Highlights d Flow cytometry, RNA-seq, and protein and image analyses reveal brain TME complexity d Glioma IDH mutation status and brain metastasis primary tumors shape the brain TME d Microglia and monocyte-derived macrophages exhibit multifaceted activation d TME immune cells show disease-and cell-type-specific expression patterns

In normal tissue repair, macrophages exhibit a pro-inflammatory phenotype (M1) at early stages and a pro-healing phenotype (M2) at later stages. We have previously shown that M1 macrophages initiate angiogenesis while M2 macrophages promote vessel maturation. Therefore, we reasoned that scaffolds that promote sequential M1 and M2 polarization of infiltrating macrophages should result in enhanced angiogenesis and healing. To this end, we first analyzed the in vitro kinetics of macrophage phenotype switch using flow cytometry, gene expression, and cytokine secretion analysis. Then, we designed scaffolds for bone regeneration based on modifications of decellularized bone for a short release of interferon-gamma (IFNg) to promote the M1 phenotype, followed by a more sustained release of interleukin-4 (IL4) to promote the M2 phenotype. To achieve this sequential release profile, IFNg was physically adsorbed onto the scaffolds, while IL4 was attached via biotin-streptavidin binding. Interestingly, despite the strong interactions between biotin and streptavidin, release studies showed that biotinylated IL4 was released over 6 days. These scaffolds promoted sequential M1 and M2 polarization of primary human macrophages as measured by gene expression of ten M1 and M2 markers and secretion of four cytokines, although the overlapping phases of IFNg and IL4 release tempered polarization to some extent. Murine subcutaneous implantation model showed increased vascularization in scaffolds releasing IFNg compared to controls. This study demonstrates that scaffolds for tissue engineering can be designed to harness the angiogenic behavior of host macrophages towards scaffold vascularization.

Colony-stimulating factor 1 (CSF1) is a key regulator of monocyte/macrophage differentiation that sustains the protumorigenic functions of tumor-associated macrophages (TAMs). We show that CSF1 is expressed in human melanoma, and patients with metastatic melanoma have increased CSF1 in blood compared to healthy subjects. In tumors, CSF1 expression correlated with the abundance of CD8 + T cells and CD163 + TAMs. Human melanoma cell lines consistently produced CSF1 after exposure to melanoma-specific CD8 + T cells or T cell-derived cytokines in vitro, reflecting a broadly conserved mechanism of CSF1 induction by activated CD8 + T cells. Mining of publicly available transcriptomic data sets suggested co-enrichment of CD8 + T cells with CSF1 or various TAM-specific markers in human melanoma, which was associated with nonresponsiveness to programmed cell death protein 1 (PD1) checkpoint blockade in a smaller patient cohort. Combination of anti-PD1 and anti-CSF1 receptor (CSF1R) antibodies induced the regression of BRAF V600E-driven, transplant mouse melanomas, a result that was dependent on the effective elimination of TAMs. Collectively, these data implicate CSF1 induction as a CD8 + T cell-dependent adaptive resistance mechanism and show that simultaneous CSF1R targeting may be beneficial in melanomas refractory to immune checkpoint blockade and, possibly, other T cell-based therapies.