br Pre clinical combination studies using CSF CSF
Pre-clinical combination studies using CSF-1/CSF-1R inhibitors In preclinical models, the CSF-1R pathway can be blocked by using either small molecule kinase inhibitors (GW2580 , PLX3397 , Ki20227 , BLZ945  and CYC11645 ), antibody-mediated inhibition of CSF-1 using 5A1  or anti-CSF-1R roscovitine such as AFS98 , M279  and 2G2  (Figure 1B). However, CSF-1R blockade alone has only marginal therapeutic benefit resulting at best in a delay of tumor growth. Therefore, various combination partners for CSF-1R-mediated TAM depletion are currently under investigation (Figure 2). DeNardo reported for the first time that CSF-1R pathway inhibition via PLX3397 or 5A1 antibody treatment resulted in enhanced efficacy in the transgenic breast cancer model MMTV-PyMT, when combined with paclitaxel . The authors provided evidence that this efficacy was mediated by increased CD8+ T cells numbers. In a subsequent study the underlying mechanism of the enhanced efficacy of this combination was attributed to TAM-derived IL-10, suppressing DC-mediated T cell activation . Mitchem described that the chemotherapeutic agent gemcitabine in combination with GW2580 increased the efficacy of controlling pancreatic carcinoma growth compared to GW2580 alone : When the tumor cells were co-cultured with TAM, they were found to be less sensitive to gemcitabine. In turn, gemcitabine induced increased CSF-1 release from tumor cells and thereby recruited immunosuppressive myeloid cells into the tumor which limited the activity of CD8+ T lymphocytes . Among the clinically established therapeutic modalities, which have been evaluated in combination with TAM targeting agents, irradiation showed enhanced efficacy in a prostate cancer model in combination with PLX3397 . A similar mechanistic explanation of increased CSF-1 expression derived from the irradiated tumor cells followed by enhanced recruitment of TAM and myeloid-derived suppressor cells (MDSC) was provided by the authors. The combination of PLX3397 together with the mTOR inhibitor rapamycin showed higher efficacy than each monotherapy in malignant peripheral nerve sheath tumor cell lines and xenograft model . TAM, particularly the perivascular Tie2 expressing subpopulation, have been implicated in angiogenesis and evasion of anti-angiogenic therapies [41, 42]. Treatment of an osteosarcoma model characterized by high CSF-1 secretion with the anti-mouse CSF-1R antibody AFS98 reduced the number of peritumoral macrophages associated with decreased angiogenesis and tumor growth . When the CSF-1R tyrosine kinase inhibitor GW2580 was combined with an anti-VEGFR-2 blocking antibody in the syngeneic 3LL model, Priceman demonstrated that abrogation of TAM and monocytic MDSC recruitment effectively prevented the tumor from evading anti-angiogenic therapy . A mechanistic insight was provided by the Eubank team showing that CSF-1 mediates increased Tie2 expression in human monocytes, which secrete higher levels of VEGF, and expands the Tie2+ macrophage population in PyMT tumor bearing mice . The recent approval of ipilumumab, nivolumab and pembrolizumab based on durable responses in malignant melanoma patients in conjunction with a reasonable safety profile has led to great enthusiasm in the immunotherapy field [45••, 46]. One major obstacle limiting the efficacy of immune checkpoint inhibitors is the local immunosuppressive milieu within the tumor. The immunosuppressive activity of TAM has been well documented and includes i) the regulation of T cell proliferation by depriving T cells of tryptophan via indoleamine dioxygenase (IDO), ii) from l-arginine required for T cell receptor expression via arginase I, iii) secretion of T regulatory cell recruiting chemokines and immunosuppressive cytokines IL-1Ra and IL-10 [47, 48, 49]. Hence, immunotherapies represent an attractive combination partner for CSF-1R inhibitors to achieve tumor rejection in immunocompetent mouse models. Till date, the combination of CSF-1R kinase inhibitors with PD-1 and CTLA-4 blocking antibodies as well as adoptive T cell transfer have been published [50, 51, 52••]. Zhu reported that CSF-1R kinase inhibitors preferentially depleted the mannose receptor CD206 expressing and immunosuppressive macrophage population while the remaining TAM increased their antigen presenting capacity. These changes in the macrophage infiltrate led to superior efficacy of the triple combination of immune checkpoint and CSF-1R kinase inhibitors together with gemcitabine in transplanted pancreatic cancer models .