Investigation of the role of mesenchymal stromal cells in modulating macrophage phenotype and function in the colorectal tumour microenvironment

Colorectal cancer (CRC) is the third most commonly occurring cancer. While there have been improvements in treatment options for patients, it remains the second leading cause of cancer-related deaths worldwide. CRC develops in a complex multicellular microenvironment, with patients diagnosed with mesenchymal-rich tumours having the lowest disease-free survival. However, the role mesenchymal stromal cells (MSCs) play in tumour promotion and immune invasion has yet to be fully elucidated. Macrophages are the most abundant immune cell type found in CRC, and following the promising results from T cell targeting immunotherapies, macrophages are now under investigation for their potential to be target therapeutically in CRC. The focus of my PhD project was to investigate the role of the inflammatory tumour microenvironment on stromal cell-mediated modulation of macrophage phenotype and function. Inflammatory tumour conditioned media (TCM) generated from CRC cells treated with TNF-α increased the mRNA expression of immunomodulatory factors and pathways related to the regulation of immune cell function in Balb/c MSCs. Inflammatory tumour conditioned MSCdisplayed increased surface expression of the immunomodulatory markers PD-L1 and CD47. CD47 expression was found to be higher on MSC than on cancer cells, while PD-L1 levels were comparable. TCM generated from treating CRC cells with low dose cyclophosphamide (CTX) significantly increased the expression of MSC PD-L1 and CD47. These induced changes in the MSCs were mediated by changes in the cancer cell secretome. Bioinformatic and immunohistochemical analysis of CRC tumours revealed an association between MSCs and macrophages in the tumour microenvironment. Macrophages that were co-cultured with control, TCM or inflammatory TCM treated MSCs showed reduced expression of SIRPα, MHC II, arginase1 and IL-10 while increasing CD206 expression and a suppression of macrophage phagocytic capacity. Targeting PD-1 and SIRPα with receptor blocking antibodies was used to assess if MSC PD-L1 or CD47 were modulating the changes in macrophage phenotype and function. Neither targeting PD-1 nor CD47 altered MSC mediated changes in macrophage expression profile. However, blocking both PD-1 and SIRPα showed a strong trend towards the restoration of macrophage phagocytosis. Furthermore, blocking SIRP-α led to reduced TNF-α secretion following co-culture of macrophages with inflammatory conditioned MSCs. Following from this, a viable 3D hydrogel system containing CRC cells, MSCs and monocytes was developed to better study the complex tumour microenvironment in a more physiologically relevant model. The addition of monocytes and MSCs to the culture system alters the mRNA expression of ECM remodelling proteins fibronectin 1 and MMP2 and altered cancer cell expression of PD-L1, CD47 and EGFR. The presence of MSCs in the 3D model increased the expression of tumour promoting molecules such as serpin E1, CXCL12 and macrophage inhibitory factor. The secretome was further altered by treatment with TNF-α, resulting in the induction of different cytokines and chemokines including IL-6 and GM-CSF. The treatment of the 3D CRC model with PD153035, an EGFR inhibitor, revealed that hydrogels containing MSCs responded differently to this treatment. Taken together, this data demonstrates the role of inflammation on the ability of CRC cells to modulate the mesenchymal stromal compartment of the tumour and how stromal cells, in particular, MSCs, can alter macrophage phenotype and function. We have demonstrated that targeting the PD-L1/PD-1 or CD47/SIRPα signalling axis in MSC-rich inflammatory CRC tumours may aid the restoration of macrophage phagocytosis. Finally, we have a developed a 3D model to study the complex interactions that occur in CRC and that can be used to identify novel targets and test novel therapies. Overall, this project demonstrates that stromal cells in the inflammatory tumour microenvironment can influence macrophage function through the expression of PD-L1 and CD47. We propose that these factors could represent novel therapeutic targets in stromal rich CRC.

Publisher

NUI Galway

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Attribution-NonCommercial-NoDerivs 3.0 Ireland
CC BY-NC-ND 3.0 IE

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University of Galway Theses (PhD Theses)