Microenvironment In Cancer Progression Research Articles

Abstract PO007: Role of PI3K pathway in reprogramming the tumor niche of angiosarcoma

Abstract Angiosarcoma (AS) is a highly aggressive, rare soft-tissue sarcoma that forms malignant blood vessels. Approximately half of patients have metastatic or unresectable disease with a median overall survival of less than 6 months, and tumor-related mortality is high. Inflamed tissue is frequently seen in angiosarcomas, and immune cells are key components that contribute to the landscape of the tumor microenvironment. Hemangiosarcoma (HSA) occurs commonly in companion dogs, and it shares clinical and morphological features with human AS. Recurrent mutations in TP53 and genes involved in PI3K pathway such as PIK3CA, and PIK3R1 were observed in both human AS and canine HSA, and the mutational landscape was associated with distinct molecular tumor subtypes; in particular, differing immunophenotypes. Recently, we have shown that human AS and canine HSA establish convergent transcriptional programs driven by angiogenic fusion genes and TP53 mutation. However, these vascular tumors are genomically complex, and molecular mechanisms that establish the tumor microenvironment are incompletely known. In this study, we analyzed RNA-seq transcriptomic data generated from human AS tissues (n=13) and canine HSA tissues (n=76) and HSA cell lines (n=11). The data showed that the tumors of both species enriched gene signatures associated with maintenance of hematopoietic stem cells. Then, we found that canine HSA cells supported expansion and differentiation of human CD34+ umbilical cord blood cells using long-term culture-initiating cell and colony-forming unit assays. We also developed xenograft models of canine HSA that facilitate myelopoiesis and macrophage infiltration in the tumor tissue. Our data showed that gene signatures of monocytes and naïve macrophages were detectable in canine HSA cells. In human ASs, macrophage-associated genes, in particular M2 macrophage signatures were significantly enriched, compared to normal tissues. Collectively, our data suggest that human AS and canine HSA have cell-autonomous capacity to govern hematopoietic progenitors and immune cells, potentially establishing the tumor immune niche. We engineered HSA cells to induce homozygous H1047R mutations in PIK3CA gene using CRISPR-Cas9 system. Our ongoing work is to determine if regulating PI3K pathway in the tumor cells contributes to the molecular programs that create the tumor niche of the vascular malignancy. Citation Format: Sophia Wenthe, Ashley J. Schulte, Mathew G. Angelos, Dan S. Kaufman, Jaime F. Modiano, Jong Hyuk Kim. Role of PI3K pathway in reprogramming the tumor niche of angiosarcoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO007.

Abstract LT012: The prolyl isomerase PIN1 plays a critical role in fibroblast plasticity to impact pancreatic cancer

Abstract PIN1 is a phosphorylation-directed prolyl isomerase that alters the conformation and, therefore, the function of many proteins. Due to its role in activation and stabilization of many oncogenes, we hypothesized that targeting PIN1 in pancreatic ductal adenocarcinoma (PDA) would slow tumor growth. We tested this hypothesis in vitro and in vivo with PIN1 inhibitors and/or genetic model systems. Pancreatic cancer cell lines knocked down for PIN1 or treated with PIN1 inhibitors showed decreased proliferation, invasion, and anchorage independent growth compared to control lines. Consistent with these in vitro results, treatment of pancreatic cancer xenografts or genetically engineered p48-Cre; LSL-KrasG12D; p53R172H (KPC) mice with PIN1 inhibitors decreased tumor growth and extended overall survival. Similar results were seen in KPC mice that were crossed into a full body PIN1 knockout (PIN1−/−). Further analysis of KPC PIN1−/− tumors revealed not only reduced size of pancreatic tumors, but also decreased alpha-SMA expression and decreased ECM deposition in the stroma surrounding the tumors. PDA is characterized by a dense, desmoplastic tumor stroma that contributes to tumor growth, metastasis, and therapeutic resistance. Pancreatic stellate cells (PSCs) that are activated in the tumor microenvironment play a major role in the deposition of ECM and secrete growth factors to support tumor cell proliferation and survival. To interrogate a direct role for PIN1 in the stroma, we first orthotopically injected a KPC cell line into syngeneic PIN1+/+ or PIN1−/− mice and found dramatic reduction of tumor cell growth in PIN1−/− hosts. Next, we analyzed PSCs in vitro and found that loss of PIN1 reduces their proliferation and alters their secretion of paracrine factors that support oncogenic phenotypes. For example, PSCs with loss of PIN1 have reduced expression of HGF and increased expression of SPINT1 and SPINT2, inhibitors of HGF activation. Conditioned media from control PSCs, but not from PSCs lacking PIN1 expression, activates the MET receptor on cancer cell lines, resulting in altered cancer cell phenotypes. In addition, we show that loss of PIN1 in PSCs inhibits TGF-beta induced stellate cells activation into a myofibroblast phenotype. Single cell ATAC-seq analysis demonstrated that a subset of TGF-beta responsive chromatin changes are impaired in the absence of PIN1. Our ongoing work utilizes 2D co-cultures, heterotypic 3D bioprinted tissues, and in vivo mouse models to interrogate the mechanisms by which fibroblast phenotypes and the tumor-stromal crosstalk is impacted by PIN1. Citation Format: Ellen M. Langer, Isabel A. English, Kayleigh M. Kresse, Kevin MacPherson, Brittany L. Allen-Petersen, Colin J. Daniel, Andrew Adey, Rosalie C. Sears. The prolyl isomerase PIN1 plays a critical role in fibroblast plasticity to impact pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT012.

Abstract PO006: Heating up the fight against cancer using 3D bioprinting

Abstract Tumor growth is regulated by complex interactions of tumor cells with the tumor microenvironment (TME) which consists of cancer associated fibroblasts (CAFs), various immune cells including tumor infiltrating lymphocytes (TILs) and extracellular matrix components (1). Accurately modeling the TME in vitro is challenging as there is significant genetic (2, 3) and spatial (4) heterogeneity within TME components. Triple-negative breast cancer (TNBC) patients that have tumors with TILs restricted to the margins are called immune “cold” and have poor prognosis (5, 6). Interfering with the mechanisms that exclude TILs from these tumors may improve immunotherapy response and TNBC patient outcome. A major barrier to anti-cancer drug development is the availability of in vitro models that accurately reflect the in vivo TME. Our goal is to establish and validate a 3D bioprinted tumor model that reproduces the spatial relationship between tumor, CAFs and TILs. We have generated a biobank of TNBC patient-derived organoids and tissue-matched TME components. The tumor immune microenvironment molecular subtypes were assigned to each tumor sample using transcriptomic data and immunohistochemistry analysis. We have used microfluidic 3D bioprinting (RX1™ bioprinter, Aspect Biosystems) to print a core-shell fibre design that recapitulates the immune “cold” TME and permits monitoring of immune cell infiltration into the cancer cell core of the fibre. Clinically proven checkpoint inhibitor therapy will be applied to our model to validate its biological function. This innovative bioprinted tumor model will enable phenotypic screening to efficiently identify new targets that transform hard-to-treat immune “cold” tumors into a state where immune cells can infiltrate the tumor. Using patient-derived tissue-matched TME components increases the clinical relevance of the bioprinted tumor model and enables personalized anti-cancer drug screening in vitro to accurately predict patient response to therapy. 1. Balkwill FR, Capasso M, Hagemann T. The tumor microenvironment at a glance. J Cell Sci. 2012; 125: 5591-5596. 2. Finak G, Bertos N, Pepin F, et al. Stromal gene expression predicts clinical outcome in breast cancer. Nat Med. 2008; 14: 518-527. 3. Lehmann BD, Bauer JA, Chen X, et al. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest. 2011; 121: 2750-2767. 4. Gruosso T, Gigoux M, Manem VSK, et al. Spatially distinct tumor immune microenvironments stratify triple-negative breast cancers. J Clin Invest. 2019;129(4):1785-1800. 5. Ali HR, Provenzano E, Dawson SJ, et al. Association between CD8+ T-cell infiltration and breast cancer survival in 12,439 patients. Ann Oncol. 2014; 25: 1536-1543. 6. Salgado R, Denkert C, Demaria S, et al. The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014. Ann Oncol. 2015; 26: 259-271. Citation Format: Anne-Marie Fortier, Erin Bedford, Salvador Flores Torres, Spiro Getsios, J. Matt Kinsella, Sam Wadsworth, Morag Park. Heating up the fight against cancer using 3D bioprinting [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO006.

Abstract IA018: Linking obesity-associated inflammation with cancer metastasis

Abstract Obesity is associated with increased incidence and mortality of multiple tumor types, and competes with smoking tobacco as the leading preventable risk factor for all cancer-related deaths. In breast cancer, epidemiological studies have shown that obesity is linked to enhanced metastasis to lung and liver. However, it is not known how to improve cancer outcomes in the obese patient population as the mechanistic link between obesity and cancer progression is currently lacking. Using genetic and diet-induced obesity mouse models, it was previously shown that obesity promotes pulmonary metastasis of breast cancer by inducing lung neutrophilia via a IL5–GM-CSF signaling axis. We have now determined that neutrophils are reprogrammed by obesity within the lung to produce elevated levels of reactive oxygen species (ROS) and release neutrophil extracellular traps (NETs). This in turn weakens endothelial barrier integrity, leading to enhanced microvascular permeability and tumor cell extravasation into the lung parenchyma. This effect can be reversed by targeting ROS via pharmacological or genetic approaches, or by preventing NETosis using PAD4 inhibitors. To confirm the translational relevance of our findings, we performed imaging mass cytometry on lung metastasis samples from cancer patients, and observed a significant correlation between body mass index and NETosis, neutrophil oxidative burst, and ROS status. Given the high prevalence of obesity on an international scale, our data provide insight into mechanisms of cancer progression for a significant proportion of the adult population, including (potentially) those who are metabolically obese but normal weight. Citation Format: Sheri A. McDowell, Robin Luo, Jonathan D. Spicer, Andrew J. Dannenberg, Logan A. Walsh, Daniela F. Quail. Linking obesity-associated inflammation with cancer metastasis [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr IA018.

Abstract PO017: Role of NF-κB and epiregulin in tumor-associated macrophages promoting therapeutic resistance to JAK inhibition

Abstract Breast cancer is the second leading cause of cancer-related mortalities in women. Triple-negative is the most lethal type of breast cancer with poorest survival rates. Triple-negative breast cancers do not respond to conventional hormone receptor targeted therapies and thus inhibitors for other overactivated signaling pathways are being investigated. The Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) is an oncogenic transcription factor pathway expressed in tumor and stromal cell populations. Inhibitors of this pathway such as ruxolitinib are being studied for treatment of triple-negative breast cancer but their efficacy in patients is limited. A significant barrier to the success of these inhibitors in the clinic is the lack of information about mechanisms of resistance. Tumor-associated macrophages (TAMs) are important components of the immune-suppressive environment within the tumor and are associated with poor outcome. We found that JAK inhibition in TAMs leads to induction of tumor-promoting genes that mediate therapeutic resistance of the tumor to JAK inhibition. A subset of the activated tumor promoting genes are known transcriptional targets of the NF-κB transcription factor pathway. We hypothesize that JAK inhibition activates tumor-promoting pathways in TAMs through activation of the NF-κB pathway and targeting these tumor-promoting pathways in combination with JAK inhibition will be beneficial over single therapy. We aim to elucidate activation of NF-κB in TAMs, to identify the mechanism of the observed pro-tumor response. In addition, we aim to target epiregulin, an EGFR ligand which was observed to be one of the most significantly upregulated genes in TAMs treated with JAK inhibitor. We hypothesize that targeting effects of macrophage-derived epiregulin in combination with JAK inhibition will overcome macrophage-mediated therapeutic resistance. This work will provide useful information about novel pathways that can be targeted to combat the compensatory negative effects of TAMs on tumor-targeted therapies. The results will also pave the way for development of novel inhibitor combinations targeting the tumor microenvironment in addition to targeting the tumor, and ultimately benefit patients who do not respond to single therapies. Citation Format: Aditi S. Bapat, Emily Jesser, Chelsea Lassiter, Kaylee Schwertfeger. Role of NF-κB and epiregulin in tumor-associated macrophages promoting therapeutic resistance to JAK inhibition [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO017.

Abstract PO030: A novel integrin-targeted therapeutic agent for prostate cancer

Abstract Disintegrins are disulfide-rich peptides, many containing an Arg-Gly-Asp (RGD) motif. They hold significant translational potential as anti-cancer agents based on their high-affinity/high-specificity interaction with tumor integrins and desirable pharmacological attributes. The integrin-binding activity of disintegrins depends on a mobile 11-amino acid loop displaying the RGD tripeptide motif or some other tripeptide motif. They bind only to the activated conformation of integrins on motile cancer cells and angiogenic endothelial cells. We designed a sequence-engineered RGD-disintegrin, vicrostatin (VCN), that can be produced at ~200mg/L from cell lysates of a bacterial recombinant system. VCN, a monomer (MW=7146), inhibits tumor cell adhesion and dissemination, and angiogenic endothelial cell invasion and tube formation. Integrins are cell surface adhesion receptors (containing a and b chains) operating at the interface between the extracellular matrix and cytoskeletal apparatus. A sub-group of integrins recognize the RGD sequence present in key extracellular matrix proteins involved in prostate cancer (PC) growth and dissemination. Integrins are involved in bi-directional signaling interactions that alter cellular functions. Among these interactions are those involved in PC growth, metastasis and angiogenesis. Integrin avb3 plays an important role in the development of bone metastases, supporting our belief that it, as well as other RGD-integrins, are important therapeutic targets in advanced PC. We examined the effect of treatment of a xenograft model of human PC using a liposomal formulation of VCN (LVCN). PC3 cells were implanted subcutaneously in 5-week old male nude mice. LVCN and VCN (100µg VCN equivalent per dose) administration was initiated IV when tumors became palpable (14 days). Tumor size was measured weekly by caliper in a blinded study. There was a 75% reduction in tumor growth in LVCN-treated animals compared to controls. VCN displayed little anti-tumor activity most likely due to its lack of availability to the PC cells. We also examined the anti-angiogenic activity of LVCN in this model using CD31/PECAM immunohistochemistry and observed a significant decrease in tumor-associated microvessel density. We then evaluated therapeutic efficacy of LVCN in treating bone metastases. CWR22rV1 PC cells were suspended in a solution of matrigel and injected into a drilled hole in the proximal left tibia of immunodeficient mice (100,000 cells/10µL). Following injection, the diameters of both tibias were measured by caliper twice weekly. Since CWR22rV1 cells are androgen dependent, animals received daily injections of testosterone. Tumors grew slowly initially, but at 20 days tumors were evident and treatment began. LVCN and VCN were administered twice weekly via IV injection (100µg per dose) for 5 weeks. LVCN displayed ~81% inhibition of tumor growth compared to VCN, PBS or empty liposomes. Our findings provide strong support for the effectiveness of LVCN as an inhibitor of PC growth and metastases. Citation Format: Francis S. Markland, Stephen Swenson, Radu Minea, Jacek Pinski. A novel integrin-targeted therapeutic agent for prostate cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO030.

Abstract PO021: Carcinoma-associated mesenchymal stem/stromal cells enhance ovarian cancer metastasis and increase cancer cell clonal heterogeneity through direct mitochondrial transfer

Abstract Ovarian cancer is the most deadly gynecologic cancer largely due to early, diffuse metastatic spread. The ovarian cancer tumor microenvironment (TME) significantly impacts the metastatic capacity of ovarian cancer. We recently demonstrated that a critical stromal progenitor cell within the TME, termed carcinoma-associated mesenchymal stem/stromal cells (CA-MSCs), dramatically enhance the metastasis of ovarian cancer cells. CA-MSCs directly bind to cancer cells forming heterocellular units which co-metastasize. The goal of this work is to understand the mechanism driving CA-MSC-mediated ovarian cancer metastasis. We hypothesize that CA-MSCs increase the metastatic potential of ovarian cancer cells during the process of co-metastasis. Ovarian cancer uses both hematogenous and transcoelomic routes of metastasis therefore we used two ovarian cancer murine models to evaluate both modes of metastasis. We used a genomic barcode system to assess the clonal patterns of metastasis. Ovarian cancer cells with one unique barcode per cell were grown with or without CA-MSCs. Sites of metastasis were quantified and cancer cells were harvested from each site. Barcodes were sequenced to determined clonal representation at each metastatic site. We found CA-MSCs increased the number of metastatic sites in both the hematogenous and transcoelomic mouse models. Interestingly, CA-MSCs also increased the clonal heterogeneity at all metastatic sites (2.5 - 5 fold increase in blood, lung and liver). To investigate the mechanism enabling CA-MSC-mediated enhancement of clonal heterogeneity, we studied primary patient derived CA-MSCs and ovarian cancer cells. The process of metastasis poses unique metabolic stresses on cancer cells and normal mesenchymal stem cells are known to donate mitochondria to damaged epithelial cells. We therefore assessed if CA-MSCs use a similar mechanism to enhance survival of metastatic cancer cells. We stably labeled CA-MSC mitochondria with COX8-GFP via lentiviral transduction to enable the visualization and quantification of potential mitochondrial transfer. We demonstrate that CA-MSCs actively transfer mitochondria to cancer cells. This transfer is dependent on the physical interaction of CA-MSCs with cancer cells and is increased 2-4 fold when CA-MSCs and cancer cells form metastatic heterocellular units under non-adherent conditions. We tested the functional consequences of CA-MSC to cancer cell mitochondrial transfer and demonstrate cancer cells which receive mitochondria have increased chemotherapy resistance, increased sphere forming capacity and stem-like properties. Collectively, our results indicate CA-MSCs are a significant driver of ovarian cancer metastasis by forming heterocellular metastatic units which enable CA-MSC to cancer cell mitochondrial transfer. This leads to increased cancer cell survival and tumor initiation properties and represents a novel method of maintaining cancer cell heterogeneity during metastasis. Citation Format: Catherine A. Pressimone, Leonard G. Frisbie, Alexander Pearson, Lan G. Coffman. Carcinoma-associated mesenchymal stem/stromal cells enhance ovarian cancer metastasis and increase cancer cell clonal heterogeneity through direct mitochondrial transfer [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO021.

Abstract PO036: CXCL12 embedded-Hyaluronate based 'pseudo niche': a new device for CTCs/DTCs capturing and characterization

Abstract Aim: Tumor cells can prime distant microenvironment building a pre-metastatic niche (PMN) where circulating tumor cells (CTCs) will home. 0.01% of CTCs, endowed with the capability to extravasate and infiltrate distant sites, will develop distant metastasis. In this process, a crucial role is played by chemokine gradients. Highly tumorigenic CTCs express the chemokine receptor CXCR4. CXCR4 ligand, CXCL12, attracts bone marrow-derived cells and CXCR4+ cancer cells in pre-metastatic sites to form a PMN. A CXCL12-loaded-hydrogel (CLG) was developed with the commercially available Belotero Hyaluronic gel dermal filler to develop a “pseudo niche” attracting immune and CTCs-CXCR4+cells. Aims: 1. Biological characterization of CTCs, 2. CTC/tumor microenvironment interplay 3. feasibility for clinical CTC isolation and characterization. Materials and methods: Human renal cancer cells SN12C (100 and 500) and A498 (500 and 1000) were seeded on 150ul Empty Gels (EG) or CLG [300ng/ml CXCL12], and allowed to migrate within gels, then fixed and DAPI-stained for enumeration or recovered by gel digestion and grown in complete media for 6 days. CXCR4 expression and colonies formation capability were evaluated. 100 SN12C or HT29, human colon cancer cells, were spiked in 7cc HD blood, ficoll-paqued and the mixture seeded on CLG. Isolated cells were fixed and stained with anti-hCD45-FITC, panCK-594 and DAPI (cancer cells identified as DAPI+/pan-CK+/CD45-). 7cc blood derived from 15 metastatic cancer patient’s (colon, lung, ovary, endometrial, kidney and glioblastoma) will be processed as previously described (CTCs identified as DAPI+/pan-CK+/CD45-); as comparison, CTCs will be isolated and enumerated using the commercially available Screen Cell™ filters according to manufacturer’s instructions. Results: A498 and SN12C cells efficiently infiltrate CLG as compared to empty gel (EG) (CLG/EG fold increase 1.6-1.9). SN12C and HT29 EG/CLG-recovered cells developed colonies after 6 days of culture. HT29-CLG recovered cells overexpressed CXCR4 compared to plastic grown cells and to EG cells and originates a higher number of colonies compared to plastic grown cells (171±21 for CLG VS 131±8 for plastic grown). SN12C and HT29 cells were successfully recovered in 7cc HD blood (recovery rate 20 and 14% respectively). Clinical trial on feasibility in isolating CTC cells on CLG recently started patient’s recruitment in ovarian, colon, lung and renal cancer metastatic patients. Conclusion: CLG-device mimics a PMN for capturing CTCs able to extravasate and infiltrate distant tissue, and potentially, more metastatic. CLG-isolated cells highly express CXCR4, develop higher number of colonies and migrate toward CXCL12. Moreover, the device allows cancer cell recovery from HD blood being potentially clinically useful for CTCs identification, characterization and trapping in cancer patients. Citation Format: Luigi Portella, Caterina Ieranò, Giulia Bertolini, Crescenzo D'Alterio, Giuseppina Rea, Sara Santagata, Anna Maria Trotta, Gelsomina Monaco, Roberto Pacelli, Stefania Scala. CXCL12 embedded-Hyaluronate based 'pseudo niche': a new device for CTCs/DTCs capturing and characterization [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO036.

Abstract LT017: ECM mechanical and metabolic architecture during early ductal invasions: integrating in silico modeling, histology-based machine learning and mechanobiology

Abstract Progression from a ductal carcinoma in situ (DCIS) to an invasive tumor is a major step initiating a devastating and often lethal metastatic cascade. One sentinel event that initiate this process is the development of ductal microinvasions, i.e., small cohorts of tumor cells that breach the basement membrane surrounding the duct and migrate through the extracellular matrix (ECM) leading to irreversible changes in tumor and stromal architecture. We used a combination of advanced image analysis techniques applied to patients’ histology data to extract features which identify specific properties of individual tumor cells inside the duct and on the invasive front. By integrating these histology-based data with a hybrid agent-based mathematical model, we investigated the biomechanical interactions between the cells and the ECM fiber architecture, and microenvironmental physical and metabolic features that define tumor niche prone to microinvasions. The identified physical properties of the matrix elucidate conditions that can facilitate or prevent the progression of such microinvasions. The presented method provides also a tool for quantifying morphological and immunohistochemical properties of individual cells within the mammary ducts and ductal microinvasion, as well as for testing biomechanical hypotheses of tumor cell-tumor matrix interactions. These findings can be compared to the patient histology samples and help define criteria for identification of changes in tumor architecture and future development of new diagnostic methods. Citation Format: Katarzyna A. Rejniak, Sharan Poonja, Shreya Mathur, Jessica Kingsley, Marilyn Bui. ECM mechanical and metabolic architecture during early ductal invasions: integrating in silico modeling, histology-based machine learning and mechanobiology [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT017.

Abstract IA008: Age-related stromal changes drive tumorigenesis

Abstract The stromal compartment plays a pivotal role in tumor progression. This is best illustrated in breast cancer bone metastases, where the stromal compartment supports tumor growth, albeit through poorly defined mechanisms. p38MAPKa is frequently expressed in tumor cells and surrounding stromal cells and its expression levels correlate with poor prognosis. Using a clinically relevant breast cancer metastasis model, we show that orally administered, small-molecule inhibitors of p38MAPKa or its downstream kinase, MK2, each limited outgrowth of metastatic breast cancer cells in the bone and visceral organs. Further we find that this effect is primarily mediated by inhibition of the p38MAPKa pathway within the stromal compartment. Beyond effectively limiting metastatic tumor growth, we also find that these inhibitors reduce tumor-associated and chemotherapy-induced bone loss, which is a devastating comorbidity for cancer patients that drastically impact their quality of life. Interestingly we find that p38MAPKa’s ability to limit tumor growth within the bone is dependent on CD4 T cells while MK2 does not require these cells to limit tumor progression, demonstrating that it is not a straightforward pathway. To determine which cell types within the bone contribute to tumor growth and bone loss, we have carried out single cell RNA-sequencing of stromal cells located within the bone metastatic lesion and cells outside the lesion. We find numerous differences that could explain how the stromal compartment contributes to tumor progression and bone loss and how inhibition of p38MAPKa or MK2 contribute. These data underscore the vital role stromal-derived factors play in tumor progression and identify the p38MAPK-MK2 pathway as a promising therapeutic target for metastatic disease and prevention of tumor-induced bone loss. Citation Format: Sheila A Stewart. Age-related stromal changes drive tumorigenesis [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr IA008.

Abstract PR001: The splanchnic mesenchyme during fetal development is the major source of pancreatic cancer associated fibroblasts

Abstract In pancreatic ductal adenocarcinoma (PDAC), cancer associated fibroblasts (CAFs) play critical and complex roles in the tumor microenvironment. CAFs are also a major cell type in the desmoplastic stroma in PDAC and may account for half of the entire tumor tissue. Multiple subtypes of CAFs have been suggested, but the tissue origin(s) of CAF subtypes are unknown and genetic tools to robustly target them in vivo are lacking. Here we aimed to examine three potential tissue sources of CAFs: the pancreatic epithelium (through epithelium-to-mesenchyme transition), the bone marrow (through circulation), and the pancreatic mesenchyme or tissue resident fibroblasts (TRFs) in the normal pancreas (through proliferation). We utilized a genetically engineered mouse model of PDAC, where Kras and p53 mutations were engineered in the pancreatic epithelium using an Flp-Frt system. To determine whether the pancreatic epithelium gives rise to CAFs, we permanently labeled the pancreatic epithelium with a GFP reporter and traced their cell descendants by GFP expression. Despite robust GFP labeling of the epithelium, GFP expression was rarely identified in CAFs, suggesting little contribution of epithelium to the CAF pool. To determine whether the bone marrow gives rise to CAFs, we transplanted donor bone marrow carrying a ubiquitously expressed GFP reporter allele to GFP-negative recipient mice. We found that only a small proportion of pancreatic CAFs were tagged with GFP, suggesting their bone marrow origin. Lastly, to determine whether pancreatic TRFs give rise to CAFs, we used an inducible CreER-LoxP system to allow for permanent Tomato labeling in TRFs progenitors, the splanchnic mesenchyme, during mid-gestation. Lineage tracing in PDAC showed that the vast majority of CAFs were labeled with Tomato expression, suggesting their splanchnic origin. Furthermore, certain splanchnic gene expression signatures were persistent in subsets of CAFs in both the PDAC mouse model and human patient samples. In summary, we found that bone marrow contributes to a small proportion of CAFs in PDAC, and the pancreatic epithelium contributes even less. Meanwhile, pancreatic TRFs are derived from the splanchnic mesenchyme during fetal development and they expand to contribute to the vast majority of CAFs in PDAC. Moreover, the persistence of splanchnic signature defines subtypes of CAFs. This study provides approaches to robustly target CAFs in vivo and novel insights into CAF heterogeneity in PDAC. Citation Format: Lu Han, Yongxia Xu, Sean Sweeney, Ulyss Roesner, Melodie Parrish, Khushbu Patel, Xuezhong Yu, Michael Ostrowski, Gustavo Leone. The splanchnic mesenchyme during fetal development is the major source of pancreatic cancer associated fibroblasts [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PR001.

Abstract PO035: Engineered glioblastoma tumor models reveal extracellular matrix signals influence the efficacy of targeted inhibitors

Abstract Biomaterial-based ex vivo models of healthy and diseased brain tissue are among those tools that may help study the complexity of cerebral tissue to better diagnose, prevent, and treat brain diseases. We have established engineered brain tumor biomaterials based on methacrylamide-functionalized gelatin hydrogels and used microfluidic-forming techniques to generate platforms that combine transitions of biophysical and biomolecular properties found in the glioblastoma tumor microenvironment, from the core to the tumor margins (1). Moreover, this biomaterial approach is able to monitor the response of patient-derived xenograft (PDX) cell populations with different molecular signatures; in particular, we have analyzed the amplified and the constitutively activated vIII mutant EGF receptor. We have also characterized PDX response to targeted inhibitors, such as erlotinib, a tyrosine kinase inhibitor that specifically blocks EGFR (2). Patients with glioblastoma (GBM) exhibit poor survival rates, tied both to the significant intra and interpatient heterogeneity of the tumor as well as the complex signaling pathways underlying malignancy. Spatial and temporal gradients regulate cell proliferation, migration, and differentiation during cancer. Therefore, the use of a microfluidic approach to fabricate patterned biomaterials, have the ability to examine transitions between defined environments (e.g., glioma core, periphery and neural tissue). Cells within these structured materials can be fully analyzed by imaging, secretomic and gene expression analyses. Cell morphology evolved to a more spread shape from low to high crosslinking density of gelatin and hypoxic levels depended on cell concentration. Using patient derived GBM tumor samples we are able to generate a miniaturized tumor tissue analog to examine how the heterogeneities within the tumor microenvironment impact glioma malignant phenotype, growth, and therapeutic efficacy. The ability to manipulate tumor extracellular matrix (ECM) can improve therapeutic outcomes and restrict glioma infiltration. Our in vitro tumor model is able to analyze the relationship between HA and the invasive phenotype of GBM tumor cells. We show that cell growth, motility and proteomic responses of GBM cells within our platform were significantly altered by HA molecular weight in response to a tyrosine kinase inhibitor. These results provide additional insights regarding the importance of extracellular microenvironment in the invasive potential of GBM tumors. We have demonstrated that repeated dosing of erlotinib promotes expression of PDGFRb in EGFR vIII mutant cells and that extracellular HA plays a favorable role in the inhibition of EGFR, through STAT3 deactivation (3). (1) Pedron S et al. (2015), Adv. Mater., 27: 1567; (2) Pedron S. et al. (2017), Adv. Healthcare Mater., 6: 1700529; (3) Pedron S, et al. (2019), Biomaterials, 219: 119371. Citation Format: Sara Pedron, Jann N. Sarkaria, Brendan A.C. Harley. Engineered glioblastoma tumor models reveal extracellular matrix signals influence the efficacy of targeted inhibitors [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO035.

Abstract PO039: Novel in vivo imaging method to evaluate "Don't eat me" signal of tumor against microglia

Abstract Tumor formation is promoted by increased expression of “Don’t eat me” signals such as CD47 in cancer cells that inhibit phagocytic action of macrophages. However, the signals can regulate generation of brain metastasis is still unclear. In the brain, microglia and perivascular macrophages are the only resident immune cells of the brain parenchyma. Here, we have developed an in-vivo imaging method that can allow us to evaluate microglial phagocytosis of tumor and to detemine the fates of cancer cells in the brain. The skull of the CX3CR1-EGFP mice, in which microglia is specifically labeled with EGFP, was replaced with a glass coverslip, and the fluorescently labeled lung cancer cells (CMT167-mCherry) were injected via the internal carotid artery of the mice. The fate of arrested cancer cells in the brain vascular was monitored for 12-16 days. Of the 324 cancer cells (4 mice), 49.8% (N=138) of cells displaced from their original site, 40.4% (N=112) of cells developed micro-metastasis, and 6.9% (N=27) of cells were processed by microglia resulting in cell death. To evaluate the “Don’t eat me” signal against microglia, a CD47 gene knockout cell line was monitored as well, and these cells developed significantly less micro-metastasizes compared with CD47 positive cells. This in vivo imaging of the brain metastasis potentially provide a method for monitoring of tumor fate and validation of crucial target. Citation Format: Takahiro Tsuji, Hiroaki Wake, Mariko Shindo, Daisuke Kato, Hiroaki Ozasa, Toyohiro Hirai. Novel in vivo imaging method to evaluate "Don't eat me" signal of tumor against microglia [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO039.

Abstract PO050: EphA2 signaling segregation and modulation by diverse tumor microenvironment components differentially regulates cancer cell invasion and survival

Abstract The receptor tyrosine kinase EphA2 is overexpressed in many solid cancers and associated with disease aggressiveness and poor clinical outcome. EphA2 signaling mechanisms are complex, context-dependent and still not completely understood. In several cancers, EphA2-ephrinA1 interaction is associated with cell repulsion and growth suppressive signaling. Yet, this receptor can also be activated in a ligand-independent manner by crosstalk with growth factor receptors and adhesion molecules, and its signaling can be further regulated via proteolytic cleavage by the matrix metalloproteinase MT1-MMP (MMP14). Studying how such proteolytic events, duality in signaling modes and repulsive responses are co-regulated and coexist in cancer could reveal potential therapeutic opportunities. To this goal, we have here used human breast, ovarian and prostate cancer cells grown in 2D and 3D extracellular matrices (ECM), as mono- and co-cultures to mimic the confines provided by the tumor microenvironment. Our results revealed segregation of EphA2 signaling within cancer colonies, including EphA2-MMP14 co-internalization at the cancer cells confronting ephrinA1-expressing cells, in contrast to the stabilization of the receptor and induction of non-canonical signaling in the colonies. This separation of the ligand-mediated and non-canonical EphA2 signaling was dependent on MMP14 and affected platinum chemotherapy-induced apoptosis. By a mass spectroscopy-based screen for EphA2 interacting proteins in the different signaling contexts we identified regulators of proteolysis as well as endocytic pathways with differential interactions upon the ligand-dependent and independent activation states of this receptor. Our studies will help to understand the diverse EphA2 signaling modes and their impact on the regulation of cell-cell and cell-ECM communication affecting cell invasion and survival upon anti-cancer drug treatments. Citation Format: Lidia Moyano-Galceran, Pauliina S. Turunen, Nami Sugiyama, Pernilla von Nandelstadh, Erika Gucciardo, Markku Varjosalo, Kaisa Lehti. EphA2 signaling segregation and modulation by diverse tumor microenvironment components differentially regulates cancer cell invasion and survival [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO050.

Abstract IA014: Regulation of disseminated tumor cell dormancy in brain by the perivascular niche

Abstract The brain provides compelling evidence that tumour dormancy is not simply an intriguing biological phenomenon. Given effective therapy, it may emerge as a culprit of lethality. To date, the mechanism by which the brain microenvironment drives disseminated tumor cells (DTCs) into a dormant state remain elusive. Here, I describe our studies leveraging serial intravital imaging to demonstrate that a large fraction of disseminated breast tumor cells within brain persist as single cells, and that every one of these cells is physically associated with microvasculature. Despite occupying a vascular niche, however, the burden for enforcing dormancy does not lie with endothelium. Instead, perivascular astrocytes are the dominant effector. Specifically, we show that astrocytic laminin-211 signals through DTC dystroglycan to suppress proliferation. Transcriptomic data suggest quiescence may ultimately be driven by sequestration of the Hippo effector yes-associated protein. We view these findings as a first step towards therapies that maintain DTC dormancy to prevent brain metastasis. Citation Format: Cyrus M. Ghajar. Regulation of disseminated tumor cell dormancy in brain by the perivascular niche [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr IA014.

Abstract PO015: JAGGED-1 as a novel metastatic extravasation promoter of triple negative breast cancer cells

Abstract Though survival is improving for breast cancer patients, clinical outcomes remain poor for patients with aggressive molecular subtypes. Triple negative breast cancer (TNBC) is considered the most aggressive breast cancer subtype, and despite having a 90% five-year relative survival rate for localized disease, that number drops to as low as 11% for patients with distant TNBC metastasis. The poor clinical outcomes for TNBC patients are largely due to lack of clinically actionable molecular targets and pathways compared to other breast cancer subtypes. Given the lack of effective treatment strategies for advanced TNBC, there is an urgent and unmet need to understand the means through which tumor cells disseminate and metastasize to distant tissues in hopes of identifying new targets for therapeutic intervention. For tumor cells to metastasize, they acquire complex series of pathophysiological phenotypes, starting with invasion into surrounding tissue and intravasation into systemic lymphatic or blood vascular systems. Only a small fraction of tumor cells can survive in circulation, with a smaller fraction able to extravasate from vessels and establish distant metastases. While some cancer cells partially coopt the mechanisms that leukocytes use to extravasate, circulating TNBC cells also use distinct mechanisms to breach an endothelial barrier. However, the mechanism(s) of TNBC extravasation remains largely unknown, underlying the need to study further. High expression of Notch ligand JAGGED-1 (JAG1) is associated with increased metastasis and mortality in patients with breast cancer. While JAG1 enhances tumor angiogenesis and tumor growth, the means through which JAG1 promotes tumor cell dissemination is not clear. Our preliminary data supports a role for JAG1 as a key regulator of tumor cell interactions with the endothelium, controlling tumor cell adhesion, transendothelial migration (TEM), and transcription of critical paracrine-acting extravasation proteins, such as SPARC and ICAM1. Using both JAG-specific ligand traps as well as CRISPR/Cas9 mediated deletion of JAG1, we show that JAG1 promotes TEM in static transwell models, promotes primary tumor growth, and directs a tumor cell transcriptional program that upregulates cell surface interaction pathways distinct from global Notch inhibition. It is our central hypothesis that JAG1 promotes TNBC migration across the vascular endothelium, thereby allowing disseminated cancer cells to exit from circulation and enhancing TNBC metastasis. To define the JAG1-mediated elements of extravasation, we will examine five CRISPR/Cas9 JAG1 knockout lines in a cutting-edge flow system that recapitulates the vascular shear stress microenvironments where extravasation is thought to occur in vivo. We will evaluate multiple steps of extravasation including tumor cell binding, rolling, and TEM under various flow parameters. Moreover, we will study in vivo lung extravasation using a murine tail vein injection model and interrogate transcriptional candidates downstream of JAG1 as identified by our RNAseq data. Citation Format: Benjamin Gordon, Reyhaan A. Chaudhri, Tim Sargis, L.A. Naiche, Jan K. Kitajewski. JAGGED-1 as a novel metastatic extravasation promoter of triple negative breast cancer cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO015.

Abstract PR008: Tumor-cell-intrinsic transcriptional and epigenetic regulation of EGFR underlies the heterogeneity of immune infiltration and response to immunotherapy in pancreatic cancer

Abstract Resistance to immunotherapy is a major problem of current clinical care for cancer patients. While T cell abundance is essential for tumor responsiveness to immunotherapy, factors that dictate T cell infiltration in tumor microenvironments are not fully understood. To understand the tumor-cell-intrinsic factors underlying the heterogeneity of tumor immunity and sensitivity to immunotherapy, we established a new experimental system by generating a library of congenic pancreatic tumor cell clones from a genetic mouse model driven by mutant Kras and p53. These tumor cell clones robustly formed implanted tumors that recapitulated T-cell-inflamed and non-T-cell-inflamed tumor microenvironments, associated with distinct patterns of infiltration by T cells and myeloid cells. We found that the non-T-cell-inflamed phenotype was dominant over the T-cell-inflamed phenotype in the local tumor microenvironment. An integrated transcriptomic and epigenetic analysis revealed that tumor-cell-intrinsic expression of CXCL1, EPHA2, PTGS2, and USP22 as determinants of the non-T-cell-inflamed microenvironment, and ablation of tumor-cell-intrinsic CXCL1, EPHA2, PTGS2, or USP22 promoted T cell infiltration and sensitivity to a combination of chemotherapies, CD40 agonist, and checkpoint blockades. Furthermore, we performed an in vivo CRISPR-based genetic screen to identify tumor cell intrinsic epigenetic regulators of anti-tumor immunity and discovered novel therapeutic opportunities to improve the efficacy of currently developed immunotherapy for pancreatic cancer. Specifically, we identified lysine demethylase 3A (KDM3A) as a potent epigenetic regulator of immunotherapy response in PDA. Mechanistically, KDM3A acts through Krueppel-like factor 5 (KLF5) and SMAD family member 4 (SMAD4) to regulate the expression of the epidermal growth factor receptor (EGFR). Ablation of KDM3A, KLF5, SMAD4, or EGFR in tumor cells altered the immune TME and sensitized tumors to combination immunotherapy, while treatment of established tumors with an EGFR inhibitor erlotinib prompted a dose-dependent increase in intratumoral T cells. This study defines an epigenetic-transcriptional mechanism by which tumor cells modulate their immune microenvironment and highlights the potential of EGFR inhibitors as immunotherapy sensitizers in PDA. Together, these results demonstrated that heterogeneity of tumor immune phenotypes is driven by tumor-cell-intrinsic factors, including epigenetic factors, that can be manipulated to influence the outcome of immunotherapies. Citation Format: Jinyang Li, Salina Yuan, Robert Norgard, Fangxue Yan, Andres Blanco, Ben Stanger. Tumor-cell-intrinsic transcriptional and epigenetic regulation of EGFR underlies the heterogeneity of immune infiltration and response to immunotherapy in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PR008.

Abstract PO019: Classification of tumors by collagen expression reveals genotype-tumor ECM interactions

Abstract Although the relationship between genotype and the tumor environment remains unclear, tumors evolve in specific contexts such that the genotypes and tumor environment shape each other to create an ecosystem. We aim to improve classification of tumors by considering the whole tumor ecosystem by combining genotypes with components of the extracellular matrix (ECM) to develop new prognostic markers and improve targeting of treatments. Collagens are major components of the tumor environment that do far more than just form structures. The function of the full panoply of 43 collagen genes in tumors remains underappreciated. Using the TCGA dataset, we find that mRNA expression of the 43 collagen genes classifies tumors by their cell of origin very similarly as published reports. K-means clustering in each tumor type by collagen mRNA expression revealed classifications strongly associated with overall survival, specific pathways, and immune cell signatures. The collagen defined groups were strongly associated with specific somatic mutations, copy number changes, ploidy and aneuploidy levels. The collagen clusters also revealed specific immunoenvironments, suggesting which tumors were most likely to respond to immunotherapy. To further evaluate these enrichments, we developed a machine learning model to predict which tumors have high or low aneuploidy and specific gain or loss of chromosome arms based on collagen expression, highlighting the connection between collagen expression and specific cancer genomes with areas under the curve above 0.8 for many tumor types including all the GI tumors. Notably, clusters with high total collagen were typically associated with lower aneuploidy levels and tumors with high aneuploidy were grouped in clusters with a mix of minor collagens, and lower collagen type I. These data support a model where tumors with high collagen type I environments have lower aneuploidy and ploidy levels compared to tumors with higher expression of tissue specific minor collagens. The classifier is driven by expression of minor, non-collagen type I, collagens that typically have very specific expression in normal tissue and become dysregulated in many tumors. These findings strongly suggest minor collagens are critical components defining disease progression, the cancer genome and should be included in pre-clinical studies to model the actual human tumor environment and to improve drug development. Preliminary proof-of-concept data for one such minor collagen, COL7A1, will be presented. In sum, these findings demonstrate how classification of tumors by collagens identified strong links between specific cancer genomes and the tumor ECM. Citation Format: Alexander Brodsky, Kevin S. Guo, Amanda Khoo, Vahid Agbortoko, Dongfang Yang, Elizabeth Y. Wu, Ian Y. Wong. Classification of tumors by collagen expression reveals genotype-tumor ECM interactions [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO019.

Abstract PO037: Oxidized phosphatidylcholines induce tumorigenesis and metastasis through autophagy

Abstract Increased levels of oxidized phosphatidylcholines have been observed in many diseases and act as damage-associated molecular patterns, mediating immune responses. However, the roles of oxidized phospholipids in tumor metastasis have not been entirely elucidated. By using western blotting and confocal fluorescence imaging of human breast cancer cells (MCF7) and hepatocellular carcinoma cells (HepG2), we found that 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) induces changes in EMT-marker expression with increased expression of relative transcription factors to promote the EMT process. POVPC treatment significantly increased the migration and invasion capabilities in cancer cell lines, including HepG2 cells, MCF7 cells, murine skin melanoma cells (B16F10), human colon cancer cells (HCT116), and murine mammary carcinoma cells (4T1). Oxidized phospholipids such as POVPC, 1-palmitoyl-2-glutaroyl-sn-glycero-phosphocholine (PGPC), and 1-palmitoyl-2-(5-keto-6-octenedioyl)-sn-glycero-3-phosphocholine (KOdiA-PC) treatment induced autophagic flux, while nonoxidized phospholipids do not have such abilities. To identify whether POVPC regulates cancer metastasis via autophagy pathways, cells were treated with the pharmacological inhibitors of autophagy, 3-MA or CQ, followed by treatment with POVPC. Autophagy inhibitors decreased pro-metastatic ability compared to POVPC alone. Because ATG5 or ATG7 are critical components of autophagy pathway, cells were transfected with siATG5 or siATG7, followed by treatment with POVPC. Knockdown of ATG5 or ATG7 restored the epithelial markers and decreased both migration and invasion capabilities compared to POVPC alone. Using an LC-MS/MS analysis, the levels of oxidized phosphatidylcholines including POVPC and PGPC did not change in 18 of patient-derived intrahepatic cholangiocarcinoma tissues compared to 18 of corresponding adjacent nontumor tissues; however, POVPC and PGPC levels were greatly higher at stage II of intrahepatic cholangiocarcinoma tissues. In 16 of human breast tumor tissues, the levels of PGPC were higher than those in 16 of adjacent nontumor tissues and 4 of benign tumor tissues. In patient-derived intrahepatic cholangiocarcinoma tissues and breast cancer tissues, the protein levels of autophagy markers were significantly higher compared to nontumor tissues throughout all stages. Together, these results show that oxidized phosphatidylcholines accumulated in the tumor microenvironment associate with the regulation of tumorigenesis and metastasis through autophagic flux. Citation Format: Jin Kyung Seok, Eun-Hee Hong, Joo Young Lee. Oxidized phosphatidylcholines induce tumorigenesis and metastasis through autophagy [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO037.

Abstract LT010: Pleiotrophin drives pro-metastatic immune niche within breast tumor microenvironment

Abstract Background: Pleiotrophin (PTN), a heparin binding cytokine important during embryonic development, is expressed at elevated levels in several cancers, including breast cancer. High PTN expression in serum and tumor tissues of breast cancer patients correlates with worse outcome. Previous studies indicate PTN contributes to angiogenesis, hematopoiesis, and inflammation; however, the function of PTN in breast cancer progression and metastasis has not been elucidated. Methods: The effect of anti-PTN therapy (mAb; 3B10) on breast tumor progression and pulmonary metastasis was evaluated in four preclinical models of breast cancer: 4T1, E0771, Met-1, MMTV-PyMT. In addition, the effect of global loss of PTN using Ptn-deficient mice was evaluated in two murine models of breast cancer. Tissues from these experiments were analyzed by RNAseq and histology for changes in angiogenesis, hematopoiesis, immune cell infiltration, and cytokine/chemokine expression. Findings: TCGA data shows high PTN expression correlates with poor survival of Stage IV breast cancer patients. Furthermore, our data indicate that: a) blocking PTN pharmacologically or genetically results in a significant reduction of lung metastasis in multiple preclinical mouse models of breast cancer, b) scRNAseq and dual FISH-IHC (fluorescence in-situ hybridization and immunohistochemistry) demonstrates that endothelial cells and select population of tumor cells express PTN in the tumor microenvironment (TME), c) inhibition of PTN results in a decrease in overall inflammation with a particular decrease in neutrophil populations at primary and secondary tumor sites, d) in a genetically engineered mouse model (GEMM) of breast cancer, MMTV-PyMT, PTN expression is enriched at the metastatic site. Conclusion: Our data suggests that PTN is important in driving a pro-metastatic immune niche within the TME that promotes tumor cell escape from the primary tumor and survival of metastatic cancer cells in secondary sites. Additionally, our studies highlight that inhibition of PTN has potential to reduce metastatic burden in breast cancer and suggest that future studies testing the combination of PTN inhibition with standard chemotherapy or immune therapy are warranted. Citation Format: Debolina Ganguly, Noah Sorrelle, Adrian Dominguez, Jason Toombs, Marcel Schmidt, Fa Valdes Mora, David Gallego Ortega, Anton Wellstein, Rolf A. Brekken. Pleiotrophin drives pro-metastatic immune niche within breast tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT010.