The evolving tumor microenvironment: From cancer initiation to metastatic outgrowth.

For many years, cancer researchers focused on the attributes of tumour cells that lead to life-threatening malignancy. These studies have provided a comprehensive understanding of the role of oncogenes and tumour suppressor genes, as well as their associated signal transduction pathways, in cancer [1]. In recent years, it has become clear that a complete understanding of the many steps and processes that occur during cancer progression must include the response of the tissues in the immediate vicinity of the tumour, as well as the systemic changes that occur in the bone marrow, circulation and sites of metastasis. Tumours are complex tissues that contain extracellular matrix (ECM), activated fibroblasts, immune cells, pericytes, adipocytes, epithelial cells, glial cells and vascular and lymphatic endothelial cells. Collectively, this tissue is referred to as the tumour microenvironment (TME). The tumour stroma has been likened to the granulation tissue that forms during wound healing [2]. This process involves the temporal orchestration of resident and surrounding uninjured cells, the coagulation system and the immune system, as well as the recruitment of various types of cells that produce and remodel the ECM. Whereas wounds usually heal with time, the signals that initiate the formation of the tumour stroma persist, leading to the description of tumours as ‘wounds that do not heal’[2]. It has become clear that the non-cancerous cells that comprise the TME are not innocent bystanders; rather, they are conscripted to promote tumour progression. The TME contains multiple types of immune cells, which are recruited or activated by the chemokines and cytokines that are secreted by tumour cells [3]. In addition, cell death and necrosis drive the recruitment and activation of macrophages in the TME. Tumour-associated macrophages are a rich source of pro-angiogenic factors and they have been shown to promote metastasis [4, 5]. Various types of immune cells also facilitate tumour cell intravasion and promote the formation of premetastatic niches [6]. Cancer-associated fibroblasts (CAFs) also promote primary tumour growth and metastasis [7–9]. Multiple origins of CAFs have been proposed, including adjacent tissue, the bone marrow and endothelial cells [10, 11]. These cells express markers that are associated with an activated phenotype, such as stromal cell-derived factor-1 (SDF-1), fibroblast activation protein and α-smooth muscle actin [7–9]. Vascular and lymphatic endothelial cells are recruited to the TME by the members of the VEGF family that are secreted by tumour and stromal cells [12]. In 1975, Judah Folkman proposed that tumours cannot grow beyond a relatively small size without stimulating a vascular system to supply them with nutrients [13]. Subsequent studies have validated this hypothesis and have identified key stimulators and inhibitors of angiogenesis. The data indicate that the relative concentrations of the stimulators and inhibitors determine endothelial cell phenotype, with the change from a quiescent to angiogenic phenotype being referred to as the ‘angiogenic switch’[14]. This switch corresponds to the transition of a poorly vascularized tumour to one that is well vascularized. Thus, the presence of endogenous inhibitors of angiogenesis in the TME is probably central to tumour dormancy [15]. Of the endogenous inhibitors, thrombospondin-1 (TSP-1) is highly expressed in the TME by stromal fibroblasts and immune cells [16]. The importance of TSP-1 is underscored by the fact that oncogenes suppress TSP-1, whereas tumour suppressor genes stimulate TSP-1 [17, 18]. Tumour cells have also been reported to instruct stromal cells to decrease their expression of TSP-1, thus, further decreasing the local barriers to angiogenesis [19]. This type of cross-talk between the tumour cells and stromal cells may facilitate co-evolution of the various cell types that comprise tumour tissue. The ECM is a key component of the TME that has both positive and negative effects on tumour growth. Many of the endogenous inhibitors of angiogenesis are derived from ECM proteins [20]. However, the components of the ECM, including its associated growth factors and the cellular proteases that modify structure and function of the ECM, also promote tumour growth and metastasis [21–23]. Through its interaction with integrins, proteogly-cans and other receptors, the ECM serves to support cytoskeletal organization, and cell adhesion, migration and invasion [24, 25]. Taken together, the data support the hypothesis that dynamic and reciprocal interactions between the tumours cells and their neighbouring stromal cells within the TME determine the course of tumour progression. These studies also indicate that the constituents of the TME are central to metastasis. In this review series, we will explore the function of the various cellular and protein constituents of the TME. This journey will begin with a discussion of the role of lymphangiogenesis and cancer metastasis. Mumprecht and Detmar summarize data showing that the members of the VEGF family stimulate lymphangiogenesis within tumour tissue and distant lymph nodes. Expansion of the lymphatic vasculature in sentinel lymph nodes creates a premetastatic niche that favours future tumour cell growth. Subsequent reviews will discuss the role of CAFs, vascular endothelial cells, pericytes, immune cells, adipocytes, glial cells and ECM in the TME. They will also explore exciting new therapeutic opportunities that target the constituents and processes that are essential to TME structure and function. In this context, anti-angiogenic therapeutics, such as Avastin, are already demonstrating significant efficacy [26].

The tumor microenvironment (TME) comprises an assortment of immune and non-immune cells. The interactions between the cancer cells and their surrounding TME are known to be a cardinal factor in all stages of cancer progression, from initiation to metastasis. Tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) are considered two of the most abundant TME members associated with poor prognosis in various cancer types. Intercellular communication between the cancer cells and TME cells might occur via direct cell-cell contact or achieved through secreted factors such as cytokines, growth factors and extracellular vesicles (EVs). EVs are released by almost every cell type and by cancer cells in particular. EVs are loaded with unique molecular cargos that might include DNA, proteins, RNA and lipids, commonly reflecting the physiological traits of their donor cells. Once released, EVs are capable of initiating short- and long-distance communication in an autocrine, paracrine and endocrine fashion. The molecular cargos within the EVs are able to impart phenotypic changes at the receiving end thus allowing EV-releasing cancer cells to deliver messages to TME cells and tighten their grasp over the cancerous tissue. In this concise review, we aim to document the bidirectional EV-based communication between cancer cell, TAMs and CAFs, tilting the balance in favor of cancer progression and metastasis.

The Tumor Microenvironment: A Druggable Target for Metastatic Disease?

Background: Pancreatic ductal adenocarcinoma (PDAC) accounts for ~95% of pancreatic cancer cases and is currently the 3rd leading cause of adult cancer deaths in the US. Due to the lack of methods for early detection and limited treatment options, PDAC has a dismal 5-year survival rate at just <9%. The desmoplastic PDAC tumor microenvironment (TME) is complex and heterogeneous. With recent advances in technology, the characterization of tumors to investigate cellular diversity and evolution in cancer is rapidly accelerating. Here we employed single cell RNA sequencing (scRNA-Seq) to profile transcriptomes of individual cells from dissociated pancreatic tumors isolated from either patients or transgenic mice developing spontaneous PDAC (KPC). This approach elucidated the cellular makeup of individual tumor to reveal distinct cell types like epithelial, endothelial and immune cells, cancer associated fibroblasts (CAFs) and cells undergoing epithelial-to-mesenchymal transition (EMT). Deregulated pathways specific to distinctly identified cell populations were also mapped. Methods: Freshly harvested human or mouse PDAC tumors were mechanically and enzymatically dissociated to single cells using a Miltenyi gentleMACS Tissue Dissociator. The 10X Genomics Chromium Single Cell 3′ Solution was employed for capture, amplification and labeling of mRNA from single cells and for library preparation. scRNA-Seq was performed on Illumina HiSeq 2500. Custom R packages were used for clustering and bioinformatics data analyses. Results: Viability of single cell suspensions used for library preps from all samples was >81%. By unsupervised clustering of the scRNA-Seq matrix and using known signature genes for various cell types we revealed the cellular heterogeneity in a human PDAC to identify distinct cell types. These included epithelial tumor cells (EPCAM+, KRT19+); CD133+ tumor stem cells; CAFs (COL5A1+, COL6A2+, SPARC+); endothelial cells CDH5+ VWF+; and CD45 (PTPRC)+ immune cells also positive for CD3D, CD2, FCGR2A; and cells undergone EMT (CDH2+, ITGA5+, SNAI2+). Using a similar strategy, mouse PDAC diversity was represented by epithelial and EMT tumor cells, CAFs, endothelial cells, macrophages and MDSCs. KRAS and glycolysis signaling pathways in epithelial tumor cells, while hypoxia in the EMT cluster were up regulated. KPC tumors differed in the extent of immune cell infiltration, with a low influx in the smaller versus large tumor, both perhaps representing distinct stages of tumor progression. Conclusions: Our data supports high throughput scRNA-Seq of solid tumors like PDAC as a powerful method to probe and elucidate the underlying tumor heterogeneity. Understanding diversity and complexity of the PDAC TME in individual tumors may help identify unique therapeutic targets and potentially inform treatment/maintenance strategies for patients with advanced disease. Citation Format: Pawan Noel, Wei Lin, Erkut Borazanci, Albert Amini, Ruben Muñoz, Emily Rodela, Serina Ng, Daniel Von Hoff, Haiyong Han. Single cell RNA sequencing of pancreatic ductal adenocarcinoma reveals tumor heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5353.

Background. Immune checkpoint inhibitors (ICIs), particularly targeting the PD-1 pathway, are promising in treating hepatocellular carcinoma (HCC). However, their variable effectiveness among individuals calls for a better understanding of the tumor microenvironment (TME) and reliable predictive biomarkers. Here, we employ spatial transcriptomics (ST) to develop a deep-learning model that aims to investigate the TME in HCC using Hematoxylin and eosin (H&E) staining images. Our focus is on identifying cell types within the TME that are linked to the response to ICI in a cohort of patients treated with nivolumab. Methods. Our study leveraged 21 Visium ST datasets from HCC to develop a model aimed at inferring the cellular composition of TME from H&E images that matched with ST data. The comprehensive TME cell types enrichment score, encompassing Tumor Endothelial Cells (TECs), Tumor-Associated Macrophages (TAMs), B cells, T cells, and Cancer-Associated Fibroblasts (CAFs) in each spot, was calculated by the CellDART algorithm, a method that maps cell type by integrating with a reference scRNA-seq data. The model was trained in a patch-wise manner, utilizing a pre-trained ResNetRS50 as the backbone model. For the internal validation, Spearman correlation coefficients were evaluated for predicted cell types using H&E, and the trained model was correlated with cell type enrichment scores estimated by ST datasets of 4 samples independent of the training set. The model was applied to H&E images of 16 patients with HCC who underwent nivolumab treatment. The model predicted the 5 cell types enrichment score in the tumor and tumor-adjacent normal liver tissues. Results. The internal validation set results demonstrate robust Spearman correlations between our model predictions and actual cellular compositions in the TME. Specifically, we observe strong correlations for T cells (rho = 0.66), TAMs (rho=0.46), CAFs (rho=0.32), B cells (rho=0.30), and TECs (rho=0.24). When we applied the model to the H&E images of the cohort of patients who underwent nivolumab, immunotherapy non-responders showed a significantly higher predicted enrichment score of CAFs in tumor-adjacent regions (Mann-Whitney test, p<.01). Upon stratifying patients based on stromal CAF prediction, those with low stromal CAF levels exhibited better overall survival (Log-rank test, p<.05). Conclusions. We present a deep learning model to analyze TME in HCC solely on H&E images trained by ST data. Our findings showed a potential relationship between CAFs in tumor-adjacent regions and non-responders to nivolumab treatment in HCC. These insights underscore the potential to predict nivolumab treatment responders using H&E images combined with the deep learning model. This approach could provide a significant advancement in personalized treatment strategies for HCC patients. Citation Format: Dongjoo Lee, Haenara Shin, Seungho Cook, Daeseung Lee, Hongyoon Choi, Won-Mook Choi, Changhoon Yoo, Kwon Joong Na. Unveiling the tumor microenvironment of hepatocellular carcinoma using AI trained by spatial transcriptomics: A preliminary study to predict response to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7398.

Simple SummaryThe tumor microenvironment (TME) is composed of various cell types and extracellular components and plays an important role in cancer development and progression, as well as in therapeutic resistance and metastasis. Cancer-associated fibroblasts (CAFs) are a key cell type in the TME and interact with cancer cells as well as with other TME cells, such as immune cells. Immunocompetent mouse cancer models that recapitulate the interactions of cancer cells with the TME have provided a better understanding of the TME network and support the development of new therapeutic strategies. In this review, we focus on cancer cell–TME interactions in heterogeneous tumor tissue, and we provide an overview of therapeutic strategies that target the TME.The tumor microenvironment (TME) plays a key role in cancer development and progression, as well as contributes to the therapeutic resistance and metastasis of cancer cells. The TME is heterogeneous and consists of multiple cell types, including cancer-associated fibroblasts (CAFs), endothelial cells, and immune cells, as well as various extracellular components. Recent studies have revealed cross talk between cancer cells and CAFs as well as between CAFs and other TME cells, including immune cells. Signaling by transforming growth factor-β, derived from CAFs, has recently been shown to induce remodeling of tumor tissue, including the promotion of angiogenesis and immune cell recruitment. Immunocompetent mouse cancer models that recapitulate interactions of cancer cells with the TME have provided insight into the TME network and support the development of new anticancer therapeutic strategies. Recent studies based on such models have revealed that the antitumor action of molecularly targeted agents is mediated in part by effects on the tumor immune environment. In this review, we focus on cancer cell–TME interactions in heterogeneous tumor tissue, and we provide an overview of the basis for anticancer therapeutic strategies that target the TME, including immunotherapy.

Background: Metastatic disease is the foremost cause of breast cancer (BC) related mortality in women. One of the crucial challenges in reducing metastasis-related mortality is in identifying and understanding why certain BCs metastasize and recur. Although cancer cell-intrinsic factors are a key determinant in BC metastasis, host-intrinsic factors like the cells of the tumor microenvironment (TME), such as cancer-associated fibroblasts (CAFs) are what might be driving certain BCs to metastasize and recur. We recently showed that CAFs from the primary (TME) enter into circulation as cCAFs, form heterotypic clusters with tumor cells, and arrive at metastatic sites. This interaction between CAFs and tumor cells is crucial in furthering the metastatic cascade. However, the functional heterogeneity of the different CAF phenotypes and how that impacts BC metastasis is unknown. Stromal derived factor-1 (SDF-1/CXCL12) is an important chemokine that is known to be involved in promoting tumor cell invasion and metastasis. In this study, we examine the functional differences between CAF isolated from different molecular subtypes of BC. Furthermore, we elucidate the role of CAF secreted SDF-1 as being one of the mechanisms by which a subset of CAF cells permanently reprograms poorly-metastatic MCF-7 cells to augment EMT-driven genes and become metastatic in vivo. Methods: We used primary CAF cell lines derived from BCs of different molecular subtypes and examined their global gene expression profiles using RNAseq. We used NSG mice xenografted with MCF-7 BC cells and primary CAF cells to evaluate the contribution of two different CAF phenotypes in promoting BC progression and metastasis. We developed dissociated tumor cell lines from CAF co-injected MCF-7 xenografts to determine the molecular changes that CAFs impart to MCF-7 cells in vivo. Using limiting dilution tumor xenograft assays and gene expression profiles we examined the molecular pathways, BC stemness markers, and tumor-initiating capacity of the dissociated tumor cell lines. By long-term SDF-1 treatment of MCF-7 cells in vitro and using these cells in various RNASeq, in vitro, and in vivo assays we determined the role of SDF-1 in altering the phenotype of MCF-7 cells. Results: We found that CAFs from different molecular subtypes have a differential effect on tumor initiation, progression, and metastasis in xenograft assays. We also found that a subset of CAFs has the ability to make the poorly-metastatic MCF-7 cells metastatic in vivo. We also found that this subset of CAF cells has a higher expression of SDF-1 and the tumors formed with the co-injection of MCF-7 cells with this subset of CAF cells are enriched in cancer stem cell-like metastasis initiating cells. We also found that upon serial transplant, the tumors formed by CAF-reprogramed MCF-7 cells have a gene expression profile that shows enrichment for EMT-driven genes and these tumors are robustly metastatic. Furthermore, we found that the SDF-1/CXCR4 axis is one of the critical mechanisms by which this subset of CAF cells permanently reprograms the phenotype of the MCF-7 cells. Conclusions: CAFs are a highly heterogeneous population of cells in the breast TME. This study examines the contribution of the functional heterogeneity of the CAF cells in BC metastasis. We have found that a unique subset of CAF cells has the ability to reprogram a poorly metastatic BC cell to not only become metastatic but induce a permanent EMT-driven phenotypic shift in the BC cells. SDF-1 may be one of the mechanisms that drive this CAF-induced change and understanding the CAF functional landscape and its role in BC metastasis might be crucial to developing new therapeutic modalities to abrogate BC metastasis. Citation Format: Utsav Sharma, Jun Sun, Kelsie Medina-Saenz, Susan Bare, Philip Miller, Manuel Picon-Ruiz, Joyce Slingerland, Dorraya El-Ashry, Marc Lippman. Triple-negative breast cancer CAFs induce a metastatic phenotype in MCF-7 cells via the SDF-1/CXCR4 axis [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-08.

Cancer-associated fibroblast-derived exosomes in cancer progression: A focus on hepatocellular carcinoma.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) remains challenging to treat in part due to the dense desmoplastic stroma characteristic of the tumor microenvironment (TME). Often, less than 50% of the tumor mass is composed of malignant ductal epithelial cells. The relationship between the TME and the ductal tumor is important in tumorigenesis, metastasis, and treatment resistance. Cancer associated fibroblasts (CAFs) are an abundant cell type in the TME and have a dynamic role in both promoting and inhibiting tumor progression. Efforts to target fibroblast function therapeutically have yielded mixed results. While pre-clinical data have generated some optimism that CAF inhibition may improve clinical outcomes, this has not translated to the clinical setting. One likely reason for these inconsistent findings is the evolving understanding of CAF heterogeneity. Expanding our understanding of the interactions between the epithelial tumor and CAFs may identify new modalities for modulating the TME to augment treatment efficacy and improve patient outcomes. This requires the generation of human model systems to better interrogate intercellular interactions. Methods: We have developed an ex vivo model system to investigate interactions between ductal epithelial tumor cells and CAFs using matched patient-derived organoids (PDOs) and CAFs extracted following dissociation of surgical specimens. Flow cytometry was used to evaluate the viability of both cellular components as well as associated proliferation and cell identity. Moreover, we have used immunohistochemistry to further characterize this system while preserving the spatial relationship of the cell populations. Most recently, we employed the MULTI-seq multiplex single cell RNA sequencing (scRNA-seq) pipeline to explore the transcriptional dynamics of this system at intervals over 96 hours by examining 4 conditions with 1,500 cells sequenced per condition per time point. Results: Flow cytometry demonstrated that both PDOs and CAFs remain viable in this co-culture construct and that both cell types can be reliably identified using epithelial markers to identify organoid cells and an exclusion panel to identify the CAFs. We have further validated these findings using immunohistochemistry and demonstrated that in co-culture both PDOs and CAFs remain viable and continue to proliferate. We have also demonstrated successful extraction of both cell types from the Matrigel scaffold in which they are grown for use in downstream high dimensional genomics assays. Conclusions: We have previously demonstrated the utility of a PDO model to explore clinically relevant biological mechanisms in PDAC. However, the addition of CAFs to this model provides a more comprehensive representation of the in vivo tumor. This ex vivo system is viable and flexible for multiple end point assays and can be used to explore relevant cellular interactions and mechanisms. Our ongoing investigation will specifically focus on PDO-CAF interactions and transcriptional dynamics over time as we interrogate the scRNA-seq data. Citation Format: Jacquelyn W. Zimmerman, Genevieve Stein-O'Brien, Richard A. Burkhart, Elana J. Fertig, Elizabeth M. Jaffee. Patient-derived organoids and cancer associated fibroblasts as a co-culture model to explore cell type interactions in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-080.

Frequent relapse and development of chemoresistance are major causes of poor survival in ovarian cancer. Cancer stem cells (CSCs) consist of a small subpopulation in the tumor that are capable of initiation and maintenance. CSCs are typically resistant to cytotoxic chemotherapy and are a potential cause of relapse and chemoresistance. Most studies on CSCs focus on cancer cells alone while CSCs exist in a complex microenvironment in tumors. This microenvironment or CSC niche provides optimal conditions to maintain their tumor initiating potential. Hence, understanding the crosstalk between CSCs and the tumor microenvironment can potentially provide effective therapeutic targets to prevent chemoresistance and relapse. Cancer associated fibroblasts (CAFs) are a major constituent of the ovarian cancer tumor microenvironment and are highly enriched in the residual tumors following chemotherapy. Therefore, we studied the role of CAFs in promoting ovarian cancer chemoresistance and disease relapse through providing an optimal microenvironment for CSCs. CAFs isolated from ovarian cancer patient tumors were used in heterotypic 3D or 2D coculture systems with high grade serous ovarian cancer cell lines or with patient-derived ovarian cancer cells to study their effect on CSCs and chemoresistance. Matched pre- and post-chemotherapy patient tumors were used to confirm our findings. CAFs significantly increased resistance to carboplatin and enriched CSCs by increasing their symmetric division as well as dedifferentiation of bulk ovarian cancer cells. Pre-coculture with CAFs increased in vivo tumor initiation capacity of the ovarian cancer cells 10-fold. The CSC-CAF crosstalk responsible for CSC induction was found to be mediated by Wnt signaling. Inhibition of Wnt secretion by CAFs could block their effect on CSCs. Wnt5a was the most highly expressed Wnt in CAFs, which was further induced by ovarian cancer cells. CRISPR knockdown of Wnt5a in CAFs or treatment with a specific Wnt5a inhibitor abrogated the induction of CSCs by CAFs. Wnt5a was found to signal through a non-canonical Wnt pathway in the CSCs involving the coreceptor ROR2, protein kinase C (PKC), and CAMP Responsive Element Binding Protein 1 (CREB1). Inhibition of each of them prevented CSC induction and functional rescue experiments confirmed the sequence of the Wnt5a-ROR2-PKC-CREB1 axis. Treatment of mouse xenografts established by co-injection of CAFs and ovarian cancer cells, with the Wnt5a inhibitor sensitized them to carboplatin, and eliminated the CSCs in the residual tumors. Our results indicate that CAF-derived Wnt5a is instrumental in ovarian cancer CSC growth and maintenance. In the long term, our studies will broaden the understanding of the mechanism of CSC maintenance by the tumor microenvironment and contribute towards the development of novel therapeutic approaches to prevent ovarian cancer chemoresistance and relapse. Citation Format: Yiming Fang, Xue Xiao, Ji Wang, Subramanyam Dasari, Kenneth P. Nephew, Dmitriy Zamarin, Anirban K. Mitra. Cancer associated fibroblasts in the tumor microenvironment maintain ovarian cancer stem cells through non-canonical Wnt5a signaling [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 LT020.

Decision letter: Neutrophil-mediated fibroblast-tumor cell il-6/stat-3 signaling underlies the association between neutrophil-to-lymphocyte ratio dynamics and chemotherapy response in localized pancreatic cancer: A hybrid clinical-preclinical study

Calcitriol (1,25-dihydroxycholecalciferol), the most active form of vitamin D, has selective anti-proliferative effects on tumor-derived endothelial cells (TDEC) compared with Matrigel-derived endothelial cells (MDEC). Although both cell types have an intact vitamin D receptor-signaling axis, this study demonstrates that upon treatment with calcitriol, 24-hydroxylase (CYP24) mRNA, protein and enzymatic activity were markedly induced in MDEC in a time-dependent manner but not in TDEC. Furthermore, treatment of MDEC with a CYP24 small interfering RNA restored sensitivity to calcitriol. To investigate the lack of CYP24 induction in TDEC, we examined methylation patterns in the promoter regions of the CYP24 gene in these two cell types. We identified two putative CpG island regions located at the 5' end. Using methylation-specific PCR and bisulfite sequencing, we determined that these CpG islands were hypermethylated in TDEC but not in MDEC. These data may explain the recruitment of vitamin D receptor to the promoter region in MDEC but not TDEC, as revealed by chromatin immunoprecipitation analyses. Treatment of TDEC with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine restored calcitriol-mediated induction of CYP24, which led to loss of sensitivity to calcitriol growth inhibitory effects. CYP24 promoter hypermethylation was also observed in endothelial cells isolated from other tumors but not in endothelial cells isolated from normal mouse tissues. These observations indicate that the methylation status of the CYP24 promoter differs in endothelial cells isolated from different microenvironments (tumor versus normal) and that methylation silencing of CYP24 contributes to selective calcitriol-mediated growth inhibition in endothelial cells.

Background: Cancer associated fibroblasts (CAFs) are the most abundant non-cancerous cell component of the breast cancer (BC) TME and have been shown to participate in most all steps of metastasis. We have previously shown that CTCs cluster with circulating CAFs (cCAFs) in BC patients at all disease stages, and this observation is recapitulated in the mouse PYMT BC model. Our central hypothesis is that cCAF/CTC co-clusters comprise the functional metastatic unit, and here we sought to determine the role of cCAFs in co-clusters on CTC extravasation and metastatic seeding. Methods: Xenografts: In vitro co-clusters were produced by culturing human CAF23 cells and MDA231(Ffluc) cells in ULA plates for 18 hrs. Clusters were injected via tail vein into NSG mice and metastatic growth was monitored by BLI. Endothelial binding assays: In vitro clusters, single CAFs or single BC cells were labeled with Cell Tracker dyes, pipetted onto confluent HUVEC monolayers, and allowed to bind for 30 minutes before gentle washing. Bound cells were counted on a fluorescent microscope. Ex vivo extravasation assays: Cell Tracker labeled co-clusters were injected via tail vein into FVB mice. One hour before sacrifice, FITC-lectin was injected via tail vein to label lung endothelia. Lungs were then extracted and imaged by confocal microscopy. Results/Discussion: Xenograft experiments demonstrate that within the first week, CAF23/MDA231 co-clusters produce durable, proliferative lung metastases faster than MDA231 monoclusters. This early difference in metastatic growth suggests that CAFs help BC cells establish metastases faster, possibly by aiding extravasation into the lung parenchyma. Using our ex vivo model of lung extravasation, we saw that significantly more CAF23/MDA231 co-clusters than MDA231 monoclusters were in the process of and/or had extravasated into the lung parenchyma at 8 and 24 hrs post-injection. In vitro binding experiments revealed that HUVEC endothelial cells bind more CAF23/BC co-clusters than BC monoclusters. Similarly, HUVEC cells bound significantly more single CAF23 or CAF19 cells than BC cells. CAF binding increased when endothelial e-selectin expression was induced with IL1-b; binding to e-selectin being the first step in cancer cell rolling and extravasation. Immunofluorescence and flow cytometry showed that CAFs express the CA19-9 antigen, sialyl-Lewis A (sLeA), which acts as an e-selectin ligand. Tannic acid, an inhibitor of the chemokine SDF1/CXCL12, decreased binding, as did 5mM EDTA. CD44 siRNA had no effect on binding whereas depletion of hyaluronin, a ligand of CD44, had a significant effect on CAFs but not on BC cells. Together, these experiments point to CAFs in co-clusters as critical enhancers of endothelial binding and extravasation, which may ultimately result in faster establishment of metastases and enhanced survival of CTCs. Future investigations will focus on therapeutic targeting of the mechanisms we have identified to block cCAF/CTC extravasation and metastasis. Citation Format: Angela Spartz, Benjamin Troness, Utsav Sharma, Geneva Taylor, Dorraya El Ashry. Circulating cancer associated fibroblasts (cCAFs) enhance CTC extravasation and establishment of metastases [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 PO038.

The 2-year survival rate for patients with central nervous system (CNS) metastases (CMs) remains below 2%. With the aim to define new therapeutic options, researchers have investigated how interactions between CM cells and the surrounding tumor microenvironment (TME) support metastatic progression. As an important component of the TME, cancer associated fibroblasts (CAFs) have largely been reported as tumor supportive. But new evidence for the high heterogeneity of the CAF compartment, including in vivo studies from our lab, showed that certain CAF subpopulations could exhibit tumor inhibitory capabilities. Interestingly, recent reports revealed that CAFs showing high expression of Immunoglobulin Superfamily containing Leucin Rich Repeat (ISLR) could show tumor inhibitory capabilities in both pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC). Yet, despite the invaluable clinical potential of tumor inhibitory CAFs, the CAF compartment in the TME of CMs is still largely under described. Using CM patient tissue and CM patient-derived cell lines established in our lab, we performed single cell RNA sequencing (scRNA-seq) to define CAF subpopulations present in the TME of CMs. We identified distinct clusters characterized by differential expression of ISLR and tumor supportive CAF marker Actin Alpha 2, Smooth Muscle (ACTA2/α-SMA). CAF subpopulations were thus defined as follows: ISLRhigh/α-SMAlow, ISLRlow/α-SMAhigh, ISLRlow/α-SMAlow. Altogether, we hypothesized that ISLRhigh/α-SMAlow CAFs could act to restrain tumor progression in the TME of CMs, as opposed to potentially tumor supportive ISLRlow/α-SMAhighCAFs. ISLRhigh and ISLRlow CAFs were obtained through fluorescence-activated cell sorting (FACS) performed on one of our patient-derived CAF cell lines represented in all defined scRNA-seq clusters. ISLRhigh CAFs were validated to express lower levels of α-SMA (ISLRhigh/α-SMAlow CAFs) compared to ISLRlow cells (ISLRlow/α-SMAhigh CAFs). This mutually exclusive expression of ISLR and α-SMA in different CAF subpopulations is in accordance with recent data in PDAC and CRC and could indicate common underlying mechanisms driving the phenotype of each subpopulation. Further in vitro and in vivo experiments are currently underway in our lab to determine the tumor supportive capabilities of both ISLRhigh/α-SMAlow and ISLRlow/α-SMAhigh CAFs. Ultimately, we expect the current study to unravel the diverse and dynamic nature of CAFs in the TME of CMs, which is still largely unknown. Citation Format: Thomas Simon, David Buckley, Bodour Salhia. Tumor inhibitory CAFs in CNS metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3195.

ABSTRACTBackgroundMetastatic colorectal cancer (mCRC) is the main cause of CRC mortality, with limited treatment options. Although immunotherapy has benefited some cancer patients, mCRC typically lacks the molecular features that respond to this treatment. However, recent studies indicate that the immune microenvironment of mCRC may be modified to enhance the effect of immune checkpoint inhibitors. This study aimed to explore the metastatic tumor microenvironment (TME) by comparing cell populations in colorectal liver (CLM), lung (mLu), and peritoneal (PM) metastases.MethodsRNA isolated from 20 CLM, 15 mLu, and 35 PM samples was subjected to mRNA sequencing and explored through TME deconvolution tools, consensus molecular subtyping (CMS), and differential gene expression and gene set enrichment analysis, with respect to the metastatic sites. Clinical data and KRAS/BRAF hotspot mutation status were also obtained for all the cases.ResultsThe cell type fractions in the TME were relatively similar between the metastatic sites, except for cancer‐associated fibroblasts (CAFs), B cells, endothelial cells, and CD4+ T cells. Notably, PM showed enrichment for CAFs and endothelial cells, consistent with distinct pathways associated with metastatic growth and progression in the peritoneal cavity. PM with the mesenchymal subtype, CMS4, had increased CAFs, endothelial cells, and macrophages, along with up‐regulated genes related to TNF‐α signaling via NF‐κB, EMT, and angiogenesis.ConclusionsTumor samples from different metastatic sites exhibited a broadly similar TME in terms of immune cell composition, with some intriguing differences. Targeting CAF‐associated pathways, macrophages, and TNF‐α signaling through NR4A could represent potential novel therapeutic approaches in CMS4 PM.