Cancer Research Research Articles

Abstract C047: AGX101: Preclinical evaluation of a TM4SF1-directed tubulin inhibitor conjugate with ongoing first-in-human trial

Abstract Introduction: TM4SF1 (Transmembrane-4 L-Six-Family-Member-1) is an endothelial marker with critical roles in angiogenesis, as well as a tumor cell antigen that contributes significantly to invasion and metastasis. TM4SF1 is upregulated 20-fold in angiogenic tumor vascular endothelium compared to normal vasculature endothelium and exhibits a unique nuclear internalization pathway. AGX101 is a novel tubulin inhibitor conjugate specifically directed against TM4SF1, delivering a potent maytansinoid payload directly to the nucleus of cells within the tumor microenvironment. Delivery to both the vascular and tumor cell compartments of the tumor results in three mechanisms of action (MoAs): (1) activation of tumor immune surveillance, (2) tumor blood supply deprivation, and (3) direct tumor cell killing. Methods: TM4SF1 expression was assessed across solid tumor samples using immunohistochemistry. Safety and pharmacokinetics of AGX101 were evaluated in non-human primates, with escalating doses to determine the highest non-severely toxic dose (HNSTD). Efficacy studies were also conducted in mouse models, enabling assessment of the minimum effective dose (MED) needed to engage each of the three MoAs. The combination of HNSTD and MED enables calculation of a therapeutic index (TI). Preclinical efficacy studies used either AGX101 (in human tumor xenograft models, to study the tumor cell killing MoA) or AGXB01, a rodent surrogate of AGX101, to study vascular and immune MoAs and all three MoAs in syngeneic mouse cancer models. The potential for synergy with immune checkpoint inhibitors (ICIs) was also investigated. A first-in-human study of AGX101 in patients with advanced solid tumors with the primary objective of safety and dose-limiting toxicities (DLTs) has initiated. Results: TMFS41 is broadly expressed across solid tumors with increasing scoring intensity in higher grade tumors. In non-human primates, AGX101 exhibited a favorable safety profile, being tolerated at relatively high doses and with dose dependent, monitorable, and reversible effects. In preclinical efficacy studies, AGXB01 and AGX101 as monotherapies demonstrated robust efficacy through each of the three MoAs in multiple syngeneic, xenograft, and orthotopic tumor models in mice and rats as well as non-tumor models of angiogenesis. Measured by exposure, TI was large. Additionally, AGXB01 was shown to potentiate the effects of ICIs in syngeneic mouse models (CT26, Renca, and B16-F10), suggesting a potential synergistic approach in cancer therapy. The first dose level of AGX101 monotherapy in humans has cleared without DLTs. Conclusion: AGX101, targeting the TM4SF1 antigen, represents a promising new approach in cancer therapy. The preclinical data suggest that AGX101 could provide a significant therapeutic benefit by novel and differentiated mechanisms of action, namely selectively targeting the tumor vasculature and potentiating immune-based therapies. Further clinical development of AGX101 is ongoing. Citation Format: Ildefonso Ismael Rodriguez Rivera, Meredith S. Pelster, Shou-Ching Jaminet, Paul Jaminet, Glen J. Weiss. AGX101: Preclinical evaluation of a TM4SF1-directed tubulin inhibitor conjugate with ongoing first-in-human trial [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C047.

Abstract B006: Role of the BAF complex in pancreatic cancer-associated fibroblast function

Abstract Fibroblasts comprise the majority cell type within connective tissue and play an important role in the support and repair of every organ. This homeostatic support occurs via the activation of fibroblasts (e.g. myofibroblasts) that then secrete growth factors, cytokines, and components of the extracellular matrix (ECM) that can further modulate residential cells. It has been demonstrated that cell-derived ECMs (CDMs) alone, generated in vitro, are sufficient to activate or maintain naive fibroblasts depending on the activation status of the fibroblasts that generated the CDM. Using this CDM system, we published that Netrin G1 is a regulator of pancreatic cancer-associated fibroblast (CAF) function that is pathologically expressed, and that its expression is dependent on the ECM. The mechanistic details behind how the ECM regulates fibroblasts’ gene expression to alter its function and activation status, however, is currently unknown. We previously showed that loss of the β5 integrin subunit negatively impacts the ability of pancreatic CAFs to maintain a constitutively activated state and dampens their pro- tumorigenic functions. RNA sequencing of these β5-deficient CAFs showed altered expression of Netrin G1, as well as members of the BAF (BRG/BRM-associated factor) family of chromatin remodelers. The goal of this project is to elucidate the role of the BAF complex in mediating the outside-in signaling from the ECM. To determine the role of the BAF complex on fibroblast activation and function, knockdown (KD) cell lines for the ATP-subunits Brg1 and Brm1 were generated in patient-derived pancreatic CAFs. Akin to β5 KD CAFs, loss of both Brg1 and Brm1 decreased the expression of published TGFβ-dependent markers of pancreatic CAF activation. However, unlike β5 KD CAFs that are unable to generate substantial, aligned CDMs, Brg1 and Brm1 KD CAFs had no effect on matrix deposition or alignment; like Netrin G1 KD CAFs that have decreased pro-tumor functions. Indeed, loss of Brm1 appears to decrease Netrin G1 akin to β5 KD CAFs. Expression levels of Netrin G1 were not restored in β5 nor Brm1 KD CAFs treated with recombinant TGFβ-1 during CDM production, whereas the TGFβ-dependent activation markers were. Collectively, these data begin to suggest that pro-tumorigenic ECM- mediated signaling may alter fibroblast gene expression via the BAF complex in a TGFβ dependent manner, opening the door to epigenetic drug targets as a new therapeutic access point to normalize the tumor-enabling microenvironment in pancreatic cancer patients. Citation Format: Jaye C. Gardiner, Khoa A. Tran, Janusz Franco-Barraza, Edna Cukierman. Role of the BAF complex in pancreatic cancer-associated fibroblast function [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B006.

Abstract C031: Targeting colorectal cancer-associated fibroblasts with all-trans retinoic acid to reprogram the tumor microenvironment

Abstract Cancer-associated fibroblasts (CAFs) are a prominent component of the tumor microenvironment (TME) and are known for their role in supporting tumor growth and progression through interactions with cancer cells and other stromal elements. Liver metastatic CAFs (lmCAFs), in particular, play an important role in establishing the secondary niche that facilitates tumor metastasis. Reprogramming the stroma, particularly targeting the pro-tumorigenic functions of lmCAFs, has emerged as a promising therapeutic intervention to disrupt the supportive environment tumors rely on for progression. An initial drug screen of FDA-approved oncology therapies was conducted on patient-derived colorectal cancer (CRC) CAFs and tumor organoids (PDTOs) to identify compounds that may be re-purposed as stroma targeting agents. All-trans retinoic acid (ATRA) was shown to specifically inhibit the proliferation of CAFs without affecting CRC-PDTOs. ATRA is an active metabolite of Vitamin A that exerts its canonical biological effects by binding to nuclear retinoic acid receptors (RAR). The differential RAR expression in multiple CAFs and PDTOs was confirmed with bulk RNA sequencing, with higher expression found in the CAF lines. Subsequent analysis included the qPCR assessment of glycolysis enzyme levels, CAF activation markers, and ATRA signaling in lmCAFs following treatment with ATRA for 5 days. Protein expression of key components of the TGF-b signaling pathway, including SMAD 2/3, Akt, and b-catenin, were also analyzed. Treatment of lmCAFs with ATRA induced expression of RARb and ALDH1A1, biomarkers associated with ATRA signaling pathways. In one patient-derived CAF line, ATRA treatment exhibited a decrease in CAF activation markers (e.g. aSMA) and glycolytic enzymes (e.g. lactate dehydrogenase). On the other hand, another CAF line showed increased fibroblast activation and a shift away from metabolic dependency on glycolysis upon ATRA treatment. Additionally, ATRA targeted TGF-b signaling through the pSMAD 2/3 and Akt pathways, highlighting the multifaceted impact on CAF function and heterogeneity. ATRA effectively reprograms some CAFs by inhibiting their proliferation and altering key signaling pathways, thereby disrupting the pro-tumorigenic environment of the TME. The results of this study suggest that ATRA holds potential as a therapeutic strategy to target and reprogram the CAFs within the permissive stromal components essential for tumor progression. Citation Format: Brandon Choi, Yuyuan Zhou, Seungil Kim, Shannon M. Mumenthaler. Targeting colorectal cancer-associated fibroblasts with all-trans retinoic acid to reprogram the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C031.

Abstract C023: P21-positive senescent stromal cells promote prostate cancer immune suppression and progression that can be reversed by senolytic therapy

Abstract Cellular senescence is historically viewed as a tumor-suppressive cell cycle arrest program to limit outgrowth of pre-malignant cells. However, emerging evidence suggests senescent cell persistence contributes to immune suppression and cancer progression. Using prostate cancer patient samples and murine models, we find that p16+ senescent cells accumulate throughout malignant progression and are associated with immune suppression and poor survival. p16 ablation in mice eliminated senescent epithelial cells and reversed immune suppression mediated by immature myeloid cells but not prostate tumorigenesis. Single cell sequencing revealed an additional population of senescent p21+ fibroblasts and myeloid cells. Targeting BCL-xL or PD-L1 induced during senescence could remove both p16+ epithelial and p21+ stromal senescent cells, reactivate T cell immunity, and block tumor initiation as well as progression in advanced prostate cancer models. Our findings demonstrate that targeting heterogenous senescent populations can not only prevent malignant transformation but also yield therapeutic benefits in advanced prostate cancers through activating anti-tumor immunity. Citation Format: Lin Zhou, Kelly D. DeMarco, Katherine C. Murphy, Zhenpeng Wu, Jinping Li, Junhui Li, Calvin Johnson, Zhong Jiang, Shi Bai, Tianyi Ye, Karl Simin, Lihua Julie Zhu, Jason R Pitarresi, Hong Wu, Marcus Ruscetti. P21-positive senescent stromal cells promote prostate cancer immune suppression and progression that can be reversed by senolytic therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C023.

Abstract C017: Exploring the composition of and interaction of the tumor microenvironment in Langerhans cell histiocytosis

Abstract Langerhans cell histiocytosis (LCH) is a rare inflammatory hematological neoplasm, characterized by the accumulation of LCH cells in various tissues and organs. These LCH cells originate from myeloid progenitors that carry a MAPK-pathway mutation. Once these mutated cells enter the tissue from the periphery, they differentiate into so-called LCH cells. In addition to LCH cells, the tumor microenvironment (TME) consists of a wide variety of immune cells. However, the interactions between these cells within the TME and their contribution to disease progression remain poorly understood. In this study, we focus on understanding the role of lymphocytes in LCH lesions. We used single-cell RNA sequencing to comprehensively profile the immune populations in LCH lesions on the transcriptomic level, which will be supplemented with spatial profiling. Our analysis identified a wide variety of infiltrating immune cells within LCH lesions with high cellular heterogeneity among patients. LCH- and T cells are the most prevalent cell types, suggesting a key role in lesion development and maintenance. Furthermore, the immune landscape includes several dendritic cell subsets – such as plasmacytoid DCs (pDCs), cDC1, and mature regulatory dendritic cells (mregDCs) – alongside tumor-associated macrophages (TAMs), monocytes, NK cells, B cells, and plasmablasts. Among T cells, regulatory T cells (Tregs) are particularly abundant and appear to be heavily influenced by LCH cells and other myeloid cells via the TNF-TNFR2 and CD86-CTLA4 pathways. Additionally, LCH cells express high levels of TGFβ – a known regulator of T cell activity – and MMP9 – which might hamper cytotoxic T cell infiltration. This suggests that LCH cells actively contribute to creating an immunosuppressive environment. Surprisingly, pDCs seem to be a major immune regulator within lesions and are likely to interact with T cells via TNFSF9 and Granzyme B. These insights into the immunosuppressive environment within LCH lesions highlight the critical role of Tregs as central regulators within the tumor microenvironment. The implications of these findings will be further explored and validated through in-vitro experiments and high-plex spatial protein profiling. Ultimately, our work may pave the way for developing targeted therapies aimed at modulating the TME, potentially enhancing anti-tumor immunity, and offering new strategies for treating LCH. Citation Format: Wouter van Midden, Raphaela Schwentner, Sebastian Eder, Philipp Ben Soussia-Weiss, Giulio Abagnale, Caroline Hutter. Exploring the composition of and interaction of the tumor microenvironment in Langerhans cell histiocytosis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C017.

Abstract C007: Identifying the role of Interleukin-17 signaling in intestinal stem cells during pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC), the most aggressive type of pancreatic cancer, is predicted to become the second leading cause of cancer related deaths by 2030. Interleukin 17 (IL-17) signaling can promote tumor growth. In pancreatic cancer, IL-17 signaling in cancer cells is capable of triggering the formation of neutrophil extracellular traps (NETs) which is detrimental to the anti-tumor effect of immunotherapy. IL-17 signaling has been shown to have critical roles in maintaining gut homeostasis. Intestinal epithelial cells directly interact with the gut microbiome and are actively involved in infection control, injury repair, immune responses, and the production of antimicrobial peptides and cytokines, and need to be constantly replenished by intestinal stem cells (ISCs). Our lab’s study has previously shown that deletion of Interleukin 17 receptor A (IL-17RA) in the entire gut epithelium induces pro-tumorigenic IL-17 signaling due to the disruption of gut homeostasis, suggesting that immune responses induced by gut IL-17/IL-17RA signaling can affect the behavior of distant tumors. However, the mechanism of IL-17 signaling within specific gut epithelial compartments such as ISCs, enterocytes, secretory cells, etc. and their role in maintaining gut homeostasis and cancer risk are not well-studied until now. Thus, we aimed to identify the importance of IL-17 signaling in ISCs during pancreatic cancer. We hypothesized that disruption of IL-17/IL-17RA signaling within ISCs impacts tumor growth. Our preliminary data shows that IL-17 signaling in enterocytes maintains intestinal homeostasis and disruption of this axis triggers systemic increase in B cells that promotes tumor growth by increasing IL-17 levels in circulation. To identify the function of IL-17/IL-17RA signaling in ISCs, we utilized Leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5), a lineage marker for ISCs, to drive Cre enzyme tissue specific expression to specifically knockout Il17ra in gut ISCs - an inducible transgenic mouse model Il17ra fl/fl ;Lgr5- EGFP-CreERT2 (cKO). We used multiplex staining and imaging to validate the cell type specific loss of IL-17RA expression in tamoxifen treated cKO mice. We used implantable pancreatic tumor models and performed immunoprofiling using flow cytometry. We found that the pancreatic tumor growth was altered by the remote signal from gut ISCs in cKO mice. Furthermore, we performed RNAseq in both tumor and gut tissue which identified that the major changes in gene expression were associated with both innate and adaptive immune pathways, especially B cell activation. We conclude that the IL-17/IL-17RA signaling in gut ISCs changes intestinal immune responses locally that can affect distant tumor immunity. Citation Format: Haoyue Liu, Vidhi Chandra, Le Li, Olivereen Le Roux, Virginia Tahan, Florencia McAllister. Identifying the role of Interleukin-17 signaling in intestinal stem cells during pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C007.

Abstract C036: Pharmacological modulation of glutamine metabolism alters macrophage activity and tumor immune responses

Abstract As a critical component of innate and adaptive immunity, macrophages play an important role in shaping the tumor microenvironment (TME), which affects tumor initiation, progression, and response to treatment. Tumor-associated macrophages can display anti-tumor or pro-tumor activity, a cell fate attributed to macrophage polarization that depends on metabolomic regulation. Targeting macrophage metabolism could be a promising approach for cancer treatment by converting a pro-tumor TME into an anti-tumor TME. To examine in vivo mechanisms behind glutamine metabolism on macrophages, we treated immunocompetent murine ovarian cancer models with a clinical-grade glutamine prodrug, JHU083, and performed single-cell RNA sequencing (scRNAseq). In addition to the significant anti-tumor efficacy of JHU083, we found that JHU083 specifically suppressed the M2-like tumor macrophages while sparing M1-like macrophages. To better understand the molecular mechanism of how JHU083 causes macrophage polarization shift, we cultured an M1-like macrophage cell line with DON (6-Diazo-5-oxo-L-norleucine), the active component of JHU083, and performed bulk RNA sequencing. Using bulk RNAseq and scRNAseq transcriptome analyses on M1-like macrophages, glutamine inhibitor treatment significantly enhanced MHC class II genes, including CD74, H2-Aa, H2-Eb1, H2-Ea, H2-Ab1, and TNF family genes including Tnfsf12, and Tnfrsf14 which may promote T cell trafficking and cytotoxicity through reversing immunosuppressive tumor microenvironment. Moreover, DON-treated bone marrow-derived M1 macrophages promoted CD4+ T cell expansion. On the other hand, DON treatment significantly suppressed bone marrow-derived M2 macrophages. In conclusion, our findings suggest that glutamine metabolism reprogramming can promote anti-tumor functions in tumor macrophages. Therefore, targeting glutamine metabolism to regulate macrophage function and polarization can be a promising direction for future cancer therapy. Citation Format: Jiawei Fan, Sumeng Qi, Ie-Ming Shih, Tian-Li Wang. Pharmacological modulation of glutamine metabolism alters macrophage activity and tumor immune responses [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C036.

Abstract C041: MYC and p53 alterations cooperate through VEGF signaling to repress cytotoxic T cell and immunotherapy responses in prostate cancer

Abstract Patients with castration-resistant prostate cancer (CRPC) are generally unresponsive to tumor targeted and immunotherapies. Whether genetic alterations acquired during the evolution of CRPC impact immune and immunotherapy responses is largely unknown. Using our innovative electroporation-based mouse models, we generated distinct genetic subtypes of CRPC found in patients and uncovered unique immune microenvironments. Specifically, mouse and human prostate tumors with MYC amplification and p53 disruption had weak cytotoxic lymphocyte infiltration and an overall dismal prognosis. MYC and p53 cooperated to induce tumor intrinsic secretion of VEGF, which by signaling through VEGFR2 expressed on CD8+ T cells, could directly inhibit T cell activity. Targeting VEGF-VEGFR2 signaling in vivo led to CD8+ T cell-mediated tumor and metastasis growth suppression and significantly increased overall survival in MYC and p53 altered CPRC. VEGFR2 blockade also led to induction of PD-L1, and in combination with PD-L1 immune checkpoint blockade produced anti-tumor efficacy in multiple preclinical CRPC mouse models. Thus, our results identify a genetic mechanism of immune suppression through VEGF signaling in prostate cancer that can be targeted to reactivate immune and immunotherapy responses in an aggressive subtype of CRPC. Citation Format: Katherine Murphy. MYC and p53 alterations cooperate through VEGF signaling to repress cytotoxic T cell and immunotherapy responses in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C041.

Abstract A021: HRI inhibition by hemin as a novel targeted therapy for glioblastoma via the integrated stress response

Abstract The treatment of glioblastoma (GBM), a highly malignant brain tumor, is critically hindered by the ineffectiveness of current modalities such as surgery, radiation, and chemotherapy. These traditional methods fail to completely remove the tumor mass and lack the ability to discriminate between cancerous and normal brain cells, often resulting in damage to healthy tissue and recurrence of the disease. This underscores an urgent necessity to develop novel therapeutic strategies that can target tumor cells with precision, offering hope for improved survival rates and quality of life for GBM patients. This study investigates targeted therapy, focusing on the Integrated Stress Response (ISR) that cancer cells harness to survive hypoxic stress. Specifically, it demonstrates that EIF2AK1, which encodes Heme-regulated eIF2α kinase (HRI), is activated under hypoxia and co-expressed with the glioma stem cell marker SOX2, which specifically happens in glioma cells, increasing the targeted accuracy of the repurposing drug. This correlation, indicating hypoxia-driven stemness, is confirmed at both the genetic level and through Gene Set Enrichment Analysis (GSEA). Furthermore, GSEA in Spatial Transcriptomics shows hypoxia-induced glycolysis, disrupting the tumor microenvironment and causing necrotic cell death. Stemness phenotype is induced in the peripheral cells due to the unfavorable hypoxic environment. Hemin, an HRI inhibitor, has been repurposed to inhibit ISR and mitigate hypoxia. Treatment with Hemin on the U87 cell line resulted in IC50 values of 23.50 µM and 52.46 µM at 24 and 48 hours, respectively, surpassing Temozolomide's efficacy. A decrease in HRI expression after the Hemin treatment suggests the and ISR activity and, potentially, hypoxia. This would reverse the unfavorable microenvironment so that the stemness phenotype doesn’t spread. Potentially, invasiveness and recurrences of GBM in clinic situation would decrease, thus potentially improving patient prognosis. The therapeutic potential of Hemin is enhanced by its ability to kill glioma cells directly and accurately in the glioma cell in original TME when cells are proliferating with adequate oxygen. Therefore, this study demonstrates the therapeutic potential of repurposing Hemin, an HRI inhibitor, to precisely target hypoxia-induced glioma stem cells in glioblastomas, disrupting the aggressive tumor microenvironment to potentially improve patient prognosis. Citation Format: Xingchuan Ma. HRI inhibition by hemin as a novel targeted therapy for glioblastoma via the integrated stress response [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A021.

Abstract C046: PARP inhibitors as immune modulators in metastatic ovarian cancer treatment

Abstract Epithelial ovarian carcinoma (EOC) is one of the top five causes of cancer-related death in women. Most patients with EOC achieve initial remission after primary or interval cytoreductive surgery combined with platinum-taxane chemotherapy. However, mutations in BRCA1 or BRCA2 genes, which lead to homologous recombination (HR) defects, play a crucial role in platinum sensitivity and justify the use of poly (ADP-ribose) polymerase (PARP) inhibitors as maintenance therapy, commonly linked to extended progression-free survival (PFS). Besides their direct cytotoxic and cytostatic effects, PARP inhibitors (PARPi) have shown significant immunostimulatory properties by disrupting DNA repair in cancer cells, and opening possibilities for synergy with immune checkpoint inhibitors (ICIs). In this study, we investigate the immunomodulatory effects of PARPi using multiparametric flow cytometry, multiplexed immunolabeling, single-cell transcriptomics, and functional assays in an experimental BR5Brca1-/- syngeneic mouse model and human EOC tumor samples. We examine the molecular and cellular mechanisms that can be exploited to develop more effective combination therapies. PARPi may increase the mutational burden in EOCs due to unresolved DNA damage and the release of damage-associated molecular patterns (DAMPs), thereby increasing T-cell infiltration. In addition, PARPi appear to promote potent type I interferon (IFN) secretion through the activation of cGAMP synthase and the stimulator of the interferon genes (STING) pathway. In combination with ICIs, PARPi showed a beneficial effect on the balance between adaptive anti-tumor immunity and innate myeloid components, leading to an improved cytotoxic T-cell response, as observed in mouse models and HGSOC tumor samples. These observations emphasize the role of strategically designed combinations of PARPi and immunotherapeutic agents, which could be the key to overcoming immunosuppression in the EOC microenvironment, thereby improving clinical outcomes. Citation Format: Sarka Vosahlikova, Peter Holicek, Irena Moserova, Michal Hensler, Romana Mikyskova, Lenka Kasikova, Josef Pasuvka, Jana Drozenova, Katerina Mojzisova, Marek Kovar, Iain McNiesh, Michael Halaska, Lukas Rob, Sandra Orsulic, Milan Reinis, Lorenzo Galluzzi, Radek Spisek, Jitka Palich Fucikova. PARP inhibitors as immune modulators in metastatic ovarian cancer treatment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C046.

Abstract B034: Engineering the tumor microenvironment: Patient-specific microphysiological systems of the head and neck cancer tumor microenvironment

Abstract Introduction: Survival rates in advanced head and neck cancer (HNC) remain poor with no clinically approved biomarkers to predict treatment efficacy. HNC is a highly heterogenous disease and the lack of available models that capture patient heterogeneity and the complexity of the tumor microenvironment (TME) contributes to these poor patient outcomes. Microphysiological systems (MPS) are complex multicellular in vitro models that can recapitulate three-dimensional organ structure and function. The low cell number requirements for MPS permits the creation of patient-specific TME models, where all the cell types can be isolated from a single piece of tumor tissue. These patient-specific MPS represent a promising strategy for elucidating the contribution of the TME to treatment resistance, predicting the best treatment for each patient and improving patient outcomes in HNC. Here, we report a patient-specific MPS that contains multiple TME compartments and recapitulates patient heterogeneity. Methods: Tumor tissue was collected from patients with oral cavity carcinoma undergoing standard of care surgery followed by radiation or chemoradiation. Tissue from each patient was digested and tumor infiltrating leukocytes (TIL) were isolated using CD45 magnetic beads, while epithelial cells and fibroblasts were expanded in culture using specific media formulations. Tissue was also snap frozen for single cell RNA sequencing analysis. To build the MPS, a custom designed microfluidic device was filled with a hydrogel matrix containing patient-matched TIL, fibroblasts and epithelial spheroids to mimic the solid tumor. Lumens molded from the hydrogel were seeded with blood or lymphatic endothelial cells to create vasculature. Multiple replicate MPS can be built from each patient, which are cultured in an optimized media formulation to support the multiple primary cell types. Patient-specific MPS were treated with radiation or cisplatin + radiation to mimic the treatment the patient received. Multiple orthogonal endpoints were measured including tumor cell migration and viability, cytokine secretion and angiogenesis. Single cell sequencing was also performed on the original tumor tissue and matched MPS. Results: We have successfully created patient-specific models of the HNC TME. Single cell RNA sequencing analysis of the MPS suggests that cellular heterogeneity is being retained. Patient characteristics varied between MPS with some patient TME MPS showing highly migratory tumor cells or angiogenic responses. Treatment responses were measured for each patient-specific MPS and ongoing work will characterize the contributions of the TME to treatment resistance. These data demonstrate the feasibility of using patient-specific MPS to investigate the contribution of the TME to treatment response and resistance and lays important groundwork for using MPS to identify patient treatment responses. Citation Format: Adeel Ahmed, Marcos Lares, Fauzan Ahmed, Adam R. Burr, Paul M. Harari, David J. Beebe, Sheena C. Kerr. Engineering the tumor microenvironment: Patient-specific microphysiological systems of the head and neck cancer tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B034.

Abstract A018: Endothelial pyrimidine synthesis deficiency promotes tumor growth

Abstract The tumor microenvironment plays a crucial role in cancer progression and treatment response. Systemically administered metabolic cancer therapies target not only malignant but also stromal cells, including immune cells - the only stromal population previously addressed in this context. However, how such treatments impact other non-malignant cell types in tumors remains poorly understood. Here we show that inhibition of de novo pyrimidine synthesis in endothelial cells accelerates tumor growth and alters tumor immune repertoire. We found that whole-body de novo pyrimidine synthesis deficiency in mice, caused by the inducible whole-body ablation of DHODH, accelerates the growth rate of orthotopic lung tumors. Single-cell transcriptomic analysis of tumor-bearing lungs revealed that DHODH deficiency in the stroma impacts multiple cell populations, including immune and, surprisingly, endothelial cells. To explore the endothelial-specific effects, we generated a mouse model with inducible DHODH deficiency restricted to endothelial cells. The endothelium-specific model recapitulates the accelerated lung tumor growth observed in the whole-body DHODH deficiency model. Single-cell transcriptomics analysis of tumor-bearing lungs in the endothelium-specific model pointed to changes in the immune repertoire, particularly an enrichment of monocytes. We confirmed these results on the protein level using spectral flow cytometry, and we are currently in the process of uncovering the mechanism by which endothelial deficiency of pyrimidine synthesis affects the immune landscape of tumors. Inhibitors of pyrimidine de novo synthesis have been tested in clinical trials but failed due to their low efficiency. Our findings indicate that systemic treatment targeting pyrimidine synthesis may be hampered by its pro-tumorigenic effects in the endothelium, highlighting the unexpected role of endothelial metabolism in this context. Citation Format: Petra Hyrossova, Isidora Milisav, Silvia Novais, Mirko Milosevic, Jakub Rohlena, Katerina Rohlenova. Endothelial pyrimidine synthesis deficiency promotes tumor growth [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A018.

Abstract C028: Decreased heterogeneity in immune interactions in oral cavity squamous cell carcinoma patients with recurrence

Abstract There have been improvements in clinical risk stratifications for oral cavity squamous cell carcinoma (OSCC) with the AJCC 8th edition staging system. Other studies suggest the addition of a metric of specific immune interactions in and around the tumor may be helpful to further stratify patients, with mixed findings. The advent of spatial imaging has allowed a global view for assessing tumor features in the context of all surrounding cell interactions. We hypothesize that the lack of architectural information could contribute to varied clinical outcomes in previous studies, such as tumor recurrence. This highlights how spatial technologies could aid in understanding the role of tissue architecture and its influence on biology and medicine. To study tissue remodeling spatially, we focused on multiplexed-ion beam imaging (MIBI), a spatial proteomics platform to visualize 40 immuno-onco proteins simultaneously. Our banked patient cohort consists of 20 recurrent and 49 non-recurrent OSCC primary surgery samples. Clinical metadata capturing pathologic stage, alcohol use, smoking history, survival data, and more were also collected. With our pathologist, we curated 290 field-of-views (FOVs) of matched primary tumors, uninvolved lymph nodes, and metastatic lymph nodes (if any). Downstream computational analysis using existing MIBI codebases augmented with in-house spatial analytics has revealed a robust spatial dataset. After using machine-learning algorithms to cell segment our images, we validated these assigned cells and marker staining to further annotate cell populations. When we analyzed direct cell interactions, there was a decrease in heterogeneity in immune interactions within recurrent patients compared to non-recurrent patients. To ascertain how the loss of these interactions affected recurrence prognosis, we investigated their prevalence within the tumor border vicinity. We hypothesize that the proximity and cell-cell interactions near the border will promote a tumor-suppressive environment. Utilizing centroid-to-centroid distance measurements, we distilled the top 30 cell-cell interactions within a 100-micron distance from the tumor border. Cell pairs involving macrophage tissue-resident memory cells and CD8 T memory resident cells were more abundant in recurrent patients, while pairs involving CD4 T regulatory and CD8 cells were found in non-recurrent patients. Reassuringly, cell pairings involving myeloid cells were upregulated in the recurrent cohort as expected. Interestingly, cancer-associated fibroblast cell pairings were upregulated indiscriminately in both patient sub- cohorts. This suggests that the impact of certain known immunosuppressive changes may contribute in varying amounts to disease control. Our data demonstrates differences in the architecture of tumors with known divergent outcomes, which suggests global tissue-level changes could be a predictive factor. Clinically, interrogating these interactions of interest could guide treatment escalation or de-escalation in the future. Citation Format: Connie J. Zhou, Jane Lee, Rebecca Jaszczak, Steven R. Long, Patrick K. Ha, Matthew H. Spitzer. Decreased heterogeneity in immune interactions in oral cavity squamous cell carcinoma patients with recurrence [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C028.

Abstract A042: Desmosome mutations in the microenvironment regulate melanoma proliferation

Abstract Desmosomes are transmembrane protein complexes critical for cell-cell adhesion in epithelial tissues and other cell types. In skin cancers like melanoma, epithelial cells called keratinocytes are the predominant cells in the primary melanoma microenvironment, yet it remains largely unknown whether this cell type is subject to genetic alterations during melanoma development and its role in early melanoma progression. In this study, using an integrative analysis of tumor mutations and protein biophysical interactions, we identify a high frequency of genetic alterations in desmosome-related genes in human cancers, with the highest occurrences in cutaneous melanoma. Notably, over 70% of cutaneous melanoma cases exhibit mutations in desmosome genes and desmosome gene mutations are associated with lowered expression of the desmosome complex in melanoma. Using spatial transcriptomics and immunohistochemistry analyses, we find that the decrease in desmosome gene expression predominantly occurs in keratinocytes within the tumor microenvironment rather than in the melanoma cells themselves. To study the functional significance of this alteration in melanoma development, we used a human melanoma/keratinocyte co-culture system using primary melanoma cell lines. Our studies show that knockdown of desmosome genes in keratinocytes markedly increased proliferation of adjacent melanoma cells in melanoma/keratinocyte co-cultures. Additionally, melanoma cell proliferation is enhanced when exposed to media preconditioned by desmosome-deficient keratinocytes. These observations suggest that the gradual accumulation of mutations in desmosome genes within neighboring keratinocytes may create a conducive environment that primes melanoma cells for neoplastic transformation. This study underscores the critical role of genetic alterations in the tumor microenvironment in melanoma progression and highlights the potential for targeting desmosome-related pathways in therapeutic strategies against melanoma. Citation Format: Mohita Tagore. Desmosome mutations in the microenvironment regulate melanoma proliferation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A042.

Abstract A038: A targetable tumor-induced wound-healing response is essential for osteosarcoma lung metastasis

Abstract Lung metastasis is responsible for nearly all deaths in children and teenagers affected by osteosarcoma, the most common pediatric sarcoma. The mechanisms by which malignant bone cells manipulate the lung microenvironment to promote the colonization and growth of metastatic tumor cells remain unclear but represent a potential therapeutic opportunity for preventing and treating metastasis. In this study, we sought to understand how osteosarcoma cells educate the lung microenvironment during metastatic progression. To address this gap, we generated several time series datasets of niche-labeled, single-cell RNA-seq of developing metastatic lesions and then used these to characterize the changes occurring in tumor and tumor-associated stromal cells during colonization. After identifying candidate mechanisms, we tested several therapeutic interventions to evaluate their effects on metastasis development in vitro using a tumor-on-lung co-culture system and in vivo using our animal models. Osteosarcoma cells induced acute epithelial injury upon lung dissemination. Shortly after dissemination, a paracrine loop established between a hyper-secretory, hypo-proliferative subpopulation of tumor cells and alveolar epithelial cells triggers the adoption of a chronic, non-resolving wound phenotype within the surrounding tissues that culminates in extensive tissue fibrosis with persistent extracellular matrix deposition, especially fibronectin. Cellular changes observed during this process include the proliferation of type 2 alveolar epithelial cells, the accumulation of highly fibrogenic epithelial intermediates (analogous to those observed in pulmonary fibrosis), and the emergence of a large population of scar-promoting macrophages. The formation of this matrix seems essential for lesion formation—disruption of the matrix-tumor cell interaction eliminated pseudonodules in vivo. Inhibition of pro-fibrotic pathways using nintedanib, an FDA- approved drug used to treat pulmonary fibrosis, or of tumor-epithelial paracrine signals with anakinra, an antagonist of IL1 signaling, effectively blocked metastatic lesion growth in both murine and human xenograft models, showing evidence of microenvironment reprogramming in the single cell data. Combining these therapies, which target primarily the hyper-secretory, hypo-proliferative “anchor” cells, with conventional therapies, which target proliferating tumor cells, proved highly effective in eliminating established metastatic lesions. Our work demonstrates that disseminated osteosarcoma cells trigger an aberrant, non-resolving wound-healing response in tumor-associated lung tissues, similar to that observed in non-malignant pulmonary fibrosis, and that this pathologic wound-healing response promotes metastatic growth. Therapeutic disruption of fibrosis-associated signaling prevents metastasis and treats established metastatic disease in multiple osteosarcoma models. Citation Format: James B. Reinecke, Leyre Jimenez Garcia, Amy C. Gross, Matthew V. Cannon, Yogesh Budhathoki, Ryan D. Roberts. A targetable tumor-induced wound-healing response is essential for osteosarcoma lung metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A038.

Abstract PR004: Age-induced chronic accumulation of glucocorticoids drives therapy resistance in lung cancer

Abstract Non-small cell lung cancer (NSCLC), a highly aggressive cancer, is the leading cause of cancer- related deaths in the U.S. While chemotherapy and targeted therapy are the main treatment modalities in the clinic, many patients do not respond to these treatments. Strikingly, the median age for lung cancer diagnosis is 71. Yet, preclinical and clinical research have largely been skewed towards young mice and patients. Here, we address this gap in knowledge and evaluate if old age affects the response to standard of care NSCLC therapies in KRAS-driven NSCLC. Using human sera from young and old healthy subjects to treat NSCLC cell lines, we show that age-driven circulatory changes are sufficient to drive a state of profound resistance to both chemotherapies as well as KRASG12C targeted therapies. We confirmed the relevance of our findings in vivo, by transplanting cancer cells derived from the K-rasLSL-G12D/+; p53fl/fl genetically engineered mouse model of lung cancer into young and old C57BL/6 mice. Using this model we showed that unlike young lung tumor-bearing mice on chemotherapy, old tumor-bearing mice do not respond to treatment and have a poor prognosis. Mechanistically, we found that old age drives a transcriptional reprogramming that favors drug resistance and identified the glucocorticoid receptor as a major driver of these transcriptional changes. We traced this to an age-induced chronic accumulation of glucocorticoids in circulation and within the tumor interstitial fluid. Chronic treatment with age-relevant concentrations of cortisol alone was sufficient to drive glucocorticoid receptor activation and induce resistance to therapies mirroring the effects of old age. Further supporting a driving role for glucocorticoids in driving age- induced drug resistance, blocking the glucocorticoid receptor genetically or pharmacologically rescues the drug resistance state imposed by old age in NSCLC cells. Lastly, using TCGA patient data we show an inverse correlation between glucocorticoid receptor activation (GR score) and progression-free survival of lung cancer patients. Together, our work demonstrates a role for age-induced glucocorticoids in circulation in promoting therapy-resistance in NSCLC patients and supports the use of therapeutic agents that block the glucocorticoid receptor as a strategy to increase therapy efficacy in NSCLC patients. Citation Format: Devesh Raizada, Felicia Lazure, Stanislav Drapela, Nadir Sarigul, Joanne Tejero, Didem Ilter, Ana Da Silva Gomes. Age-induced chronic accumulation of glucocorticoids drives therapy resistance in lung cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR004.

Abstract B024: Deciphering the neurogenic tumor microenvironment: A novel gene network driving innervation in metastatic melanoma offers prognostic and therapeutic insights

Abstract Melanoma, a highly aggressive and life-threatening cancer, poses a significant therapeutic challenge, particularly for patients with distant metastasis, who face a 5-year survival rate of only approximately 22.5%. This underscores the urgent need for innovative therapeutic approaches. Emerging evidence suggests that sensory and parasympathetic innervation within the tumor microenvironment (TME) plays a crucial role in driving metastasis and immune evasion, partly through the promotion of M2 macrophages and cancer stem cells. To uncover the mechanisms behind tumor-specific innervation in metastatic melanoma, we employed a systems biology approach integrating bulk RNAseq from diverse melanoma cell lines, TCGA-SKCM data analysis, single-cell RNAseq, and spatial transcriptomics. This comprehensive analysis led us to identify a previously uncharacterized gene regulatory network (GRN) orchestrating tumor innervation. This GRN is driven by axon guidance cues (Netrin, Slit, Semaphorin), their receptors (DCC, Robo, Neuropilin, ITGB1), and transcription factors (RAS, RND1, TFAP2, CAMKII). We further discovered that the pioneer factor FOXC2, known for its association with metastasis, plays a critical role in opening the promoters of innervation-specific genes. Additionally, we identified Ephrin and CXCR4-mediated crosstalk between neurons and tumor cells. Machine learning models validated the accuracy of this GRN in predicting melanoma metastasis, and a neural network-based model confirmed the enrichment of nerve cells near metastatic tumor cells within the TME in TCGA immunohistochemistry data. The functional relevance of this network was further supported by CRISPR-mediated knockout studies, which disrupted tumor-specific signaling pathways. Notably, we also observed the enrichment of genes linked to mRNA processing bodies and neuron-specific membrane-less condensates in metastatic melanoma. These findings highlight the critical role of neuronal infiltration in melanoma metastasis. Currently, therapeutic strategies targeting this aspect of the TME remain limited. Our identified gene set offers potential as a metastatic predictor, and targeting the neural component of the TME represents a promising new therapeutic avenue for melanoma. Citation Format: Anjana Goli, Subhajit Dutta. Deciphering the neurogenic tumor microenvironment: A novel gene network driving innervation in metastatic melanoma offers prognostic and therapeutic insights [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B024.

Abstract PR010: TRPV1+ sensory innervation as a novel driver of ovarian cancer progression

Abstract Introduction: A primary challenge currently faced in ovarian cancer (OVCA) treatment is that patient prognoses remain poor despite current therapeutic interventions. Overall survival for OVCA patients with advanced disease is <30%, and most patients recur within 5 years. As such, there is a critical need for new therapies that can overcome OVCA chemoresistance and produce meaningful increases in patient survival. Recent efforts that have targeted the tumor microenvironment (TME), such as immune checkpoint inhibitors, have been successful in some cancers but have limited therapeutic benefit when used to treat OVCA. Thus, new TME targeting therapies must be developed to effectively treat OVCA patients. A novel TME component ripe for therapeutic targeting are the nerve fibers that infiltrate tumors. Recent studies in several cancers have shown that tumor innervation can promote tumor growth/metastasis. However, in ovarian cancer the role of innervation in promoting cancer progression remains a gap in knowledge. Here we show that Transient Receptor Potential Cation Channel Subfamily V Member 1 (TRPV1)+ sensory innervation plays a significant role in driving ovarian cancer progression. Methods: We performed IHC staining to determine the extent of TRPV1+ nerve infiltration in OVCA vs normal reproductive tissue in patients. We examined the ability of OVCA cells to promote tumor innervation using a neurite outgrowth assay. We utilized both syngeneic and patient derived xenograft (PDX) mouse models of ovarian cancer metastasis, where mouse or human OVCA cells were injected intraperitoneally, to explore the functional role of TRPV1+ sensory nerves in OVCA. TRPV1+ sensory nerves were ablated genetically using TRPV1 driven expression of diphtheria toxin alpha (DTA) or chemically using resiniferatoxin. TRPV1+ sensory nerves were activated pharmacologically using capsaicin. Results: Analysis of patient samples showed that TRPV1+ sensory innervation is much higher in ovarian tumors vs benign reproductive tissue. We found that conditioned media from OVCA cells was able to promote neurite outgrowth of dissociated mouse sensory neurons. Ablation of TRPV1+ sensory nerve fibers in vivo caused reduced tumor burden and prolonged survival in our syngeneic and PDX OVCA mouse models. Stimulation of TRPV1+ sensory nerves with capsaicin accelerated OVCA growth and decreased survival in tumor bearing mice. Conclusions: Our results establish TRPV1+ sensory innervation as a novel driver of OVCA growth/metastasis and a potential therapeutic target for OVCA treatment. Significance to tumor microenvironment field: Our data add to a growing body of evidence that sensory nerves are pro-tumorigenic in multiple cancer types and could be therapeutically targeted for cancer treatment. Citation Format: Matthew Knarr, Katherine Cummins, Dusan Racordon, Hunter Reavis, Timothy Lippert, Ryan Hausler, Priyanka Rawat, Jamie Moon, Dave S.B. Hoon, Roger Greenberg, Paola Vermeer, Ronny Drapkin. TRPV1+ sensory innervation as a novel driver of ovarian cancer progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR010.

Abstract B001: Intra- and inter-tumoral heterogeneity of melanoma across different metastatic sites

Abstract Melanoma, a highly metastatic skin cancer, exhibits variations in prognosis and response to therapy based on the site of metastasis. Despite the success of immunotherapy and targeted therapies in melanoma, over half of metastatic melanoma patients will experience disease progression due to therapy resistance. The heterogeneity and plasticity of melanoma cells contribute to metastatic dissemination and therapy resistance. Our aim is to determine whether distinct clones and/or their transcriptional cell states can predict the formation of tumors in various organs and assess how these clones change over time. To identify melanoma clones across different metastatic sites, NOD scid gamma (NSG) mice and C57BL/6 mice were injected subcutaneously, intravenously or intracranially with the same pool of cells of barcoded luciferase expressing YUMMER1.7 murine melanoma cells. Transduction conditions ensured that one DNA barcode integrated into cell genomes at one barcode per cell, serving as a lineage tag. Bioluminescence imaging was performed once a week to monitor tumor growth of mice injected intravenously and intracranially. Subcutaneous tumors were measured by calliper. Mice were euthanized at different time points; day 8, 15, 22, 29 post-implantation and at ethical endpoints. Tumors were harvested and DNA sequencing was performed to identify barcodes expressed by the tumor cells. All mice developed tumors, with 100% penetrance in NSG mice. However, in C57BL/6 mice, 10% of mice intravenously injected and 27% of subcutaneously implanted mice showed complete lesion regression, suggesting that the immune system may be responsible for tumor regression. Analysis of barcodes allowed us to assess the heterogeneity of melanoma tumors at different metastatic sites and their evolution over time. Lineage tracing and clonal heterogeneity will be evaluated using the state of art technology, SPLINTR (Single-cell Profiling and LINeage Tracing), enabling us to match a cells evolution with changes in transcriptional states. Barcode analysis performed before implanting the cells and, at different timepoints in subcutaneous and lung tumors showed that dominant subclones at the baseline were also dominant in subcutaneous and lung tumors in immunocompromised mice. In contrast, dominant subclones in immunocompetent mice was those present in lower proportion at baseline. Additionally, greater variability of subclones was observed especially in lung and brain tumors, across immunocompetent mice, likely as a mechanism of resistance, enabling these tumors to overcome immunoediting. Understanding the variability of clonality between different metastatic sites and over time will improve our comprehension of the role of different subclones in organ- specific metastasis and their transcriptional cell states. Citation Format: Veronica L. Aedo Lopez, Reem Saleh, Benjamin Blyth, Xin Du, Dane Vassiliadis, Katie Fennell, Davide Moi, Roberta Mazzieri, Riccardo Dolcetti, Karen E. Sheppard, Grant A. McArthur. Intra- and inter-tumoral heterogeneity of melanoma across different metastatic sites [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B001.

Abstract PR015: Sepsis-induced inflammation alters natural killer cell-mediated surveillance of liver metastasis

Abstract A majority of pancreatic ductal adenocarcinoma (PDAC) patients who undergo surgery for primary tumor resection will develop lethal metastases, despite having no evidence of metastasis at the time of their initial diagnosis. Liver metastases constitute nearly half of recurrences detected within the first six months following PDAC resection. Several clinical and experimental studies provide evidence that perioperative inflammation, including infectious complications like sepsis, positively correlates with liver metastasis across several malignancies. Despite these observations, it is still unknown whether there is a causal link between perioperative inflammation and PDAC liver metastasis. To gain mechanistic insight into the regulation of PDAC liver metastasis in the context of perioperative inflammation, we established mouse models that combine experimental PDAC liver metastasis with either the cecal ligation and puncture (CLP) model of polymicrobial sepsis or the lipopolysaccharide (LPS) model of acute sterile endotoxemia. Mice challenged with CLP prior to seeding PDAC cells in the liver had increased metastatic burden at endpoint compared to sham laparotomy controls. In contrast, mice challenged with LPS prior to PDAC cell seeding had reduced metastatic burden at endpoint compared to PBS-injected controls. We hypothesized that these opposing pro- and anti-metastatic effects of CLP and LPS, respectively, were due to qualitatively distinct inflammatory responses elicited in the liver. Our profiling of the host response to either CLP or LPS revealed shared systemic changes, including activation of the acute-phase protein response. However, LPS-associated inflammation uniquely upregulated a distinct set of interferon-regulated genes in the liver important for modifying immune cell activity (i.e., Ccl2, Ccl5, Cxcl9, Cxcl10). Accordingly, immunostaining and flow cytometry of the liver revealed that LPS uniquely increased the abundance of activated natural killer (NK) cells and MPO+ myeloid cells. We utilized Tlr4 -/- mice, which lack the primary receptor for LPS, to determine that the anti-metastatic effect of LPS required a host-facilitated response downstream of TLR4-mediated signaling. Through a series of genetic models and short-term depletion experiments, we determined that NK cells restrict PDAC cell seeding and outgrowth in our experimental liver metastasis model, and that this protective effect is further enhanced by activation of type-I interferon signaling in the host. Our findings confirm prior work identifying NK cell-mediated surveillance of disseminated cancer cells as an important mechanism in protecting against liver metastasis and show sterile endotoxemia and its associated inflammation establish a protective ‘anti-metastatic niche’ marked by enhanced NK cell activity. Further insights into the establishment of this niche may reveal potential therapeutic strategies to reduce the incidence of liver metastasis among resected PDAC patients. Citation Format: Nicole Sivetz, Patrick J. Cunniff, Christopher R. Vakoc, Semir Beyaz, Mikala Egeblad. Sepsis-induced inflammation alters natural killer cell-mediated surveillance of liver metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR015.