Stromal Cancer-associated Fibroblasts Research Articles

Abstract 1580: Investigating the tumor microenvironment of pancreatic ductal adenocarcinoma with BRCA2 mutation

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and deadliest cancers with a survival rate of less than 12% at 5 years. It is estimated that around 14% of human PDAC contain mutations in genes involved in DNA Damage Repair (DDR), including BRCA1/2, ATM, and others. Additionally, PDAC has an extensive network of extracellular matrix components surrounding the tumor in the stroma, such as cancer-associated fibroblasts, which promote cancer cell growth. The interactions of the tumor with its tumor microenvironment and stroma can bring vulnerabilities that could be targeted if identified. New therapeutic strategies to those targets can be developed for specifically PDAC with DDR mutations. Using publicly available RNA-seq dataset of PDAC patients from The Cancer Genome Atlas (TCGA), I computationally estimated the infiltration of immune and stromal cell types into the tumor. From the analyses, I found that there was a significant difference in the migration of Common Lymphoid Progenitor (CLP) cells into PDAC with mutations to DDR genes. CLP cells usually divide into T-cells, B-cells, and NK cells, suggesting that DDR mutations could cause a more immunogenic environment. There was also a decrease in the infiltration of stromal elements, mainly fibroblasts, into PDAC with mutations to DDR genes. In the experiment I am currently working on, I hypothesize there are also in vivo differences in the infiltration rates of certain immune cell types and fibroblasts into BRCA2-mutated PDAC, since BRCA2 is the most commonly mutated DDR gene in PDAC. For my current experimental design, the mice in the experimental group will be orthotopically implanted with PDAC with Brca2 knockout. The control group will have mice orthotopically implanted with PDAC without Brca2 knockout. After letting the tumors grow for 3 weeks, the mice will be sacrificed and the pancreatic tumors will be collected and stained through trichrome staining. Trichrome staining probes for the stromal cancer-associated fibroblasts. Furthermore, immunohistochemistry (IHC) staining will be done to look for specific populations of cells, such as specific types of fibroblasts, effective and immunosuppressive immune cells including T-cells, B-cells, and NK cells. The aim is to see whether the computational findings in regards to the tumor microenvironment are also reflected in vivo. I will have the data from this experiment by the time of the conference in April 2024. Overall, my project is about investigating the tumor microenvironment and fibroblast abundance in PDAC with BRCA2 knockout mice, using computational data, IHC staining and trichrome staining. Citation Format: Fatimah Al-Musawi, Chang-il Hwang. Investigating the tumor microenvironment of pancreatic ductal adenocarcinoma with BRCA2 mutation [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 1580.

Abstract B062: CRO67 has therapeutic potential against pancreatic tumor cells and cancer associated fibroblasts

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to therapy. Stromal cancer-associated fibroblasts (CAFs) play a key role in promoting tumor progression and chemoresistance, by creating a fibrotic microenvironment that impedes drug access and feeds PDAC cells nutrients and pro-tumor signals. CRO-67 (Patent PCT/AU2023/050505) is a novel drug developed with Noxopharm Ltd using a rational medicinal chemistry design to improve bioavailability of chromans which have potent anti-cancer activity. Using our patient-derived PDAC tumor in a dish model (explants; maintain multicellular architecture and fibrosis) we previously showed that CRO-67 has both anti-tumor and CAF reprogramming capacity in 4 patient explants [Cancer Res (2022) 82 (22_Supplement):C073]. Aims: 1) To expand evaluation of CRO-67 in additional patient-derived PDAC tumor explants given PDAC heterogeneity. 2) Assess the effect of CRO-67 on PDAC cell and CAF function. 3) Validate the therapeutic potential of CRO-67 on PDAC growth in vivo. Methods: 1) PDAC tumor samples collected from 7 patients undergoing pancreatic resection. Tumor explants (1–2mm diameter) were cultured on gelatin sponges, treated with CRO-67 (0–50μg/mL) every 3 days and fixed on day 12. Therapeutic response assessed by immunohistochemistry for cytokeratin (PDAC cells), α-smooth muscle actin (CAFs), bromodeoxyuridine (proliferation) and TUNEL (cell death). 2) PDAC cells (MiaPaCa-2) and patient-derived CAFs were treated with CRO-67 (1.5μM) for 48h before proliferation analysis (IncuCYTE S3) and for 24h before apoptosis (Annexin V/DAPI staining; flow cytometry) and cell cycle (DAPI staining; flow cytometry) analysis. 3) Subcutaneous PDAC (BxPC3 human cells) tumors in mice were treated with CRO-67 (2.5mg/kg Intraperitoneally, twice a day) for 21 days and tumor volume measured (calipers). Results: 1) CRO-67 treatment decreased tumor and CAF cell frequency in 6/6 (no quantifiable tumor in patient 7 explants) and 7/7 patient PDAC explants, respectively. It also decreased explant cell proliferation and increased cell death versus controls. 2) CRO-67 completely abolished proliferation of MiaPaCa-2 and reduced the average growth rate of CAFs by 81.3±8.8% (p=0.0025; n=5), increased apoptosis in MiaPaCa-2 by 455.7±21.4% (p=0.003; n=3) and in CAFs by 172.6±13.1% (p=0.0067; n=5) and increased the fraction of cells in G2/M cell cycle phase by 163.8±12.8% (p<0.0001; n=3) in MiaPaCa-2 and by 74.6±8.6% (p=0.0015; n=5) in CAFs versus controls. 3) CRO-67 reduced PDAC tumors in vivo by 56.7±6.6% (p=0.0013; n=9 mice/group) versus controls. Conclusions: CRO-67: 1) reduced PDAC cells and CAFs in human PDAC explants via reduced proliferation and increased death; 2) reduced proliferation of PDAC cells and CAFs in vitro, increased apoptosis and disrupted cell cycle progression through G2/M phase; 3) reduced PDAC tumor growth in vivo. Implication: We predict CRO-67 is a potential ‘dual cell’ therapy with direct anti-tumor effects and CAF reprogramming capacity, warranting further investigation in vivo. Citation Format: Keilah Garcia Netto, Shannon Chiang, John Kokkinos, Koroush S. Haghighi, Aparna Raina, Omali Pitiyarachchi, Janet Youkhana, Quach Truong, Daniel Wenholz, Xiang Li, Olivier Laczka, Naresh Kumar, John Wilkinson, David Goldstein, George Sharbeen, Phoebe A. Phillips. CRO67 has therapeutic potential against pancreatic tumor cells and cancer associated fibroblasts [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B062.

Histologic, immunohistochemical, and in situ hybridization study of myxoid stroma in feline oral squamous cell carcinoma.

Oral squamous cell carcinoma (oSCC) is a highly invasive malignant neoplasm in cats. Recently, tumor stroma, known as tumor microenvironments, have been considered to play an essential role in tumor progression. However, their role in feline squamous cell carcinoma (SCC) remains unclear. This study aimed to reveal the cancer microenvironment of feline oSCC and evaluate the pathological mechanisms of progression. We used 19 samples from 17 cats with oSCC, which were examined using light microscopy, immunohistochemistry, and in situ hybridization (RNAscope®). Feline oSCCs had two types of stroma, namely fibrotic and myxoid stromal reaction patterns, which were easily distinguished using hematoxylin-eosin staining. The myxoid stroma was rich in hyaluronic acid, which seems to be produced by neoplastic cells. Furthermore, the presence of myxoid stroma was correlated with histological parameters, including the appearance of cancer-associated fibroblasts and tumor budding. Periostin protein expression was also frequently observed in the stroma of feline oSCC and was significantly more common in the myxoid stromal reaction pattern group than in the fibrotic group. Positive signals for periostin mRNA were detected in stromal cancer-associated fibroblasts. This study indicates that the interaction between neoplastic cells and stromal reaction pattern components, such as hyaluronic acid and periostin, may be involved in tumor malignancy. Therefore, we propose that focus be placed not only on the tumor tissue but also on the characterization of the stroma for analyzing feline oSCC.

Analysis of stromal PDGFR-β and α-SMA expression and their clinical relevance in brain metastases of breast cancer patients

BackgroundBreast cancer brain metastasis (BCBM) is a growing therapeutic challenge and clinical concern. Stromal cancer-associated fibroblasts (CAFs) are crucial factors in the modulation of tumorigeneses and metastases. Herein, we investigated the relationship between the expression of stromal CAF markers in metastatic sites, platelet-derived growth factor receptor-beta (PDGFR-β), and alpha-smooth muscle actin (α-SMA) and the clinical and prognostic variables in BCBM patients.MethodsImmunohistochemistry (IHC) of the stromal expression of PDGFR-β and α-SMA was performed on 50 cases of surgically resected BCBM. The expression of the CAF markers was analyzed in the context of clinico-pathological characteristics.ResultsExpression of PDGFR-β and α-SMA was lower in the triple-negative (TN) subtype than in other molecular subtypes (p = 0.073 and p = 0.016, respectively). And their expressions were related to a specific pattern of CAF distribution (PDGFR-β, p = 0.009; α-SMA, p = 0.043) and BM solidity (p = 0.009 and p = 0.002, respectively). High PDGFR-β expression was significantly related to longer recurrence-free survival (RFS) (p = 0.011). TN molecular subtype and PDGFR-β expression were independent prognostic factors of recurrence-free survival (p = 0.029 and p = 0.030, respectively) and TN molecular subtype was an independent prognostic factor of overall survival (p < 0.001).ConclusionsExpression of PDGFR-β in the stroma of BM was associated with RFS in BCBM patients, and the clinical implication was uniquely linked to the low expression of PDGFR-β and α-SMA in the aggressive form of the TN subtype.

Abstract 72: Ablation of WNT10B alters the tumor microenvironment in highly metastatic breast cancer, altering paclitaxel response

Abstract Triple-negative breast cancer (TNBC) has the worst survival outcome and the greatest incidence of metastasis among breast cancer subtypes. We have shown that the WNT10B network, composed of β-CATENIN, HMGA2, and EZH2, is predictive of higher rates of TNBC metastasis and poorer overall survival in TNBC. The stroma-tumor microenvironment (TME) interactions in metastatic breast cancer remain poorly defined, and prior to our work, the role of WNT10B signaling in the TME was unknown. Global gene expression profiling of the Wnt10bKO total mammary glands demonstrated a gene signature consistent with altered metabolism and markers promoting adipocyte differentiation. Wnt10b ablation in the mammary gland increases adipogenic differentiation and decreases the number of mammary gland terminal-end buds, and epithelia to stroma/fibroblast cell ratios. Therefore, we questioned the role of WNT10B in stromal cancer-associated fibroblast (CAFs) and cancer-associated adipocytes (CAAs) in the TME of transformed mammary glands. By backcrossing our Wnt10bKO mice to the highly metastatic MMTV-PyMT (PyMT) transgenic mammary tumor model, we demonstrated alteration in the age of tumor onset, changes in the incidence of transformation of thoracic and abdominal-inguinal tumors, and impacts on survival rates. Moreover, adipogenic markers are higher and persist through transformation in PyMTWnt10bKO mice compared to PyMT mice. Proliferation, migration, and invasion are also impacted in the PyMTWnt10bKO CAFs when co-cultured with PyMT tumor cells. The ability of PyMTWnt10bKO CAFs to alter cellular plasticity and generate CAAs is higher than PyMT CAFs. Additionally, PyMTWnt10bKO CAFs lose expression of canonical Wnt markers DVL and LRP and gain non-canonical Wnt signaling via WNT5B/ROR pathways that persist in the KO CAAs. We also observed alterations in chemoresistance in KO tumor epithelium. The resistance to paclitaxel from enriched PyMT epithelia from thoracic tumors was higher than epithelia enriched from the abdominal-inguinal tumors. Interestingly, the Wnt10bKO TME inverted the response to paclitaxel and the abdominal-inguinal epithelial tumor cells were more resistant than those from the thoracic epithelium. Furthermore, resistance to paclitaxel was associated with higher responses to PRI-724, a CBP-β-CATENIN inhibitor. Neither WT nor KO epithelial subtypes were resistant to olaparib exposure. Therefore, we conclude that Wnt10b is essential to educate highly metastatic breast cancer stromal cells to promote growth, metastatic colonization, and resistance to paclitaxel. Citation Format: Hannah R. Kelso, Rachel S. Perkins, Ikbale El Ayachi, Jackelyn A. Alva-Ornelas, Tiffany N. Seagroves, Victoria L. Seewaldt, Susan A. Krum, Gustavo Adolfo Miranda-Carboni. Ablation of WNT10B alters the tumor microenvironment in highly metastatic breast cancer, altering paclitaxel response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 72.

Interplay Between Extracellular Matrix Remodeling and Angiogenesis in Tumor Ecosystem.

Studying the complex mechanisms of tumorigenesis and examining the interactions of neoplastic cells within tumor ecosystem are critical to explore the possibility of effective cancer treatment modalities. Dynamic tumor ecosystem is constantly evolving and is composed of tumor cells, extracellular matrix (ECM), secreted factors, and stromal cancer-associated fibroblasts (CAF), pericytes, endothelial cells (EC), adipocytes, and immune cells. ECM remodeling by synthesis, contraction, and/or proteolytic degradation of ECM components and release of matrix-sequestered growth factors create a microenvironment that promotes EC proliferation, migration, and angiogenesis. Stromal CAFs release multiple angiogenic cues (angiogenic growth factors, cytokines, and proteolytic enzymes) which interact with ECM proteins, thus contribute to enhance proangiogenic/promigratory properties and support aggressive tumor growth. Targeting angiogenesis brings about vascular changes including reduced adherence junction proteins, basement membrane and pericyte coverage, and increased leakiness. This facilitates ECM remodeling, metastatic colonization and chemoresistance. Owing to significant role of denser and stiffer ECM in inducing chemoresistance, direct or indirect targeting of ECM components is being reported as major axis of anticancer treatment. Exploring the agents targeting angiogenesis and ECM in a context specific manner may lead to reduced tumor burden by promoting conventional therapeutic effectiveness and overcoming the hurdles of therapy resistance.

Secretome of Stromal Cancer-Associated Fibroblasts (CAFs): Relevance in Cancer.

The cancer secretome reflects the assortment of proteins released by cancer cells. Investigating cell secretomes not only provides a deeper knowledge of the healthy and transformed state but also helps in the discovery of novel biomarkers. Secretomes of cancer cells have been studied in the past, however, the secretome contribution of stromal cells needs to be studied. Cancer-associated fibroblasts (CAFs) are one of the predominantly present cell populations in the tumor microenvironment (TME). CAFs play key role in functions associated with matrix deposition and remodeling, reciprocal exchange of nutrients, and molecular interactions and signaling with neighboring cells in the TME. Investigating CAFs secretomes or CAFs-secreted factors would help in identifying novel CAF-specific biomarkers, unique druggable targets, and an improved understanding for personalized cancer diagnosis and prognosis. In this review, we have tried to include all studies available in PubMed with the keywords "CAFs Secretome". We aim to provide a comprehensive summary of the studies investigating role of the CAF secretome on cancer development, progression, and therapeutic outcome. However, challenges associated with this process have also been addressed in the later sections. We have highlighted the functions and clinical relevance of secretome analysis in stromal CAF-rich cancer types. This review specifically discusses the secretome of stromal CAFs in cancers. A deeper understanding of the components of the CAF secretome and their interactions with cancer cells will help in the identification of personalized biomarkers and a more precise treatment plan.

Cancer-Associated Fibroblast Diversity Shapes Tumor Metabolism in Pancreatic Cancer

Despite extensive research, the 5-year survival rate of pancreatic cancer (PDAC) patients remains at only 9%. Patients often show poor treatment response, due partly to a highly complex tumor microenvironment (TME). Cancer-associated fibroblast (CAF) heterogeneity is characteristic of the pancreatic TME, where several CAF subpopulations have been identified, such as myofibroblastic CAFs (myCAFs), inflammatory CAFs (iCAFs), and antigen presenting CAFs (apCAFs). In PDAC, cancer cells continuously adapt their metabolism (metabolic switch) to environmental changes in pH, oxygenation, and nutrient availability. Recent advances show that these environmental alterations are all heavily driven by stromal CAFs. CAFs and cancer cells exchange cytokines and metabolites, engaging in a tight bidirectional crosstalk, which promotes tumor aggressiveness and allows constant adaptation to external stress, such as chemotherapy. In this review, we summarize CAF diversity and CAF-mediated metabolic rewiring, in a PDAC-specific context. First, we recapitulate the most recently identified CAF subtypes, focusing on the cell of origin, activation mechanism, species-dependent markers, and functions. Next, we describe in detail the metabolic crosstalk between CAFs and tumor cells. Additionally, we elucidate how CAF-driven paracrine signaling, desmoplasia, and acidosis orchestrate cancer cell metabolism. Finally, we highlight how the CAF/cancer cell crosstalk could pave the way for new therapeutic strategies.

Abstract C073: CRO-67 is a novel therapeutic for pancreatic cancer: Implications for tumor and stromal reprogramming

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies and new therapeutic strategies are urgently required. A key drug delivery barrier and promoter of tumor progression is the fibrotic and heterogeneous microenvironment of PDAC which is mainly orchestrated by stromal cancer-associated fibroblasts (CAFs). Thus, any novel therapeutic strategy should not only target PDAC cells but should also aim to inhibit the pro-tumor properties of the PDAC stroma. However, to accurately evaluate the clinical potential of novel PDAC therapeutics, preclinical models are required that closely mimic the heterogeneity and multicellular architecture of human disease. Aim: We aimed to investigate the therapeutic potential of novel functionalized benzopyrans (CRO-05 &amp; CRO-67) in a patient-derived PDAC tumor explant model. Methods: We collected PDAC tumor samples from patients undergoing resection of their primary tumor. We cultured whole tumor explants (1-2mm diameter) on gelatin sponges. This model accurately maintains the multicellular tumor and stromal architecture of human PDAC [Kokkinos et al, Scientific Reports, 11,1941 (2021)]. Tumor explants were treated every 3 days with the racemic compound CRO-05 or its active enantiomer CRO-67 and explants were fixed in paraformaldehyde at day-12. Therapeutic response was assessed by immunohistochemistry for markers of tumor cells (cytokeratin), CAFs (α-Smooth Muscle Actin), cell-proliferation (Bromodeoxyuridine), and cell-death (TUNEL). Results: Treatment of human PDAC explants with CRO-05 resulted in reduced tumor cell frequency in 3/4 patients and in 4/4 patients, reduced CAF frequency, decreased cell-proliferation, and increased cell-death relative to controls. CRO-67 treatment in explants from 4/5 patients led to reduced tumor cell frequency, and in 5/5 patients, reduced CAF frequency, decreased cell-proliferation, and increased cell-death relative to controls. Conclusions: This work identifies the functionalized benzopyrans CRO-05 and CRO-67 as novel dual-cell therapeutics that potently inhibit both PDAC tumor cells and their surrounding CAFs and provides support for their clinical development as novel PDAC therapeutics. Citation Format: John Kokkinos, George Sharbeen, Koroush S. Haghighi, Janet Youkhana, Aparna Raina, Omali Pitiyarachchi, Quach Truong, Daniel Wenholz, Olivier Laczka, Naresh Kumar, David Goldstein, Phoebe A. Phillips. CRO-67 is a novel therapeutic for pancreatic cancer: Implications for tumor and stromal reprogramming [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C073.

Abstract A054: Activating transcription factor 3 promotes KRAS-mediated pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) has ~10% survival 5 years after diagnosis often due to lack of chemotherapeutic response. Unlike many other tumors, pancreatic tumors have a high stromal content and cancer associated fibroblasts (CAFs) play a role in response. To improve patient outcome, we need to determine how tumor and non-tumor cell interactions contribute to PDAC progression. Our laboratory recently showed global deletion of Atf3 in mice expressing oncogenic KRAS (Atf3-/-Ptf1acreERT/+KRASG12D; referred to as APK) reduced preneoplastic lesion progression. However, APK mice also showed increased fibrosis and altered immune cell infiltration and single cell RNA-seq revealed ATF3 expression in tumor and non-tumor cells. These findings suggest ATF3 may affect multiple cell types in PDAC. The goal of this study was to determine the epithelial-specific role ATF3 plays in PDAC initiation. We hypothesize that ATF3 promotes PDAC initiation and progression through epithelial and non-epithelial cell mechanisms. Methods: Mice allowing cre recombinase-inducible KRASG12D activation with (Ptf1acreERT/+KRASG12D) or without Atf3 (APK mice) or combined with Atf3 deletion specific to pancreatic acinar cells (AacinarPK mice) were generated. 2-4 month-old mice from all genotypes were gavaged with tamoxifen to induce cre-mediated recombination. Ten and twelve days following cre activation, pancreatic injury was induced by cerulein. Two weeks post injury, general morphology, tissue histology, and gene expression was compared. In some cases, cells were isolated from pancreatic tissue and prepared for 3-dimensional organoid cultures. Results: Histological analysis indicated AacinarPK mice had fewer high-grade PanIN lesions compared to both APK or Ptf1acreERT/+KRASG12D mice with reduced lesion size, based on the PanIN marker CK19, and pERK accumulation, a downstream mediator of KRAS. In addition, Ptf1acreERT/+KRASG12D, APK, and AacinarPK show differences in stromal makeup. While analysis of the stromal compartment shows no change in overall CAF accumulation, IHC for a-SMA showed reduced accumulation of myCAFs in APK tissue. There is also a reduction in immune cell infiltration when ATF3 is deleted, based on CD45 staining. Analysis of organoid cultures showed loss of ATF3 reduced compact morphology of organoid structures and reduced mesenchymal cell transformation. Conclusion and Future Directions: This analysis supports an epithelial-specific role for ATF3 in KRASG12 -mediated PDAC. Since APK and AacinarPK mice do not show the same phenotype, our data also suggests ATF3 has additional roles in nonepithelial cells. Therefore, cell-specific targeting needs to be considered in treating PDAC. Future experiments will examine ATF3-regulated pathways in the various cell populations found in PDAC. Citation Format: Mickenzie B. Martin, Ye Shen, Christopher Pin. Activating transcription factor 3 promotes KRAS-mediated pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A054.

CD9 mediates the uptake of extracellular vesicles from cancer-associated fibroblasts that promote pancreatic cancer cell aggressiveness.

In pancreatic ductal adenocarcinoma (PDAC), signaling from stromal cells is implicated in metastatic progression. Tumor-stroma cross-talk is often mediated through extracellular vesicles (EVs). We previously reported that EVs derived from cancer-associated stromal fibroblasts (CAFs) that are abundant in annexin A6 (ANXA6+ EVs) support tumor cell aggressiveness in PDAC. Here, we found that the cell surface glycoprotein and tetraspanin CD9 is a key component of CAF-derived ANXA6+ EVs for mediating this cross-talk. CD9 was abundant on the surface of ANXA6+ CAFs isolated from patient PDAC samples and from various mouse models of PDAC. CD9 colocalized with CAF markers in the tumor stroma, and CD9 abundance correlated with tumor stage. Blocking CD9 impaired the uptake of ANXA6+ EVs into cultured PDAC cells. Signaling pathway arrays and further analyses revealed that the uptake of CD9+ANXA6+ EVs induced mitogen-activated protein kinase (MAPK) pathway activity, cell migration, and epithelial-to-mesenchymal transition (EMT). Blocking either CD9 or p38 MAPK signaling impaired CD9+ANXA6+ EV-induced cell migration and EMT in PDAC cells. Analysis of bioinformatic datasets indicated that CD9 abundance was an independent marker of poor prognosis in patients with PDAC. Our findings suggest that CD9-mediated stromal cell signaling promotes PDAC progression.

Vasopressin Receptor Type-2 Mediated Signaling in Renal Cell Carcinoma Stimulates Stromal Fibroblast Activation.

Vasopressin type-2 receptor (V2R) is ectopically expressed and plays a pathogenic role in clear cell renal cell carcinoma (ccRCC) tumor cells. Here we examined how V2R signaling within human ccRCC tumor cells (Caki1 cells) stimulates stromal cancer-associated fibroblasts (CAFs). We found that cell culture conditioned media from Caki1 cells increased activation, migration, and proliferation of fibroblasts in vitro, which was inhibited by V2R gene silencing in Caki1 cells. Analysis of the conditioned media and mRNA of the V2R gene silenced and control Caki1 cells showed that V2R regulates the production of CAF-activating factors. Some of these factors were also found to be regulated by YAP in these Caki1 cells. YAP expression colocalized and correlated with V2R expression in ccRCC tumor tissue. V2R gene silencing or V2R antagonist significantly reduced YAP in Caki1 cells. Moreover, the V2R antagonist reduced YAP expression and myofibroblasts in mouse xenograft tumors. These results suggest that V2R plays an important role in secreting pro-fibrotic factors that stimulate fibroblast activation by a YAP-dependent mechanism in ccRCC tumors. Our results demonstrate a novel role for the V2R-YAP axis in the regulation of myofibroblasts in ccRCC and a potential therapeutic target.

Abstract 5556: Monotherapy of anti-CXCL16 antibody is a potential therapeutic for macrophage-enriched triple-negative breast cancer by modulating tumor immune microenvironment and glutamine metabolism

Abstract CXCL16, as a chemotactic cytokine, has been observed in various human solid cancers. The effects of CXCL16 on tumor behavior is controversial and showed tissue-specific manners. In breast cancers, previous studies have showed that CXCL16 mainly originated from mesenchymal stromal cells or cancer-associated fibroblasts and played pro-tumorigenic roles. CXCL16 is highly expressed in triple-negative breast cancers (TNBC) in TCGA dataset for breast cancer. This study aimed to investigate the therapeutic effects of CXCL16 neutralizing antibody and its mechanisms in TNBC. To establish a xenograft mouse model of TNBC, MDA-MB-231 cells were injected subcutaneously to athymic nude mice, and mice were randomized into anti-CXCL16 and control groups. When measurable tumors were established, anti-CXCL16 neutralizing antibody (CLS-A101, cellus inc.) or control IgG were intravenously injected. Tumor growth was significantly delayed in the anti-CXCL16 group than the control group by 67% for 3 weeks. Flow cytometric analysis showed that macrophages (CD11b+F4/80+ cells) and myeloid derived suppressor cells (MDSC, CD11b+GR1+ cells) were decreased in the anti-CXCL16 group than the control group. Histologic examination showed that anti-CXCL16 antibody significantly reduced tumor vessel formation and decreased macrophage infiltration in both intra- and peri-tumoral area. Collectively, treatment of anti-CXCL16 antibody reduced tumor growth by inhibiting macrophage and MDSC infiltration following reduced tumor angiogenesis. To explore the mechanistic insight into the monotherapy of anti-CXCL16 antibody, we further analyzed the metabolic changes. In human tumor tissues, TNBC harboring high expression of CXCL16 showed significant shift of glutamine metabolism pathway-related genes (upregulation of GLS and downregulation of GLUL, GLUD1, and GLUD2) toward accumulating glutamate. For cellular experiments, CXCL16-enriched cancer/macrophage co-cultured conditioned medium (co-CM) or cancer single-cultured conditioned medium (s-CM) were treated to cancer cells, and glutamine and glutamate levels were measured. In MDA-MB-231 cells, co-CM treatment significantly enhanced glutamate/glutamine ratio and anti-CXCL16 antibody significantly decreased it. Consistently, glutamate measures in tumor lysates from TNBC xenograft were lower in the anti-CXCL16 group than the control group. In conclusion, anti-CXCL16 antibody (CLS-A101) inhibited immune suppressive myeloid cell infiltration followed tumor angiogenesis and glutamate productions in cancer cells. These dual actions on both tumor immune microenvironment and cancer cell metabolism enables anti-CXCL16 antibody to be a potential monotherapy for TNBC. Citation Format: Min Joo Kim, Hyun Jin Sun, Geun-Hyung Kang, Seong Keun Kim, Sun Wook Cho. Monotherapy of anti-CXCL16 antibody is a potential therapeutic for macrophage-enriched triple-negative breast cancer by modulating tumor immune microenvironment and glutamine metabolism [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 5556.

Abstract 2878: Mediating NK cell function against solid tumors, via targeting of tumor stroma, using a TEM8-targeting Tri-specific Killer Engager (TriKE®)

Abstract Immunotherapies for solid tumors need to overcome barriers within the solid tumor microenvironment (TME), including stromal cells, cancer-associated fibroblasts, endothelial cells, pericytes, and immune cells. These can comprise a significant portion of the tumor mass in many common carcinomas and contribute to immune cell dysfunction within the tumor. Stromal cells support cancer cells through several mechanisms and can promote metastasis. Tumor vasculature supplies cancer cells with nutrients and oxygen necessary for tumor growth and dissemination. Thus, targeting these components of cancer can enhance tumor rejection directly and increase sensitization of surviving tumors to immune infiltration and chemotherapeutic treatments through weakening of the tumor architecture. While there are current antiangiogenic therapies being tested clinically, they have associated toxicities in normal angiogenic processes, limiting clinical efficacy. Tumor Endothelial Marker 8 (TEM8, encoded by the ANTXR1 gene) is a highly conserved integrin-like adhesion molecule that was discovered on the endothelium of colorectal cancer, and subsequent studies identified its expression on multiple stromal cells—endothelial cells, fibroblasts, and pericytes—in the tumor microenvironment of diverse human cancer types. Little to no expression has been demonstrated in normal human stroma, indicating that this would be a good target for immunotherapy. Using a mammalian expression system, we designed and expressed a TEM8 targeting TriKE (cam1615TEM8) consisting of a humanized anti-CD16 single domain antibody, a wild-type IL-15 moiety, and an anti-TEM8 scFv. cam1615TEM8 induces NK cell degranulation and cytokine production against TEM8+ tumor and stromal cell lines (endothelial cells and fibroblasts). Using TEM8- cancer lines and a TEM8-CRISPR knockout version of A549 lung cancer lines, we show that cam1615TEM8 only activates NK cells in the presence of tumor antigen. cam1615TEM8 also preferentially stimulates TEM8+ tumor spheroid killing. Moreover, the camelid anti-CD16 nanobody selectively enhances signaling by the IL-15 moiety to NK cells, specifically promoting NK cell survival and proliferation in vitro and in vivo. In an in vivo xenogeneic mouse model, containing human NK cells and tumor, cam1615TEM8-treated mice had enhanced NK cell tumor infiltration, significantly decreased tumor growth, and enhanced survival when compared to IL-15 treated mice. Since TEM8 is highly conserved, and the scFv within the TriKE is reactive to mouse TEM8, we were able to show reduced tumor endothelial density in the tumors of mice treated with cam1615TEM8. Given the findings of this study cam1615TEM8 could be leveraged in several clinical settings by inducing NK cell specific targeting of the tumor itself and/or the tumor endothelium and stroma. Citation Format: Martin Felices, Michael Kaminski, Peter Hinderlie, Rachel Hopps, Laura Bendzick, Melissa Geller, Jeffrey S. Miller. Mediating NK cell function against solid tumors, via targeting of tumor stroma, using a TEM8-targeting Tri-specific Killer Engager (TriKE®) [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 2878.

Abstract SY23-03: Mesenchymal lineage heterogeneity as a determinant of matrix composition and tumor progression

Abstract The mechanisms underlying evolution of tumor-associated stroma remain poorly understood. In solid tumors featuring a prominent stromal reaction, an improved understanding of the functions and origins of abundant stromal cell types may facilitate the development of new and effective therapies. Pancreatic ductal adenocarcinoma (PDAC) is the quintessence of a fibro-inflammatory malignancy, with 50-90% of tumor volume occupied by a dense, desmoplastic stroma. Cancer-associated fibroblasts (CAFs) are the key cell type which drives the stromal reaction in PDAC, and recent reports suggest that stromal CAFs represent a heterogeneous population of cells from diverse origins, potentially including cell types which support and others which suppress tumor growth. Pancreatic stellate cells (PSCs) are lipid-storing cells in healthy pancreas which can transdifferentiate to an activated CAF phenotype. PSCs have been suggested as the predominant source of fibroblasts in the PDAC tumor microenvironment. However, proper lineage tracing studies have never been performed, such that the relative contribution and specific functions of PSCs in the tumor microenvironment are unknown. We have developed a novel mouse model to track PSC differentiation and function during pancreatic tumor progression in vivo. This model revealed that PSC-derived CAFs in fact give rise to a numerically minor but functionally significant subset of PDAC CAFs, and may represent a viable therapeutic target. We adapted our mouse model to enable targeted ablation of PSCs and derivative CAFs within their host tissue for the first time. Functional studies in this model reveal non-redundant functions for PSC-derived CAFs in shaping the PDAC microenvironment, and highlight the significance of mesenchymal lineage heterogeneity for pancreatic tumorigenesis. Further, we find that tumor genotype with respect to p53 status dictates stromal evolutionary routes with respect to cell of origin, a finding that may extend to additional cancer cell-intrinsic genomic alterations. Together, these findings shed light on mechanisms that shape the tumor microenvironment during pancreatic cancer progression, and may hold relevance in additional solid tumor types. Citation Format: Mara H. Sherman. Mesenchymal lineage heterogeneity as a determinant of matrix composition and tumor progression [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 SY23-03.

Identification of Prognostic Biomarkers Originating From the Tumor Stroma of Betel Quid-Associated Oral Cancer Tissues

BackgroundPartial epithelial-mesenchymal transition (p-EMT) is a distinct clinicopathological feature prevalent in oral cavity tumors of The Cancer Genome Atlas. Located at the invasion front, p-EMT cells require additional support from the tumor stroma for collective cell migration, including track clearing, extracellular matrix remodeling and immune evasion. The pathological roles of otherwise nonmalignant cancer-associated fibroblasts (CAFs) in cancer progression are emerging.MethodsGene set enrichment analysis was used to reveal differentially enriched genes and molecular pathways in OC3 and TW2.6 xenograft tissues, representing mesenchymal and p-EMT tumors, respectively. R packages of genomic data science were executed for statistical evaluations and data visualization. Immunohistochemistry and Alcian blue staining were conducted to validate the bioinformatic results. Univariate and multivariate Cox proportional hazards models were performed to identify covariates significantly associated with overall survival in clinical datasets. Kaplan–Meier curves of estimated overall survival were compared for statistical difference using the log-rank test.ResultsCompared to mesenchymal OC3 cells, tumor stroma derived from p-EMT TW2.6 cells was significantly enriched in microvessel density, tumor-excluded macrophages, inflammatory CAFs, and extracellular hyaluronan deposition. By translating these results to clinical transcriptomic datasets of oral cancer specimens, including the Puram single-cell RNA-seq cohort comprising ~6000 cells, we identified the expression of stromal TGFBI and HYAL1 as independent poor and protective biomarkers, respectively, for 40 Taiwanese oral cancer tissues that were all derived from betel quid users. In The Cancer Genome Atlas, TGFBI was a poor marker not only for head and neck cancer but also for additional six cancer types and HYAL1 was a good indicator for four tumor cohorts, suggesting common stromal effects existing in different cancer types.ConclusionsAs the tumor stroma coevolves with cancer progression, the cellular origins of molecular markers identified from conventional whole tissue mRNA-based analyses should be cautiously interpreted. By incorporating disease-matched xenograft tissue and single-cell RNA-seq results, we suggested that TGFBI and HYAL1, primarily expressed by stromal CAFs and endothelial cells, respectively, could serve as robust prognostic biomarkers for oral cancer control.

Exploiting the STAT3 Nexus in Cancer-Associated Fibroblasts to Improve Cancer Therapy.

The tumor microenvironment (TME) is composed of a heterogenous population of cells that exist alongside the extracellular matrix and soluble components. These components can shape an environment that is conducive to tumor growth and metastatic spread. It is well-established that stromal cancer-associated fibroblasts (CAFs) in the TME play a pivotal role in creating and maintaining a growth-permissive environment for tumor cells. A growing body of work has uncovered that tumor cells recruit and educate CAFs to remodel the TME, however, the mechanisms by which this occurs remain incompletely understood. Recent studies suggest that the signal transducer and activator of transcription 3 (STAT3) is a key transcription factor that regulates the function of CAFs, and their crosstalk with tumor and immune cells within the TME. CAF-intrinsic STAT3 activity within the TME correlates with tumor progression, immune suppression and eventually the establishment of metastases. In this review, we will focus on the roles of STAT3 in regulating CAF function and their crosstalk with other cells constituting the TME and discuss the utility of targeting STAT3 within the TME for therapeutic benefit.

Abstract 2414: HMGA1 induces FGF19 to drive tumor progression and recruit cancer associated fibroblasts in pancreatic adenocarcinoma

Abstract Pancreatic ductal adenocarcinomas (PDACs) are highly lethal tumors characterized by a dense desmoplastic stroma comprised of cancer associated fibroblasts (CAFs) and fibrotic scar tissue. While stromal CAFs act as a barrier to therapy and release signals promoting tumor growth and invasion, the stroma restricts tumor growth in mouse models. The High Mobility Group A1 (HMGA1) protein is an oncofetal protein and epigenetic regulator that amplifies signals from the microenvironment to foster stem cell properties within intestinal epithelium. Although HMGA1 is highly expressed in embryonic and adult stem cells, it is silenced postnatally in most differentiated cells. HMGA1 becomes aberrantly re-expressed in diverse tumors where high levels predict adverse clinical outcomes. In PDAC, HMGA1 is detected only in late-stage precursor lesions or invasive tumors, but not in normal pancreas nor in early precursor lesions. Moreover, high HMGA1 nuclear staining associates with poor differentiation status and decreased patient survival. Here, we discovered a novel epigenetic program whereby HMGA1 recruits stromal CAFs and drives tumor progression by inducing FGF19. Silencing HMGA1 in PDAC cell lines slows proliferation, disrupts oncogenic properties in vitro (migration, invasion, clonogenicity, 3D sphere formation), and depletes tumor initiator cells in xenograft assays. RNA sequencing revealed transcriptional networks up-regulated by HMGA1 that function in cell signaling and proliferation; we focused on FGF19 as a potential mediator of tumor-stromal crosstalk. HMGA1 binds directly to the FGF19 promoter and recruits active histone marks (H3K4me2, H3K27Aac) to induce gene expression and protein secretion from PDAC cells. Silencing FGF19 recapitulates effects of HMGA1 silencing, disrupting oncogenic and cancer stem cell properties in vitro while depleting tumor initiator cells in vivo. In co-culture experiments, FGF19 is required for CAF migration across a membrane towards PDAC cells. Silencing HMGA1, FGF19, or treatment with FGF19 inhibitors disrupts CAF recruitment. Silencing HMGA1 or FGF19 in PDAC cells also decreases both the desmoplastic stroma and tumor growth in mouse xenografts. In primary tumors, co-expression of HMGA1 and FGF19 predict decreased survival. Together, our results reveal a novel paradigm whereby tumor cells collaborate with CAFs via HMGA1 and FGF19 to drive progression, thus illuminating FGF19 as a rational therapeutic target for PDACs overexpressing HMGA1 and FGF19. Citation Format: Lionel Chia, Shuai Shuai, Jung-Hyun Kim, Woo Jung Sung, Ruitao Zhang, Tait Huso, Leslie Cope, Karen Reddy, Linda M. Resar. HMGA1 induces FGF19 to drive tumor progression and recruit cancer associated fibroblasts in pancreatic adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2414.

Abstract PR004: NetrinG1’s pro-tumor role on stroma-derived extracellular vesicles in pancreatic cancer

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is a devastating disease which is, in part, driven and supported by changes in its microenvironment, or stroma. These changes occur early, yet PDAC is usually undetected until the disease has metastasized, causing survival rates to drop dramatically. Therefore, there exists a grave importance to detect this disease as early as possible and understand its progression. This project dissects the intercellular communication that exists between the primary stromal component, cancer-associated fibroblasts (CAFs), and PDAC cells. Specifically, we focus on how CAF-secreted extracellular vesicles (EVs) promote PDAC progression, with an additional goal to identify biomarkers suitable to generate a non-invasive “liquid biopsy” test for early PDAC detection and prognosis. PDAC communicates with its microenvironment, in part, through the exchange of specific types of EVs, which include exosomes and recently characterized “ectosomes.” We observe distinct types of CAF-derived EVs containing unique surface receptors. One novel surface protein, NetrinG1, is expressed on the plasma membrane of pancreatic CAFs, but not their normal/healthy counterparts. Further, PDAC cells, but not healthy pancreatic epithelial cells, upregulate NetrinG1’s lone binding partner, suggesting a role for these factors in PDAC-selective EV uptake. Functional assays designed to test PDAC viability in nutrient deprivation show that CAF-EVs are capable of protecting PDAC cells from the induction of programmed cell death. Further, we show NetrinG1’s expression in CAFs is necessary for this EV-mediated survival effect. We also determine that NetrinG1 localizes to the novel “ectosome” EV sub-population, suggesting it possesses unique cargo and is packaged into EVs and secreted through a yet-unknown mechanism separate from canonical exosome trafficking. We also determine, that sub-populations of EVs can be “filtered” locally by the extracellular matrix, based on various EV surface markers. This suggests that the extracellular matrix can play an important role in determining the fate of secreted EVs; which has significant implications for what sub-populations maybe be found circulating systemically in blood, or acting locally in the tumor microenvironment. Pursuing our biomarker goal, we confirm stromal NetrinG1 expression precedes tumorigenesis and are currently seeking to validate the prognostic potential of NetrinG1(+)-EVs in blood of PDAC patients. Altogether, this research shines light on a novel mechanism of tumor-stroma communication, and introduces EV biomarkers potentially capable of identifying both early PDAC occurrences and predicted efficacy of certain adjuvant interventions. Citation Format: Kristopher Raghavan, Ralph Francescone, Janusz Franco-Barraza, Edna Cukierman. NetrinG1’s pro-tumor role on stroma-derived extracellular vesicles 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 PR004.

CaMKII Mediates TGFβ1-Induced Fibroblasts Activation and Its Cross Talk with Colon Cancer Cells.

Cancer-associated fibroblasts (CAFs), as the activated fibroblasts in tumor stroma, are important modifiers of tumor progression. TGFβ1 has been the mostly accepted factor to fuel normal fibroblasts transformation into CAFs. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is thought to play an important role in fibroblasts activation induced by TGFβ1. The aim of this study is to investigate the potential role of CaMKII in TGFβ1-induced fibroblasts activation and CAF-like differentiation. Cross talk between CaMKII-dependent fibroblasts and colon cancer in colon cancer progression also was addressed RESULTS: Immunostaining demonstrated that in colon cancer stroma, CaMKII overexpressed in stromal CAFs. In vitro, TGFβ1 increased CAF markers expression in human colon fibroblasts CCD-18Co, but not in CaMKII depletion fibroblasts. CaMKII knockdown by CaMKII shRNA significantly inhibited TGFβ1-induced fibroblasts activation and CAF-like differentiation. Smad3, AKT, and MAPK were targeted in TGFβ1-CaMKII-mediated pathway. Human colon cancer cell line HCT-116 activated fibroblasts directly, whereas CaMKII depletion dragged CCD-18Co fibroblasts undergoing CAF-associated trans-differentiation. Furthermore, increased proliferation, migration, and invasion of colon cancer cells were stimulated when co-cultured with normal fibroblasts, but not with CaMKII depletion fibroblasts. These findings provide evidence that CaMKII is a critical mediator in TGFβ1-induced fibroblasts activation and is involved in the cross talk with colon cancer cells. CaMKII is a potentially effective target for future treatment of colon cancer.