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Molecular mechanisms regulating the accumulation and functional activity of tumour-associated macrophages

University of British Columbia

Most solid tumour masses also contain a significant number of macrophages and other hostderived immune cells. In previous studies, such infiltrating cells have been shown to exhibit a wide range of biological activities in vitro, some of which, if they were also to occur in vivo, could have a profound effect on tumour growth and/or metastasis. At present, the molecular mechanisms that regulate the accumulation and functional activity of tumour-associated macrophages remain poorly defined. One would anticipate, however, that factors which enhance the recruitment of monocytes or other more primitive macrophage precursor cell types into a tumour site from the peripheral blood, or which enhance the survival, proliferation and/or differentiation of these cells within the tumour microenvironment, might function to increase the total number of macrophages present within a tumour. The work described in this thesis was designed to test this hypothesis through the identification of specific tumour cell-derived factors that could play a role in regulating the accumulation of tumour-associated macrophages. Macrophage progenitor cells with extensive proliferative capacity are known to circulate and have been identified within various histologically distinct tumour types. As a first step towards trying to define the mechanisms that lead to an accumulation of macrophages in tumours, an effort was made to determine whether tumour cells alone are able to provide all of the signals necessary to support the proliferation and differentiation of myeloid progenitor cells in vitro. Using nylon wool non-adherent bone marrow cells as a source of progenitors, tumour cells derived from the murine fibrosarcoma Fsa-N were shown to be fully capable of maintaining the output of clonogenic cells for up to 4 weeks in vitro in the absence of any other exogenous source of growth factors active on these cells. This activity was, however, not restricted to tumour cells as nontransformed fibroblasts derived from a number of non-hemopoietic tissues had a similar supportive capacity. In order to identify some of the factors that may play an important role in this process, fibroblastoid cell lines derived from a number of mutant mouse strains were also tested for their myelosupportive capacity. Interestingly, no difference was observed in the output of clonogenic cells in cultures containing embryonic Sl/Sl and littermate-derived +/+ fibroblasts indicating that Steel factor is not necessary for the sustained production of the progenitor cell type measured in this assay system. In contrast, fibroblastoid cells derived from macrophage-colony stimulating factor (M-CSF)-deficient op/op mice were found to be compromised in their supportive capacity relative to equivalent cells derived from phenotypically normal op/+ littermate controls. Taken together, the data from these experiments suggest that tumour cells and other non-transformed fibroblastoid cells are capable of maintaining myeloid progenitor cells in vitro and that M-CSF can play an important role in this process. In order to define the contribution of M-CSF to the accumulation and functional activity of tumour-associated macrophages in vivo, fibroblastoid cell lines derived from both M-CSFdeficient op/op mice and their phenotypically normal op/+ littermate controls were transformed with a retroviral vector encoding Polyoma large T. The lines produced, designated op/opPy and op/+Py, were then inoculated subcutaneously into SCID mice and both the proportion and phenotype of the macrophages present within the tumours that developed was determined. Since tumours derived from both cell types were found to contain a similar percentage of macrophages it would appear that M-CSF does not play an important role in determining the macrophage content of at least these particular tumours. M-CSF does, however, play a role in regulating their functional activity. Thus, while the macrophages present in M-CSF-deficient op/opPy tumours expressed the same level of 114/A10 mRNA as macrophages present within op/+Py tumours, they expressed much lower levels of Interleukin-1β (IL-1β), Tumour Necrosis Factor-a (TNF-a) and Fc Receptor (FcR)yll mRNA. The molecular mechanisms responsible for maintaining the viability of tumour-associated macrophages in the absence of M-CSF remain to be determined; while preliminary evidence was obtained to indicate that op/op and op/opPy cells may elaborate a soluble factor distinct from M-CSF and GM-CSF that can promote monocyte survival in vitro. Finally, experiments to define the molecular mechanisms that regulate the production and functional activity of TNF-a within the tumour microenvironment were initiated. Two fibroblastoid tumour cell lines, Fsa-R and Fsa-N, were inoculated subcutaneously into syngeneic LPS hyporesponsive (Lps[sup d]) C3H/HeJ and normal (Lps[sup n]) C3H/HeN mice and tumour growth, macrophage content, and the production of TNF-α by tumour-associated macrophages was determined. Fsa-N and Fsa-R tumours grew equally well in both mouse strains and contained almost exactly the same proportion of macrophages. However, the macrophages present within the tumours grown in C3H/HeJ mice produced 5-10 fold less TNF-a than equivalent cells present within tumours grown in C3H/HeN mice. These data suggest that the mechanisms that operate within the tumour microenvironment to induce the production of TNF-a act, at least in part, via the same signal transduction pathway that is defective in Lps[sup d] C3H/HeJ mice. Moreover, it appears that the differences in the level of TNF-a produced within tumours grown in C3H/HeN and C3H/HeJ mice, while seemingly dramatic, are insufficient to alter either tumour growth rate or macrophage content.

Thesis/Dissertation

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2009-06-04

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http://hdl.handle.net/2429/8781

Pathology

University of British Columbia

UBCV

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