To uncover why anti-PD-1 therapy fails in the majority of melanomas, Neubert et al. investigated the role of colony-stimulating factor 1 (CSF1) in creating a protumorigenic, macrophage-rich microenvironment. CSF1 is an important regulator of both monocytes and macrophages and controls proliferation and survival of macrophages from their precursors. The results of their experiments, published in Science Translational Medicine, indicate the impact of CSF1 expression and its role in limiting the effectiveness of immune checkpoint blockade therapy via enhancing function and accumulation of M2-like tumor-associated macrophages (TAMs).
Neubert et al. began by observing that the levels of CSF1 were higher in peripheral blood of melanoma patients than healthy volunteers and further elevated in Stage IV than Stage IIIB patients. To uncover a direct relationship between CSF1 and melanoma cells, four low passage melanoma cell lines were tested for CSF1 production in vitro. Media conditioned by any of the four melanoma cell lines showed no or low CSF1 expression levels, however after co-culturing the cell lines with MelanA-specific cytotoxic CD8+ T lymphocyte (CTL) clones, CSF1 could be detected in the conditioned media from each line. Additionally, intracellular CSF1 expression increased in the melanoma cells upon encountering melanoma-specific CTLs, while non-melanoma-specific CTLs didn’t cause secretion of CSF1 from the cell lines. Staining studies of eight human melanoma specimens demonstrated that approximately 80% of melanoma cells expressed CSF1, which correlated with regions of high CD8+ T cell density.
Stimulation was independent of direct cell contact between CTLs and melanoma cells and further experiments determined that the soluble factors derived from CTLs able to induce CSF1 expression in melanoma cells were TNFα and IFNγ. Each cytokine was able to induce CSF1 expression alone, and their combination strongly increased CSF1 expression in all fifteen cell lines tested, with peak concentration of T cell-released IFNγ correlating with the onset of CSF1 expression in cell supernatant. Based on these in vitro studies, TNFα and IFNγ were concluded to be CTL-derived factors able to induce CSF1 expression in melanoma cell lines. The team then utilized a large data set of human metastatic melanoma patient information from The Cancer Genome Atlas (TCGA) to demonstrate that expression levels correlated between CSF1, IFNγ, and TNFα.
TCGA data was also used to examine the relationship between CD8+ T cell and TAM levels and responsiveness to anti-PD-1 blockade. Analysis of the expression of pretreatment tumor biopsies labeled either as responders or nonresponders demonstrated that nonresponders had a strong association between high amounts of CD8+ T cells and expression of CSF1+, CSF1R+, and CD163. These results led the team to conclude that CD8+ T cell presence in human melanoma initiated infiltration of TAMs driven by CSF1, which was produced by melanoma cells following exposure to CTL cytokines, suggesting that cascade of events limited antitumor response. A high ratio of CD8A/CSF1R, indicative of TAM infiltration being less extensive, was found to be indicative of improved overall survival.
The importance of CSF1 was next demonstrated in murine models to “reverse translate” the human observations. The BRAFV600E mutant ovalbumin-expressing SM1 cells (SM1-OVA), which secrete high levels of CSF1 and produce highly macrophage-infiltrated tumors, were monitored for tumor growth inhibition following both single agent anti-PD-1 or anti-CSF1R treatments or combination therapy. The combination therapy proved most effective, with total regression of all tumors after 17 days of treatment and increased CD4+ and CD8+ T cells in the spleens of the mice. A second model, Yummer1.7, which better approximated the high mutational load of human melanoma, demonstrated that combination of anti-CSF1R and anti-PD-1 therapy eradicated the majority of their tumors and significantly extended mouse survival after therapy. In a third transgenic model, iBIP2, combination therapy was unable to inhibit melanoma progression due to an inability to eliminate/repolarize TAMs with the anti-CSF1R antibody. Interestingly, infiltration of multiple myeloid cells was significantly higher in iBIP2 than SM1-OVA tumors; expression analysis indicated high expression of the chemokines Ccl2 and Cxcl12, and the cytokine IL-4 genes, potentially abrogating the impact of the single or combined therapies.
The work presented by Neubert et al. demonstrates the importance of CSF1 in metastatic melanoma, in vitro and in vivo. Anti-CSF1R combination therapy with anti-PD-1 significantly improved tumor regression and overall survival in two of three mouse models, indicating its potential for use in clinical trials. The authors indicate their intention to use CSF1R inhibition with anti-PD-1/PD-L1 in melanomas stratified for an abundance of M2-like TAMs in the tumor microenvironment.
by Brynn Vessey
