Cancer Research Research Articles

Abstract A033: Integrating spatial transcriptomics and second harmonic generation imaging to identify targets of supermere-associated Discoidin Domain Receptor 1 involved in collagen alignment

Abstract Immune checkpoint blockade has been shown to be an effective treatment for colorectal cancers (CRCs) highly infiltrated by CD8+ T cells. However, there is a paucity of CD8+ T-cell infiltration in the majority of CRCs. We previously identified the collagen-activated receptor Discoidin Domain Receptor 1 (DDR1) as a member of a four-gene signature associated with T-cell exclusion in CRC. The cleaved ectodomain (cECD) of DDR1 induces collagen alignment, leading to T-cell exclusion in a breast cancer model. We found that almost all DDR1 cECD is found in supermeres, a novel secreted nanoparticle identified by our lab, and that DDR1 cECD is required for supermere signaling in recipient cells. We hypothesized that supermere-associated DDR1 cECD signals to CRC stromal cells, leading to collagen alignment. To test this hypothesis, we developed a method to determine which genes are associated with collagen alignment and DDR1 expression. To do this, 30 μm thick CRC sections are imaged using a Nikon AX multiphoton microscope by second harmonic generation imaging, which permits label-free imaging of collagen fibers. Spatial heatmaps of the collagen alignment are generated from the resulting images. Spatial transcriptomics is performed on a serial tumor section using the Visium v2 platform and the gene expression matrix is integrated with the collagen alignment heatmap data. As proof-of-principle, two CRC tumors have been analyzed using this method. Highly aligned regions were found to be associated with expression of genes involved in extracellular matrix organization, keloidal collagen and specific fibroblast subtypes. Genes which may be regulated by DDR1 cECD and be involved in collagen alignment will be confirmed at the protein level by multiplexed immunofluorescence on serial sections and in vitro experiments. This method will provide a rich dataset to elucidate the roles of supermeres and DDR1 cECD in T-cell exclusion and collagen fiber alignment and may support the discovery of new strategies to overcome immune exclusion in CRC. Citation Format: Maxwell S. Hamilton, Oleg Tutanov, Harsimran Kaur, Alan J. Simmons, Clark Massick, Kasey Vickers, Ken S. Lau, Ambra Pozzi, Robert J. Coffey. Integrating spatial transcriptomics and second harmonic generation imaging to identify targets of supermere-associated Discoidin Domain Receptor 1 involved in collagen alignment [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 A033.

Abstract B010: Improving pancreatic cancer image classification with transfer learning: CD45 vs. PANCK

Abstract Pancreatic ductal adenocarcinoma (PDAC) is known for its high aggressiveness and is the fourth most common cause of death of cancer-related death in the United States. It is highly malignant and often goes undiagnosed until advanced stages due to its late-onset symptoms, resulting in poor prognosis. Using quantitative analyses of histological features, such as the expression of prognostic biomarker, can provide valuable insight to guide treatment decisions, monitor disease progression and predict outcomes. Previous researchers have found that CD45 and PANCK play a huge role in characterizing the tumor microenvironment of PDAC. One study using single-cell RNA sequencing compared the microenvironments of healthy pancreata and pancreatic tumors, revealing differences in cellular composition. Tumor samples showed a higher proportion of immune cells, including CD45+ cells, compared to heathy samples and utilized PANCK to differentiate between epithelial and non-epithelial cells. This study aims to enhance the accuracy of classifying pancreatic cancer pathological images based on prognostic markers using transfer learning techniques. We curated and pre-processed a dataset of pancreatic cancer pathological images with different prognostic markers. Using the pre-trained convolutional neural network (CNN) model VGG16, we fine-tuned it with our dataset to better differentiate between two prognostic markers, CD45 and PANCK. The model's performance was evaluated using a similarity matrix to illustrate distinctions among the two prognostic markers. Our results demonstrate that the transfer learning approach significantly improves classification accuracy, achieving confusion matrix validation results of 90% between CD45 and PANCK prognostic markers. SSIM values were relatively low, highlighting the ability of our model to differentiate between the two prognostic markers. The low SSIM value also shows variability within the two prognostic markers, indicating heterogeneity in structural changes due to pancreatic cancer. These findings indicate that transfer learning can be a powerful tool for improving the classification of pathological images and that there is notable variability in the structural features associated with different prognostic markers in pancreatic cancer. Citation Format: Miracle Thomas. Improving pancreatic cancer image classification with transfer learning: CD45 vs. PANCK [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 B010.

Abstract IA027: Age-related stromal changes drive breast cancer tumor progression

Abstract Age is the single largest risk factor for the development of cancer, but how age impacts the molecular mechanisms that drive cancer remain poorly understood. While it is clear that age-related accumulation of cell autonomous mutations contributes to tumorigenesis, the central role age-related changes in the tumor microenvironment play in the transformation process is becoming more fully appreciated. Underscoring the importance of an aged microenvironment in cancer development are findings that senescent fibroblasts, which accumulate with age, directly stimulate preneoplastic and neoplastic cell growth and tumor progression. Investigations into how senescent fibroblasts promote tumorigenesis revealed that they express a plethora of growth factors, extracellular matrix remodeling enzymes, chemokines, and cytokines collectively referred to as the senescence associated secretory phenotype (SASP). Chemotherapy induces similar changes that can negatively impact a patient’s quality of life. We will discuss how these changes impact tumor progression and therapy-induced bone loss. Citation Format: Sheila A. Stewart. Age-related stromal changes drive breast cancer tumor 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 IA027.

Abstract C027: The role of Liver Receptor Homolog 1 (LRH-1) in regulating cancer progression by modulating the immune response

Abstract Breast cancer is a significant global health concern, being the second most common type of cancer and the second leading cause of cancer-related deaths among women in the U.S. Metastatic breast cancer is responsible for most of these deaths, and current FDA-approved immunotherapies are effective only for a small subset of patients with PD-L1-positive tumors. Thus, there is a critical need for new therapeutic targets to enhance the immune response against breast cancer. Elevated plasma cholesterol concentrations are associated with poor breast cancer outcomes. Our previous studies indicated that myeloid immune cells are highly sensitive to changes in cholesterol levels and other proteins involved in regulating cholesterol homeostasis. However, the role of Liver Receptor Homolog 1 (LRH-1), a key regulator of cholesterol homeostasis, in myeloid cells is not well understood. Notably, higher expression of LRH-1 mRNA in breast tumors is associated with longer time to recurrence and improved overall survival. Therefore, we aimed to investigate the role of LRH-1 in myeloid immune cells and its impact on breast cancer progression. Initial analysis indicated that LRH-1 is expressed in myeloid cells, with particularly high levels observed in neutrophils. Therefore, we examined how LRH-1 regulates neutrophil functions important to cancer biology, including migration, NETosis, and phagocytosis, as well as its influence through neutrophils on T cells. We found that LRH-1 inhibited neutrophil migration toward cancer cells. Since tumor infiltrating neutrophils are generally associated with poor prognosis, LRH-1 reducing neutrophil migration could indicate a decreased ability of these immune-suppressive cells to infiltrate tumors. Importantly, neutrophils treated with an LRH-1 agonist showed reduced NETosis, while antagonist led to increased NETosis. LRH-1 agonist treatment also significantly reduced neutrophil phagocytosis, whereas an antagonist had no notable effect. Furthermore, T cells exhibited increased expansion when co-cultured with neutrophils treated with an LRH-1 agonist, and reduced proliferation with neutrophils pretreated with an LRH-1 antagonist or inverse agonist, particularly in CD4+ helper T cells and later divisions. Since neutrophils were previously implicated in invoking recurrence from dormant lesions, we evaluated LRH-1 in this context. The treatment of mice harboring dormant D2.0R lesions with an LRH-1 inverse agonist resulted in sooner recurrence and metastatic outgrowth compared to vehicle treated mice. In summary, our findings suggest that LRH-1 plays a crucial role in regulating various neutrophil functions, including migration, NETosis, phagocytosis, and T cell expansion. The ultimate consequence of these regulations are control of dormant mammary cancer lesions. These results highlight LRH-1 as a potential therapeutic target for cancer treatment or prevention of metastatic recurrence. Funding: National Cancer Institute (ERN: R01CA234025) Department of Defense (ERN: BCRP Era of Hope Award) Citation Format: Yu Wang, Bryan Duong, Natalia Krawczynska, Shruti V. Bendre, Claire P. Schane, Erin Weisser, Lara Kockaya, Yifan Fei, Anasuya Das Gupta, Hashni E. Vidana Gamage, Adam T. Nelczyk, Dhanya Pradeep, Erik R. Nelson. The role of Liver Receptor Homolog 1 (LRH-1) in regulating cancer progression by modulating the immune 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 C027.

Abstract C043: Alprazolam abrogates the secretion of proinflammatory cytokines in PDAC CAFs

Abstract The present study sets out to determine how commonly prescribed triazolobenzodiazepines (triazoloBZDs) modulate the tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC), and how this can be leveraged therapeutically to improve patient outcomes. PDAC is one of the most lethal cancer subtypes with a 5-year survival rate of ∼13%. The poor prognosis of this disease is, in part, attributable to the dense, fibrotic tumor microenvironment (TME), and lack of durable responses to standard-of-care treatment options. A recent study from our lab reported that over 40% of PDAC patients at Roswell Park are prescribed benzodiazepines (BZDs) to manage anxiety, nausea, and other disease-related side effects. Importantly, survival outcomes in these patients varied in a compound-specific manner, as the usage of the n-unsubstituted BZD lorazepam was associated with poorer outcomes, while usage of the n-substituted triazoloBZD alprazolam was associated with improved progression-free survival. In vitro and in vivo data from the aforementioned study suggested that these findings may be a result of BZD-specific regulation of interleukin-6 (IL6), a proinflammatory cytokine with a known role in PDAC pathogenesis. More specifically, alprazolam-treated cancer-associated fibroblasts (CAFs) exhibited a potent reduction in IL6 transcription and secretion, while lorazepam treatment increased IL6 production. Based on these data, we hypothesized that triazoloBZDs may uniquely reduce intratumoral inflammation to alleviate immunosuppression in PDAC. We now find that triazoloBZDs such as alprazolam suppress the secretion of several proinflammatory cytokines (IL6, CCL2, CXCL12, IL8) by CAFs, the stromal components of the PDAC TME responsible for fibrosis and exclusion of immune infiltrates. Furthermore, we have observed that this phenotype is likely a result of triazoloBZD-mediated inhibition of the platelet- activating factor (PTAFR) signaling cascade. Investigations are currently underway to determine the impact of triazoloBZDs on regulating the immune landscape of the PDAC TME in vivo. Altogether, these findings provide a basis to inform clinical selection of BZDs for symptom management and present a novel therapeutic opportunity to repurpose FDA-approved compounds for treatment of PDAC. Citation Format: Hunter D. Reavis, Arwen A. Tisdale, Kathryn E. Maraszek, Michael E. Feigin. Alprazolam abrogates the secretion of proinflammatory cytokines in PDAC CAFs [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 C043.

Abstract A017: HER2 overexpression initiates breast tumorigenesis non-cell-autonomously by altering energy metabolism in the tissue microenvironment

Abstract Breast cancer is the most common cancer globally, with approximately 70% of pre-malignant breast tumors, known as ductal carcinoma in situ (DCIS), and 20% of invasive breast tumors overexpressing human epidermal growth factor receptor 2 (HER2). While the biological role of HER2 in invasive breast cancer has been extensively studied, its impact on breast cancer stem cells (BCSCs) and early-stage tumorigenesis, particularly within the tumor microenvironment, remains unclear. Here, we explore the role of HER2 overexpression in driving cellular events within the tumor microenvironment during the premalignant stage of breast tumorigenesis, using in vitro, in vivo, and ex vivo models. We conducted multi-omics analyses on mammary ducts from a HER2+ breast cancer mouse model at the pre-cancerous stage. Our results showed that HER2/Neu overexpression alters glucose and lipid metabolism and pentose phosphate pathway in mammary epithelial cells. Ex vivo organ culture of mammary ducts confirmed that these metabolic changes lead to increased reactive oxygen species (ROS) levels within the tumor microenvironment suggesting that oxidative stress and subsequent DNA damage are early events in HER2-driven tumorigenesis. Immunostaining of histological sections from mouse mammary glands further revealed that elevated oxidative stress and DNA damage not only in HER2+ cells but also in neighboring HER2- cells. Moreover, whole genome sequencing of tumor cells confirmed that both HER2+ and HER2- tumor cells have similar mutational burden. This suggests a non-cell-autonomous effect, where HER2 overexpression in one cell population affects the tumor microenvironment and adjacent cells, contributing to early-stage cancer development. To investigate the involvement of BCSCs during tumor initiation, we performed mammosphere assays on primary cells from HER2+ mouse models and clinical DCIS samples. Our findings showed that neither BCSCs nor their cells of origin express HER2 in both DCIS samples and murine mammary glands and tumors. This suggests that HER2 overexpression may not directly drive BCSC formation but rather creates a genotoxic microenvironment that facilitates the transformation of HER2- cell populations through a non-cell-autonomous mechanism. In summary, our findings indicate that HER2 overexpression contributes to the creation of a genotoxic tumor microenvironment by altering cellular energy metabolism and increasing ROS levels. This, in turn, may lead to the transformation of BCSCs in HER2- cell populations during early breast tumorigenesis. Understanding the role of HER2 overexpression in shaping the tumor microenvironment could provide new insights into therapeutic and preventive strategies against breast cancer. Citation Format: Sevim B. Gurler, Oliver Wagstaff, Lili Dimitrova, Fuhui Chen, Robert Pedley, William Weston, Ian J. Donaldson, Brian A. Telfer, David Novo, Kyriaki Pavlou, George Taylor, Yaqing Ou, Kaye Williams, Andrew Gilmore, Keith Brennan, Ahmet Ucar. HER2 overexpression initiates breast tumorigenesis non-cell-autonomously by altering energy metabolism in the tissue 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 A017.

Abstract B008: Therapy-protective peristromal niches mediate positive ecological interaction between therapy-sensitive and therapy-resistant cells, altering the evolutionary dynamics of acquired targeted therapy resistance in lung cancers

Abstract Pharmacological inhibitors of oncogenic signaling, such as drugs that target ALK kinase (ALKi) in ALK+ lung cancers, can induce strong and durable clinical responses. However, targeted therapies are not curative in advanced cancers and even strong responders eventually develop resistance. While therapy resistance is typically attributed to cell-intrinsic (epi)mutational characteristics of tumor cells, it can also result from interactions of tumor cells with the tumor microenvironment (TME). In contrast to the detailed elucidation of the molecular mediators of therapy resistance, our knowledge of the evolutionary dynamics underlying the resistance emergence, including the impact of TME, remains poorly defined. A key open question in understanding the evolutionary dynamics of therapy resistance is whether relapse results from an expansion of pre-existing therapy-resistant subpopulations or from a bona fide gradual evolutionary process. To elucidate this question, we integrated experimental mouse studies with mathematical modeling, interrogating therapeutic responses of therapy-naïve experimental xenograft ALK+ tumors, spiked-in with differentially labeled resistant cells. We found that ALKi treatment induced a rapid expansion of resistant cells, which drastically reduced the magnitude and duration of remissions. Surprisingly, our in silico analyses pointed to the existence of a strong positive ecological interaction between therapy-resistant and therapy-sensitive competitors. Using a combination of spatial analyses, experimental studies, and mathematical modeling, we found that this interaction was mediated by peristromal niches that protected therapy-sensitive tumor cells. Specifically, by limiting tumor regression, the therapy- induced expansion of resistant cells limited the loss of protective peristromal niches, while the subsequent resumption of tumor growth created new peristromal niches capable of supporting the survival and proliferation of therapy-sensitive cells. While this niche-mediated interaction had only a marginal impact on the transition from remission to relapse, enhanced survival of therapy-sensitive cells potentiated their ability to adapt to ALKi, leading to a higher diversity of resistance phenotypes. In summary, our study has revealed a new type of indirect, niche-mediated ecological interaction between therapy-resistant and therapy-sensitive cells. The rapid expansion of rare pre-existent resistant cells and fast transition to relapse challenge a common assumption of the pre-existence of therapy resistance. Finally, our results highlight the essentiality of TME considerations in understanding the evolutionary dynamic underlying the development of therapy resistance. Citation Format: Mark Robertson-Tessi, Bina Desai, Tatiana Miti, Pragya Kumar, Sagnik Yarlagadda, Rishi Shah, Robert Vander Velde, Daria Miroshnychenko, David Basanta, Alexander Anderson, Andriy Marusyk. Therapy-protective peristromal niches mediate positive ecological interaction between therapy-sensitive and therapy-resistant cells, altering the evolutionary dynamics of acquired targeted therapy resistance in lung cancers [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 B008.

Abstract PR012: KRAS mutation-specific effects on the tumor immune microenvironment drive tumor progression in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with abysmal survival rates. The highly immunosuppressive microenvironment of PDAC poses a major barrier to effective therapy. KRAS mutations are near-ubiquitous in PDAC, and studies indicate clinical outcomes vary depending on the specific KRAS genotype. Given that tumor genotype can shape the immune cell composition of tumors, we examined the immune cell composition of pancreatic tumors with specific KRAS mutations, focusing on KRASG12D mutations (representing 40% of PDAC cases which have the worst prognosis) and KRASG12R mutations (which make up 20% of PDAC cases and have a better prognosis). Orthotopically implanted Kras G12R/+ ;Trp53 KO mouse pancreatic tumors have a distinct immune profile in comparison to Kras G12D/+ ;Trp53 KO tumors, characterized by an influx of intratumoral CD3+ T cells and mature dendritic cells. Selective depletion of CD4+ T cells induces a rapid progression in KRASG12R, but not KRASG12D mouse tumors. RNA sequencing of KRASG12R tumor cells revealed enrichment of Type I interferon (IFN) pathways accompanied by related chemokines, CXCL9 and CXCL10 in comparison to KRASG12D tumors. Analysis of publicly available and real-world human PDAC datasets and enrichment of dendritic cells and other immune related cell types in KRASG12R tumors. Collectively, these data link the KRASG12R mutation to a unique immunogenic role. Citation Format: Andrew Wenger, Tal Baron, Erika Hissong, Rohit Chandiwani, Lukas Dow, Despina Siolas, Katherine Bacchi, Huanhuan Sun, Erika Hissong, Adrian Vega, Whitney Sisso, and Kawther Abdilleh. KRAS mutation-specific effects on the tumor immune microenvironment drive tumor progression in 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 PR012.

Abstract B041: Investigating the impact of extracellular matrix stiffness on macrophage function and organoid growth in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of only 11%. Current treatments mostly target the cancer cells themselves and have not been able to significantly improve survival rates, meaning there is an urgent need for novel therapies. PDAC is unique among solid tumors because it exhibits extensive desmoplasia that is characterized by an abundance of extracellular matrix (ECM) components. In recent years, an increasing body of work has shown that cancer tissue is stiffer than normal tissue by a factor of 3 or more. This increase in ECM stiffness has been shown to play an important role in affecting cell-to-cell communication as well as cancer progression and chemoresistance . However, how ECM stiffness affects tumor-associated macrophages, an important component of the pancreatic tumor microenvironment (TME), is still understudied. Previous studies have demonstrated that macrophages are highly responsive to mechanical cues, with the mechanical environment influencing macrophage polarization and function in a context- and disease-dependent manner. Elucidating these potential changes in PDAC can provide us with insights on how stiffness affects therapeutic outcome. Healthy pancreatic tissue typically exhibits a storage modulus of approximately 0.5-1 kPa, whereas PDAC tissue can range from 4-10 kPa. To study macrophage responses to varying stiffness, we cultured RAW264.7 mouse macrophage cell lines on 2D substrates with stiffness levels of 0.2, 2, and 8 kPa. We observed that macrophages cultured on stiffer matrices exhibited a trend toward a pro-inflammatory phenotype, with increased expression of M1 markers such as iNOS, IL-1β, and IL-6, compared to those cultured on softer matrices. However, conditioned media from macrophages cultured on both soft and stiff substrates similarly reduced the size of PDAC organoids, indicating a lack of functional difference between these groups. These findings suggest the need for more sophisticated models that better replicate the PDAC TME to fully capture the complex cell-to-cell interactions that are not evident in 2D cultures. To address this limitation, we developed a 3D tri-culture tunable biomimetic mouse model comprising PDAC organoids, host-matched cancer-associated fibroblasts (CAFs), and macrophages. This novel model allows us to modulate the stiffness of the 3D co-culture system through the addition of collagen I and CAFs. Rheometry data confirmed that the stiff tri-culture model had a significantly higher storage modulus than the soft tri-culture model. Using qPCR, we investigated the role of mechanosensitive ion channels, specifically Transient Receptor Potential Vanilloid-type 4 (TRPV4) and Piezo1, in mediating macrophage mechanosensing in response to varying levels of matrix stiffness. This study highlights the importance of 3D models that can recapitulate biophysical cues of cancer and will allow us to better understand how macrophages respond to stiffness, which may give rise to new therapeutic targets and development of new treatments in the future. Citation Format: Bayan Mahgoub, Weikun Xiao, Eileen Fung, Shannon Mumenthaler, Reginald Hill. Investigating the impact of extracellular matrix stiffness on macrophage function and organoid growth in pancreatic ductal adenocarcinoma [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 B041.

Abstract C009: MK2 knockout and radiation enhances immune cell influx in a mouse model of HPV+ head and neck squamous cell carcinoma

Abstract Introduction: HPV mediated head and neck squamous cell carcinoma (HNSCC) has an immunosuppressive microenvironment which contributes to local and distant failures. We have previously shown that MAPKAPK2 (MK2) inhibition (MK2i) in HNSCC results in improved response to irradiation (RT). MK2i was synergistic with RT resulting in better control than monotherapy. To understand the mechanism behind the improvement of RT in MK2i treated mice we tested the hypothesis that MK2 ablation can enhance the radiotherapy mediated immunogenic response in a solid tumor. Methods: We used CRISPR gene editing to knockout the MK2 gene in the highly metastatic murine cell line, MLM3 (KO). We implanted tumors into c57bl6 and NSG mice and compared tumor growth compared to wild type (WT) with and without radiation. At end point, tumors were excised, digested and immunophenotyped utilizing labelled antibodies and flow cytometry. Before immunophenotyping, 8 mgs of tumor were cut off and incubated in media for 24 hours to assess cytokine release by cytokine array analysis. Results: We found that the differences in tumor growth between WT and KO cells in c57bl6 mice were significantly greater than those seen in NSG mice. Survival studies with c57bl6 mice implanted with WT or KO tumors with and without RT showed that MLM3 KO implanted mice had significantly greater survival (58.5 days) compared to WT (35.5 days). WT tumors treated with RT improved median overall survival with the WT + RT group surviving longer than WT (56 days) and the MK2 KO + RT had the longest survival overall (65 days). Immunophenotyping WT v KO tumors grown in c57bl6 mice showed an increase in CD45 cells in the KO tumors compared to WT. The cells that were increased in number in the KO tumors were labelled by Ly6G (neutrophils), F4/80 (macrophages), CD14+MD11c (dendritic cells), CD4 (Th), CD8 (Tc), CD3+NK1.1 (NKt) and NK1.1 (NK cells). Irradiating the tumors accentuated the influx of immune cells causing a greater influx of CD45 cells at 3 days post-RT which included increases in F4/80+CD80 (M1 macrophages), CD14 (monocytes), CD11c+MHCII, NK1.1, NK1.1+CD3 and CD4 cells. However, the influx of CD8 cells were reduced at 3 days. At 8 days post RT, many cell types remained elevated in the KO tumors compared to wildtype and there was a partial recovery of CD8 cell influx. However, there was also an increase in immune suppressor cells into the tumors including Ly6C+arginase (M-MDSCs), Ly6C+arginase (PMN-MDSCs) and CD4+T-bet (Th1) cells. The cytokine analysis showed that six cytokines were differentially secreted from tumors: Dkk-1, IL-2, IGFBP-5, IL-3, IL-17a and CD14. All these cytokines were suppressed in the KO with respect to the WT tumors. Conclusion: Here we show evidence that genetic ablation of MK2 in a mouse model of HPV mediated HNSCC results in an increase in immune cells infiltrate into tumors. Irradiation enhances this difference by stimulating a greater influx of immune cells. These data suggest that tumoral MK2 may aid in the immunosuppression of tumors in HNSCC. Citation Format: Deri Morgan, Colby Spiess, Alyssa Schmidt, Dakota D.D. Okuwone, Harmony Saunders, Chris E. Lominska, Mary A. Markiewicz, Yelder Tonia, Devin Shrock, Yuting Lin, Hao Gao, Gregory N Gan. MK2 knockout and radiation enhances immune cell influx in a mouse model of HPV+ head and neck squamous cell carcinoma [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 C009.

Abstract C022: Exercise reduces pancreatic tumor in a gut microbiome dependent manner

Abstract Preclinical studies suggest that aerobic exercise influences the tumor microenvironment by normalizing vasculature and reducing immunosuppression in Pancreatic Ductal Adenocarcinoma (PDAC). Gut microbes can influence PDAC progression by modulating metabolic and immune pathways. However, a knowledge gap remains regarding the role of the gut microbiome in the anti-tumor effects of exercise. We hypothesized that the exercise-induced anti-tumor effect in PDAC is regulated by gut microbiome. We compared the effect of treadmill running on growth of orthotopic PDAC in C57BL/6 mice from Taconic Biosciences (Tac) and Jackson Laboratories (Jax), which have different microbiomes. Tumor-bearing mice performed treadmill exercise (ex, 12 meters/minute (m/min) for 45 minutes (min) daily) or were non-exercised controls (no-ex). Exercise reduced tumor weight compared to no-ex controls in Jax (no-ex, 0.72±0.05 grams (g); ex, 0.54±0.04g; p=0.04) but not in Tac mice (no-ex, 0.72±0.05g; ex, 0.78±0.05g; p=0.47). Interestingly, in Tac mice treated with antibiotics (ATBx) ad libitum, treadmill exercise reduced tumor growth (no-ex, 0.74±0.04 g; ex, 0.53±0.03g; p= 0.006). Taken together, this data indicates that the anti-tumor effect of exercise is affected by gut microbiome. We considered potential mechanisms by which exercise suppresses tumor growth. Tumor infiltrating immune cells were measured by flow cytometry and immunohistochemistry. No significant differences in the tumor infiltrating immune cells were observed between no-ex and ex mice from Tac or Jax, regardless of ATBx, indicating that gut microbiome mediated anti-tumor effect is independent of immune pathways. Previous studies have demonstrated that exercise reduces tumor growth in an intensity dependent manner. Therefore, to determine exercise intensity as percentage of Peak Velocity (VPeak), exercise capacity (ExCap) test was performed. VPeak (m/min), time to exhaustion (min), and distance to exhaustion (m) were used as indicators for treadmill ExCap. Treadmill ExCap was significantly lower in tumor bearing Tac mice (24.86±3.55m/min, 12.56±1.79min, 217.17±31.02m) compared to Jax mice (30.50±3.81m/min; 16.58±2.07min; 320.15±40.02m; p=0.01). However, ATBx treatment did not change the treadmill ExCap in the Tac mice (25.50±3.19m/min; 12.62±1.58min; 209.26±26.16m; p=0.97) despite significantly changing the effect of exercise on tumor growth, indicating that ExCap may not regulate differences observed in the tumor weight. Overall, these results indicate that gut microbiome regulates anti-tumor effects of exercise, that are independent of tumor infiltrating immune cells or exercise intensity. Analysis of serum and tumor metabolites, 16s rRNAseq to evaluate microbial differences, and bulk RNAseq on tumors, is underway with the goal of identifying microbial-derived metabolites which mediate the anti-tumor effect of exercise. Understanding how the gut microbiome mediates the efficacy of exercise to suppress tumor growth will be critical for maximizing the benefits of exercise for patients with PDAC. Citation Format: Sumedha Pareek, Vidhi Chandra, Le Li, Hetal Patel, Truong Lam, Olivereen LeRoux, Haoyue Liu, Jonghae Lee, Florencia McAllister, Keri Schadler. Exercise reduces pancreatic tumor in a gut microbiome dependent manner [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 C022.

Abstract B042: The impact of opioids on the pancreatic tumor immune microenvironment

Abstract Pancreatic cancer is a highly lethal malignancy with a 5-year survival rate of 12.8%. Due to the high prevalence of cancer-related pain, 75% of pancreatic cancer patients are prescribed opioids. Opioids, which signal through the G protein-coupled receptor (GPCR) mu-opioid receptor (MOR), have peripheral effects outside of their analgesic function. Importantly, several studies have correlated opioids with worsened survival of patients receiving immunotherapy. However, the mechanism behind this is unknown. Immunotherapy is largely ineffective in pancreatic cancer, due in part to the tumor microenvironment (TME) characterized by dense, desmoplastic stroma, with cancer-associated fibroblasts (CAFs) comprising up to 90% of the tumor volume. CAFs play a large role in immunosuppression and therapy resistance, such as through promoting infiltration of immunosuppressive cells, resulting in reduced presence of antitumor immune cells and impaired immunotherapy efficacy. While the role of CAFs in promoting immunosuppression in the tumor microenvironment is well characterized, the effect of opioids on this phenomenon is unknown. We now provide evidence that MOR directly regulates CAF function. Specifically, knockdown of MOR in CAFs reduces gene expression of alpha-smooth muscle actin (aSMA) and leukemia inhibitory factor. MOR knockdown in CAFs also decreases protein expression of aSMA and type 1a collagen. We hypothesize that opioids promote MOR-dependent CAF activation and secretion of pro-inflammatory cytokines, resulting in a more immunosuppressive TME, and thus impaired immunotherapy response. The long-term goal of this work is to determine the role of opioids on CAFs in the pancreatic TME, as well as the role of MOR in promoting immunosuppression and the resulting value of MOR as a target to improve immunotherapy efficacy. This research is clinically significant because it could uncover a novel mechanism driving immunosuppression caused by a medication commonly prescribed to almost all pancreatic cancer patients. Citation Format: Kathryn Maraszek, Michael Feigin. The impact of opioids on the pancreatic tumor immune 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 B042.

Abstract B029: Mapping the vascular walls of colorectal cancer

Abstract Introduction: Properties of tumor angiogenesis impact on tumor growth and composition of the tumor microenvironment. Tumor vessels are composed of pericytes and other mural cells. Detailed characterization of the cellular composition of the microvasculature can suggest functional vessel and case properties relevant for outcome, response to treatment and immune surveillance. Results: Based on single cell RNA-seq from three human stage II/III colon cancers and pilot in-situ multi-antibody staining of six tumors two main clusters of perivascular cells were identified; B1 (MCAM+, MYH11-) and B2 (MCAM+, MYH11+) cells. B1 cells displayed pericyte features including expression of RGS5, whereas B2 cells showed high expression of smooth muscle cell markers like ACTG2 and Desmin. Desmin-high B2 cells were predominantly located in muscular layers, whereas Desmin-low/MYH11-high B2 cells showed perivascular enrichment. An extended novel analyses workflow of digital image analysis was used to profile peri-endothelial composition in 19 intervals; 11 1-µm intervals from 0 to 10 µm and 8 5-µm intervals from 10 to 50 µm from endothelial areas. Each of these expansion areas was quantitated regarding density of PDGFRB+/- cells including B1/B2 cells, and a MYH11+/MCAM- cells. Density of macrophage subsets (CD68-positive subsets defined by CD163 and CD11c) were also quantitated. In general, B1 cells showed stronger spatial association with endothelial cells than B2 cells, and this spatial association was stronger for the PDGFRB+ B1 subset. Survival analysis indicated that prognostic relevance was strongest when PDGFRB+ (favorable prognosis) cells were closest to endothelial cells. Regarding perivascular macrophage density, M0 and transit M1/M2 macrophages were predominantly enriched in the peri-endothelial regions, whereas density of M1 and M2 increased gradually as distance from vessels increased. M2 density was overall associated with poor prognosis, whereas high density of other macrophage subsets overall was associated with good prognosis. Ongoing studies are using a clustering-based approach to identify vessel subsets, and prognosis associations are being consolidated in additional cohorts. Summary: This study provides a novel approach for high resolution profiling of perivascular status in CRC, and possibly other tumor types, that could be of specific importance to identify tumor features associated with response to angiogenesis-directed therapies. Citation Format: Linglong( 凌 龙 ) Huang( 黄 ), Mercedes Herrera, Jonas Sjölund, Vladimir Chocoloff, Simon Joost, Rasul M. Tabiev, Lina Wik Leiss, Carina Strell, Luis Nunes, Artur Mezheyeuski, David Edler, Anna Martling, Fredrik Pontén, Bengt Glimelius, Tobias Sjöblom, Maria Kasper, Kristian Pietras, Arne Östman. Mapping the vascular walls of colorectal 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 B029.

Abstract B011: Sensory neurons regulate intracellular and intercellular mitochondrial dynamics in breast cancer cells

Abstract Crosstalk between cancer and the nervous system regulates tumor progression. These interactions occur at both the local (i.e., within the tumor parenchyma) and systemic (e.g., endocrine signaling) levels. More recent work has determined that cancer cells not only interact with their local environment via secreted molecules, but in some cases, via acquiring proteins or organelles from other cells within the tumor microenvironment (TME). Here, we demonstrate that breast cancer cells acquire mitochondria from neurons. Breast cancer cells with higher metastatic potential acquire more mitochondria in NGF dependent manner compared to the cell line with lower metastatic potential. These results suggest that in addition to their known roles in electrochemical and paracrine signaling, tumor-innervating nerves can also enhance cancer cell fitness via transfer of mitochondria. We are now characterizing the mechanisms underlying this transfer, determining the fate of transferred mitochondria in recipient cells, and defining how transferred mitochondria drive cancer development, progression, and metastasis. Further, sensory neurons at the tumor microenvironment dictate the intracellular mitochondria dynamics affecting morphology and function. The change in mitochondria dynamics enhances cancer cell survival and metastasis, we are uncovering how sensory neuron induced mitochondria dynamics drives tumor progression. Citation Format: Ankit Tiwari, Yue Wu, Jeremy C. Borniger. Sensory neurons regulate intracellular and intercellular mitochondrial dynamics in breast cancer cells [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 B011.

Abstract PR007: Pancreatic cancer cachexia is mediated by tumor-derived PTHrP

Abstract Purpose: Our prior work has established that metastasis is initiated by PTHrP-driven mechanisms in pancreatic ductal adenocarcinoma (PDAC). PTHLH (the gene encoding the PTHrP protein) is directly adjacent to and co-amplified along with KRAS, the major oncogenic driver in PDAC patients. The KRAS-PTHLH amplicon is a marker of the highly aggressive squamous/quasi-mesenchymal/basal-like PDAC patient subtypes, is associated with increases in metastasis and cancer cachexia, and correlates with decreased overall patient survival. We have deleted Pthlh in the autochthonous Kras- and Tp53-driven pancreatic cancer mouse model (i.e. KPC mice) and found that the resulting KPC-Pthlh LoxP mice (herein KPC-PthrpcKO) live nearly twice as long as KPC controls. Intriguingly, recent evidence has emerged for PTHrP’s role in cachexia-associated adipose tissue wasting and we posit that the dramatic survival extension in KPC-PthrpcKO mice may be due to reduced cachexia. Results: In PDAC patients, PTHrP is co-amplified along with KRAS and correlates with significantly decreased overall survival. We generated KPC-PthrpcKO mice and showed that they have reduced tumor burden and dramatically increased overall survival relative to KPC controls. In parallel experiments, we treated mice with an anti-PTHrP neutralizing monoclonal antibody, which similarly extended survival. Upon further analysis, we observed that the overall body condition of KPC-PthrpcKO mice (and anti- PTHrP treated KPC mice) was greatly improved, with less loss of adipose and muscle tissue. Mechanistic studies revealed that tumor cell-derived PTHrP signaling to adipocytes in white adipose tissue depots mediates cachexia. Specifically, we found that adipose tissue wasting and lipolysis were greatly reduced upon deletion or pharmacological inhibition of PTHrP. In the same vein, RNA-seq of cachectic adipose tissue revealed that PTHrP mediates adipose wasting by turning off de novo lipogenesis (DNL), a process critical for the generation of fatty acids in adipocytes. PTHrP binds to its cognate receptor, PTH1R, on adipocytes and blocks fatty acid synthesis by turning off the essential DNL transcription factors CHREBP and SREBP, likely through a PTH1R-PKA-CREB1 signaling axis. Thus, the genetic deletion and pharmacological inhibition of PTHrP in vivo led to a profound reduction in cachexia-related adipose tissue wasting and muscle atrophy. Re-introduction of PTHrP into a PDAC cell line with low cachexia-inducing potential (and low baseline PTHrP) dramatically increased the degree of cachexia observed upon orthotopic implantation, leading to a reduction in overall survival. Therefore, PTHrP is both necessary and sufficient to induce cachexia in pancreatic cancer. Conclusions: This work has demonstrated the importance of the previously unappreciated roles of PTHrP signaling in driving pancreatic cancer cachexia and adipose tissue remodeling, and future studies will look to translate anti-PTHrP therapy into clinical trials. Citation Format: Jason Pitarresi. Pancreatic cancer cachexia is mediated by tumor-derived PTHrP [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 PR007.

Abstract C029: PM2.5-induced drug resistance in lung cancer

Abstract Long-term exposure to PM2.5 air pollution is a significant contributor to lung cancer incidence. Our previous studies have shown that such exposure promotes lung cancer progression by activating the EGFR (epidermal growth factor receptor) and AhR (aryl hydrocarbon Receptor) pathways. We observed that in lung cancer cells with EGFR mutations, prolonged PM2.5 exposure induces EGFR activation, resulting in accelerated tumor cell growth both in vitro and in vivo. This suggests that PM2.5 may play a role in the resistance to EGFR-TKI (tyrosine kinase inhibitor) therapies, raising concerns about treatment efficacy. To explore this, we exposed the EGFR-mutant lung cancer cell line to PM2.5 for 90 days. Our results revealed that long-term exposure not only reduced the therapeutic effect of EGFR-TKIs but also increased the expression of cMyc, a gene linked to poor prognosis and drug resistance. We further confirmed that PM2.5-induced EGFR-TKI resistance is driven by the AhR-cMyc pathway. AhR, activated by environmental toxins in PM2.5, increases cMyc expression, leading to drug resistance. When we administered cMyc inhibitors, the PM2.5-exposed cells regained sensitivity to EGFR-TKI therapy. These findings indicate that targeting the AhR-cMyc pathway could be a promising approach to overcome PM2.5-induced resistance in lung cancer treatment. Further research is needed to explore the broader implications of environmental pollution on cancer therapy and to develop strategies to counteract the effects of pollutants like PM2.5. Citation Format: Chi-Yuan Chen, Chieh-Wen Chan, Tong-Hong Wang. PM2.5-induced drug 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 C029.

Abstract C006: Tumor-associated macrophages drive prostate cancer progression via IL-1β signaling

Abstract Inflammation is linked to prostate cancer progression. Inflammatory cell infiltrates are commonly observed in prostate biopsies, and inflammation-induced lesions (proliferative inflammatory atrophy, PIA) are precursors of prostate cancer. However, the mechanism by which inflammation impacts prostate cancer progression is poorly understood. Here, we investigated the significance of inflammation on prostate cancer progression using a cMyc-driven prostate adenocarcinoma mouse model (Hi-Myc). We observed a robust tumor-associated macrophage (TAM) infiltrate early during progression of Hi-Myc tumors. Depleting TAMs led to a decrease in both tumor weight and invasive area, demonstrating the functional importance of TAMs in tumor maintenance. To elucidate the molecular basis of how TAMs influence tumor progression, we collected Hi-Myc tumors throughout cancer development from the precursor stage of prostatic intraepithelial neoplasia to prostate adenocarcinoma and performed single-cell RNA sequencing (scRNA-seq). Our study revealed that a gene signature of strong IL-1β signaling activation was observed in TAMs from Hi-Myc tumors, but not in macrophages from wild-type prostates. Importantly, IL-1β neutralization led to delayed tumor progression with reduced tumor weight and invasive area. Furthermore, blocking IL-1β signaling decreased TAM infiltration, suggesting a positive feedback loop created by TAMs. To understand the effect of IL-1β on cancer cell invasion, we used a protease-dependent fluorescent probe to investigate the activity of major extracellular matrix degraders and found that IL-1β neutralization impairs MMP activity, likely through loss of expression by tumor cells and macrophages. To further investigate the targets of IL-1β signaling, we analyzed Il1r1 expression levels across all cell types and found the highest levels in cancer-associated fibroblasts (CAFs). Moreover, CAFs expressed elevated levels of the myeloid chemokines Ccl2, Csf1, Cxcl1, Cxcl2 as well as Il6 compared to fibroblasts from wild-type prostates. In vitro studies of wild-type prostate fibroblasts treated with recombinant IL-1β confirmed that IL-1β directly upregulates expression of these inflammatory cytokines and chemokines. In addition, IL-1β directly promotes proliferation of tumor-derived prostate cancer organoids in vitro. Overall, our study suggests that TAMs and CAFs cooperatively drive pro-tumorigenic IL-1β signaling in prostate cancer, demonstrating a direct mechanistic link between inflammation and prostate cancer progression. Citation Format: Young Sun Lee, Jimmy L. Zhao, Max Land, Joseph Chan, Perianne Smith, Roshan Sharma, Sanjay Kottapalli, Linda Fong, Zhenghao Chen, Cathy Wang, Jesse Kirkpatrick, Ava Soleimany, Samir Zaidi, Kayla Lawrence, Amanda Kulick, Teng Han, Zhen Sun, Philip Watson, Anuradha Gopalan, Ojasvi Chaudhary, Tianhao Xu, Ignas Masilionis, Ronan Chaligne, Dana Rathkopf, Michael Morris, Sangeeta Bhatia, Michael Haffner, Dana Pe'er, Charles Sawyers. Tumor-associated macrophages drive prostate cancer progression via IL-1β signaling [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 C006.

Abstract C050: Leveraging ECM deposition by CAF-like Ewing sarcoma tumor cells to target micrometastases with matrix-binding VHHs

Abstract Ewing sarcoma (EwS) is an aggressive bone and soft tissue tumor and EwS patients often succumb to their disease years after initial treatment due to relapses at metastatic sites. Novel therapeutic strategies are needed that can successfully eliminate microscopic residual disease foci that persist at the end of primary therapy. Extracellular matrix (ECM) proteins in the tumor microenvironment (TME) provide critical pro-survival and pro-invasion signals to tumor cells. Our published and unpublished data show that the glycoprotein tenascin-C (TNC) is enriched in EwS metastases and that TNC and other pro-tumorigenic ECM proteins are deposited by subpopulations of CAF-like tumor cells that are activated in response to tumor and TME-derived TGF-beta ligands. TNC is abundantly produced by multiple human tumors of epithelial and non-epithelial lineage but is otherwise rarely expressed outside of development and wound healing. Importantly, TNC is specifically enriched in metastatic lesions where it has been implicated as a mediator of metastatic competence and treatment resistance. In the current work we have tested whether the TNC-rich TME of disseminated EwS tumor foci could be leveraged to direct therapies to sites of residual micrometastatic disease. To achieve this, we generated monovalent and bivalent anti-human TNC VHHs (hTNC-VHH) using a mammalian expression platform. Camelid-derived hTNC-VHH sequences were sourced from published literature. VHHs are single domain heavy chain only antibody fragments with higher stability and tissue penetration than conventional monoclonal antibodies. Using EwS tumor spheroids in collagen-rich 3D culture, hTNC-VHH penetrated dense ECM matrices and bound through multiple cell layers in under 10 minutes. hTNC-VHH accumulated in TNC-positive but not TNC-knockout EwS spheroids and were retained beyond 96 hours. To assess their potential to drive protein and immune therapies to TNC-rich TMEs, we fused hTNC-VHH to immune modulating CD3- and CD28-activating VHHs. These hTNC-VHH-CD3/CD28-conjugates promoted immune cell activation and tumor cell death in PBMC-tumor co-culture assays as determined by CD25 flow cytometry and fluorescence microscopy, respectively. To assess in vivo localization, fluorescently-tagged hTNC-VHH were intravenously injected into mice that had been xenografted with EwS cells. Tissue microscopy confirmed selective binding of hTNC-VHH to small lung and liver EwS micrometastases in less than 4 hours. VHHs were not retained in non-tumor bearing organs including the brain and heart or in tumor-free lung and liver regions. Experiments are ongoing to assess anti-tumor efficacy and pharmacodynamics of hTNC-VHH conjugates in EwS xenografts. Together these findings suggest that the ECM-remodeling properties of CAF-like EwS cells can be exploited to recruit novel ECM-targeting protein therapeutics to micrometastases. If successful, this innovative approach could eradicate microscopic residual disease and prevent metastatic EwS recurrence. Citation Format: Emma D. Wrenn, Jason P. Price, Raymond O. Ruff, Nicolas M. Garcia, James M. Olson, Elizabeth R. Lawlor. Leveraging ECM deposition by CAF-like Ewing sarcoma tumor cells to target micrometastases with matrix-binding VHHs [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 C050.

Abstract B019: Regulatory macrophages contribute to tumor cell plasticity in colorectal cancer metastases

Abstract Metastatic disease is the common cause of death in colorectal cancer. To better understand the cellular landscape of colorectal metastasis, we profiled primary tumors and liver, lung, and peritoneal metastases using single-cell RNA sequencing. Our analysis revealed a subset of tumor epithelial cells with a highly plastic transcription profile and, inversely, low expression of stemness markers. Tumor cells with plasticity markers were spatially enriched at the tumor- stroma interface with exposure to tissue microenvironmental factors. Cell-free malignant ascites, which recapitulates the tumor microenvironment, impaired expression of proliferation and stem- like markers in favor of plasticity markers and enhanced migration of tumor cells. Furthermore, Co-culture of colorectal tumor cells with CD11b+ macrophages isolated from malignant ascites from patients was sufficient to recapitulate tumor cell plasticity. Profiling of tumor-associated macrophages identified enrichment for a lipid-handling features and high spatial correlation with plastic tumor cells. Taken together, we demonstrate that tumor-associated macrophages contribute to disease progression through development of pro-malignant features. Citation Format: Matthew A. Cottam, Muhammad B. Mirza, Emily N. Arner, Marium Siddiqui, Clare Lipscombe, Zaid Hatem, Katy E. Beckermann, Kamran Idrees. Regulatory macrophages contribute to tumor cell plasticity in colorectal cancer metastases [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 B019.

Abstract B040: Investigating the impact of tumor EMT states on immune cell infiltration in breast cancer

Abstract Breast cancer is one of the most frequently diagnosed cancers and the second leading cause of women’s cancer death. Although the five-year survival rate for women in the United States with nonmetastatic invasive breast cancer is 90%, this rate drops to less than 30% for those with tumors that have metastasized to distant organs. Epithelial-to-Mesenchymal Transition (EMT) plays a critical role in tumor metastasis by enabling the loss of epithelial characteristics and the gain of mesenchymal traits, leading to increased cell motility, invasion, and resistance to therapy. Consequently, targeting the EMT process may prevent tumor cell migration and improve patient outcomes. Rather than being a binary switch from an epithelial to a mesenchymal state, cells undergoing EMT can assume a spectrum of intermediate states, some of which may reshape the tumor microenvironment by recruiting pro-tumor immunosuppressive cells, such as regulatory T cells, M2-like macrophages, and myeloid-derived suppressor cells. However, the impact of EMT on the immune composition of the tumor microenvironment and the causative mechanisms remain poorly understood. Using multi-omics approaches in a mouse model of triple-negative breast cancer, we assessed the interaction between tumor and immune cells in the tumor microenvironment driven by the EMT. A heterogeneous mouse breast cancer cell line (4T1) was used to isolate five distinct single-cell-derived clonal populations residing in distinct states within the EMT spectrum. These EMT clones, along with the parental 4T1 cell line, were orthotopically transplanted into the mammary fat pads of BALB/c mice. Primary tumors were subsequently harvested and dissociated for single-cell RNA-sequencing (scRNA-seq) and DNA methylation analysis. Lungs were histologically evaluated for metastases. Tumors derived from an intermediate EMT clone exhibited increased tumor growth kinetics and a higher incidence of metastasis compared to epithelial or mesenchymal clones. In scRNA-seq data, tumors derived from different EMT clones largely retained their original EMT states in vivo and recruited distinct immune cell profiles. DNA methylation analysis further revealed that these tumors displayed unique methylation patterns in EMT-related CpGs. This study introduced a model that captures the complexity of the tumor microenvironment across the EMT spectrum. This was accomplished through immunophenotyping using scRNA-seq and identifying unique epigenetic patterns through DNA methylation. Our findings highlight the potential for distinct immune cell populations to be recruited to tumors in different EMT states, thereby influencing survival and metastasis outcomes. Furthermore, our results open new opportunities for developing cancer combination treatment strategies that target specific tumor-immune cell interactions. Citation Format: Hanxu Lu, Meisam Bagheri, Todd Miller, Diwakar Pattabiraman, Brock Christensen. Investigating the impact of tumor EMT states on immune cell infiltration in breast 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 B040.