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Emerging pre-clinical data suggests that radiation both stimulates and suppresses tumor immunity. The role of tumor associated macrophages (TAMs) has been extensively investigated in the non-irradiated tumor microenvironment. We aimed to investigate the role of macrophages in the immune response to radiation. Single dose 10Gy irradiation to tumors generated from colorectal (MC38) and pancreatic (KPC) cell lines induced Colony Stimulating Factor (CSF-1). Coincident with the elevation in CSF1, significant increases of CD11b+ F480+ macrophages within the tumor microenvironment peaked five days following irradiation returning to baseline during tumor regrowth. These TAMs were skewed toward an immunosuppressive phenotpye (CD206hi iNOSlo) compared to those from controls. Furthermore, the TAMs from irradiated tumors were more effective in suppression of CD8 T cell expansion in ex vivo assays. As a result of these data, we investigated the effect of anti-Colony Stimulating Factor (αCSF-1), which reduced macrophages in both control and irradiated tumors. αCSF had no effect on the growth of control tumors, yet led to a significant enhancement of tumor growth delay after radiation. TAMs sorted from irradiated tumors after aCSF treatment had reduced inflammatory gene expression and increased expression of some genes associated with immune suppression (Arg-1, IL-10, CCL2). Following aCSF, there was a significant increase in CD8 T lymphocytes with greater expression of cytotoxic markers. Radiation is also immune-stimulatory to tumors consistent with enhanced recognition of tumor cell antigens by T cells isolated from irradiated tumors. CD8 depletion however abrogated the growth delay from aCSF further documenting the immunosuppressive effect of the macrophages within irradiated tumors. aCSF1 was given to mice bearing two tumors, one of which was irradiated. An increased growth delay was only observed in the irradiated tumours. These data document the immunosuppressive nature of macrophages generated in irradiated tumors. TAMs expressed high levels of PD-L1 and aCSF treatment resulted in a reduction in tumor PD-L1. As a result we determined the effect of checkpoint inhibition using anti-PD-L1 in combination with aCSF following 10Gy irradiation. Whilst combination treatment did not augment outcomes in mice bearing MC38 tumors, there was complete tumor regression in a minority of mice bearing KPC tumors. These results suggest that infiltrating macrophages limit the adaptive immune response initiated following of radiation. Whilst it has been demonstrated that tumor cell PD-L1 expression plays an important role in checkpoint blockade, our data suggests macrophages are an important additional source. This is of particular importance in the context of radiation which can elicit a potent immune response but in which the TAMs from irradiated tumors also suppress this in part via altered PD-L1 expression. Citation Format: Keaton I. Jones, Jiske Tiersma, Jon Buzzelli, Alex Gordon-Weeks, Arseniy Yuzhalin, Jaehong Im, Ruth J. Muschel. Radiation-induced immunosuppressive macrophages limit CD8 T-cell mediated tumor killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 998.

Host immune cells in the tumor microenvironment play a vital role in tumor development and metastasis. We previously reported that accumulation of CD11b+ Gr1mid- and Gr1low-expressing myeloid cells is important for metastatic progression and growth in a mouse model of liver metastasis. However, little is known about the mechanisms whereby these cells support metastatic development. To characterize the molecular mechanisms by which CD11b+ Gr1mid and Gr1low cells contribute to liver metastasis, MC38 tumor cells were isolated after CD11b+ cell depletion and analyzed using whole-genome expression profiling. CD11b+ Gr1mid- and Gr1low cells accumulate in liver metastases generated by splenic injection of MC38 colon carcinoma and Lewis lung carcinoma cells. The Gr1mid population is recruited to liver metastases where they differentiated into Gr1low cells. This differentiation is stimulated by tumor cells in tissue culture. CD11b+ cells were depleted by DT administration in CD11b-DTR mice, resulting in markedly smaller metastases, decreased proliferation of the tumor cells and greatly reduced metastasis-associated blood vessels. However, depletion had only a negligible effect on tumor cell apoptosis and necrosis. CD11b+ Gr1mid and Gr1low cells enhanced MC38 and LLC tumor cell proliferation, migration and invasion in in vitro co-culture systems. These responses were dependent on ERK1/2 activation and its inhibition in tumor cells minimized the pro-tumorigenic effects of Gr1mid and Gr1low cells. Stimulation of ERK1/2 by Gr1mid and Gr1low cells may explain why depletion of CD11b+ cells diminished proliferation of liver metastases in the murine model. Gene expression analysis of MC38 cells in liver metastases after CD11b+ cell depletion revealed upregulation of ANGPTL7, an angiopoietin-like protein. To test the functional significance of this upregulation, we overexpressed ANGPTL7 in MC38 tumor cells. Overexpression of ANGPTL7 resulted in a striking reduction in tumor burden and tumor-associated vessels. Our findings show that CD11b+ Gr1mid and Gr1low myeloid cells augment proliferation and the migratory and invasive potential of tumor cells in liver metastases by stimulating ERK1/2 signaling. They further suggest that these myeloid cells mediate angiogenesis through upregulation of ANGPTL7 in tumor cells. These studies reinforce the importance of host myeloid cells in sustaining liver metastasis and identify potential targets for therapeutic manipulation. Citation Format: Su Yin Lim, Alex Gordon-Weeks, Lei Zhao, Veerle Kersemans, Danny Allen, Sean Smart, Ruth J. Muschel. Accumulation of CD11b+ Gr1 myeloid cells in liver metastases stimulates tumor growth and angiogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1087. doi:10.1158/1538-7445.AM2014-1087

Metastatic colorectal cancer (CRC) is one of the most prominent causes of cancer-related mortality. However, therapeutic options for patients with metastatic CRC are limited and development of new and more effective therapeutics aimed at downstaging the disease and prolonging survival is urgently required. Primary tumor dissemination to secondary sites, predominantly the liver, is a dynamic process dependent on the interactions between tumor cells and the host tissue microenvironment. Immune cells in the tumor microenvironment have been shown to play pivotal roles in the metastatic process but their pro-metastatic functions in CRC liver metastasis remains incompletely understood. To simulate liver metastasis, MC38 colon carcinoma cells were injected into the spleen of C57BL/6 mice. A population of CD11b/Gr1mid cells, distinct from other metastasis-associated myeloid cells previously described, increased dramatically during establishment of liver metastases. Adoptive transfer of bone marrow cells confirmed that these CD11b/Gr1mid cells were derived from bone marrow progenitor cells. MC38 tumor cells expressed high levels of CCL2 and serum levels were elevated in tumor-bearing mice compared to controls. Inhibition of tumor-derived CCL2 using shRNA, and absence of its cognate receptor in CCR2 KO mice significantly diminished CD11b/Gr1mid cell infiltration into tumor-bearing livers. Importantly, inhibition of CD11b/Gr1mid cell recruitment led to a marked reduction in tumor burden. Moreover, when CD11b/Gr1mid cells were depleted in a transgenic CD11b-DTR mouse model, a striking decrease in tumor vessel density was observed, with a concomitant reduction in tumor cell proliferation. Serum CCL2 concentrations were similarly elevated in CRC patients, including those who present metastasis, compared to normal controls. Furthermore, CD11b/CCR2 myeloid cells, analogous to the CD11b/Gr1mid population described in our mouse model, were identified in liver metastases of several CRC patients. Together, our studies demonstrate the importance of a distinct CD11b/Gr1mid myeloid subset in sustaining CRC liver metastasis and implicate a CCL2/CCR2-dependent pathway in their recruitment to metastatic sites. Our findings reinforce how tumor cells utilize host systems for successful propagation and identify a potential target for therapeutic manipulation. Citation Format: Su Yin Lim, Lei Zhao, Alex Gordon-Weeks, Thomas Tapmeier, Ruth Muschel. Tumor-derived CCL2 promotes recruitment of CD11b/Gr1mid myeloid cells to sustain colorectal cancer liver metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2871. doi:10.1158/1538-7445.AM2013-2871