Metastatic Research Articles

Abstract A006: Investigating the effects of fibroblasts derived from the primary versus the metastatic organ on 3D biomimetic models of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer death in the United States with a 5-year survival rate of only 13%. Early diagnosis is very difficult and thus 53% of patients are diagnosed after metastasis has already occurred, with the liver being the most frequently affected site. Both primary tumors and metastases have dense desmoplastic stroma due to a high percentage of fibroblasts within the tumor microenvironment (TME). Previously, we developed a tunable 3D biomimetic model of the liver metastatic niche (LMN) to investigate how local fibroblasts affect tumor growth and chemoresistance in PDAC liver metastasis. This study aims to investigate the contributions of fibroblasts at the primary versus metastatic sites. We used matched primary PDAC and liver metastatic (LM) organoids derived from the Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre (“KPC”) genetically engineered mouse model. This allows us to compare the effects of primary PDAC cancer associated fibroblasts (CAFs) and liver metastasis associated fibroblasts (MAFs) on tumor cell growth and chemoresistance. Rheometry analyses were conducted to study the impact of local fibroblasts on the extracellular matrix (ECM) stiffness. Our results showed that both storage and loss moduli were enhanced only when PDAC CAFs were present in the co-cultured gels compared to gels with MAFs. Additionally, the 3D biomimetic model showed fibroblasts increased tumor growth and chemoresistance to gemcitabine, only when they were derived from the same site as the tumor cells. This highlighted the importance of studying the organ-specific microenvironment to develop better treatment options for patients with metastatic PDAC. To delve deeper into the molecular basis of these observations, we conducted RNA-sequencing to identify and compare the gene expression profiles in primary and metastatic tumor organoids exposed to conditioned media from CAFs and MAFs using a transwell membrane co-culture model. Our results showed significant differences in gene expression based on the type of fibroblasts co-cultured with the organoids. Moreover, we investigated the role of MAF-derived exosomes on LM growth and chemoresistance using exosome depletion and direct exosome addition to LM organoids. Lastly, we performed exosomes size and distribution characterizations using dynamic light scattering (DLS) analysis. Taken together, these findings highlight the critical role of the organ-specific microenvironment in tumor progression and the necessity of targeted therapies to improve patient outcomes in metastatic PDAC. Our results underscore the importance of developing a tunable 3D biomimetic model of the LMN, as it enables the testing of novel therapeutic strategies designed to disrupt interactions between MAFs and tumor cells, and potentially advancing treatments for metastatic PDAC. Citation Format: Mahsa Pahlavanneshan, Weikun Xiao, Eileen Fung, Vaidhyanathan Mahaganapathy, Chae Young Eun, Chang-Il Hwang, Shannon Mumenthaler, Reginald Hill. Investigating the effects of fibroblasts derived from the primary versus the metastatic organ on 3D biomimetic models of 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 A006.

Abstract C015: IL-27R signaling modulates protective versus pathogenic T cell responses in hepatocellular carcinoma

Abstract Liver cancer is the 3rd leading cause of cancer death. Hepatocellular carcinoma (HCC) is the major form of primary liver cancer, with a growing incidence rate estimated to surge by 50% in the coming twenty years. Although some success has been achieved with immune checkpoint blockade inhibitors, many HCC patients do not respond well to the treatments. Therefore, it is critical to better understand immune mechanisms regulating HCC to identify new biomarkers and develop novel immunotherapies for HCC patients. HCC is driven by chronic liver diseases and is frequently associated with chronic inflammation in the liver. Cytokines are small mediators of inflammation that play an essential role in inflammatory diseases and tumorigenesis. IL27 is a member of the IL6/IL12 superfamily with context-dependent roles in various inflammatory disorders and cancer. IL27 receptor (R) is expressed by most immune cells and several non-immune cells and plays pivotal roles in the regulation of immune responses. Recent work from our lab shows that IL27R signaling suppresses anti-cancer immune response in HCC, acting via the control of innate cytotoxic cells, and Il27ra -/- mice develop significantly less HCC. However, how IL27R signaling regulates T cell subsets in HCC in vivo remains elusive. Here, using diethylnitrosamine (DEN)-induced mouse model of HCC we found a reduction of regulatory T cells (Tregs) in tumor-bearing Il27ra-/-mice along with lowered HCC burden. ScRNA sequencing of CD45+ cells isolated from HCC tumors indicated a less proliferative phenotype of Tregs and more cytotoxic and less exhausted CD8 T cells in mice with Il27ra deficiency. Pathway analysis suggested that IL27R-deficient Tregs were more quiescent, as characterized by the downregulation of various metabolic pathways. Our newly generated Foxp3 cre Il27ra flox mice show a significant reduction of DEN-driven HCC accompanied by an increase of tumor-infiltrating activated CD8 T cells compared to IL27R sufficient controls. Treg-specific deletion of IL27R caused the upregulation of TCF1 in Tregs which has been recently demonstrated to control Treg immune-suppressive functions in colorectal cancer. Meanwhile, tumor-infiltrating effector CD8 T cells were increased in Foxp3 cre+ Il27ra flox mice relative to their Foxp3 cre- Il27ra flox littermate controls. Interestingly, CD8 T cell specific deletion of IL27R did not impact HCC growth in CD8 cre Il27ra flox mice. Overall, our data suggest that IL27R signaling suppresses anti-HCC immunity by enhancing the pro-tumorigenic Tregs and suppressing anti-tumor effector CD8 T cell functions. Citation Format: Zhengzheng Shi, Jiani Zhu, Aleksandra Mazitova, Anastasiia Marchenko, Sergei Grivennikov, Ekaterina Koltsova. IL-27R signaling modulates protective versus pathogenic T cell responses in hepatocellular 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 C015.

Abstract A034: Single cell RNA sequencing of triple negative breast cancer patient-derived xenograft model identifies CRABP1 of cancer-associated fibroblast as a key regulator of breast cancer metastasis

Abstract Triple-negative breast cancer (TNBC) is associated with a high risk of distant metastasis, particularly to the lung and liver. Both intrinsic characteristics of cancer cells and the tumor microenvironment (TME) influence TNBC growth and metastasis. The TME, comprising stromal, immune, and endothelial cells, exhibits heterogeneity both between and within cell types. Identifying the molecular characteristics of TME cells that affect cancer progression is crucial. We aim to investigate this using single-cell RNA sequencing of patient-derived xenograft (PDX) models to explore TME cells and genes involved in TNBC metastasis. We established PDX models by transplanting tumor tissues from 26 TNBC patients into immunodeficient mice. These models were categorized based on their metastatic potential and patient outcomes: non-metastatic (n=5) and metastatic (n=4). Single-cell RNA sequencing of nine PDX tumors revealed significant differences in gene expression of murine stromal cells between metastatic and non-metastatic models. We identified ten genes (Serpinb2, Spp1, Tnc, Thbs1, Timp1, Il11, Mt2, Crabp1, Cck, Mt1) that were highly expressed in the stromal cells of metastatic PDX models. To test if TNBC cells influence these fibroblast genes, we exposed NIH3T3 fibroblasts to conditioned media from the 4T1 TNBC cell line. In three independent experiments, only Crabp1 expression in NIH3T3 cells was consistently increased by 4T1- conditioned media. CRABP1, a retinoic acid-binding protein, is known for its role in differentiation and proliferation by regulating MAPK signaling. Although Crabp1-positive cancer-associated fibroblasts have been noted, its role in TNBC metastasis was previously unexplored. We investigated CRABP1 function using a CRABP1 knockdown NIH3T3 cell line. CRABP1 knockdown led to reduced invasion and migration of 4T1 cells in trans-well assays and decreased invasiveness of 4T1 spheroids in a collagen matrix. Additionally, CRABP1 knockdown NIH3T3 cells showed reduced proliferation in vitro and fewer alpha-SMA positive cancer-associated fibroblasts in co-injected 4T1 tumors in BALB/C mice. In summary, CRABP1, identified through single-cell RNA sequencing of stromal cells in TNBC PDX models, is a potential regulator of TNBC metastasis, affecting cancer cell migration, invasion, and fibroblast proliferation. Citation Format: Woohang Heo, Yujeong Her, Sieun Yang, Dakyung Lee, Rokhyun Kim, Jong-Il Kim, Hyeong-Gon Moon. Single cell RNA sequencing of triple negative breast cancer patient-derived xenograft model identifies CRABP1 of cancer-associated fibroblast as a key regulator of breast cancer 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 A034.

The Role of Urinary Extracellular Vesicles in Kidney Cancer: Diagnostic and Therapeutic Potential

Renal cancer ranks as the 14th most common cancer globally, with renal cell carcinoma (RCC) being the primary variant, arising from renal tubular epithelial cells; clear cell RCC constitutes about 80% of cases. Despite their limitations, surgery and targeted therapy remain the mainstays of RCC treatment. Regardless of advancements in RCC research, substantial obstacles continue to exist, such as delayed diagnosis, advanced distant metastasis, and drug resistance. As urine is an easily accessible biofluid, the identification of EVs has paved the way for novel biomarker research. Urinary extracellular vesicles (uEVs) are a novel source of biomarkers with potential applications in cancer detection and management, utilizing a less invasive approach. New data indicate that uEVs are crucial in several areas of RCC, containing tumor development, metastasis, immune evasion, and response to drugs. These vesicles facilitate intercellular communication by transporting a variety of bioactive substances, including RNA, DNA, proteins, and lipids, and are released into the extracellular space by the majority of cell types. uEVs RNAs and proteins are presently being investigated for their possible application as diagnostic biomarkers for different types of kidney cancer. This review summarizes the most recent research examining the potential of uEVs cargo as a biomarker for the diagnosis, prognosis, and treatment of renal cancer.

Abstract A008: Microenvironmentally released cytokines mediate organ specific transcriptional profiles for metastatic colorectal cancer

Abstract Metastatic disease represents a complex series of interaction between tumors and their surrounding microenvironment. Here we utilize model systems to define organ specific transcriptional signatures present in metastatic seeding. We have identified unique epigenetic programing necessary for the colonization of the liver or the lung by colorectal cancer. We have shown that manipulation of master regulators of these signatures in vitro and in vivo changes the metastatic potential of the tumor. In specific, we have found that native cytokines within the liver and lung are engaged by the metastatic tumor cells to promote this reprograming. In the case of the liver, we have identified hepatocyte released CCL2 acts on metastasizing tumors to promote colonization. Through in vitro and in vivo models, we showed that CCL2 engagement drives upregulation of the transcription factor TCF7 and subsequent enhancer reprograming to promote survival of the metastatic cells in the liver. In the lung, we have identified additional, unique secreted cytokines which lead to the upregulation of other TCF family members. Together this data identifies an important signaling nodes in colorectal cancer. Here we propose a model in which secreted factors by the host organ, such as CCL2, reprogram genes within the tumor cell to promote growth and survival of the metastatic cell in foreign microenvironments such as the lung or liver. Targeting these proteins may kill existing metastatic lesions as well as block new metastases from forming which will be essential in improving the outcomes of patients with oligometastatic and premetastatic disease. Citation Format: Charlie Niesen, Nathan Wu, Jonathan Rennhack. Microenvironmentally released cytokines mediate organ specific transcriptional profiles for metastatic 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 A008.

Abstract B023: Regulatory T (Treg) cell contributes to tumor cell dissemination via extracellular matrix (ECM) remodeling, which is driven by TAMs in an IFNg-dependent manner

Abstract Background: The extracellular matrix (ECM) is a highly organized non-cellular network consisting of structural proteins, growth factors, cytokines and other secreted molecules. The ECM is highly dynamic and is a critical player in the regulation of local invasion and metastasis. The ECM network is ever-changing and an important immunosuppressive component of the tumor microenvironment (TME). However, the reciprocal effects of the immune system on the tumor-associated ECM are elusive and largely unexplored. Regulatory T (Treg) cells function to enforce peripheral tolerance and are potent suppressors of tumor immunity. We have previously shown that Treg cells promote tumor growth in murine breast cancer models by favoring alternative activation of tumor-associated macrophages (TAMs) via suppression of IFN-g. Aims: In this work, we sought to assess whether Treg cell-mediated immune suppression is associated with changes in the ECM, and whether those ECM changes functionally impact metastatic dissemination. Further, we assessed the requirement of TAMs and IFN-g in the ECM remodeling. Design Methods: In this study, we used spontaneous and transplantable models of breast cancer susceptible to genetic ablation of Treg cells. We used a combination of histopathological analysis, tumor decellularization, bioengineering 3D-chip modeling, in vivo manipulations and bioinformatic analysis, to evaluate remodeling of the ECM, cancer cell invasive behavior and metastatic dissemination. Results Summary: Treg cell ablation resulted in significant ECM remodeling, with reduced amounts and organization of collagen fibers, and increased amounts of fibronectin and laminin. Tumor cells seeding on Treg cell ablated tumor- derived decellularized ECM matrices results in reduced epithelial-mesenchymal transition (EMT) transcription factors, and profound impairment of collective migration. In vivo, Treg cell ablation in a neo-adjuvant setting followed by primary tumor resection resulted in significant reduction of circulating tumor cells (CTC) and impairment of lung metastatic disease. Importantly, we demonstrated that Treg cell ablation-driven changes in the matrisome correlate with long-term improved survival in a cohort of breast cancer patient samples. Through genetic knock-out models and in-vivo neutralization, we observed that Treg cell-dependent changes in ECM-related phenotypes are dependent on IFN-g-signaling. Single-cell RNA-sequencing (scRNA-seq) of murine breast tumors revealed that the matrisome signature is highly upregulated among TAMs, compared to other TME cell types. Lastly, using conditional genetic knock-out models we show these IFN-g-dependent changes are mostly driven by TAMs. Conclusions: Altogether, we identified a novel metastasis-promoting effect of Treg cells in the breast cancer microenvironment through regulation of ECM dynamics beyond their described effects on primary tumor growth. Citation Format: Ailen D. Garcia-Santillan, Jessanne Y. Lichtenberg, He Shen, Jasmine M Rodriguez, Jonathan Barra, Nicholas M Clark, Wei Du, Leandro M Martinez, Ashley D Hadjis, Taylor Calicchia, Mikhail G Dozmorov, Jose J Bravo-Cordero, Amy L Olex, Priscilla Y Hwang, Paula D Bos. Regulatory T (Treg) cell contributes to tumor cell dissemination via extracellular matrix (ECM) remodeling, which is driven by TAMs in an IFNg-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 B023.

Abstract PR017: Anti-metastatic immunotherapy discovery using ex vivo lung tissue cultures and high-throughput single-cell chemical transcriptomics

Abstract Introduction: Tumors systemically remodel the immune system during metastasis. Developing anti-metastatic immunotherapies that target these tumor-immune interactions to make the metastatic niche hostile colonization would represent a paradigm shift in cancer treatment. However, traditional high-throughput screening (HTS) platforms that use simple read-outs and contrived in vitro systems are ill-suited to identify such therapies. In contrast, HTS platforms that accurately model the metastatic niche and use high-dimensional read-outs such as multiplexed single-cell RNA-sequencing (scRNA-seq) can accurately profile nuanced perturbation responses in individual immune cell types and therefore have the potential to discover anti-metastatic immunotherapies. In this study, we describe the development of an ex vivo lung tissue culture HTS platform that preserves immune gene expression signatures observed in the in vivo metastatic niche and is amenable to single-cell chemical transcriptomic analysis using MULTI- seq (McGinnis et al., Nature Methods, 2019). Inspired by our recent description of myeloid cell TLR-NFκB inflammation during breast cancer lung metastasis (McGinnis et al., Cancer Cell, 2024), we then performed the first anti-metastatic immunotherapy drug screen and identified TLR-NFκB inhibitors that effectively operate on all desired myeloid cell types in the lung metastatic niche. Methods and Results: Building upon existing techniques for culturing precision-cut lung slices (Wu et al., Journal of Experimental Medicine, 2024) and patient- derived tumor fragments (Voabil et al., Nature Medicine, 2021), we established and optimized an ex vivo lung tissue culture system that enables 48-hour culturing of lung slices isolated from 4T1 tumor-bearing mice. scRNA-seq profiling revealed successful capture of all relevant immune cell types and retention of metastasis-associated gene expression programs (e.g., myeloid TLR-NFκB inflammation and neutrophil degranulation) following culture. We then scaled our platform to address how myeloid cells in the lung metastatic niche respond to perturbations targeting every component of the canonical TLR-NFκB signaling cascade. Cell-type-specific perturbation modeling and quantification of TLR-NFκB signaling inhibition identified compounds that optimally perturb all intended myeloid cell types (e.g., tissue-resident macrophages, neutrophils, and monocytes) and represent prioritized candidates for future in vivo validation studies to assess impact on metastatic disease progression. Conclusions: In this study, we describe the development and application of a novel HTS platform that couples ex vivo lung tissue cultures with single-cell chemical transcriptomics to identify anti-metastatic immunotherapy candidates. We demonstrate how our platform can identify TLR-NFκB signaling inhibitors that optimally operate in the lung metastatic niche, paving the way for future interrogation of additional metastasis-associated immune cell signaling pathways in different disease and tissue backgrounds. Citation Format: Chris McGinnis, Winnie Yao, Ansuman Satpathy. Anti-metastatic immunotherapy discovery using ex vivo lung tissue cultures and high-throughput single-cell chemical transcriptomics [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 PR017.

Abstract C010: Tumor MK2 drives tumor progression and an immunosuppressive microenvironment in head and neck squamous cell carcinoma

Abstract Background: Head and neck squamous carcinoma (HNSCC) results in poor patient outcomes driven primarily by locoregional and distant metastatic tumor spread. We have demonstrated that highly phosphorylated/active MAPKAPK-2 (MK2) is a poor prognostic factor in HNSCC patients. MK2 is associated with the expression of pro-inflammatory cytokines that are linked with creating a pro-tumorigenic microenvironment. Nevertheless, how MK2 contributes to HNSCC development has not been established. In this study, we aimed to identify the effect of MK2 on cancer progression, immune infiltration, and cytokine secretion. Method: To assess the effect of tumor MK2 in vivo, we used CRISPR-Cas9 to knock out (KO) MK2 in a metastatic murine HNSCC cell line, Ly2, and orthotopically implanted wildtype (WT) or MK2 KO cells into the floor-of-mouth of Balb/c mice. At the endpoint of the experiment, the tumors were excised, and myeloid and lymphocyte immune profiling was done via flow cytometry. Mouse lungs and cervical lymph nodes were dissected, paraffin-fixed, sectioned, and H&E stained, and the presence/quantity of metastatic foci was evaluated by a blinded board-certified pathologist. We broadly evaluated the tumor secretome via a 105-cytokine dot blot array using 8 mg of the same resected tumor sample and performed densitometric analysis comparing WT and KO tumors. Results: Loss of MK2 significantly reduced primary tumor growth, as well as lymph node and lung metastasis, compared to WT tumors. Tumor immunophenotyping by flow cytometry revealed that MK2 KO tumors had increased overall immune cell (CD45+) infiltration, but decreased quantities of immunosuppressive neutrophils (CD11b+Ly6G+Arg1+Ly6C-) and a shift in macrophage polarity favoring M1 (anti-tumor) macrophages (CD11b+Ly6G-F4-80+CD80+). In addition, there were increased quantities of anti-tumor immune cells including natural killer (NK)-T, CD4+, and CD8+ T cells in the MK2-KO tumors, while the proportion of CD4+CD25+ Tregs and exhausted CD8 T cells (CD8+PD1+) was decreased compared to WT tumors. Furthermore, cytokine array analysis revealed that secreted levels of several cytokines, including CXCL1, CXCL2, GM-CSF, G-CSF, IL-1alpha, and IL-6, were significantly lower in MK2 KO tumors compared to WT tumors. Conclusion: These results show that tumoral MK2 signaling promotes tumor growth, metastasis, immunosuppressive inflammation, and pro-tumorigenic cytokine release which substantiates future therapeutic targeting of this pathway. Citation Format: Dakota D.D. Okwuone, Deri Morgan, Alyssa Schmidt, Devin Shrock, Yuting Lin, Hao Gao, Sufi M. Thomas, Gregory N. Gan. Tumor MK2 drives tumor progression and an immunosuppressive microenvironment in 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 C010.

Abstract C013: Single cell analysis reveals tumor size-driven immune changes in primary breast tumors and corresponding lymph nodes

Abstract Introduction A sentinel lymph node is the primary node draining the tumor and is likely affected early in the metastatic process. The sentinel node holds a key position in the immune response against tumor in breast and represents a unique connection between the tumor and the host immune response. Detection of lymph node metastasis is important for staging a patient’s disease and is closely associated with prognosis. Although the clinical significance is debated, isolated tumor cells (<0.2 mm) appear to have no impact on survival while macro-metastasis (>2 mm) are linked to a poorer prognosis. Alterations in the immune response in lymph nodes with large versus small metastases have been observed, but little is known about the relationship between the immune response in the primary tumor and the metastatic status of the corresponding lymph nodes. Methods We analyzed single-cell suspensions from primary tumors and lymph nodes of 38 patients with early, operable breast cancer enrolled in the OSLO2 clinical observational trial using mass cytometry with a 47- antibody immune panel (including PanKeratin and EpCAM). In total 3.7 million single cells were analyzed and clustered into 40 metaclusters using FlowSOM. A second set of 22 metastatic lymph nodes from the OSLO2 cohort was used to validate results form the metastatic tumor cells. Additionally, immunofluorescence with E-cadherin and PanKeratin was applied, and intensity measured in 4 metastatic lymph nodes and imaging mass-cytometry was applied on one metastatic lymph node Results We found that the immune microenvironment in primary tumor was dominated by CD4 and CD8 T cells, particularly of exhausted and memory phenotype and with a higher frequency of activated regulatory T cells (Treg). In contrast, the immune composition in lymph nodes was predominantly composed of naturally occurring naive B and T cells. Notably, in line with previous publications we observed a skewing towards memory and exhausted phenotype in T cells, along with increased abundance of activated Treg in lymph nodes with larger metastasis to those with smaller or no metastasis. Furthermore, there were no correlation between the immune profile in the primary tumor and corresponding lymph node. The immune profile in the primary tumor was influenced by tumor size, with larger tumors showing a higher frequency of activated Tregs and exhausted CD8 T cells. Furthermore, In situ analysis revealed that the microenvironment in smaller metastase had an mesenchymal phenotype, while larger tumor deposits exhibited a more epithelial-like phenotype. ConclusionThe immune profile of the primary tumor did not predict the immune profile or metastatic status of the corresponding lymph node. Tumor size in both primary tumors and metastatic lymph nodes is the main drivers of changes in immune cell composition. Tumor cells from smaller metastases exhibited an mesenchymal phenotype, while larger metastasis displayed an epithelial phenotype. Citation Format: Inga Hansine H. Rye, Marit Otterlei Fjørtoft, Inger Riise Bergheim, Karin Teien Lande, Kanutte H. Huse, June Helen H. Myklebust, Ole Christian H. Lingjærde, Øystein. Garred, Jon H. Lømo, Hege H. Russnes. Single cell analysis reveals tumor size-driven immune changes in primary breast tumors and corresponding lymph nodes [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 C013.

Abstract A002: Dissecting the brain metastatic microenvironment to uncover immune and molecular correlates of response to immunotherapy

Abstract Despite promising results with immune checkpoint blockade (ICB) therapy, brain metastases (BrM) remain a deadly complication for cancer patients. Understanding the highly specialized brain metastatic tumor microenvironment (BrTME) is crucial for identifying determinants of response, however, progress has been hindered by limited access to patient samples and scarcity of relevant preclinical models. Here, we developed two mouse melanoma BrM models by intracardiac injection of cells derived from our previously established M4-B2905 melanoma mouse cell line. We characterized their mutational landscape and performed single-cell phenotypic and transcriptomic analysis, demonstrating that these models recapitulate the cellular and molecular features of human melanoma BrMs. Comparative and interactome analysis of our models revealed key factors contributing to ICB response and resistance. We found that the responsive model (BR1) was characterized by tumor cells that polarize microglia toward reactive states via inflammatory programs and response to IFN signaling. These microglia express high levels of antigen presentation and immunostimulatory molecules, triggering T cell recruitment and activation and promoting ICB therapy response. In contrast, in the resistant model (BR3), the tumor cells express neurological molecular signatures and ligands that sustain microglia homeostasis. This results in poor T cell infiltration predominantly composed of naïve cells, evading immune reaction and promoting ICB resistance. While we showed that systemic ICB therapy induced BrTME changes in both models, differences between responder and resistant models were maintained, emphasizing the importance of tumor cell-intrinsic programs in dictating the BrTME. We validated the translational relevance of our findings and demonstrated that BR1 signatures positively correlate with T cell infiltration and are associated with improved patient outcomes, whereas BR3 signatures negatively correlate with T cells and are found in patients with worse prognosis. Here we address an important challenge in the field by providing clinically relevant BrM models that recapitulate both the cellular and molecular features of human disease and the variability of ICB response seen in patients. We further reveal mechanistic insights into BrM ICB response and identify potential therapeutic targets to modulate the BrTME. Citation Format: Amelie Daugherty-Lopes, Eva Perez-Guijarro, Vishaka Gopalan, Jessica Rappaport, Quanyi Chen, April Huang, Khiem C. Lam, Sung Chin, Jessica Ebersole, Emily Wu, Gabriel Needle, Isabella Church, George Kyriakopoulos, Shaojun Xie, Yongmei Zaho, Charli Gruen, Antonella Sassano, Romina E Araya, Andres Thorkelsson, Cari Smith, Maxwell P. Lee, Sridhar Hannenhalli, Chi-Ping Day, Glenn Merlino, Romina S. Goldszmid, Shaojun Xie. Dissecting the brain metastatic microenvironment to uncover immune and molecular correlates of response to immunotherapy [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 A002.

Abstract C034: Investigating residual disease in its spatial context in BRCA1 p53-deficient mammary tumors

Abstract Although various effective anti-cancer treatments have become available over the last decades, resistance to all available therapies remains the major cause of death of cancer patients with disseminated tumors. Striking examples are patients with triple-negative breast cancer (TNBC), which are frequently defective in the repair of DNA double strand breaks, e.g. due to loss of BRCA1 function. Because of this defect, the patients initially respond very well to DNA damage-inducing chemotherapy. Unfortunately, disseminated tumors are usually not eradicated and resistant tumor cells are eventually selected from residual primary or metastatic tumor sites. It is therefore crucial to understand the molecular mechanisms underlying the drug tolerance of the residual tumor cells. To study residual disease, we used the K14cre;Brca1 F/F ;p53 F/F (KB1P) mouse model for hereditary breast cancer, which provides the unique opportunity to explore and target those mechanisms in a fully immunocompetent model. The mammary tumors that spontaneously develop highly resemble their human counterparts, both morphologically and in their therapy response. For example, tumors shrink in response to poly(ADP-ribose) polymerase inhibition, platinum-based treatment or the commonly used doxorubicin, docetaxel and cyclophosphamide (TAC) combination therapy. But despite repeated drug sensitivity, the KB1P mammary tumors are not eradicated, not even by a frequent dosing schedule. By combining single-cell RNA sequencing, spatial transcriptomics and imaging mass cytometry, we dissected the intratumoral heterogeneity and alterations in the tumor microenvironment of residual disease. We identified specific subpopulations of tumor cells that have a survival benefit after treatment, and these occurred together with an altered microenvironment characterized by a highly reactive stroma infiltrated with both anti-inflammatory and pro-tumoral immune cells. Interestingly, those structural and transcriptional changes are reversed in the relapsed tumors, highlighting the plasticity of drug tolerance. To investigate the mechanisms of the altered tumor-stroma interactions that are relevant for drug tolerance, we have designed a custom-made CRISPR/Cas9 library based on differential gene expression of the residual tumor cells. This library enables us to functionally test relevant mechanisms of drug tolerance in vivo and we expect that these analyses will provide useful insights into the tumor-stroma interactions that contribute to residual disease. Taken together, our detailed analyses demonstrate the substantial remodeling of tumor cells in their microenvironment upon treatment. To develop new therapeutic approaches to eradicate drug-tolerant tumor cells and thereby circumvent tumor relapse, it is essential to better understand the heterogeneous cellular composition of residual tumors in its spatial context. With this project, we hope to provide comprehensive data to develop better therapeutic strategies that target the tumor-stroma interaction and eradicate residual tumors. Citation Format: Morgane Decollogny, Demeter Túrós, Astrid Chanfon, Myriam Siffert, Ismar Klebic, Sven Rottenberg. Investigating residual disease in its spatial context in BRCA1 p53-deficient mammary tumors [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 C034.

Abstract A035: Infection of primary mammary fibroblasts with cytomegalovirus coordinately increases autotaxin-lysophosphatidate-inflammatory signaling, which could increase breast cancer metastasis

Abstract Background: Metastatic breast cancer is the most prevalent cause of mortality by cancer-related deaths in women. Our group showed that the 63% of Canadian breast cancer patients who were infected with CMV were 5.6-times more likely to develop Stage IV cancers with reduced relapse-free survival. Also, the metastatic phenotype of breast tumors and the number and sizes of lung metastases were increased in mice with latent infections. Fibroblasts are important sites of CMV infection. Cancer-associated fibroblasts (CAFs) are also major sites of autotaxin (ATX) secretion in breast tumors, whereas ATX production and CMV infection in breast cancer cells is negligible. ATX produces lysophosphatidate (LPA), which increases inflammatory cytokine production that stimulates further ATX secretion in a feedforward cycle. LPA promotes breast tumor growth and metastasis by signaling through six G protein-coupled receptors. Signaling is terminated by lipid phosphate phosphatases (LPPs)-1 and -3, which degrade LPA and are decreased in breast tumors. Hypothesis: Breast cancer metastasis could be increased by active CMV infection of CAFs through increased inflammation and LPA signaling. Results: Quiescent fibroblasts isolated and cultured from mammary tissue of non-tumor bearing mice (n>3) produced negligible quantities of ATX. LPA signaling was coordinately promoted in these primary fibroblasts infected for 48 h with 1 MOI of mouse CMV (n=3) with a 5-fold increase in mRNA for ATX (p=0.008), increased mRNAs for LPA2,3,4 receptors (p<0.05) and decreased mRNA expressions for LPP1 and LPP3 (p<0.05). An increase in the secretion of granulocyte monocyte-colony stimulating factor of 548 pg/ml (p=0.01) and Interleukin-6 by 8307 pg/ml (p=0.03) was observed post infection (n=5), thus creating a pro-inflammatory environment. Increased mRNA expression of fibroblast markers, including alpha-smooth muscle actin (p=0.005), fibroblast-associated protein (p=0.007), epidermal growth factor receptor (EGFR) (p=0.001) and transforming growth factor- ß receptor 1 (TGF-ßR1) (p=0.03) was also observed post-infection (n=3). The results were analyzed by student’s t-test. Assessment of these factors in breast cancer patients (n=1100) using the Tumor Immune Estimation Resource Analysis showed a positive correlation in the expression of EGFR and TGF-ßR1 with the LPA3,4 receptors (p<0.05). Conclusion: The changes in expression of ATX, LPA receptors and LPPs in CMV- infected fibroblasts are consistent with increased metastasis. Our results show that mCMV infection of primary mammary fibroblasts dramatically reprograms them to express CAF-like properties and promotes ATX-LPA-inflammatory signaling. CAFs are a major constituent of the tumor stroma. They promote tumor progression by secreting factors such as ATX, which increases metastasis, fibrosis and favors immunosuppression. Our results support the use of pharmacological agents to block signaling by the ATX-LPA-inflammatory cycle to treat breast and other cancers, especially for the majority of patients who are infected with CMV. Citation Format: Humayara Khan, Xiaoyun Tang, Frank Wuest, David N. Brindley, Denise G. Hemmings. Infection of primary mammary fibroblasts with cytomegalovirus coordinately increases autotaxin-lysophosphatidate-inflammatory signaling, which could increase breast cancer 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 A035.

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 C045: The extracellular matrix glycoprotein periostin supports chemotherapy resistance by modulating macrophage recruitment and phagocytosis in triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) accounts for approximately 15–20% of all breast cancer cases and is notoriously aggressive, often developing resistance to standard chemotherapy thus leading to a high rate of metastatic relapse. Evidence suggests that this resistance is linked to changes in the extracellular matrix (ECM) composition, particularly an increase in periostin (POSTN) expression, which is also associated with poor prognosis in breast cancer. Our recent publication showed that the ECM alone can influence macrophage phenotypes. Furthermore, another study from our lab has demonstrated that POSTN mediates resistance to anti-angiogenic therapy by recruiting macrophages in pancreatic neuroendocrine tumors. Notably, POSTN deletion reduced monocyte/macrophage recruitment and enhanced cytotoxic CD8+ T-cell infiltration. Based on these findings, we hypothesized that paclitaxel (PTX), a widely used chemotherapeutic drug for the management of TNBC, may stimulate POSTN production by fibroblasts leading to the recruitment of pro-tumoral macrophages that facilitate tumor growth resulting into therapy resistance. In this study, we showed that PTX induces increased POSTN expression in mouse models of TNBC through immunohistochemistry, and in both 2D and 3D in vitro models via interactions between TNBC cell lines and mammary fibroblasts, as demonstrated by immunofluorescence. An in vitro migration assay confirmed that PTX-induced POSTN enhances the recruitment of human monocytes and macrophages, which display a mixed immunosuppressive and inflammatory phenotype, as revealed by flow cytometry. Not only did these recruited macrophages exhibit a reduced phagocytic capacity against labelled TNBC cells in an in vitro phagocytosis assay, but we also found that POSTN plays a crucial role in inducing SIRPα expression, that may diminish macrophage phagocytic potential. Increased sialylation and Siglec-1 expression are known to be linked to poor prognosis and polarization towards a more immunosuppressive macrophage phenotype in TNBC. Interestingly in our model, we observed that higher concentrations of recombinant human POSTN induce increased Siglec-1 expression on macrophages, a receptor that interacts with sialic acids in the tumor ECM. Further work using a decellularized tissue model that preserves only the ECM from patient-derived tumor biopsies pre- and post-chemo, aims to further explore the correlation between PTX-induced POSTN, ECM sialylation, and Siglec-1 expression on macrophages. Additionally, we have developed POSTN-knockout murine cancer cells and fibroblasts using CRISPR/Cas9, allowing us to investigate how POSTN influences macrophage phenotypes and chemotherapy responses in vivo. Understanding the mechanisms behind chemotherapy resistance in TNBC is crucial for developing effective treatments for this aggressive cancer subtype. By elucidating the intricate interplay between POSTN, macrophages, and chemotherapy response, we aim to identify new therapeutic strategies to improve outcomes for TNBC patients. Citation Format: Ludovica Tarantola, Ioanna Keklikoglou, Oliver M. Pearce. The extracellular matrix glycoprotein periostin supports chemotherapy resistance by modulating macrophage recruitment and phagocytosis in triple negative 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 C045.

Abstract C018: Awakened dormant cancer cells undergo highly mesenchymal to quasi- mesenchymal transition and acquire stemness

Abstract The awakening of dormant disseminated cancer cells is likely responsible for the clinical relapses of patients whose primary tumors have been cured months and even years earlier. In the present study, we demonstrate that dormant breast cancer cells lodged in the lungs reside in a highly mesenchymal, non-proliferative phenotypic state. The awakening of these cells does not occur by a cancer cell-autonomous process. Instead, inflammation and wound healing of the surrounding tissue microenvironment causes them to shift from a highly mesenchymal to a quasi-mesenchymal phenotypic state in which they acquire stemness and proliferative ability. Once awakened, these cells can stably reside in this quasi-mesenchymal state and maintain their stemness, doing so without ongoing heterotypic signaling from the lung microenvironment. EGFR ligands released by the cells of the injured tissue microenvironment, including notably M2 type macrophages, promote dormant cancer cells to move toward this quasi-mesenchymal state, a transition that is essential for the awakening process. An understanding of the mechanisms of metastatic awakening may lead in the future to treatment strategies designed to prevent such awakening and resulting metastatic relapse. Citation Format: Jingwei Zhang, Robert Weinberg. Awakened dormant cancer cells undergo highly mesenchymal to quasi- mesenchymal transition and acquire stemness [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 C018.

Abstract A032: Uncovering the impact of current therapies on LUAD dormant metastasis

Abstract Despite recent therapeutic advances, Lung Adenocarcinoma (LUAD) remains a leading cause of mortality worldwide, with most deaths attributed to metastasis. Metastasis is mediated by disseminated metastasis initiating cells (MICs) that can persist in a dormant state in distant sites for months to years and ultimately drive metastatic relapse, which is clinically incurable. Adjuvant therapy seeks to prevent cancer relapse, however, efforts to improve adjuvant treatments are hindered by an insufficient understanding of the molecular mechanisms supporting the long-term viability of dormant MICs. We have established that primitive LUAD cells characterized by the spontaneous expression of the pluripotency transcription factor SOX2 can enter an immune-evasive dormant state with downregulation of immune response mediators (STING, NK ligands, MHC-I) to evade immune surveillance during dormancy. Now, we hypothesize that dormant and immune evasive progenitor MICs may also escape therapies targeting growth pathways. We have generated latency competent cells (LCCs) that can remain in a latent state for months by isolating progenitor cells from the Kras LSL-G12D/+ ;Trp53 flox/flox mouse model and screening their metastatic potential and progenitor state in the B6 background. LCCs entering a slow-cycling state in response to dormancy signals remain sensitive to targeted KRAS inhibitors but contain a resistant subpopulation that survives. To specifically characterize the progenitor state in these LCC populations, we have developed and validated two strategies to fluorescently mark SOX2 expression and activity, respectively. As speculated, the resistant population is mostly composed of SOX2+ progenitor LCCs after treatment with KRAS inhibitor under dormancy signaling. Furthermore, induced-dormancy also drives an accumulation of cells in the SOX2+ progenitor state in these LCC-rich populations. Ongoing studies are focused on delineating the emergence of the SOX2+ progenitor state in LCCs during dormancy and the biological mechanisms driving their persistence and resistance to targeted therapies. We aim to define (1) the dependence of dormant LCCs on oncogenic signaling, the (2) effect of targeted therapy on MICs, and (3) strategies to overcome potential resistance mechanisms in dormant MICs. Our ultimate goal is to delineate strategies to clear latent MICs and prevent LUAD recurrence. Citation Format: Inês Godet, Zhenghan Wang, Joan Massagué. Uncovering the impact of current therapies on LUAD dormant 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 A032.

Abstract C014: Degradation of extracellular trap DNA sustains anti-tumor immune responses in breast cancer

Abstract Increased extracellular DNA (exDNA) levels in the blood are a hallmark of metastatic cancer, arising from tumor lysis, apoptosis, necrosis, and neutrophil extracellular traps (NETs) released by tumor-expanded neutrophils. NETs, web-like chromatin structures extruded by neutrophils to trap pathogens, are highly immunogenic due to their DNA and histone content and their persistent interaction with dendritic cells. Tumor cells can also release extracellular traps. In myeloid leukemia, blasts release nuclear content via traps to activate coagulation or sustain myeloproliferation. Extracellular traps have been found in NPM1 mutant AML patients, with mutant NPM co-localizing with histones along exDNA traps. Forcing trap extrusion in NPM mutant leukemia cells has been used to create dendritic cell-based vaccines that break tolerance against NPMc antigens. This suggests that NETs in the tumor microenvironment (TME) may promote tolerance, with breaking tolerance requiring consistent NET formation and dendritic cell interaction. NETs are also seen in solid tumors, such as triple-negative breast cancer (TNBC), where they aid in cancer cell migration, invasion, and awakening of circulating tumor cells. However, the capacity of tumor cells to directly extrude DNA traps is not fully understood. To investigate this, various human and murine breast cancer cell lines were seeded on poly-D-lysine coated slides, stained with DAPI and Sytoxgreen, and observed via confocal microscopy. Tumor cells extruded DNA traps within 4 hours without stimuli. Notably, trap extrusion correlated with cell line aggressiveness and was abolished by DNase, indicating dsDNA composition. The absence of traps in 24-hour cultures suggests DNA extrusion may help rare tumor cells survive and proliferate. In line, DNase treatment reduced tumor proliferation and increased apoptosis. To assess the significance of DNA traps in vivo, BALB/c mice were injected with the highly metastatic 4T1 clone 5 breast cancer model, with or without DNase treatment, and monitored starting 5 days post-injection and weekly until day 28. Histological analysis at the injection site 5 days post-injection showed that DNase treatment reduced both local trap formation and tumor cell proliferation, as indicated by BrdU incorporation. Moreover, immunohistochemistry revealed that in DNase-treated mice, initial tumor growth was followed by complete tumor regression, accompanied by local and systemic CD8 T cell activation. Correspondingly, although a few metastases were detectable at day 14 in DNase-treated mice, no metastases were observed by day 28. Although it remains unclear whether tumor-derived or immune cell-derived traps are most relevant in our model, the results demonstrate that their presence in the TME promotes tolerance despite the inherent immune adjuvant properties of the traps. This suggests that DNase treatment could be particularly effective in combating early tumor development or relapse when used as part of a combination therapy designed to sustain anti-tumor immune responses. Citation Format: Sabina Sangaletti, Paola Portararo, Laura Botti, Valeria Cancila, Claudio Tripodo, Mario P. Colombo, Claudia Chiodoni. Degradation of extracellular trap DNA sustains anti-tumor immune responses 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 C014.

Abstract A013: Microenvironment driven dynamic deposition of histone H3.3 controls entry and exit from dormancy in disseminated cancer cells

Abstract Breast cancer metastases can go undetected for long periods of time, are highly resistant to therapy, and are the cause for more than 90% of breast cancer mortality. Dormancy is a critical step in the metastatic cascade, whereby disseminated cancer cells (DCCs) enter a non-proliferative state at distal sites until they reactivate and give rise to overt metastasis, often years after disease remission. As such, dormancy represents a major clinical obstacle in breast cancer patient care. Chromatin remodeling serves as a foundation for the cellular reprogramming required to drive cell fate transitions. However, little is known about the nature of this remodeling in dormancy. Here, we hypothesize that differential deposition of histone H3.3 determines whether DCCs enter and remain dormant in secondary organs or proliferate and form overt metastases. We used a combination of H3.3 deposition sensors and genetic tools to manipulate H3.3 and HIRA in vitro and in vivo to track H3.3 deposition through the metastatic cascade and evaluate its importance for dormancy. To evaluate relevance to patient disease we developed a dormancy score to implement from publicly available RNA seq. To define mechanism of regulation, we used a combination of whole genome sequencing (ChIP-seq, ATAC-seq and RNA-seq) followed by in-depth analysis and functional validation. Our data demonstrates that H3.3 incorporation at secondary sites is heterogenous, revealing a subset of DCCs with low H3.3 in chromatin. Blocking H3.3 deposition onto chromatin leads to a reversible arrest in G0/G1 which tracks with the activation of canonical dormancy signaling. Mechanistically, our data supports a model where blocking H3.3 incorporation into chromatin directly represses the expression of SKP2, the main regulator of progression through the restriction point of cell cycle via regulation of p27 degradation. SKP2 suppression in turn leads to the accumulation of p27 thereby causing cell cycle arrest in G0/G1 and dormancy establishment. We traced this H3.3-mediated regulation of dormancy through the dynamic regulation of H3.3 histone chaperone, HIRA, whose levels inversely correlate with dormancy in experimental models as well as in patient samples. We found that HIRA levels respond to the metastatic microenvironment where fibrotic conditions induce HIRA expression and trigger metastatic outgrowth in an H3.3-dependent manner. Together, our work unveils for the first time a patient-relevant epigenetic mechanism, impinging on H3.3 deposition, as a major regulatory point of DCCs’ cell fate at secondary organs controlling both entry of DCC’s into dormant state as well as their reactivation and thriving as overt metastasis. Citation Format: Stanislav Drapela, Cristina Megino-Luque, Rojan Chimeh Rad, Devesh Raizada, Nadir Sarigul, Didem Ilter, Brian J. Czerniecki, Jose Javier Bravo-Cordero, Ana P. Gomes. Microenvironment driven dynamic deposition of histone H3.3 controls entry and exit from dormancy in disseminated 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 A013.

Abstract A010: Development and characterization of novel models of lobular breast cancer metastases

Abstract Invasive lobular carcinoma (ILC) is the most common special histological subtype of breast cancer, after No Special Type (NST, ductal carcinoma/IDC), and affects 10-15% of all breast cancer patients. ILC is characterized by E-cadherin loss, Estrogen Receptor (ER) positivity, and a distinct single file invasive growth pattern associated with a unique pattern of metastasis. ILC metastasizes to common breast cancer sites like bone and lung, but ILC also spreads to gynecologic tissues, the peritoneum, gastrointestinal tract and central nervous system - with enrichment for orbital and leptomeningeal metastases. In vitro and in vivo models of ILC metastasis have not been extensively developed. Models of spontaneous metastasis from ER+ breast cancer overall are critically limited; ER+ ILC metastases models present only micro-metastatic disease with extremely long latency (>9 months). Together these issues limit research on the unique nature of metastatic ILC. We leveraged mammary intraductal (MIND) xenografting with two ER+ ILC cell lines (MM134 and 44PE) and confirmed prior reports that these lines spontaneously metastasize to bone, brain, ovary, uterus, lung, and adrenal glands with up to 100% incidence. Importantly, we find that supplementation with low dose estradiol (E2) in drinking water rapidly advances metastatic progression, but not primary tumor growth. Using ex vivo bioluminescence imaging (BLI), we find BLI+ lesions are detectable as early as 90 days. At 150 days post-challenge, 100% of mice have BLI+ metastases at multiple sites. Beyond 22 weeks, 100% of mice present with symptomatic and ultimately lethal CNS metastases. BLI+ macro-metastatic lesions were isolated and expanded ex vivo as organotrophic cell line variants. Analysis of RNA-seq data is ongoing to evaluate 1) common factors enriched in metastatic ILC, and 2) organotrophic factors and pathways enriched in specific metastatic sites. In patients with ILC, bone metastases are typically the first site of metastatic development, occur at a higher incidence versus NST, and are associated with increased morbidity. Bone metastases from ILC patients are more osteogenic compared to the characteristically osteolytic lesions of NST. To examine how ILC cells may promote osteoblastic responses, we prepared ILC and NST conditioned media (CM) and applied it to differentiating osteoblasts (OB). We observed increased OB differentiation and increased mineral deposition with ILC CM compared to NST CM, supporting that ILC cell lines have a distinct impact on the bone microenvironment which may manifest in vivo yet be defined in vitro. Collectively, our observations support that ER+ ILC cell lines can recapitulate key features of clinical disease progression, including spontaneous metastasis mimicking clinical phenotypes. We are leveraging these models to better understand the ILC metastatic cascade and adaptation to unique metastatic niches, and to identify shared and unique attributes of ILC metastases that can be exploited for advancing therapeutics. Citation Format: Joseph L. Sottnik, Matthew J. Sikora. Development and characterization of novel models of lobular breast 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 A010.

Orbital metastases from breast cancer: three case reports

Purpose: to describe rare cases of orbital metastases from breast cancer.Material and Methods. Three cases of Luminal A(1) and B(2) breast cancer with orbital metastases were identified and studied during the period from 01.01.2018 to 01.05.2024. Metastasis to the orbit was detected 5, 2 and 1.5 years after radical mastectomy for breast cancer. Patients received chemotherapy and radiation therapy.Results. Cases of orbital metastasis originating from breast cancer were found to have a low frequency, but may be associated with a long period between cancer diagnosis and initiation of treatment. Early detection of breast cancer and its active treatment with chemotherapy and radiation therapy are important factors in preventing orbital metastasis.Conclusion. A routine follow-up surveillance after radical treatment of breast cancer is important because there is an increase risk of developing orbital metastasis. Comprehensive strategies for treating breast cancer metastases can provide long-term disease control.