Metastasis Model Research Articles

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.

Abstract B018: Multifaceted pro-metastatic role of IL-17 signaling in modulating the tumor microenvironment of colorectal cancer

Abstract Colorectal cancer (CRC) ranks second in mortality and advanced CRCs are resistant to current immunotherapies and prone to metastasis, which necessitates research into enhancing innate and adaptive anti-cancer immune responses. Chronic inflammation mediated by cytokines like IL6 and notably, IL17 activates the oncogenic pathways in cancer cells, promoting sporadic and inflammatory CRC. What is unknown, is the ability of cytokines to control tumor microenvironment (TME) via cell type specific mechanisms to increase cancer progression, metastasis and/or therapy resistance. Increased levels of IL17A in CRC portends poor prognosis indicating its potential involvement into metastasis. However, the role of IL17 signaling in TME in the process of CRC progression and metastasis remains enigmatic. We utilized different models of CRC progression and metastasis, as well as variety of genetic mice models and therapeutic interventions. First, using a model of colitis-associated cancer we found that “late” IL17A neutralization decreases tumor burden, possibly acting through regulation of ferroptosis, NK cells mediated cytotoxicity, as well as myeloid cells and chemokine network essential for the control of immunosuppressive TME. Next, models of CRC metastasis, including those based on MC38 cells and APTAK cancerous organoids, showed that IL17A neutralization reduces metastasis growth. Also, FACS analysis revealed that IL17A promotes the recruitment of tumor- associated macrophages and other key myeloid subsets into the metastatic liver environment. Further research, using mice with conditional knockout of IL17RC receptor in myeloid cells (Il17rc f/f -LysMCre) or hepatocytes (Il17rc f/f -AlbCre) also showed significance of IL17 signaling activation in CRC metastasis through two distinct mechanisms. Employing different techniques, from FACS, immunohistochemistry, to RNAseq and scRNAseq data analysis, we observed that local hepatic activation of IL17RC signaling recruits neutrophils and reduces infiltration of cytotoxic CD8+T and NK cells to the metastatic niche supporting tumor growth, while myeloid IL17RC signaling reshapes TME, including macrophages and neutrophils, enhancing CRC metastasis. Our research indicates that IL17 signaling plays a multifaceted pathogenic role in CRC progression by operating in at least several cell types, providing new insights into the basic biology of CRC metastasis and potentially illuminating the design and improvement of CRC treatment strategies. Citation Format: Katarzyna Chojnacka, Katrina Mae Reyes, Hana Tomizawa, Sergei Grivennikov. Multifaceted pro-metastatic role of IL-17 signaling in modulating the tumor microenvironment 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 B018.

Abstract A029: Integrated modeling of renal cancer bone metastasis to illuminate tumor progression and therapy response

Abstract Bone metastatic clear cell renal cell carcinoma (BM ccRCC) is a persistent clinical challenge and a source of significant morbidity and lethality for up to 40% of metastatic patients. Despite the recent approval of life-prolonging agents (antiangiogenic agents, immunotherapy) patients with BM ccRCC will eventually progress, due to emerging resistance. Bidirectional communication between tumor cells and bone stroma has emerged as a key determinant of disease progression and recurrence. However, a major challenge in addressing the black box of therapy response in bone is the lack of efficient and informative systems that allow us to analyze the heterotypic stromal-epithelial interaction, further complicated by the anatomical inaccessibility of bone. For these reasons, the evolution of the metastatic niche and its implications in therapy response remain elusive. In order to improve clinical results, it is critical to establish biologically informed pre-clinical models to provide a mechanistic understanding of tumor progression in bone and treatment outcomes. To this purpose, we have established clinically relevant models of ccRCC bone metastasis that span in vivo and ex vivo multiphoton microscopy (iMPM and eMPM), tissue engineered bone window systems, and computational oncology. iMPM displays both sensitivity and time-resolution to identify dynamic interactions between ccRCC cells and bone 3D adaptive niches, which support therapy response and resistance. However, iMPM in bone is limited due to its cortical thickness, increased light scattering, and complex topology. As a novel alternative, we created a tissue-engineered bone construct (TEBC) that, after direct implantation of cancer cells, is combined with an adjacent skin window, allowing for non- destructive intravital examination of tumor growth. To flank these in vivo dynamic analyses, we established a pipeline for ex vivo extraction of topological information related to the molecular and cellular niches involved in tumor progression and response to treatment (eMPM). This method allows reconstruction of whole lesions, including zones more distant from the bone interface, paired with an extensive panel of molecular markers. To further investigate the effects of a broad multi-parameter space on tumor regression or persistence upon treatment, in silico, we developed an in vivo-inspired agent-based model (ABM) of ccRCC in bone. A computational approach combined to ad hoc biological experimentation can refine the experimental design, test clinically relevant hypotheses (including impact of treatments on disease progression) and predict scenarios that guide biological testing towards more successful outcomes. Consequently, by integrating these approaches, we are currently studying the progression of the bone metastatic niche and its response to therapy, with the ultimate goal of overcoming therapy failure. Citation Format: Sergio Barrios, Luca Marsilio, Stefan Maksimovic, Matthew Campbell, Stefano Casarin, Eleonora Dondossola. Integrated modeling of renal cancer bone metastasis to illuminate tumor progression and therapy 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 A029.

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.

Abstract PR002: Lymph node colonization reprograms lymphocyte responses to generate systemic tolerance and promote distant metastasis

Abstract The majority of cancer-associated deaths result from distant organ metastasis, yet the mechanisms that enable this process remain poorly understood. Colonization of locoregional or distant lymph nodes (LNs) typically precedes the formation of distant organ metastases, yet it has been unclear whether LN metastasis plays a functional role in disease progression. LNs are major sites of anti-tumor lymphocyte education, including in the context of immunotherapy, yet LN metastasis frequently correlates with further disease progression. By leveraging our mouse models of LN metastasis, we find that LN colonization represents a critical step in tumor progression by inducing tumor-specific immune tolerance in a manner that promotes further dissemination of tumors to distant organs. This promotion of distant seeding by LN metastases is reliant upon adaptive immunity and is tumor specific. Using flow cytometry and single-cell sequencing to perform comprehensive immune profiling, we identify multiple cellular mediators of tolerance. LN metastases evade NK cells, induce shifts in DC and B cell populations, and promote CD8 T cell exhaustion, but of particular importance, we find that regulatory T cells (Tregs) are enriched in the LNs of mice and patients with LN metastases. LN metastatic cells exhibit an enhanced capacity to induce Treg differentiation from naïve CD4 T cells, and ablation of Tregs using FoxP3-DTR mice eliminates the ability of LN metastases to promote distant metastasis. Furthermore, adoptive transfer of Tregs from the LNs of mice bearing LN metastases to naïve mice facilitates lung metastasis in a manner that Tregs from mice without LN metastases cannot. These Tregs are induced in a tumor antigen-specific manner and TCR recognition of tumors is required for the metastasis-promoting tolerance. Using photoconvertible mice, we track the trafficking dynamics of Tregs from tumor involved LNs and demonstrate their elevated trafficking to sites of distant metastasis prior to the formation of detectable metastases in those sites. Through bulk and single-cell TCR sequencing of the Treg repertoire we reveal how LN colonization alters the clonal architecture of the Treg compartment and demonstrate how the evolution of metastatic traits coincides with shifts in the Treg repertoire. Parallel analyses in patients with melanoma and head and neck cancer reveal similar CD4 T cell dynamics in the context of LN involvement. Together, these findings demonstrate a critical role for LN metastasis in promoting tumor-specific immune tolerance and metastatic progression. Citation Format: Cort B. Breuer, Norma A. Gutierrez, Zhewen Xiong, Amanda R. Kirane, John B. Sunwoo, Nathan E. Reticker-Flynn. Lymph node colonization reprograms lymphocyte responses to generate systemic tolerance and promote distant 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 PR002.

Abstract B048: Neutrophil extracellular traps-targeting DNase I enhances the therapeutic efficacy of CAR-T cell adoptive immunotherapy in a syngeneic murine metastasis model

Abstract In recent years, chimeric antigen receptor (CAR) T cell therapy has emerged as a promising approach for treating various malignancies, most notably in hematologic cancers. However, its efficacy in solid tumors is often hindered by an immunosuppressive tumor microenvironment (TME), which, in particular, is frequently characterized by the presence of tumor-associated cell-free DNA (cfDNA), especially in the form of neutrophil extracellular traps (NETs). In adoptive immunotherapies NETs, composed primarily of neutrophil-derived DNA, histones and neutrophil granule enzymes (e.g., MPO, NE), act as physical barriers and induce the secretion of immunosuppressive factors that impair CAR T cell function. This study investigates the potential of co-administration of deoxyribonuclease I (DNase I) with CAR T cells in a syngeneic B16 melanoma murine model of lung metastasis. Bioluminescent imaging of melanoma metastatic processes has shown that a single injection of DNase I (10 mg/kg) suppresses B16-EGFR lung metastasis at early stages in comparison to the vehicle control group but does not improve survival. However, co-administration of DNase I with murine EGFR-CAR T cells significantly suppresses tumor burden, decreases the number of metastatic foci, and substantially prolongs survival compared to the CAR T cell monotherapy group. Degrading of NETs [RB1] by DNase I increases the amount of tumor-infiltrating T and CAR T cells and reduces the immunosuppressive effects of the TME. This is reflected in the CD8 population of tumor-infiltrating CAR T cells from the DNase I treated group, which have lower expression of PD-1 and TIM-3 exhaustion markers. This research highlights the critical role of the NETs in modulating CAR T cell efficacy and provides a compelling rationale for incorporating DNase I as an adjunctive treatment to improve therapeutic responses in patients undergoing CAR T cell therapy. However, further clinical studies are warranted to validate these findings and explore the translational potential of this combinatorial approach in enhancing cancer treatment. [RB1]Again - this takes the emphasis and the reader away from NETs and into the cfDNA universe. Citation Format: Alexey Stepanov, Wenjian Wang, Yingqin Hou, Ivan Chernikov Ivan Chernikov, Reid Bissonnette, George Tetz, Grigory Borisenko, Dmitry Genkin. Neutrophil extracellular traps-targeting DNase I enhances the therapeutic efficacy of CAR-T cell adoptive immunotherapy in a syngeneic murine metastasis model [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 B048.

Abstract A012: Aging drives TNBC lung metastasis dependency on FGFR-triggered metabolic reprogramming exposing an age-induced liability with therapeutic potential

Abstract The clinical manifestation of metastasis in a vital organ is the final stage of breast cancer progression and the main culprit of breast cancer related mortality. This remain especially problematic in the case of triple negative breast cancer (TNBC), the most aggressive type of breast cancer, often diagnosed in its metastatic form and for which chemotherapies are still the major therapeutic option. Typically, TNBC is seen as a disease of younger women, however, 20% of TNBCs occur in patients aged 65 and older, whose prognosis is equally staggering. Unfortunately, due to co-morbidities, tolerability of chemotherapies, as well as severe reduction in quality of life, majority of women above age 65 can’t or decide not to receive chemotherapy ultimately cutting their lives short. Metastatic outgrowth is largely dictated by the ability of TNBC disseminated cancer cells (DCCs) to thrive distal organs and is largely dependent on the physiology of the specific organs. However, how the aging process, a major driver of changes in organismal physiology, affects the interaction between the host organ and TNBC DCCs remains unknown. Here, we adapted several traditional syngeneic models of TNBC metastasis to old mice of different strains and coupled with RNA-seq to unveil molecular adaptations of TNBC DCCs to the aging lung. We show that the changes that occur in the lung with aging drives an increase in mitochondrial fitness in TNBC DCCs, an adaptation that is necessary for metastasis to thrive in the old lung environment. Mechanistically, our data show that the expression of FGF19, a gut hormone not normally expressed in TNBC DCCs, drives this increase in mitochondrial fitness in the old lung through an autocrine signaling axis impinging on activation of FGF-19 cognate receptor family, FGFRs. Inhibition of FGF19-FGFR signaling using an FDA- approved FGFR inhibitor not only blocks the age-induced increase in mitochondrial fitness but also effectively abolishes lung metastasis in old, but not in young, immune competent and immune compromised mice. We traced this to age-specific metastatic adaptation of TNBC lung metastasis to the increase in bile acids, the canonical regulators of FGF19 expression, in the old lung environment. Together, our work demonstrates an important contribution of age-driven metabolic reprogramming of the host to the traits that enable metastasis to thrive in the lung and provides rationale for the use of FDA-approved FGFR inhibitors for the treatment of breast cancer lung metastasis in the most vulnerable of stage IV metastatic TNBC patients, the elderly. Citation Format: Stanislav Drapela, Shiun Chang, Rojan Chimeh Rad, Vanessa Rubio, Didem Ilter, Paulo Rodriguez, Ana P. Gomes. Aging drives TNBC lung metastasis dependency on FGFR-triggered metabolic reprogramming exposing an age-induced liability with therapeutic potential [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 A012.

Abstract IA013: Stromal and immune plasticity shape the metastatic microenvironment

Abstract Mortality from cancer is almost exclusively due to metastasis to distant organs. Therefore, understanding the biology of tumor metastasis is the most significant challenge in cancer research today. The tumor microenvironment is central in supporting tumor growth. However, the role of the metastatic microenvironment in the multistage process of metastasis is largely unresolved. Since each metastatic microenvironment exerts specific functions that support or oppose colonization by disseminated tumor, understanding distinct organ-specific mechanisms that enable metastatic growth is of crucial importance. Utilizing mouse models of breast cancer metastasis to lungs, brain and bone, combined with clinical data from patients with metastatic disease, we uncover the mechanisms that underlie the early events of metastasis to provide the mechanistic basis for therapeutic targeting to reduce metastatic relapse. Our findings reveal organ-specific co-evolution of stromal and immune cells at distinct metastatic niches, that lead to the formation of an immunosuppressive microenvironment, permissive to metastatic growth. Analysis of human metastasis reveal that these pathways are also operative in human disease. We show that genetic and/or pharmacological targeting of specific nodes in the interactions between stromal and immune cells results in attenuated metastasis and improved survival. Our findings imply that combinatorial therapeutic strategies to target the crosstalk between stromal and immune cells at the metastatic microenvironment may be a promising treatment strategy to inhibit metastatic relapse. Citation Format: Neta Erez. Stromal and immune plasticity shape the metastatic 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 IA013.

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 A003: The role of aging in promoting immune mediated reactivation from metastatic melanoma dormancy

Abstract Metastasis is the primary cause of melanoma death. A major problem with the vast majority of previous studies is that young mice (8-weeks old, equivalent to 20 human years) are used to investigate the mechanisms of metastatic outgrowth, whereas the median age of melanoma diagnosis in humans is ∼65 years. Advanced age also correlates with metastasis, mortality, and immune dysfunction. Moreover, most studies often only focus on lung metastases, despite melanoma commonly metastasizing to sites such as the liver in patients, which are significantly more resistant to checkpoint inhibitors (ICIs) compared to patients with lung metastases. We have begun addressing these core limitations by developing novel syngeneic mouse models of melanoma metastasis and colonization in young (8-week), middle-aged (12-month), and geriatric (18-22 month) mice, in which metastatic colonization/dissemination can be targeted to either the lung or liver alone, or to both simultaneously. Our data now show that aging promotes reactivation of dormant tumor cells and subsequent outgrowth of lung and liver metastases. We have found that frontline innate lymphocytes such as NK-cells and γδ T-cells are decreased in the pre-metastatic lung and livers of aged mice relative to young mice and remain decreased during early and late-stage metastasis. Using inducible KO mouse models and antibody depletion methods in young growth restrictive mice, we find that depletion of either NK-cells or γδ T-cells is sufficient to promote reactivation from dormancy and resistance to anti-PD1 therapy. Secondly, we further delineated changes in the post-metastatic lung and liver niche. We find that PROS1 secretion by reactivated melanoma cells expressing high levels of the MER tyrosine kinase receptor (MER) in the aged metastatic lung and liver increases CD11b myeloid cells expressing the immunosuppressive factor ARG1. Within this CD11b subset, we see an increase in polymorphonuclear myeloid derived suppressor cells (PMN MDSCs). Treatment with recombinant ARG1 promotes reactivation from dormancy in young mice whilst depletion of PMN MDSCs inhibits age induced outgrowth in the lung and liver. Overall, our study highlights common immune-mediated age-related changes in the pre and post metastatic lung and liver which contribute to aggressive metastatic initiation, reactivation from dormancy, progression, and resistance to anti-PD1 Citation Format: Kelly Coutant, Jhon Passamonte, Christopher Price, Pulkit Datt, Mitchell Fane. The role of aging in promoting immune mediated reactivation from metastatic melanoma dormancy [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 A003.

Abstract C039: The impact of antiangiogenic therapy on blood vessels and CD8 infiltration in renal cancer metastases is space, time, site, dose, and drug dependent

Abstract Clear cell renal cell carcinoma (ccRCC) is the most prevalent renal neoplasm, with bone as a major site for distant spread in 35% to 40% of the patients. These metastases are typically osteolytic, resistant to treatments, hence result in a variety of skeletal-related complications strongly contributing to mortality. A key feature of ccRCC is the loss of function of the von Hippel–Lindau (VHL) protein leading to extensive angiogenesis. Consequently, different anti-angiogenic tyrosine kinase inhibitors (TKIs) are used as the first or subsequent line of treatment for these patients as single agents or in combination with immune checkpoint inhibitors. However, limited data about their efficacy in bone metastasis is available, and their impact on the immune infiltrate, including effector CD8 cells, is unclear. We hypothesized that TKIs, by inhibiting angiogenesis, significantly reprogram the microenvironment and limit immune infiltration in space, time, dose, and drug dependent fashion. To test these hypotheses, we developed an immunocompetent mouse model of bone metastasis by orthotopical intratibial implantation of luciferase-GFP-positive mouse RENCA-VHL- cell line. Interestingly, intratibial injections gave rise to metastases in lungs, creating a unique opportunity to study two metastatic sites. Three different TKIs currently used in patients were tested and compared to control: axitinib (A), cabozantinib (C), and lenvantinib (L). Outcomes were monitored by macroscopic bioluminescence detection in vivo, 3D multiparametric spatial analysis of bones and lungs, in vivo, and ex vivo. Compared to control-treated mice, high doses of A, C, and L significantly reduced tumor progression in both tumor sites. Even though these TKIs are clinically considered equal, significant differences in their efficacy were noted. Spatial analysis of tumor cells and blood vessels showed a significant decrease in blood vessels formation in both an organ- and TKI-specific pattern over time. Additionally, the reduction of neo angiogenesis in treated tumors directly limited the infiltration of CD8 cells, which localized at the interface with tumor free bone/lung tissue. Interestingly, TKI dose reduction significantly improved CD8 infiltration, while controlling tumor size. TKI withdrawal, instead, led to rapid progression of tumor in both sites and significantly shortened mice survival. In conclusion, we established bone and lung metastatic models of ccRCC and characterized and compared the effects of three antiangiogenic TKIs on angiogenesis and CD8 infiltration. TKIs reduced tumor growth and progression, because of blood vessel formation inhibition. Furthermore, TKI treatment reduced the infiltration of effector T cells in tumors in a dose-dependent manner, an outcome that can have a major impact when TKIs are combined with immunotherapy, a preferential therapeutic regimen for ccRCC patients. Overall, we believe our studies provide important insights and efficacy predictions for innovative therapeutic applications in ccRCC bone metastatic patients. Citation Format: Stefan Maksimovic, Nina Costanza Boscolo, Ludovica La Posta, Matthew T. Campbell, Eleonora Dondossola. The impact of antiangiogenic therapy on blood vessels and CD8 infiltration in renal cancer metastases is space, time, site, dose, and drug dependent [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 C039.

Abstract B031: Lean adipocyte-derived lipid decreases metastatic potential of breast cancer cells

Abstract Obesity rates are increasing globally, making it of critical importance to study obesity-linked diseases. Breast cancer is the most common cancer in women, and obesity is associated with both increased incidence and metastatic progression of breast cancer. Metastasis formation depends on a cancer cell leaving the primary site, migrating through the bloodstream, and establishing new growth in a distant site. Both metastasizing cell-intrinsic and metastatic site-dependent factors can help to regulate the effectiveness of this process; however, it is unknown whether obesity-linked metastasis depends on changes to the metastasizing cell and/or changes to the distant metastatic site. This study aims to characterize how the lean versus obese tumor microenvironment impacts breast cancer metastasis and identify adipocyte-derived molecules that modulate the metastatic potential of breast cancer cells. We and others have shown that obesity leads to increased metastasis formation in mouse models of orthotopic breast cancer. However, my preliminary data show that an obese metastatic site is not sufficient to increase metastasis formation in an intravenous injection model of metastasis, emphasizing the importance of the primary tumor microenvironment in programming breast cancer cells for metastasis. Cancer cell stemness helps to mediate metastasis formation and can be assessed in vitro with the mammosphere assay. Importantly, my data demonstrate that a secreted lipid from lean, but not obese, adipocytes potently inhibits the ability of breast cancer cells to form mammospheres. Furthermore, this inhibition induces a G1/S cell cycle arrest. Together, these data suggest that lean adipocytes in the breast cancer tumor microenvironment act in a paracrine manner to decrease the metastatic potential of breast cancer cells. My work demonstrates the role of adipocytes in the regulation of metastasis in breast cancer and provides important insight into the role of the tumor microenvironment in mediating metastasis in lean versus obese conditions. Citation Format: Abigail E. Jackson, Keren I Hilgendorf. Lean adipocyte-derived lipid decreases metastatic potential of 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 B031.

Abstract C026: TGF-β induces an atypical EMT to evade immune mechanosurveillance in lung adenocarcinoma dormant metastasis

Abstract Epithelial-to-mesenchymal transition (EMT) plays important roles during development, injury repair, cancer invasion and metastasis. The heterogeneity of EMT programs is manifest in the diverse EMT-like phenotypes occurring during tumor progression. However, little is known about the mechanistic basis and functional role of specific forms of EMT in cancer. Here we address this question in lung adenocarcinoma (LUAD) cells that enter a dormancy period in response to TGF-β upon disseminating to distant sites. Through in vivo selection, we established dormant metastasis models from human and mouse early-stage LUAD cell lines. LUAD cells with the capacity to enter dormancy are characterized by expression of SOX2 and NKX2-1 primitive progenitor markers, whereas cells derived from overt metastases express high level of the late-stage progenitor marker Sox9. Using a TGF-β signaling reporter, we showed that in the primitive progenitors, TGF-β induces growth inhibition accompanied by a classic EMT response that subsequently transitions into an atypical mesenchymal state of round morphology as the disseminated cells establish dormancy. Prolonged treatment with TGF-β in vitro recapitulates this transition in these primitive progenitors, but not in the late-stage progenitors. Given this dramatic morphological change, we focused on actin-cytoskeleton regulators and found that TGF-β induces this transition by driving the expression of the actin-depolymerizing factor gelsolin, which changes a migratory, stress fiber-rich mesenchymal phenotype into a cortical actin-rich, spheroidal state. This transition lowers the biomechanical stiffness of metastatic progenitors, protecting them from killing by mechanosensitive cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Inhibiting this actin depolymerization process clears tissues of dormant metastatic cells. Thus, LUAD primitive progenitors undergo an atypical EMT as part of a strategy to evade immune-mediated elimination during dormancy. Our results provide a mechanistic basis and functional role of this atypical EMT response of LUAD metastatic progenitors and further illuminate the role of TGF-β as a crucial driver of immune evasive metastatic dormancy. Citation Format: Zhenghan Wang, Yassmin Elbanna, Inês Godet, Lila Peters, George Lampe, Yanyan Chen, Joao Xavier, Morgan Huse, Joan Massagué. TGF-β induces an atypical EMT to evade immune mechanosurveillance in lung adenocarcinoma 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 C026.

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

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

Abstract IA016: Vascular control of metastasis

Abstract The molecular analysis of tumor vessel interactions during tumor progression and metastasis has primarily focused on the study of tumor cell-derived angiogenic and lymphangiogenic signals with the purpose to exploit such factors as therapeutic targets. Angiogenic factors activate endothelial cells in nearby blood and lymphatic capillaries to sprout towards the tumor. Tumor angiogenesis thereby not only nourishes the growing tumor, but access to the blood and lymphatic vasculatures enables cells from the primary tumor to enter the circulation to eventually form metastases at distant sites. The complex cellular interactions between tumor cells and endothelial cells have in this context mostly been studied from a tumor cell-centric perspective, i.e., the tumor cells send signals to which endothelial cells merely respond. Yet, the past decade has witnessed a fundamental change of paradigm with the discovery that the vascular endothelium does not just respond to exogenous cytokines but exerts active ’angiocrine’ gatekeeper roles controlling their microenvironment in an instructive manner. We have applied the concepts of angiocrine signaling towards the study of tumor progression and metastasis. Employing novel surgical preclinical metastasis models that better mimic tumor progression and the response to therapy as it occurs in humans, we have molecularly dissected vascular endothelial cells in progressing primary tumors as well as in the pre-metastatic and metastatic niches. These experiments were on the one hand aimed at establishing the systems map of endothelial transcriptomic changes during tumor progression and metastasis and on the other hand to identify and validate novel therapeutic targets. This presentation will review recent advances in the field of tumor microenvironment research and present novel angiocrine signaling mechanisms as promising targets of future mechanism-driven anti-metastatic therapy. Citation Format: Hellmut G. Augustin. Vascular control of 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 IA016.

Abstract A001: Reciprocal activation of breast cancer metastases and the lung epithelium during metastatic outgrowth

Abstract Introduction. During metastasis, tumor cells interact extensively with endogenous cells in distant organs. These interactions change the behavior of these surrounding cell populations, often inducing a more pro-tumor microenvironment. The lung is one of the most common sites of breast cancer (BC) metastasis, and in the lung, alveolar epithelial cells are the most common cell type. My data suggest that the lung alveolar epithelium contributes to metastatic outgrowth, and I hypothesize that BC lung micrometastases activate surrounding lung epithelial cells which, in turn, support the outgrowth of BC metastases within the lung. The overall purpose of these studies is to identify factors secreted by resident lung epithelial cells that could be used as metastasis-specific therapeutic targets and/or agents. Methods. To test this hypothesis, I utilized immunocompetent preclinical BC metastasis models to study lung metastatic outgrowth, the stage at which most patients are diagnosed with metastatic disease. A custom imaging panel was developed to quantify lung wound repair, and single-cell RNA-sequencing (scRNAseq) was performed on mouse lungs with high or low metastatic burden to identify genes in the lung epithelium that produce potentially targetable pro-metastatic secreted factors. No-contact co-culture experiments were used to directly study reciprocal paracrine interactions between lung type II alveolar epithelial (AT2) and BC cells. Results. A wound repair-related phenotype, characterized by chronic inflammation, developed within the lung microenvironment during metastatic outgrowth, including an increase in the number and activation of AT2 cells surrounding metastases as they grow. Single-cell RNA-sequencing of mouse lungs with a high vs. low metastatic burden showed that metastatic outgrowth significantly changed AT2 gene expression resulting in a modified secretome. No-contact co-culture experiments indicated that TNBC cells directly alter AT2 gene expression, while AT2 secreted factors promoted TNBC growth. I investigated the possible mechanism(s) responsible for these effects and discovered that AT2 pulmonary surfactant protein and lipid levels are altered by BC-derived secreted factors. Interestingly, treatment with the naturally derived calf surfactant Infasurf, which contains native surfactant proteins/lipids and is FDA-approved for use in premature infants, inhibited BC proliferation. Conclusion. Low levels of surfactant are commonly associated with pulmonary disease and lung cancer. My BC lung metastasis data suggest that something similar may be occurring in the lungs during metastatic outgrowth. Overall, my studies demonstrate the potential for targeting lung epithelial cells in the metastatic microenvironment, in addition to directly targeting malignant cells, as an effective way to treat and manage BC lung metastases. Citation Format: Jessica L. Christenson, Nicole S. Spoelstra, Jennifer K. Richer. Reciprocal activation of breast cancer metastases and the lung epithelium during metastatic outgrowth [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 A001.

Abstract B012: HNRNPL-mediated mRNA stability underlies metastasis triggered by resistance to radiation therapy

Abstract The high rate of locoregional/distant recurrence in triple-negative breast cancer (TNBC) is a major driver of the decreased survival observed in breast cancer patients. Radiation therapy is a reliable treatment modality to limit recurrence for many cancers, but TNBC patients have a higher chance for relapse despite receiving increased doses of radiation compared to patients with other breast cancer subtypes. This study investigated the differential pathways in radioresistant TNBC tumors that impact recurrence. We developed two radioresistant cell line models (468-RR and 4T1-RR) by administering a radiation dose of 50Gy over the course of 8 weeks to the human TNBC cell line, MDA-MB-468, and the mouse TNBC cell line, 4T1, respectively. Migration and invasion were assessed using the scratch-wound assay and transwell assay, respectively. We assessed in vivo metastasis by injecting the cells in the mammary fat pad, tail vein, or left ventricle. RNA-sequencing of the cell lines was done to identify global transcriptomic changes and regulatory pathways induced by radiation therapy. The mRNA stability of integrin b3 was assessed by inhibiting transcription using actinomycin D. Moreover, we implemented the newly developed InduroRT-mediated circRNA-sequencing (IMRT-seq) to identify the circular RNAs processed by HNRNPL that are present in radioresistant cells. We observed enhanced lung and bone metastasis, based on bioluminescent imaging, in both the spontaneous and vascular metastasis models for the radioresistant cells compared to the parental cells. Genset enrichment analysis identified mRNA stability and metabolism as upregulated pathways in radioresistant cells with HNRNPL, a ribonucleoprotein involved in mRNA stability, as one of the top genes. By knocking down HNRNPL, we observed decreased migration capacity of the radioresistant cells. Given the role of HNRNPL in integrin and extracellular matrix genes, which are key components associated with metastasis in cancer cells, we screened for these genes in our RNA-seq dataset and identified integrin b3 as a possible target. More specifically, knocking down HNRNPL decreased the mRNA stability of integrin b3 as assessed by actinomycin d treatment. By expressing integrin b3 in the HNRNPL-depleted cells, we were able to rescue the migration capacity of these cells. We believe a subset of HNRNPL-mediated circular RNAs are responsible for integrin b3 mRNA stability by sequestering miRNAs that target integrin b3. With IMRT-seq we are exploring the potential circRNA-miRNA interactions present in radioresistant TNBC responsible for its metastatic phenotype. The radioresistant cells derived from TNBC cell lines have an enhanced metastatic potential than the treatment-naive cell lines and our data suggest that RNA metabolism is pivotal to this phenotype. The upregulation of HNRNPL upon radiation by Nrf2 increases integrin b3 expression by stabilizing its transcripts and preventing its degradation. This novel mechanism of radioresistant metastasis provides a therapeutic approach to minimize TNBC recurrence. Citation Format: Ayush Kumar, Kensei Kishimoto, Christi Wisniewski, Shivam Goel, Arthur Mercurio. HNRNPL-mediated mRNA stability underlies metastasis triggered by resistance to radiation therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B012.

Charge-Stabilized Nanodiscs as a New Class of Lipid Nanoparticles.

Nanoparticles have the potential to improve disease treatment and diagnosis due to their ability to incorporate drugs, alter pharmacokinetics, and enable tissue targeting. While considerable effort is placed on developing spherical lipid-based nanocarriers, recent evidence suggests that high aspect ratio lipid nanocarriers can exhibit enhanced disease site targeting and altered cellular interactions. However, the assembly of lipid-based nanoparticles into non-spherical morphologies has typically required incorporating additional agents such as synthetic polymers, proteins, lipid-polymer conjugates, or detergents. Here, charged lipid headgroups are used to generate stable discoidal lipid nanoparticles from mixed micelles, which are termed charge-stabilized nanodiscs (CNDs). The ability to generate CNDs in buffers with physiological ionic strength is restricted to lipids with more than one anionic group, whereas monovalent lipids only generate small nanoliposomal assemblies. In mice, the smaller size and anisotropic shape of CNDs promote higher accumulation in subcutaneous tumors than spherical liposomes. Further, the surface chemistry of CNDs can be modified via layer-by-layer (LbL) assembly to improve their tumor-targeting properties over state-of-the-art LbL-liposomes when tested using a metastatic model of ovarian cancer. The application of charge-mediated anisotropy in lipid-based assemblies can aid in the future design of biomaterials and cell-membrane mimetic structures.

Targeting SPP1-orchestrated neutrophil extracellular traps-dominant pre-metastatic niche reduced HCC lung metastasis.

The mechanisms by which tumor-derived factors remodel the microenvironment of target organs to facilitate cancer metastasis, especially organ-specific metastasis, remains obscure. Our previous studies have demonstrated that SPP1 plays a key role in promoting metastasis of hepatocellular carcinoma (HCC). However, the functional roles and mechanisms of tumor-derived SPP1 in shaping the pre-metastatic niche (PMN) and promoting lung-specific metastasis are unclear. Orthotopic metastasis models, experimental metastasis models, CyTOF and flow cytometry were conducted to explore the function of SPP1 in shaping neutrophil-dominant PMN and promoting HCC lung metastasis. The main source of CXCL1 in lung tissues was investigated via fluorescence activated cell sorting and immunofluorescence staining. The expression of neutrophils and neutrophil extracellular traps (NETs) markers was detected in the lung metastatic lesions of HCC patients and mouse lung specimens. The therapeutic significance was explored via in vivo DNase I and CXCR2 inhibitor assays. SPP1 promoted HCC lung colonization and metastasis by modifying pulmonary PMN in various murine models, and plasma SPP1 levels were closely associated with lung metastasis in HCC patients. Mechanistically, SPP1 binded to CD44 on lung alveolar epithelial cells to produce CXCL1, thereby attracting and forming neutrophil-abundant PMN in the lung. The recruited neutrophils were activated by SPP1 and then formed NETs-dominant PMN to trap the disseminated tumor cells and promote metastatic colonization. Moreover, early intervention of SPP1-orchestrated PMN by co-targeting the CXCL1-CXCR2 axis and NETs formation could efficiently inhibit the lung metastasis of HCC. Our study illustrates that HCC-lung host cell-neutrophil interactions play important roles in PMN formation and SPP1-induced HCC lung metastasis. Early intervention in SPP1-orchestrated PMN via CXCR2 inhibitor and DNase I is a potential therapeutic strategy to combat HCC lung metastasis.

CSIG-12. YAP1-TEAD SIGNALING CONTRIBUTES TO LUNG CANCER COLONIZATION OF THE BRAIN

Abstract BACKGROUND Lung cancer is the most common primary tumor to metastasize to the brain. Poor understanding of the brain metastasis process limits therapeutic options. Despite emerging data on the role of YAP signaling pathway in the development of brain tumors, a gap in knowledge remains regarding its role in brain colonization. We hypothesize that YAP1 and the associated TEAD transcriptional factors are responsible for lung cancer progression and that intervention with YAP/TEAD-targeted therapy will prevent brain dissemination. METHODS Lung cancer cells at different stages of cancer progression (VMRC-LCD and NCI-H1975, lung cancer cell lines; NCI-H1915 and LN001, brain metastatic cancer cells) were genetically engineered to overexpress or silence/knockout YAP1 using lentiviral transduction of constructs expressing shRNA/sgRNA or YAP1. We tested several tumor cell capabilities using well-established in vitro assays, including invasion and migration assays, and an in vivo brain metastasis (BM) model to determine the biological effects of YAP1/TEAD suppression at different stages of lung cancer progression. Dose-response assays were performed to determine IC50 of the TEAD inhibitor, verteporfin. RESULTS YAP overexpression enhances the proliferative phenotype in metastatic and BM cell lines but not in non-metastatic cells. However, YAP overexpression only increases the migration ability of BM cell lines such as LN001 and NCI-H1915 compared to the metastatic NCI-H1975 and non-metastatic lung cancer cells. Moreover, we showed the efficacy of the YAP1/TEAD inhibitor in patient-derived lung BM cells. Finally, our in vivo preliminary data showed YAP1 involvement in lung cancer cell colonization of the brain but not in sustaining brain metastasis growth. CONCLUSIONS Our findings showed YAP1 involvement in lung cancer cell colonization of the brain, suggesting YAP1 inhibition is a clinically actionable target for preventing lung cancer dissemination and providing a strong rationale to further evaluate the benefit of a YAP1/TEAD-targeted therapy in in vivo models of BM.