Immune checkpoint inhibitors are increasingly being used in conjunction with chemotherapy regimens, but the reasons for the success or failure of these combinations remains unclear. In previous studies, we described how blocking TIM-3 promotes activation of dendritic cells through HMGB1-dependent DNA uptake, resulting in efficacy when combined with paclitaxel. Here, we show that the release of HMGB1 by tumor cells is required for the combinatorial efficacy with TIM-3 blockade observed with paclitaxel, docetaxel, fluorouracil, and irradiation. HMGB1 release during taxane therapy is an active process involving nuclear export following Toll-like receptor 4 (TLR4)-dependent reactive oxygen species production, DNA damage, and poly(ADP-ribose) polymerase activation. DNA damage promotes the accumulation of cytosolic double-stranded DNA (dsDNA), which activates the cGAS-STING pathway; however, taxanes fail to induce type I interferons. Instead, STING activation promotes endoplasmic reticulum (ER) stress and lysosomal exocytosis, driving HMGB1 secretion. Thus, non-canonical STING signaling in response to taxanes can promote the efficacy of chemoimmunotherapy.

We present a protocol to study cancer cell death in vivo using mouse tumor models by generating cell lines that allow for the induction of "pure" apoptosis or necroptosis via an inducible dimerizer system. We describe steps for lentiviral transduction and flow cytometry-based optimization of the death induction schedule. We then detail optional procedures for "intradermal vaccination" and for tracking antigen-specific CD8+ T cell responses. For complete details on the use and execution of this protocol, please refer to Hänggi et al.1.

BACKGROUND:A significant percentage of melanomas are refractory to immune checkpoint inhibitor (ICI) monotherapies and combinations. As there are currently no effective second-line therapies available for ICI-resistant patients, we sought to identify novel checkpoint inhibitor combinations for future clinical evaluation. METHODS:We used responding and resistant SM1, SW1 and B16-10 melanoma models to explore the mechanism of action of anti-programmed cell death protein 1 (PD-1)+lymphocyte activation gene (LAG-3), anti-PD-1+cytotoxic lymphocyte associated protein 4 (CTLA-4), the anti-PD-1+LAG-3+CTLA-4 triplet, the anti-PD-1+T-cell immunoglobin and mucin domain 3 (TIM-3) doublet and the anti-PD-1+LAG-3+TIM-3 triplet. Flow cytometry, tetramer binding assays, T-cell depletion and analysis of the tumor-draining lymph nodes were used to determine the mechanisms of action of the ICI combinations. Single-cell RNA sequencing (scRNA-Seq) data from patients with melanoma was additionally analyzed to determine immune checkpoint expression across T-cell subsets and their association with ICI resistance. RESULTS:The PD-1+CTLA-4+LAG-3 triplet combination had good efficacy in the SM1 and B16-F10 melanoma models and led to a more favorable immune microenvironment (increased CD69+CD8+ T cells and gp-100-tumor specific T cells, reduced exhausted T cells) than either doublet. The ICI-resistant SW1 model did not respond to the PD-1+CTLA-4+LAG-3 triplet and was characterized by an accumulation of TIM-3+terminally exhausted CD8+T cells. Targeting of TIM-3 using the anti-PD-1+LAG-3+TIM-3 triplet led to complete tumor regressions in the SW1 and SM1 models. The TIM-3-based triplet decreased exhausted CD8+T cells and regulatory T cells, increased numbers of gp-100 reactive CD8+T cells and increased progenitor-exhausted CD8+T cells (TCF7+Ki67+ TIM-3-) numbers in tumor-draining lymph nodes. scRNA-Seq analysis demonstrated that TIM-3 levels were higher in the CD8+T cells of patients with melanoma who were non-responsive to immunotherapy. PD-1 and TIM-3 expression was strongly correlated in the CD8+T cells from patients with melanoma, providing the rationale for co-targeting these checkpoints. CONCLUSION:Melanoma models characterized by high levels of terminally exhausted CD8+T cells (TIM-3+) can be successfully treated with the anti-PD-1+LAG-3+TIM-3 combination. The observation that high TIM-3 expression in CD8+T cells is associated with ICI resistance suggests the potential utility of anti-TIM-3-based combinations as a second-line treatment strategy in patients with advanced melanoma.

Prostate cancer (PC) progression is closely tied to androgen receptor activity. While androgen deprivation therapy (ADT) initially shows efficacy, 20-30% of patients develop resistance within 5 years. Previous studies in mice implicated macrophages in this resistance by transferring cholesterol, a critical molecule for androgen production. This study aims to uncover the mechanism of cholesterol transfer to identify new therapeutic targets to enhance ADT effectiveness. PC cell lines were co-cultured with primary macrophages or RAW264.7 cells labeled with fluorescent cholesterol. The transfer of cholesterol was monitored using flow cytometry and live microscopy. Within 24 hours, PC cells acquired cholesterol from macrophages in a contact-dependent manner. This process was independent of macrophage polarization or scavenger receptor SR-B1, which is often upregulated in PC. Live imaging revealed direct transfer of cholesterol-rich plasma membrane from macrophages to PC cells. This was further confirmed by labeling macrophage plasma membranes and tracking the transfer of surface receptors and vital organelles, which increased PC cell proliferation in vitro. Macrophages promote androgen production in PC cells by transferring cholesterol-rich plasma membranes via direct contact. Future research will explore the molecular drivers of this process through genomic screens and investigate its role in PC growth and resistance to ADT. Supported by Mike Slive Foundation and Miles for Moffitt. Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

The precise mechanisms by which the complement system contributes to the establishment of an immunosuppressive tumor microenvironment and promotes tumor progression remain unclear. In this study, we investigated the expression and function of complement C5a receptor 1 (C5aR1) in human and mouse cancer-associated dendritic cells (DC). First, we observed an overexpression of C5aR1 in tumor-infiltrating DCs, compared with DCs from the blood or spleen. C5aR1 expression was restricted to type 2 conventional DCs and monocyte-derived DCs, which displayed a tolerogenic phenotype capable of inhibiting T-cell activation and promoting tumor growth. C5aR1 engagement in DCs drove their migration from tumors to tumor-draining lymph nodes, where C5a levels were higher. We used this knowledge to optimize an anticancer therapy aimed at enhancing DC activity. In three syngeneic tumor models, C5aR1 inhibition significantly enhanced the efficacy of poly I:C, a Toll-like receptor 3 agonist, in combination with PD-1/PD-L1 blockade. The contribution of C5aR1 inhibition to the antitumor activity of the combination treatment relied on type 1 conventional DCs and antigen-specific CD8+ T cells, required lymphocyte egress from secondary lymphoid organs, and was associated with an increase in IFNγ signaling. In conclusion, our study highlights the importance of the C5a/C5aR1 axis in the biology of cancer-associated DCs and provides compelling evidence for the therapeutic potential of modulating the complement system to enhance DC-mediated immune responses against tumors.

Tumors growing in metabolically challenged environments, such as glioblastoma in the brain, are particularly reliant on crosstalk with their tumor microenvironment (TME) to satisfy their high energetic needs. To study the intricacies of this metabolic interplay, we interrogated the heterogeneity of the glioblastoma TME using single-cell and multi-omics analyses and identified metabolically rewired tumor-associated macrophage (TAM) subpopulations with pro-tumorigenic properties. These TAM subsets, termed lipid-laden macrophages (LLMs) to reflect their cholesterol accumulation, are epigenetically rewired, display immunosuppressive features, and are enriched in the aggressive mesenchymal glioblastoma subtype. Engulfment of cholesterol-rich myelin debris endows subsets of TAMs to acquire an LLM phenotype. Subsequently, LLMs directly transfer myelin-derived lipids to cancer cells in an LXR/Abca1-dependent manner, thereby fueling the heightened metabolic demands of mesenchymal glioblastoma. Our work provides an in-depth understanding of the immune-metabolic interplay during glioblastoma progression, thereby laying a framework to unveil targetable metabolic vulnerabilities in glioblastoma.

Immunotherapy response is associated with the presence of conventional dendritic cells (cDCs). cDC type 1 (cDC1) is critically important for CD8+ T cell activation, cDC type 2 (cDC2) regulates CD4+ T cell responses, and mature regulatory cDCs may dampen T cell responses in the tumor microenvironment (TME). However, we lack a clear understanding of cDC distribution in the human TME, cDC prevalence in metastatic sites, and cDC differences in early- versus late-stage disease. Rapid autopsy specimens of 10 patients with lung adenocarcinoma were evaluated to detect cDCs and immune cells via multiplex immunofluorescence using 18 markers and 42 tumors. First, we found that T cells, cDC1, and cDC2 were confined to stroma, whereas mature regulatory DCs were enriched in tumor, suggesting unique localization-specific functions. Second, lung and lymph node tumors were more enriched in T cells and cDCs than liver tumors, underscoring differences in the TME of metastatic sites. Third, although the proportion of T cells and cDC1 did not differ in different stages, an increase in the proportion of cDC2 and macrophages in late stage suggests potential differences in regulation of T cell responses in different stages. Collectively, these findings provide new, to our knowledge, insights into cDC biology in human cancer that may have important therapeutic implications.