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Brain metastases (BrM) remain an intractable, deadly complication for advanced melanoma patients and efficient therapeutic strategies are desperately needed. The tumor microenvironment (TME) plays an important role in response to therapy. However, studies addressing the contribution of the TME to therapy efficacy for BrM are lacking, mostly due to limited access to human samples and scarcity of appropriate preclinical models. Here, we describe two novel isogenic immunocompetent BrM models generated by intracardiac injection of UV-induced mouse melanoma cell lines, representative of mutant-RAS human melanoma subtypes. We used these models to test immune checkpoint blockade (ICB) therapy and to interrogate the role of the TME in therapeutic efficacy. To evaluate response, we developed and applied a new machine-learning method to quantify metastatic burden. We investigated the TME by high-parametric flow cytometry and single-cell RNA sequencing (scRNA-seq). We showed that the models have distinct metastatic behaviors, with BR1 being mostly brain tropic and BR3 displaying widespread metastases. Notably, BR1 BrM were sensitive to ICB with a better response to anti-PD-L1/anti-CTLA-4 combination therapy as compared to monotherapies. In contrast, BR3 BrM were resistant to both mono- and combination therapies. Interestingly, we found that ICB efficacy on extracranial BR3 metastases is organ-dependent. Characterization of the BrM immune microenvironment before and after treatment revealed dramatic differences between the models. Untreated BR1 BrM showed significant recruitment of T cells, dendritic cells, and natural killer cells, while neutrophils were enriched in untreated ICB-resistant BR3 BrM. Moreover, we uncovered phenotypically distinct microglia populations exclusively present in ICB-sensitive BR1 BrM that positively correlated with T cell infiltration. Consistent with this finding, scRNA-seq showed upregulation of genes encoding for T cell-attracting chemokines and antigen presentation uniquely in the BR1-associated microglia. Post-treatment analysis of the brain TME highlighted beneficial changes induced by ICB in the responsive BR1 model, including increased recruitment of CD8 T cells with an activated phenotype, while a mild recruitment of exhausted T cells was observed in the resistant BR3 model. Altogether, our data emphasize the importance of interrogating the BrM TME to understand therapeutic response. Our unique BrM models, mirroring the diversity of ICB response observed in patients, provide a robust platform for the much-needed mechanistic studies to optimize BrM therapy. Deciphering the contribution of the newly identified BR1 BrM-associated microglia to ICB efficacy will be crucial to the identification of novel therapeutic targets. Citation Format: Amélie Lopès, Jessica Rappaport, Eva Pérez Guijarro, Quanyi Chen, Emily Wu, Isabella Church, April Huang, Jessica Bridge, Sung Chin, Cari Smith, Charli Gruen, Khiem C. Lam, Romina E. Araya, Antonella Sassano, Chi-Ping Day, Glenn Merlino, Romina S. Goldszmid. Interrogating the role of the immune microenvironment in the response of brain metastases to immunotherapy using new preclinical melanoma models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5188.

Previous studies have shown that adoptive cell transfer (ACT) of regulatory T cells (Tregs) mitigate AAA growth in mice, however these studies have used a dissection AAA model and have not thoroughly studied Treg function in mice after ACT. Treg suppression of systemic or local inflammation has not been rigorously studied in our model. We therefore sought to evaluate the effects of Treg ACT in mice with a specific focus on Treg migration, colonization, and inflammatory changes within local tissue. Methods: AAA was induced in B57Bl/6 mice expressing the Thy 1.2 allele using a topical elastase + BAPN model (E-BAPN). Heat inactivated elastase (HiE-BAPN) was used as a control. On postoperative day (POD) 2, animals were treated with PBS (E-BAPN-PBS) injection or ACT of 2 million Tregs (E-BAPN-Tregs). CD4+ CD25+ FoxP3+ Tregs used for ACT were isolated from B57Bl/6 mice expressing the Thy 1.1 allele. Mice underwent surveillance ultrasounds (US) weekly to track AAA growth for 42 days. At specific time points mice were sacrificed and aortic diameter was recorded. Aortic tissue and regional lymph nodes were harvested for analysis. Results: Maximum aortic diameter (MAD) by both US and video microscopy was significantly higher in the AAA group (E-BAPN vs. HiE-BAPN; pThy1.1+ Tregs were detected in the tissue and regional aortic lymph nodes (LN) until day 42 with only a slight declination in the percent Thy1.1 + cells over time. Thy1.1+ Tregs retained phenotypic markers over time. E-BAPN-Treg mice had significantly altered inflammatory markers compared to E-BAPN-PBS mice including local macrophage and T-cell composition as well as inflammatory cytokines. Conclusion: Treg ACT suppress AAA growth in a topical elastase + BAPN mouse model through tissue and LN colonization and alteration of the local inflammatory milieu.

Introduction: Regulatory T cells (Tregs) suppress inflammation in atherosclerosis, and therefore have the therapeutic potential to reduce the risk of MI and stroke. However, there is currently no method to generate antigen specific Tregs that target atherosclerosis. We therefore engineered Tregs that express a Chimeric Antigen Receptor (CAR) targeting malonaldehyde-modified LDL (MDA-LDL), the most common form of oxidized LDL and a key molecular component of atherosclerosis. Methods: Novel single chain variable fragments (scFv) were synthesized using sequences from antibodies targeting human MDA-LDL. Oxidized-LDL specific CARs (ox-CARs) were subsequently engineered by fusing each scFv to an IgG4 hinge, CD28 transmembrane and CD28/CD3z cytoplasmic domains. CD4 + CD25 + CD127 low/- Tregs were purified from human blood via FACS and lentivirally transduced to express the novel ox-CARs (ox-CAR-Tregs). Human atherosclerotic plaques were obtained from patients undergoing carotid endarterectomy (CEA). Autologous ox-CAR-Tregs were analyzed for activation after ex-vivo co-culture with CEA samples. Results: A rationally designed panel of 42 ox-CARs were engineered using scFv derived from 12 antibodies targeting MDA-LDL. We first assessed CAR expression and activation in Jurkat T cells to identify promising ox-CAR variants for further evaluation in human Tregs. After culture in the presence of MDA-LDL, six ox-CAR-Treg variants consistently showed significant activation, compared to controls, based on CD71 expression, cytokine expression and proliferation in the absence of CD3/28 stimulation. Human atherosclerotic samples were identified to have substantial amounts of MDA-LDL epitopes using IHC. Autologous ox-CAR-Tregs showed a dose-dependent increase in CD71 expression after ex-vivo co-culture with atherosclerotic plaque. Conclusion: An optimized CAR targeting MDA-LDL activates Tregs when cultured with human atherosclerotic plaque ex-vivo.

Neutrophils play an important role in cancer progression and both pro- and antitumorigenic functions have been described. However, their role in response to therapy and whether environmental signals modulate their function in the tumor microenvironment (TME) remain unclear. Here, we show that neutrophil content in the TME defines the response to chemo- and immunotherapy. Neutrophil depletion or recruitment blockade impaired the response, while overexpression of neutrophil-attracting chemokines rendered non-responder tumors susceptible to therapy. Importantly, we demonstrate that tumor neutrophils are a heterogeneous and dynamic population that differ from those found in blood or bone marrow, and we identified a neutrophil subset that correlates with therapy response. Additionally, we found that mice lacking microbiota have impaired neutrophil recruitment and function associated with reduced therapy efficacy. Mechanistically, administration of a microbiota-derived NOD2 ligand to mice lacking microbiota was sufficient to restore neutrophil dynamics and response to therapy. We confirmed tumor neutrophil heterogeneity in cancer patients and showed that while total neutrophil content had no predictive value for patient’s overall survival, two specific neutrophil subsets associated with better outcome for pancreatic cancer patients. Remarkably, the subset enriched in non-responder germ-free tumors in our preclinical studies associated with a similar poor outcome in colorectal cancer patients. Collectively, our findings highlight the importance of characterizing neutrophil heterogeneity in the TME and the contribution of microbiota in shaping these cells to predict a successful therapy outcome. Supported by NIH Intramural Program

Gut microbiota impacts antitumor immunity and recent studies have shown that manipulating microbiota via fecal transplant can rescue immune checkpoint blockade (ICB) response in refractory melanoma patients. However, not all patients benefited from this approach. We hypothesize that tumor-intrinsic factors (e.g. neoantigen load and immune microenvironment) contribute to the microbiota effect on anticancer response. To test this, we performed preclinical microbiota manipulation studies using dietary intervention. Mice fed high-fiber diet (FD) had improved spontaneous tumor control and response to ICB. Mechanistically, FD-induced changes in microbiota composition skewed the tumor innate immune repertoire towards an antitumor profile marked by increased dendritic cell infiltration. Downstream of the innate compartment, FD reduced the proportion of intratumoral Tregs and exhausted CD8+ T cells. We further assessed the effects of FD across 8 tumor models including EL4 lymphoma, MC38 colon carcinoma, and 6 melanomas (M1–M6) that mimic the variety of human melanoma subtypes. Although FD had a beneficial effect overall, not all models responded to the same extent, and were accompanied by different changes in the tumor immune profile. FD delayed tumor initiation for EL4 and M4, reduced growth rate for M3 and M5, both delayed initiation and reduced growth rate for MC38, M2, and M6, but had no significant effects for M1. Additionally, we found that increasing tumor immunogenicity resulted in a synergistic effect with FD. Our data suggests that tumor-intrinsic factors influence the beneficial effect of microbiota on antitumor immunity. Identifying these factors will help refine the use of microbiota-based therapies in the clinic. This research was supported by the Intramural Research Program of the NIH (CCR-NCI). This research was supported by the Intramural Research Program of the NIH (CCR-NCI).

The work of the Scollons contains a number of conceptual developments that open new pathways towards a maturely theorized ethnography. Two such developments are discussed in this paper: the emphasis on the body, and the emphasis on space. Both developments show how the Scollons connected synchronic events with historical patterns of becoming. Their notion of the ‘historical body’ sees the body as enskilled through trajectories of learning and acquisition, and their notion of space sees space as demarcating and normative. This connection between synchrony and history enables ethnography to transcend the problem of atemporality and anecdotism.

The syndrome of cancer cachexia is currently defined as a state of ill health, malnutrition and physical wasting with marked white adipose tissue (WAT) and skeletal muscle mass wasting, representing the clinical consequence of a chronic and systemic inflammatory response. Over the last decade, WAT has been recognized as an important endocrine organ, and earning a lot of attention during cancer cachexia development. We investigated the role of microbiota along the cachexia associated cancer. We performed experiments with conventional and Germ Free mice (GF) (n=6 in each group) of 8-10 weeks old C57B/6, which were subcutaneously injected with LLC cells [4x106 cells in 0.2 mL; Tumor-bearing, (TB) or PBS control (C)]. We performed Immunohistochemistry, RT- qPCR, and Western Blot. We observed that GF Tumor-bearing mice have increased several symptoms of the cachexia compared to conventional TB mice. The WAT mass was decreased 50% in GF Tumor-bearing mice compared to all groups, which indicates a pathway related to lipolysis, as we found increased level of phosphorylated enzymes in GF Tumor-bearing mice. We also observed that GF Tumor bearing mice decreased skeletal muscle mass and gene expression that are related with atrophy were increased in GF Tumor bearing mice. Our data suggested that homeostasis of microbiota may impair the development of the cachexia syndrome. Citation Format: Rodrigo Xavier das Neves, Soumen Roy, Amiran Dzutsev, April Huang, Loretta Smith, Simone Difilippantonio, Hawes Misty, Marília Seelaender, Giorgio Trinchieri. Germ free mice accelerate cachexia-associated cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 451. doi:10.1158/1538-7445.AM2017-451

Chemotherapy induced toxicity severely affect the cancer survivors and lowers the quality of life. By 2020, there will be more than 18 million of cancer survivors all over the world. Majority of them might develop long term nephrotoxicity, ototoxicity and gut toxicity. In addition, chemotherapy may also facilitate the initiation and progression of cachexia. Recent studies have shown that gut microbiota modulates the efficacy of anti-cancer chemotherapy, however very limited knowledge is available regarding the role of gut microbiota in regulating systemic toxicity and cachexia. We hypothesized that gut microbiota modulates cisplatin induced systemic toxicity as well as cachexia. Four groups (n=10 in each group) of 6-8 weeks old C57B/6 mice were treated with cisplatin, cisplatin+antibiotics cocktails (ABX), ABX only and control. ABX cocktail contained primaxin, vancomycin and neomycin. This experiment was validated using C57B/6 germ free mice. We performed anti-p-γ-H2AX based toxicity assay for DNA damage. In addition, we performed immunohistochemistry for studying cachexia. H&E staining and DNA damage were assessed by light and super resolution confocal microscopy (Zeiss 880) and quantified by 3-D reconstruction using IMARIS. We also performed high content imaging to evaluate DNA damage in bone marrow and spleen. Body weight and kidney blood content were also analyzed to determine the degree of toxicity. Depletion of gut microbiota significantly reduced nephrotoxicity and gut toxicity. In addition, we found that depletion of gut microbiota prevents muscle and adipose tissue loss by down-regulating UCP-1and PGC-1α in adipose tissue and MURF-1 and Atrogin-1 in muscle. Experiments are confirmed by using Germ free mice. Our data suggest that modulation of gut microbiota may be utilized to reduce chemotherapy associated cachexia and systemic toxicity. Citation Format: Soumen Roy, Rodrigo Das Neves, Amiran Dzutsev, Carolyne Smith, Bathai Edwards, Miranda Dawson, Simone Difilippantonio, Loretta Smith, April Huang, Young Kim, Giorgio Trinchieri. Gut microbiota regulates cisplatin mediated cachexia and systemic toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4926. doi:10.1158/1538-7445.AM2017-4926