Effects of IL-10 and Th 2 cytokines on human Mφ phenotype and response to CSF1R inhibitor.

Tumor-associated Mφs display a plastic phenotype that is regulated by the local tumor milieu. Gene expression analysis and functional characterization of Mφs exposed in vitro to individual cytokines aids to delineate the cross-talk between defined cytokines shaping the complex Mφ phenotype. Human monocyte-derived Mφs can be differentiated in vitro with the T helper cell type 2 response cytokines IL-4 and IL-13 or the immunosuppressive IL-10. Notably, only the latter subset undergoes apoptosis when treated with the CSF 1 receptor (CSF1R) blocking antibody emactuzumab. However, under physiologic conditions, the Mφ phenotype is regulated by cytokine combination. Hence, in this study, we characterized the plasticity of IL-4 or IL-13-differentiated Mφs upon exposure to the immunosuppressive IL-10. Although IL-4-differentiated Mφs sustained their molecular phenotype in the presence of IL-10, IL-13-differentiated Mφs were skewed towards the IL-10 phenotype. Gene expression profiling revealed unique IL-4+IL-10 and IL-13+IL-10 Mφ signatures associated with up-regulation of canonical NF-κB or Wnt/ß-catenin signaling pathways, respectively. Although IL-10 was able to alter the surface marker and gene expression profile of IL-13-differentiated Mφs, addition of IL-10 did not restore emactuzumab susceptibility. Combining NF-κB and Wnt/ß-catenin signaling inhibitors with emactuzumab had no effect on viability. On average 3-5% of cancer patients overexpressed IL-4, IL-13, or IL-10 mRNA in silico. Although a small patient subset overexpressed IL-10+IL-13, IL-4+IL-10 lacked co-expression. In vitro characterization of CSF1R inhibitor-refractory Mφ phenotypes can support novel pharmacological approaches to specifically target these cells.

Macrophage Susceptibility to Emactuzumab (RG7155) Treatment.

Blockade of colony-stimulating factor-1 receptor (CSF-1R) enables the therapeutic targeting of tumor-associated macrophages (TAM) in cancer patients. Various CSF-1R inhibitors, mAbs, and tyrosine kinase inhibitors are currently evaluated in early clinical trials. Presence of an alternative survival signal, such as GM-CSF, rescues human monocyte-derived macrophages from CSF-1R inhibitor-induced apoptosis. In this study, we sought to identify additional factors that mediate resistance to CSF-1R-blocking antibody RG7155 (emactuzumab). We investigated the impact of hypoxia, macrophage-polarizing cytokines IL4 and IL10, and genetic alterations within the CSF1R locus and mitochondrial DNA. Among all investigated factors, only IL4 completely rescued viability of RG7155-treated macrophages in vitro This RG7155-resistant population was characterized by a substantially increased mannose receptor-1 (CD206) expression. Analysis of CD206 and the hemoglobin scavenger receptor CD163 expression on normal tissue allowed for discrimination of distinct macrophage populations according to localization and frequency. In emactuzumab-treated cancer patients, we found a significant reduction of CSF-1R, CD204, and CD163 mRNA levels in contrast to a less pronounced decrease of CD206 expression by transcriptome analysis of tumor biopsies. However, we detected in normal skin tissue, which shows lower IL4 mRNA expression compared with melanoma tissue, significant reduction of CD206+ dermal macrophages in RG7155-treated skin biopsies. These results suggest that in cancers where the cytokines IL4 and GM-CSF are sufficiently expressed to induce very high CD206 expression on macrophages, CSF-1R inhibition may not deplete CD206hi TAM. This observation can help to identify those patients most likely to benefit from CSF-1R-targeting agents. Mol Cancer Ther; 15(12); 3077-86. ©2016 AACR.

Fibroblasts Influence Survival and Therapeutic Response in a 3D Co-Culture Model.

In recent years, evidence has indicated that the tumor microenvironment (TME) plays a significant role in tumor progression. Fibroblasts represent an abundant cell population in the TME and produce several growth factors and cytokines. Fibroblasts generate a suitable niche for tumor cell survival and metastasis under the influence of interactions between fibroblasts and tumor cells. Investigating these interactions requires suitable experimental systems to understand the cross-talk involved. Most in vitro experimental systems use 2D cell culture and trans-well assays to study these interactions even though these paradigms poorly represent the tumor, in which direct cell-cell contacts in 3D spaces naturally occur. Investigating these interactions in vivo is of limited value due to problems regarding the challenges caused by the species-specificity of many molecules. Thus, it is essential to use in vitro models in which human fibroblasts are co-cultured with tumor cells to understand their interactions. Here, we developed a 3D co-culture model that enables direct cell-cell contacts between pancreatic, breast and or lung tumor cells and human fibroblasts/ or tumor-associated fibroblasts (TAFs). We found that co-culturing with fibroblasts/TAFs increases the proliferation in of several types of cancer cells. We also observed that co-culture induces differential expression of soluble factors in a cancer type-specific manner. Treatment with blocking antibodies against selected factors or their receptors resulted in the inhibition of cancer cell proliferation in the co-cultures. Using our co-culture model, we further revealed that TAFs can influence the response to therapeutic agents in vitro. We suggest that this model can be reliably used as a tool to investigate the interactions between a tumor and the TME.

Targeting tumor-associated macrophages with anti-CSF-1R antibody reveals a strategy for cancer therapy.

Macrophage infiltration has been identified as an independent poor prognostic factor in several cancer types. The major survival factor for these macrophages is macrophage colony-stimulating factor 1 (CSF-1). We generated a monoclonal antibody (RG7155) that inhibits CSF-1 receptor (CSF-1R) activation. In vitro RG7155 treatment results in cell death of CSF-1-differentiated macrophages. In animal models, CSF-1R inhibition strongly reduces F4/80(+) tumor-associated macrophages accompanied by an increase of the CD8(+)/CD4(+) T cell ratio. Administration of RG7155 to patients led to striking reductions of CSF-1R(+)CD163(+) macrophages in tumor tissues, which translated into clinical objective responses in diffuse-type giant cell tumor (Dt-GCT) patients.