase activity was quantified usinga SpectraMax L microplate reader (Molecular Devices,Sunnyvale, CA, USA). The results are reported as relativelight units.Therapeutic antibody productionNew Zealand White rabbits were immunized with A-172glioblastoma cells that express high levels of tumor-glycosylated human podocalyxin on their cell surface. Rabbitbeginning on day 14 after tumor injection, mice wereadministered 100 μg of Ab (4.5 mg/kg) by intraperitoneal(i.p.) injection twice weekly. Tumor dimensions were mea-sured every 3 days until the mice were killed on day 27.Histological analysisFormalin-fixed, paraffin-embedded tumor specimens wereserially sectioned. Representative sections were deparaffi-nized and stained with hematoxylin and eosin (H&E) or Ki-67 Ab (1:700; Thermo Scientific, Waltham, MA, USA)followed by donkey anti-rabbit AF488 secondary Ab (1:1,000;Invitrogen). ProLong Gold Antifade mounting compoundpoSnyder et al. Breast Cancer Research  (2015) 17:46 Page 4 of 14mAbs were rescued as previously described [25]. Briefly,individual B-cell clones were isolated from animals whosesera recognized MDA-MB-231 cell–expressed podocalyxinextracellular domain by enzyme-linked immunosorbentassay. Next, supernatants were screened against MDA-MB-231 and human embryonic kidney 293 (HEK293) cells withand without podocalyxin on their surface (both cell linesexpress endogenous podocalyxin) to ensure immunoreactiv-ity to the native protein and minimal nonspecific bindingto PODXL-deficient cells. Finally, supernatants were alsoscreened using Chinese hamster ovary (CHO) cells express-ing podocalyxin and CD34 to ensure podocalyxin specificity.By comparing binding selectivity for podocalyxin expressedon tumor and normal cells, B-cell clones that produced Abswith favorable binding profiles to tumor cells were selectedfor cloning, scale-up production and in vivo screening.Preclinical mouse model to assess anti-podocalyxintherapeutic antibody efficacyCandidate anti-PODO Abs were selected based on the levelof binding to known podocalyxin-expressing tumor celllines (Table 1). MDA-MB-231RFP tumor cells (1 × 106 cells)were incubated with 25 μg of anti-PODO Ab (PODOC1through PODOC8) or Iso Ab (anti-ovalbumin) at roomtemperature for 30 minutes. Prior to injection, the tumorcell/Ab mixture was diluted in Matrigel™ and injected s.c.into the flank region of NSG mice. For systemic therapy,Table 1 Binding selectivity (geometric mean) of candidatemAb name HUVEC MCF7 MDA-MB-231 T47DIsotype 489 657 477 697PODOC1 44,611 60,805 91,909 1,498PODOC2 3,231 1,962 26,513 2,036PODOC3 2,103 9,399 2,939 818PODOC4 113,144 15,161 143,135 34,559PODOC5 40,490 25,055 80,312 1,853PODOC6 994 1,239 4,744 2,685PODOC7 21,700 22,330 57,626 1,010PODOC8 23,207 45,707 1,950 566aHEK293, Human embryonic kidney 293 cells; HUVEC, Human umbilical vascular endwith 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI)nuclear stain (Life Technologies) was used to mount slides.H&E-stained sections were examined qualitatively for evidenceofmuscular invasion and tumor border integrity.Statistical analysisData are expressed as the mean ± standard error of themean (SEM) unless indicated otherwise. A Student’st-test was conducted for evaluation of statistical signifi-cance. Data generated from time-dependent studies wereanalyzed by two-way analysis of variance. P < 0.05 wasconsidered to be statistically significant. All data pre-sented in the figures are representative of at least two in-dependent experiments.ResultsPodocalyxin promotes tumorsphere formation in vitroTo examine the role of podocalyxin in tumor progression,we silenced expression in MDA-MB-231 human breast can-cer cells using a shRNA-containing lentivirus (shPODXL).RFP- and GFP-labeled MDA-MB-231 cells were used as amethod for subsequent tracking of knockdown cells in vivo.Gene expression analyses confirmed attenuated PODXLexpression in shPODXL cells (Figure 1A). In addition, flowcytometric analyses confirmed that cell surface podocalyxinexpression is efficiently reduced in shPODXLGFP cells(threefold decrease) compared with scrambled shRNA-docalyxin antibodies compared with isotype controlaCAOV-3 OVCAR-3 OVCAR-10 A-172 HEK293767 629 653 644 44648,817 16,502 2,834 169,600 28,22537,683 9,189 645 49,862 45910,472 8,355 1,803 181,211 3,869125,223 29,862 564 333,980 2,67931,657 9,824 1,756 172,233 27,1441,670 1,350 1,165 2,760 48537,668 11,058 2,539 278,032 13,88652,200 8,805 2,731 263,842 5,056othelial cells; mAb, Monoclonal antibody; PODOC, Podocalyxin antibody.Snyder et al. Breast Cancer Research  (2015) 17:46 Page 5 of 14infected controls (shCTRLGFP; Figure 1B, left panel). Like-wise, podocalyxin expression in shPODXLRFP cells wasreduced by approximately twofold compared withshCTRLRFP control (Figure 1B, right panel). Similarly,total levels of podocalyxin protein were reduced inshPODXL cells compared with control as shown byWestern blotting (Additional file 1).Although proliferation of MDA-MB-231 cells inmonolayer culture was unaffected by silencing PODXL(Additional file 2), the frequency of tumorsphere-forming cells in three-dimensional assays was reduced bymore than threefold in shPODXL cultures (Figure 1C). Thecomparable size (Additional file 3A, B) and morphologyFigure 1 Podocalyxin promotes tumorsphere formation. (A) PODXL gene etargeting PODXL (shPODXL) MDA-MB-231 cells as determined by quantitative PCcells (green fluorescent protein (GFP); solid green) and shPODXLGFP (dashed gree(RFP); solid red) and shPODXLRFP (dotted red) MDA-MB-231 cells (right histogramTumorsphere-forming efficiency of shCTRL (solid bar) and shPODXL (open bconditions (n = 3; **P < 0.01, ***P < 0.001). (D) Self-renewal capacity of shCTRassessed by serial passaging (P) (n = 3; ***P < 0.001 by one-way analysis of vawere cultured under anchorage-independent conditions for 7 days to asrepresentative of three experiments with similar results. Error bars indicate sof control and shPODXL tumorspheres (Additionalfile 3B) suggests podocalyxin expression alters the fre-quency of tumorsphere-initiating cells rather than quali-tatively affecting sphere formation per se. Serial passageof tumorsphere cultures is known to improve the effi-ciency of subsequent sphere formation [26,27], and boththe shCTRL and shPODXL populations exhibited anapproximately threefold increase in the frequency ofsphere-initiating cells over three passages (Figure 1D).To further confirm that podocalyxin has a causal role inpromoting tumorsphere formation in vitro, we overex-pressed PODXL in MCF7 cells (MCF7Podxl) (Additionalfile 3C), a luminal-like human breast cancer cell linexpression relative to GAPDH in scrambled shRNA (shCTRL) and shRNAR (n = 3; *P < 0.05; ***P < 0.001). (B) Podocalyxin expression on shCTRLGFPn) MDA-MB-231 cells (left histogram) and shCTRLRFP (red fluorescent protein) relative to isotype control (shaded). RFI, Relative fluorescence intensity. (C)ar) MDA-MB-231 cells cultured for 7 days under anchorage-independentLRFP (solid bars) and shPODXLRFP (open bars) MDA-MB-231 tumorspheresriance). (E) MCF7pIRES control (solid bar) and MCF7Podxl (open bar) cellssess tumorsphere-forming efficiency (n = 3; **P < 0.01). Figures aretandard error of the mean.(increased number of Ki-67-stained cells) in shCTRL tu-Snyder et al. Breast Cancer Research  (2015) 17:46 Page 6 of 14mors compared with shPODXL tumors (Figure 2C,lower panel), showing that 37.6 ± 3.9% of shCTRLtumor cells were undergoing active proliferation,whereas no Ki-67-positive cells were detectable inshPODXL tumors. We also examined the lungs of thesemice for signs of metastases 21 days after s.c. injection.Although no visible tumor nodules were observed,tumor cells were readily detectable by flow cytometry insingle-cell suspensions from the lungs. Intriguingly,there was a 12-fold decrease in the frequency of meta-static cells in the lungs of mice with establishedshPODXL-derived primary tumors compared with micewith shCTRL-derived primary tumors (Figure 2D). Wethat expresses very low levels of endogenous podoca-lyxin [10,19]. MCF7Podxl cells (Additional file 3C) exhib-ited a 30% increase in sphere-forming efficiency(Figure 1E). The results of these gain- and loss-of-function experiments suggest that podocalyxin expres-sion increases the frequency of tumorsphere-formingcells in these cell lines. Because formation of tumor-spheres in suspension culture provides an estimate ofthe frequency of tumor-initiating cells (TICs) [28-30],the observation that PODXL knockdown dampenstumorsphere formation is consistent with the notionthat podocalyxin plays a role in TIC maintenance.Podocalyxin promotes primary tumor formation andmetastasisTo examine the functional significance of podocalyxin ex-pression in vivo, immunocompromised mice (NSG strain)were given s.c. injections of shCTRL or shPODXL MDA-MB-231 cells into the flank. Palpable solid tumors weredetected 8 days after injection of shCTRL MDA-MB-231cells, and these rapidly increased in volume over time. Incomparison, growth of shPODXL MDA-MB-231 tumorswas significantly attenuated. Tumors derived fromshPODXLRFP cells were less than 250 mm3 in volume byday 21, whereas shCTRLRFP tumors reached sizes greaterthan 750 mm3 (Figure 2A, left). Similar results wereobtained in independent experiments using shCTRLGFPand shPODXLGFP MDA-MB-231 cells (Figure 2A,right). At the time the mice were killed, shCTRL tumorswere 2.6-fold greater in mass than shPODXL tumors(Figure 2B). In addition, shPODXL tumors appeared tobe more encapsulated, with little to no evidence of inva-sion into the surrounding skeletal muscle. In contrast,shCTRL tumors were highly invasive (Figure 2C, upperpanel). Ki-67 expression was used to assess the level ofproliferation of shCTRL and shPODXL tumor cells, andwe observed a significant increase in cell proliferationconclude that expression of podocalyxin enhances bothprimary tumor growth and metastasis in vivo.Podocalyxin enhances the metastatic potential of breastcancer cellsTo directly examine the functional significance of podo-calyxin expression on lung-colonizing tumor cell fre-quency and metastatic behavior, we used an in vivocompetitive assay (experimental lung metastasis). A50:50 mixture of shPODXL and shCTRL MDA-MB-231cells was injected into the tail vein of NSG mice, andrelative tumor burden was evaluated at various timepoints over the next 6 weeks by tracking GFP or RFP ex-pression (that is, tumor cells), respectively. Somewhatsurprisingly, there was no significant difference in the ra-tio of shPODXL to shCTRL cells recovered from thelung at days 3 and 7 postinjection. However, we ob-served a slight but significant reduction in the propor-tion of shPODXL cells colonizing the lung by day 14(Figure 3A). Recovered tumor cells were evaluated forpodocalyxin expression 3, 7 and 14 days after injectioninto the mice. Strikingly, all shPODXL cells began to re-express podocalyxin within 7 days of injection (likelyowing to the lack of drug selection required to maintainshRNA expression). By day 14, shPODXL cells expressedpodocalyxin protein at levels similar to those of shCTRLtumors (Figure 3B). A similar result was obtained withprimary solid tumors seeded s.c. in the flank (Additionalfile 4). Nevertheless, at 6 weeks post i.v. injection, therewas a 2.3-fold reduction in shPODXL cell-derived tumornodules on the lungs compared with competing shCTRLcell-derived tumors (Figure 3C). In addition, large, can-cerous lesions were visible in the liver of some mice,and, invariably, all visible liver tumors were derived fromshCTRL cells as determined by fluorescence microscopy(Figure 3D). Subsequent flow cytometric analyses oflung, liver and bone marrow single-cell suspensions alsorevealed a dramatic under-representation of shPODXLMDA-MB-231 cells colonizing these tissues (Figure 3E).Thus, although podocalyxin expression on MDA-MB-231 cells does not alter initial “seeding” of the lung atearly time points (up to 14 days), expression of podoca-lyxin greatly enhances subsequent establishment ofclonal tumors in the lung and other organs. The similarsize of the shCTRL- and shPODXL-derived lung nodulesin these experimental assays (Additional file 5A) sug-gests that a difference in proliferation alone cannot ac-count for the difference in the frequency of tumornodules we observed. The higher frequency of shCTRLcells in the lungs correlates (Figure 3C) with the numberof tumor nodules observed, rather than the size of thenodules.To further corroborate these findings and evaluate thefunctional significance of podocalyxin on tumors in animmunocompetent host, we used a syngeneic model oftumor growth and metastasis. Using shRNA, we silencedmouse podocalyxin surface protein expression by 6.3-Snyder et al. Breast Cancer Research  (2015) 17:46 Page 7 of 14fold in 4T1-luc mouse mammary tumor cells (BALB/cmouse-derived) (Figure 4A). Similar to our results withMDA-MB-231 cells, loss of podocalyxin expression in4T1 cells also impaired the metastatic potential of thesecells in an experimental model of lung metastasis usingimmunocompetent BALB/c mice (greater than threefoldreduction), as shown by manual counts of tumor nod-ules on the lungs (Figure 4B) and by luciferase assays oflung homogenates (Figure 4C). As in the MDA-MB-231experimental metastasis assay, the frequency of tumornodules observed in BALB/c recipients (rather than thesize of the tumor nodules) (Figure 4B and Additionalfile 5B) was affected by silencing Podxl expression inFigure 2 Podocalyxin promotes primary tumor formation, local invasioninitiated in NSG mice by scrambled shRNA control (shCTRLRFP; closed squares) on = 5 for shCTRLRFP group and n = 6 for shPODXLRFP group; growth curves aregrowth of shCTRLGFP with shPODXLGFP MDA-MB-231 cells in NSG mice (right; nfluorescent protein; RFP, Red fluorescent protein. The final volume of the excisedday 20). (B) Wet weight (g) of pooled shCTRL (solid bar) and shPODXL (open baand n = 5 for shPODXL group; *P < 0.05). Representative photograph of excisedimages of shCTRL (upper left) and shPODXL (upper right) primary tumor sectionshCTRL (lower left) and shPODXL (lower right) primary tumor sections showing(D) The number of tumor cells detected in the lungs 20 or 21 days after subcut(×103) per 106 events detected by flow cytometry (n = 11 for shCTRL group andof three independent experiments.4T1 cells. Again, the total tumor cell numbers in thelung (as measured by luciferase assay (Figure 4C)) wasproportional to the frequency of tumor nodules. Thus,in both murine and human breast cancer cell lines,podocalyxin expression enhances experimental meta-static disease.A novel podocalyxin-specific antibody prevents primarytumor growth in vivoThe finding that podocalyxin expression is capable of drivingbreast tumor progression encouraged us to evaluate the pos-sibility that mAbs targeting the extracellular domain ofpodocalyxin would prove efficacious in delaying tumorand metastasis. (A) Growth curve of subcutaneous (s.c.) flank tumorsr shRNA targeting PODXL (shPODXLRFP; open circles) MDA-MB-231 cells (left;significantly different with P < 0.001). Similarly, we compared s.c. tumor= 6; growth curves are significantly different with P < 0.05). GFP, Greentumors was measured on the dates mice were killed (left; day 21, right;r) tumors weighed immediately after excision (n = 11 for shCTRL groupshCTRL and shPODXL tumors (inset). (C) Representative H&E–staineds (scale bar = 1 mm). Representative immunofluorescence images ofKi-67-positive cells (green). DAPI stain was used as a nuclear marker (blue).aneous injection. Data shown are the number of fluorescent tumor cellsn = 5 for shPODXL group; *P < 0.05). The data shown are representativeSnyder et al. Breast Cancer Research  (2015) 17:46 Page 8 of 14growth and metastasis. Using podocalyxin-expressing A-172 glioblastoma cells as an immunogen, we generated anovel panel of anti-human podocalyxin mAbs that exhibitpreferential binding to podocalyxin expressed on humantumor cells. Of these candidates, we selected eight mAbs(PODOC1 through PODOC8) with favorable selectivityprofiles based on flow cytometry screening of tumor cellFigure 3 Podocalyxin increases the metastatic burden in lungs, liver and bscrambled shRNA control (shCTRL) and shRNA targeting PODXL (shPODXL) MDAexperimental metastasis model. Mice were killed 3, 7 or 14 days post-injection. Prthe lungs by flow cytometry (as a % of total tumor cells) (n = 6; *P < 0.05). (B) Floexpression on tumor cells in lungs isolated from NSG mice 3, 7 and 14 days postexpression in shCTRLGFP cells (solid line) compared with shPODXLRFP (dashed linein shCTRLRFP (solid line) cells compared with shPODXLGFP (dashed line) cells fromcontrol. RFI, Relative fluorescence intensity. (C) Representative fluorescence imagetumor nodules (lower left) 6 weeks post-injection. The number of fluorescent tum***P < 0.001). (D) Representative fluorescence images of livers showing shCTRLRF6 weeks post-injection. (E) Relative frequency of shCTRL and shPODXL tumorcytometry 6 weeks post-injection (n = 10; ***P < 0.001). The data shown are rpooled from mice injected with shCTRLRFP versus shPODXLGFP or shCTRLGFP vlines known to highly express podocalyxin (MDA-MB-231, CAOV-3, A-172), tumor cell lines known to expresslow levels of podocalyxin (MCF7, T47D, OVCAR-10) andnon-tumor-derived human cells known to express podo-calyxin (human umbilical vascular endothelial cells andHEK293 cells) (Table 1). Although we generated severalantipodocalyxin mAbs with affinity for podocalyxinone marrow, but not initial lung seeding. (A) A 50:50 mixture of-MB-231 cells were injected into the tail vein of NSG mice in a competitiveesented is the relative frequency of shCTRL and shPODXL cells detected inw cytometry was performed using an antibody to detect podocalyxin-injection. Histograms displaying the levels of (1) surface podocalyxin) cells from day 0 to day 14 (upper) and (2) surface podocalyxin expressionday 0 to day 14 (lower). The shaded day 0 histograms represent the isotypes of lungs showing shCTRLRFP tumor nodules (upper left) and shPODXLGFPor nodules on the lung surface was manually counted (right; n = 5;P tumor nodules (upper) and shPODXLGFP tumor nodules (lower) (n = 5)cells within the lungs, liver and bone marrow as determined by flowepresentative of three independent experiments. All data shown wereersus shPODXLRFP run in tandem experiments.r cgcysh-luunCepSnyder et al. Breast Cancer Research  (2015) 17:46 Page 9 of 14expressed on MDA-MB-231 cells, none of these exhibitedan effect on tumor cell growth in monolayers or tumor-sphere formation in vitro (data not shown).Nevertheless, because podocalyxin expression appears topredominantly affect the ability of tumor cells to colonizetissues in vivo (rather than influencing their behaviorin vitro), we went on to evaluate the effects of these anti-bodies in xenograft assays. Candidate mAbs were incubatedFigure 4 Podocalyxin enhances metastasis of 4T1 mammary tumoand shRNA targeting PODXL (shPODXL; dashed line) luciferase-expressinconjugated rat anti-mouse podocalyxin antibody and analyzed by flowused as an isotype control (shaded area). Data presented as histograms4T1-luc cells. RFI, Relative fluorescence intensity. (B) VC or shPODXL 4T1after 14 days, and visible tumor nodules on the lung were manually co***P < 0.001). (C) Luciferase expression in the lungs from noninjected, Vluminometer (**P < 0.01). The data shown are representative of two indwith tumor cells immediately before s.c. injection into theflanks of recipient mice (pretreatment screen). Oneantipodocalyxin mAb (PODOC1) inhibited MDA-MB-231 growth and dissemination for 11 days (Figure 5A),whereas the Iso and 7 other podocalyxin-binding candi-date mAbs (PODOC2 through PODOC8) failed to signifi-cantly alter tumor progression (Figure 5, B–H). Next, weassessed the ability of PODOC1 to inhibit establishedtumor growth (Figure 6). Cohorts of mice were treatedtherapeutically with PODOC1 or control Ab beginning 14days after tumor injection, when a palpable tumor hadalready formed (s.c.). We consistently found that systemictreatment of tumor-bearing mice with PODOC1 com-pletely inhibited tumor growth (Figure 6A, B) and,importantly, attenuated micrometastases to the lung(Figure 6C). Thus, we validated podocalyxin as a criticalfacilitator of tumor growth and progression and a thera-peutic target for treatment in a preclinical model. Toconfirm that PODOC1 specifically binds to podocalyxin,Western blot analysis was performed on shCTRL andshPODXL MDA-MB-231 cell lysates. PODOC1 detectedhigh levels of podocalyxin protein in shCTRL cells and sig-nificantly less in shPODXL cells (Additional file 6A). Add-itionally, in flow cytometric analyses, PODOC1 detectedextracellular podocalyxin on shCTRL and lower levels onshPODXL MDA-MB-231 cells (Additional file 6B). It wasalso important to determine whether PODOC1 was specificfor podocalyxin and did not bind to the closely relatedfamily member, CD34. Utilizing CHO cells transfectedwith human podocalyxin or CD34 (hCD34), PODOC1 wasfound to specifically detect overexpression of podocalyxin(Additional file 7B), whereas it failed to bind toells in a syngeneic mouse model. (A) Vector control (VC, black)4T1 (4T1-luc) murine mammary tumor cells were labeled with atometry to detect surface expression of podocalyxin. Rat igG2B wasowing podocalyxin expression on VC cells compared with shPODXLc cells were injected intravenously into BALB/c mice. Mice were killedted using a dissecting microscope (n = 9 and n = 8, respectively;and shPODXL 4T1-luc-injected mice was measured using aendent experiments. RLU, Relative light units.hCD34 (Additional file 7C) or mock-transfected CHO cells(Additional file 7A). Thus, our data would support theargument that the PODOC1 Ab is highly specific anddoes not cross-react with a closely related sialomucin. Weconclude that antibodies targeting the appropriate epitopeon human podocalyxin can provide therapeutic benefitin vivo.Finally, to test the potential efficacy of PODOC1 onlate-stage metastatic disease, mice were injected s.c.with shCTRLGFP MDA-MB-231 cells and tumors wereallowed to reach a size larger than 500 mm3 prior toPODOC1 therapy. At this size, we find that metastaticlesions readily develop in the lungs. PODOC1 or Iso Abwas then administered to the mice with established tumorburdens at days 20, 26, 29 and 32 (Additional file 8A).Systemic treatment with PODOC1 appeared to margin-ally slow the growth of the primary tumor, although thedifference was not statistically significant (Additionalfile 8A, B). However, PODOC1 treatment resulted in adramatic reduction in the number of tumor nodules ob-served on the lung surface (Additional file 8C) recov-ered in total lung homogenates (Additional file 8D). Weconclude that PODOC1 provides a clear therapeuticbenefit, even in late-stage metastatic disease (that is,Snyder et al. Breast Cancer Research  (2015) 17:46 Page 10 of 14when metastatic organs are already colonized withtumor cells).DiscussionAlthough podocalyxin is expressed by a minor subset of pri-mary breast tumors, these have been shown to be the mostaggressive and difficult-to-treat breast cancers [10]. Import-antly, podocalyxin expression is also a predictor of poorprognosis in many other cancers [10-15,17,18,20,31,32]. Forexample, patients with podocalyxin-positive colorectal car-cinoma (where podocalyxin-expressing cells are often lo-cated at the invasive front of the primary tumor) have aFigure 5 Pretreatment of MDA-MB-231 cells with PODOC1 delays prim(post-injection) of tumors resulting from subcutaneously injected MDA-MBnovel candidate antipodocalyxin antibodies (PODOC). (A) PODOC1 Ab (*pPODOCs (PODOC2 through PODOC8; n = 3). The data shown are represenhigher probability of lymph node and distant metastases[31]. In addition, roughly 20% of stage III colorectal carcin-omas express high levels of podocalyxin, and these representa cohort that significantly benefits from adjuvant chemo-therapy [31]. Comparatively, similar patients with low levelsof tumor podocalyxin did not appear to significantly benefitfrom chemotherapy [31]. Knowing the likelihood of successbefore accepting a treatment that is difficult for some pa-tients to tolerate has obvious decision-making benefit. Thus,podocalyxin-based “theranostic” and therapeutic strategiesmay prove to have broad applications if podocalyxin pro-motes primary tumor growth and metastasis in colorectalary tumor development. Growth curves from days 4 to 16-231 cells pretreated with isotype control (Iso; red) or one of eight< 0.05; n = 3). (B) through (H) Growth curves for other candidatetative of two independent experiments.hiof i; *PPO-potaSnyder et al. Breast Cancer Research  (2015) 17:46 Page 11 of 14carcinoma and other epithelial cancers. It is now importantto further explore the therapeutic efficacy of PODOC1 andsimilar reagents in a clinical setting. Because podocalyxin ispresent on normal human cells, including the vascularendothelium and kidney podocytes, extensive toxicologicstudies will be needed to ensure the safety of a therapeuticAb. However, we predict that because the podocalyxin-richpodocytes of the kidney are behind the blood filtration bar-rier in the urinary space, they may be spared exposure toPODOC1 therapy. Additionally, we have not observed anyadverse effect of antibodies targeting mouse podocalyxinwhen systemically administered to wild-type mice. Further-Figure 6 Systemic treatment with antipodocalyxin antibody PODOC1 inGrowth curve of tumors from mice treated intraperitoneally (i.p.) with 100 μgon day 14 and every 3 to 4 thereafter days until being killed on day 27 (n = 5administration of antibody. (B)Weight (g) of tumors treated with either Iso orPODOC1 (inset) (n = 5; *P = 0.05). (C) Number of red fluorescent protein (RFP)either Iso or PODOC1 as detected by flow cytometry (n = 5; *P < 0.05). The damore, we have found that selective deletion of Podxl frommouse endothelia is well tolerated and nontoxic in mice[33]. Thus, the data would support the argument that inter-fering with podocalyxin expression on endothelia or bindingof podocalyxin-reactive antibodies to the vasculature is un-likely to be toxic.By identifying a requirement for podocalyxin in tumori-genesis, we can now begin to characterize the key molecu-lar mechanisms by which podocalyxin promotes tumorcell growth and colonization of supportive niches. As ishighlighted by Ki-67 staining of subcutaneous tumors,one function of podocalyxin may be the promotion of pri-mary tumor cell proliferation in vivo. Intriguingly, this ef-fect was observed only in vivo because loss of podocalyxinhad no effect on the proliferation of cultured tumor cells.Notably, although silencing podocalyxin is detrimental totumorsphere-forming efficiency of MDA-MB-231 cells,the PODOC1 mAb does not appear to alter tumorsphere-forming efficiency or proliferation in vitro (not shown).Tumor formation and metastasis in vivo are dependent ona number of cellular characteristics that are difficult tomimic in vitro, including migration to and colonization ofa supportive niches, immune cell evasion, and survival ofa hypoxic environment until the establishment of an ad-equate blood supply. It is likely that podocalyxin functionsin these settings are multifactorial, and, thus far, we havebeen compelled to use in vivo models for these studies. Itis now important to evaluate the molecular pathwayspodocalyxin impinges on in vivo that lead to altered tumorcell proliferation.With regard to the early stages of tumor colonizationof tissues, it is intriguing that the bulk of our shPODXLcells begin to reexpress PODXL during the first 7 to 14days in vivo. Thus, our data would support an argumentbits primary tumor development and metastasis to the lung. (A)sotype control (Iso) or antipodocalyxin monoclonal antibody (mAb) PODOC1< 0.05 by two-way analysis of variance) (right). Orange stars indicate i.p.DOC1. Representative photograph shows tumors treated with either Iso orsitive tumor cells per 106 lung cells of mice with tumors treated i.p. withshown are representative of two independent experiments.for an important influence of podocalyxin on an earlytumor-initiating subset of cells. This notion is supportedby the fact that, in our experimental lung metastasis as-says, we found that silencing podocalyxin expression de-creased the frequency (but not the size) of tumornodules we observed. In aggregate, these data suggestthat even transient depletion of podocalyxin expressionduring the early phase of tumor establishment can havea profound effect on late-stage growth of metastases,perhaps through impaired function or decreased fre-quency of a population of cells with TIC-like properties.We do not yet know which properties of TICs are influ-enced by podocalyxin expression, but they could includeproperties that enable tumor cells to proliferate orsurvive within a metastatic niche, including invasion,migration, adhesion and recruitment of supportivevasculature. It is noteworthy that podocalyxin, and itsclose relative CD34, are well-known markers of varioussubsets of stem cells during development and play a rolein cell and tissue morphogenesis and colonization of de-veloping tissues [7,9,26,27,31]. Likewise, podocalyxinwas recently detected in an undifferentiated stemlikeSnyder et al. Breast Cancer Research  (2015) 17:46 Page 12 of 14population in glioblastoma multiforme [11], and it is awell-known marker of both embryonic stem cells andembryocarcinomas [3,6]. Thus, in both normal develop-ment and neoplastic disease, podocalyxin expression hasbeen linked to stem cell activity. Impaired tumor initiationwould be consistent with known roles for podocalyxin andCD34-type proteins in blocking cell adhesion and facilitat-ing chemokine-dependent inflammatory trafficking andhematopoietic stem cell engraftment of the bone marrowniche [34-36]. Importantly, in contrast to the wide varietyof drugs that target tumor proliferation, there is a paucityof therapeutics that target TIC activity, and therefore theAb strategy described here may be an important add-itional therapeutic avenue.In many ways, our findings are complementary tothose described in a recent publication by Lin et al.[37]. These authors provided provocative evidence thatboth podocalyxin and cortactin are important for themorphogenesis, motility, gelatin invasion and in vivometastatic potential of MDA-MB-231 cells and showedthat these proteins associate in vitro. Although they did notshow that podocalyxin is essential for cortactin-mediatedmetastasis in vivo, these data do offer a potential mechanis-tic insight into podocalyxin function through a cortactin-containing complex. Given that our present study showspodocalyxin to be functionally important for tumorsphere-forming cells in vitro and the early phases of tumorcolonization by a subset of cells in vivo, it is now importantto validate the functional significance of the cortactin andpodocalyxin interaction in this rare, but clinically critical,subset of tumor cells.ConclusionsIt has previously been shown that podocalyxin expres-sion in invasive breast carcinoma correlates with poorpatient survival and that podocalyxin enhances the mo-tility and invasiveness of breast cancer cell lines in vitro[10,19-21]. Here, using in vivo models of breast tumorgrowth and metastasis, we show that podocalyxin has acausal role in promoting the growth and proliferation ofsolid tumors and enhancing the metastasis of tumorcells to distant organs. We found that silencing podoca-lyxin expression in MDA-MB-231 cells, an aggressivetriple-negative, basal-like breast cancer cell line, se-verely impaired primary tumor growth and metastasisto the lung, liver and bone marrow in a xenograftmodel. We corroborated these results in a syngeneicmouse model using fully immunocompetent mice bysilencing podocalyxin expression in mouse mammarytumor 4T1 cells. Thus, in both mouse and human breasttumor cells, podocalyxin plays a critical role in diseaseprogression. Furthermore, we have developed a uniquemAb that targets podocalyxin and, in preclinical mousestudies, inhibits tumor growth and metastatic progression.Additional filesAdditional file 1: Podocalyxin expression can be efficiently knockeddown in MDA-MB-231 cells. shCTRL and shPODXL MDA-MB-231whole-cell lysates (5 × 104 cells) were resolved by SDS-PAGE andanalyzed by Western blotting using antipodocalyxin clone 3D3(1:4,000; Santa Cruz Biotechnologies, Santa Cruz, CA, USA). An antibodyagainst β-actin (1:10,000; Sigma-Aldrich) was used as a loading control.Additional file 2: Proliferation is not affected by podocalyxinexpression in MDA-MB-231 cells in monolayer culture. A 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium proliferation assay was performed on shCTRL and shPODXLMDA-MB-231 cells 48, 72 and 96 hours after initial seeding. Proliferationwas quantified by the amount of formazan product detected at 490-nmabsorbance using a microplate reader. The level of proliferation of shCTRLwas compared with shPODXL cells over time (nonsignificant by two-wayanalysis of variance). All values are graphed as mean ± SEM.Additional file 3: shCTRL and shPODXL cells form tumorspheresof similar size and morphology. A total of 5 × 103 shCTRL or shPODXLMDA-MB-231 cells were cultured for 7 days in MammoCult medium.(A) Tumorsphere size was calculated using ImageJ software (pixels).(B) Representative images of shCTRL and shPODXL MDA-MB-231tumorspheres. (C) Murine podocalyxin expression can be overexpressedin human MCF7 breast tumor cells. MCF7pIRES control and MCF7Podxllysates were resolved by SDS-PAGE and analyzed by Western blottingusing a goat anti-mouse podocalyxin antibody (1 μg/ml; R&D Systems).An antibody against β-actin was used as a loading control. (D) Flowcytometry was performed to detect the level of extracellular murinepodocalyxin on MCF7pIRES (left; blue) and MCF7Podxl cells (right; red). Agoat anti-mouse podocalyxin antibody (2.5 μg/106 cells; R&D Systems)and an isotype against normal goat IgG (gray) were used.Additional file 4: Podocalyxin is reexpressed in primary MDA-MB-231 tumors after 14 days in vivo. A total of 1 × 106 shCTRLGFP orshPODXLGFP MDA-MB-231 cells were injected s.c. into the right and leftflanks of NSG mice. After 14 days, mice were killed and perfused with 10ml of ice-cold PBS, and tumors were excised. (A) One-third of eachshCTRL and shPODXL tumor was processed for flow cytometric analysisusing a 2-U/ml collagenase solution for 1 hour at 37°C and stained forextracellular podocalyxin using one of two antibodies (PODOC1 or goatantihuman podocalyxin; R&D Systems). The upper two histograms displaythe level of surface podocalyxin expression in shCTRLGFP cells (left; blue)compared with shPODXLGFP cells (right, blue) as detected by PODOC1antibody. A goat antihuman AF647 (2 μg/ml; Invitrogen) secondary-onlycontrol is shown in red. The lower two histograms display the level ofsurface podocalyxin expression in shCTRLGFP cells (left; blue) compared withshPODXLGFP cells (right; blue) as detected by goat antihuman podocalyxin(2 μg/ml; R&D Systems) followed by chicken anti-goat AF647 (Invitrogen).Normal goat IgG isotype control is shown in red. (B) One-third of the tumorswere processed “fresh” for Western blot analysis by directly homogenizingthem in 500 μl of radioimmunoprecipitation assay (RIPA) lysis buffer, and thefinal third of the tumors were freeze-thawed (F/T) by snap-freezing in dryice and storing them at −80°C for 1 hour. F/T tumors were homogenized in500 μl of RIPA lysis buffer. A BCA assay was performed, and equal amountsof protein were resolved by SDS-PAGE and analyzed by Western blottingusing antipodocalyxin clone 3D3 (1:4,000; Santa Cruz Biotechnologies). Anantibody against β-actin (1:10,000; Sigma-Aldrich) was used as a loadingcontrol.Additional file 5: Metastatic lung nodules resulting from MDA-MB-231 cells or 4D1 cells are more prevalent in number, but not ofgreater size when compared with their respective podocalyxinknockdown lines. (A) NSG mice were injected (i.v.) with a 50:50 mixtureof shCTRLGFP and shPODXLRFP MDA-MB-231 cells (5 × 104 cells). After 6weeks, mice were killed and their lungs and fluorescent nodules on thelungs were imaged using a fluorescence dissecting microscope. The GFPand RFP channels were merged as a composite image to show nodulesarising from both shCTRL and shPODXL cells. (B) BALB/c mice wereinjected (i.v.) with 1 × 105 vector control (VC) or shPodxl mouse murine4D1 tumor cells. After 2 weeks, lungs were perfused with ice-cold PBS,fixed in 10% buffered formalin, embedded in paraffin and sectioned.Snyder et al. Breast Cancer Research  (2015) 17:46 Page 13 of 14Representative lung sections containing VC or shPodxl nodules werestained with H&E.Additional file 6: PODOC1 antibody specifically detects podocalyxinexpression in MDA-MB-231 cells. (A) shCTRL and shPODXL MDA-MB-231whole-cell lysates were resolved by SDS-PAGE and analyzed by Westernblotting using candidate therapeutic antibody PODOC1 (1 μg/ml). Anantibody against β-actin was used as a loading control. (B) Podocalyxinexpression on shCTRLGFP (solid) and shPODXLGFP (dashed) MDA-MB-231cells relative to secondary-only control (shaded), as detected using PODOC1(10 μg/ml) antibody followed by goat antihuman AF647 secondary antibody(2 μg/ml; Invitrogen) (right).Additional file 7: PODOC1 antibody specifically interacts withpodocalyxin and does not bind to CD34. Human CD34 (hCD34) andpodocalyxin were transiently overexpressed in CHO cells. Flow cytometrywas performed on CHO cells transfected with (A) human podocalyxin(CHOPODXL) or (B) human CD34 (CHOhCD34) or (C) mock sequence(CHOMock) and stained with either PODOC1 (left) (10 μg/ml) or mouseantihuman CD34 fluorescein isothiocyanate–conjugated antibody (right)(1:50; Invitrogen).Additional file 8: Systemic treatment with PODOC1 inhibitsmetastasis to the lung in mice with large (>500 mm3) primaryMDA-MB-231 tumors. A total of 1 × 106 shCTRLGFP MDA-MB-231 cellswere injected s.c. into the flank of NSG mice and allowed to develop intosolid tumors over 20 days. (A) Growth curve of tumors from mice treatedintraperitoneally (i.p.) with 4.5 mg/kg of isotype control (Iso) or PODOC1Ab on day 20 and at three time points until the mice were killed on day36 (nonsignificant (n.s.) by two-way analysis of variance; n = 5) (right).Orange stars indicate i.p. administration of antibody. (B) Weight (g) oftumors treated with either Iso or PODOC1 antibody (n = 5; n.s. byStudent’s t-test). (C) Representative bright-field and fluorescencemicroscopic images of lungs showing shCTRLGFP tumor nodules frommice that had been systemically treated with Iso (upper two panels) orPODOC1 (lower two panels). (D) Percentage of GFP-positive tumor cellsin the lungs of mice with tumors treated i.p. with either isotype orPODOC1 antibody as detected by flow cytometry (n = 5; *P < 0.05).AbbreviationsAb: Antibody; AF: Alexa Fluor; CHO: Chinese hamster ovary cells;DMEM: Dulbecco’s Modified Eagle’s medium;EDTA: Ethylenediaminetetraacetic acid; ER: Estrogen receptor; FBS: Fetalbovine serum; F/T: Freeze-thawed; GFP: Green fluorescent protein;H&E: Hematoxylin and eosin; HEK293: Human embryonic kidney 293;HER2: Human epidermal growth factor receptor 2; HUVEC: Human umbilicalvascular endothelial cell; IgG: Immunoglobulin G; i.p.: Intraperitoneal;Iso: Isotype; i.v.: Intravenous; mAb: Monoclonal antibody; MEP21: Myb-Etsprogenitor 21; NSG: Nonobese diabetic severe combined immunodeficiency,interleukin 2 gamma chain deficiency, NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ;PBS: Phosphate-buffered saline; PCLP1: Podocalyxin-like protein 1;PgR: Progesterone receptor; PODO: Podocalyxin; RFI: Relative fluorescenceintensity; RFP: Red fluorescent protein; RIPA: Radioimmunoprecipitation assay;RLU: Relative light units; s.c.: Subcutaneous; SEM: Standard error of the mean;shCTRL: Scrambled short-hairpin RNA control; shPODXL: Short-hairpin RNAtargeting PODXL; shRNA: Short-hairpin RNA; 4T1-luc: Luciferase-expressing4T1 cells; TIC: Tumor-initiating cell; VC: Vector control.Competing interestsThe authors (KAS, MRH, KMM, CDR, JSB, BH and MLG) declare that they havea patent pending on podocalyxin as potential therapeutic for epithelialtumors. KMM and CDR possess an awarded US patent on podocalyxin as aprognostic marker in cancer.Authors’ contributionsKS and MRH carried out all experiments, participated in the design andcoordination of the study and drafted the manuscript. BH, JB, PB, FC, KP and JSBdeveloped and produced candidate antipodocalyxin antibodies and contributedintellectually to the conception and design of the antibody studies. MET and SRMknocked down PODXL in MDA-MB-231 cells using vector provided by JAW andcontributed intellectually to all of the MDA-MB-231 studies. JAW contributed tothe experimental design of the MDA-MB-231 studies. DCH carried out xenograftassays together with KS and contributed intellectually to antipodocalyxin antibodystudies. MLG and JSN developed the podocalyxin overexpressing the MCF7 cellline and the murine 4T1 knockdown cell line and contributed intellectually to thedesign of these studies. KMM and CDR conceived of the study, participated in itsdesign and coordination and helped to draft the manuscript. All authors read andapproved the final manuscript.AcknowledgmentsWe are very grateful to Drs Martin Lopez, Megan Gilmour and Pamela Deanfor providing expert technical assistance and generating the Western blotdata. Thank you to Dr Matthew Gold for his critical reading of themanuscript. DCH received a graduate student fellowship from the Centre forBlood Research, University of British Columbia. This work was supported byan operating grant from the Canadian Institutes of Health Research (KMM,MOP# 125992), an Impact Grant from the Stem Cell Network Centre ofExcellence (KMM), and funding from National Health and Medical ResearchCouncil (SRM).Author details1The Biomedical Research Centre, University of British Columbia, 2222 HealthSciences Mall, Vancouver, BC V6T 1Z3, Canada. 2Centre for Drug Researchand Development, University of British Columbia, Vancouver, BC V6T 1Z3,Canada. 3Department of Cellular and Physiological Sciences, University ofBritish Columbia, Vancouver, BC V6T 1Z3, Canada. 4Centre for MolecularPathology, School of Molecular & Biological Science, The University ofAdelaide, Adelaide, SA 5005, Australia. 5Department of Internal Medicine,Manitoba Centre for Proteomics and Systems Biology, University ofManitoba, Winnipeg, MB, Canada.Received: 22 June 2014 Accepted: 17 March 2015References1. Weigelt B, Peterse JL, van ’t Veer LJ. 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