Cancer-associated Fibroblasts Population Research Articles

Abstract B067: Patient-specific differences in cancer-associated fibroblasts alter tumor organoid phenotype and chemosensitivity in pancreatic ductal adenocarcinoma

Abstract Pancreatic adenocarcinoma (PDAC) is the 3rd leading cause of cancer-related deaths in Canada with a 5-year survival rate ~10%. Unfortunately, only 20% of patients are eligible for surgical resection while the other 80% of patients are treated with chemotherapy (i.e. nab-paclitaxel, Gemcitabine, or FOLFIRINOX) that shows limited efficiency. Also, the tumor microenvironment (TME), and specifically cancer-associated fibroblasts (CAFs), contribute to PDAC chemoresistance. Recent studies suggest heterogeneity of CAF subtypes in patients, including myofibroblast (my), inflammatory (i), and antigen-presenting (ap) CAFs, is linked to more aggressive PDAC. While there is clear evidence CAFs effect tumor growth, chemoresistance and metastasis, the mechanisms underlying these effects are unknown. Given the involvement of environmental cues from the TME and the recent literature involving HDACs and epigenetic regulators in chemoresistance, we propose epigenetic reprogramming occurs in pancreatic cancer cells in response to CAFs, thereby affecting their response to chemotherapy. Using patient-derived tumor cells we examine the cross talk that occurs between cancer cells and CAFs in a patient-specific manner. We hypothesized that patient-specific CAF populations alter cancer cell phenotypes and chemosensitivity through epigenetic reprograming. Supported by the Baker Centre for Pancreatic Cancer, we established a living biobank of patient-derived organoids (PDO) grown in 3D cultures and CAFs grown in 2D cultures. These samples are linked to DERIVE (Determination of Response to Therapy in Individual Patients), a clinical database that includes patient response to therapy. Characterization of low passage, patient CAFs by flow cytometry shows significant heterogeneity between samples based on the proportions of myCAFs, iCAFs and apCAFs, and also identified CAFs that do not belong to any of these subtypes. Incubation of PDOs with CAF-conditioned media showed an increase in growth regardless of the CAF samples but, interestingly, myCAF-high (myCAFHI) conditioned media promoted a cystic-like phenotype in PDOs while iCAF-high conditioned media promoted a more complex PDO phenotype. myCAFHI-conditioned media also sensitized resistant PDOs to gemcitabine. Characterization of epigenetic mediators showed variable expression of histone deacetylases (HDACs), and combinatorial treatment with gemcitabine and the HDAC5 inhibitor, LMK-235 sensitized resistant PDOs to gemcitabine. We are currently performing single-cell RNA-seq, ATAC-seq and DNA methylation profiling on PDOs before and after treatment with CAF-conditioned media to identify unique CAF populations and examine the epigenetic targets underlying these outcomes. This study (1) shows CAFs present with significant heterogeneity between patients and secreted factors from CAFs induce responses in a patient-specific fashion that may change chemosensitivity, and (2) suggest targeting epigenetic mediators in PDOs and CAFs may help sensitize tumors to chemotherapy. Citation Format: Emilie Jaune-Pons, Zachary Klassen, Rachel Lu, Ye Shen, Nadeem Hussain, Michael Sey, Ken Leslie, Ephraim Tang, Anton Skaro, Matthew Cecchini, Crystal Engelage, Danielle Porplycia, Stephen Welch, Brian Yan, Christopher Pin. Patient-specific differences in cancer-associated fibroblasts alter tumor organoid phenotype and chemosensitivity in pancreatic ductal adenocarcinoma [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 B067.

Abstract C025: Fibroblast-specific IL1R1-p38 MAPK signaling sustains stromal inflammation and contributes to therapeutic resistance in pancreatic cancer

Abstract Introduction: The desmoplastic stroma of pancreatic ductal adenocarcinoma (PDAC) perpetuates therapeutic resistance by impairing drug delivery and effector immune cell infiltration and activation. We have identified tumor cell-derived interleukin-1α (IL1α) as a critical activator of the stroma, comprised of cancer-associated fibroblasts (CAF), towards a pro-inflammatory phenotype via the IL1 receptor, IL1R1, and subsequent activation of p38 MAPK. Our hypothesis is that disruption of the CAF-specific IL1α/p38 MAPK signaling axis can improve chemotherapeutic resistance by remodeling the fibrotic stromal landscape and the overall immune microenvironment in PDAC. Methods: p38 MAPK was pharmacologically inhibited with pexmetinib and genetically with an shRNA lentiviral system. Bulk RNA and ATAC sequencing (RNAseq and ATACseq) were performed on human pancreatic stellate cells (hPSC). Gene set enrichment analyses and motif enrichment analyses were performed on the RNAseq and ATACseq datasets, respectively. Tumor cells (KPC-6694), derived from LSL-KrasG12D/+; LSL-Tp53R172H/+; Pdx1Cre/+ (KPC) murine tumors, were injected orthotopically into Col1a2Cre/+; Mapk14f/f and Col1a2Cre/+; Il1r1f/f mice, and tumors were harvested for downstream analysis. Ptf1aCre/+; LSL-KrasG12D/+; Tgfbr2flox/flox (PKT) mice were treated with pexmetinib (PEX, 30mg/kg, daily PO), anakinra (AKA, 50mg/kg IP, BID) or vehicle control for 2.5 weeks prior to sacrifice for downstream analyses and single-cell RNA sequencing (scRNA) on tumors. Results: In vitro inhibition of p38 MAPK prevented CAF activation into an inflammatory fibroblast, when stimulated with IL1α, demonstrated through bulk RNAseq and ATACseq. Bulk ATACseq revealed epigenetic regulation of the inflammatory CAF phenotype by IL1-mediated activation of p38 MAPK. Stromal-specific deletion of Il1r1 or Mapk14 resulted in signification reduction of tumor weight and intratumoral myeloid-derived suppressor cells. Additionally, pharmacologic blockade of both IL1R1 (AKA) and p38 MAPK inhibition (PEX) altered intratumoral immune cell and CAF populations, by flow cytometry and scRNA. Lastly, p38 inhibition or IL1R1 blockade combined with chemotherapy significantly reduced tumor burden and favorably altered the immune landscape in a PDAC genetically engineered mouse model. Conclusions: These findings provide important mechanistic data in preclinical PDAC models to explore p38 MAPK inhibition in combination therapy to target the fibrotic stroma and immunosuppressive tumor microenvironment. Citation Format: Samara P. Singh, Austin R. Dosch, Siddharth Mehra, Iago de Castro Silva, Vanessa Tonin Garrido, Anna Bianchi, Haleh Amirian, Edmond W. Box, Jashodeep Datta, Nagaraj S. Nagathihalli, Nipun B. Merchant. Fibroblast-specific IL1R1-p38 MAPK signaling sustains stromal inflammation and contributes to therapeutic resistance in pancreatic cancer [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 C025.

Cancer-associated fibroblast spatial heterogeneity and EMILIN1 expression in the tumor microenvironment modulate TGF-β activity and CD8+ T-cell infiltration in breast cancer.

Rationale: The tumor microenvironment (TME) and its multifaceted interactions with cancer cells are major targets for cancer treatment. Single-cell technologies have brought major insights into the TME, but the resulting complexity often precludes conclusions on function. Methods: We combined single-cell RNA sequencing and spatial transcriptomic data to explore the relationship between different cancer-associated fibroblast (CAF) populations and immune cell exclusion in breast tumors. The significance of the findings was then evaluated in a cohort of tumors (N=75) from breast cancer patients using immunohistochemistry analysis. Results: Our data show for the first time the degree of spatial organization of different CAF populations in breast cancer. We found that IL-iCAFs, Detox-iCAFs, and IFNγ-iCAFs tended to cluster together, while Wound-myCAFs, TGFβ-myCAFs, and ECM-myCAFs formed another group that overlapped with elevated TGF-β signaling. Differential gene expression analysis of areas with CD8+ T-cell infiltration/exclusion within the TGF-β signaling-rich zones identified elastin microfibrillar interface protein 1 (EMILIN1) as a top modulated gene. EMILIN1, a TGF-β inhibitor, was upregulated in IFNγ-iCAFs directly modulating TGFβ immunosuppressive function. Histological analysis of 75 breast cancer samples confirmed that high EMILIN1 expression in the tumor margins was related to high CD8+ T-cell infiltration, consistent with our spatial gene expression analysis. High EMILIN1 expression was also associated with better prognosis of patients with breast cancer, underscoring its functional significance for the recruitment of cytotoxic T cells into the tumor area. Conclusion: Our data show that correlating TGF-β signaling to a CAF subpopulation is not enough because proteins with TGF-β-modulating activity originating from other CAF subpopulations can alter its activity. Therefore, therapeutic targeting should remain focused on biological processes rather than on specific CAF subtypes.

Abstract B047: Phase I trial of αvβ3 integrin cytotoxin ProAgio in patients with previously treated advanced pancreatic cancer and other solid tumor malignancies

Abstract Background: ProAgio is a pegylated peptide drug that targets and kills cells that express integrin αvβ3 including cancer associated fibroblasts (CAFs) and endothelial cells within the tumor microenvironment. ProAgio utilizes a novel binding site to directly trigger apoptosis of target cells in pre-clinical models. Here we describe the interim results of a first-in-human phase 1 trial of ProAgio in patients with advanced solid malignancies, including pancreatic cancer. Methods: The trial is enrolling adults with previously treated and histologically confirmed advanced solid malignancy for which no curative therapy exists. The dose escalation cohort uses a modified 3+3 design to determine the recommended phase 2 dose (RP2D) of ProAgio. This will be followed by a dose expansion cohort which will enroll only participants with non-neuroendocrine advanced pancreatic cancer. The primary objective of the trial is to determine the RP2D of ProAgio. Secondary objectives assess the safety (CTCAE v. 4.03), pharmacokinetics and objective response rate (ORR) of Proagio. Ascending doses of ProAgio were administered as an intravenous infusion every 1-2 weeks depending on dose level. Response is determined by serial imaging (CT or MRI) and tumor marker assessment (CA 19-9 or appropriate tumor-specific marker). Exploratory objectives include assessing biologic effect of ProAgio on pre- and on-therapy tumor biopsy specimens (apoptosis of tumor cells, integrin αvβ3 expression, CAF population, tumor vasculature and architecture), as well as identifying putative surrogate circulating biomarkers (circulating αvβ3 expressing cells among others). Results: Sixteen participants (14 with pancreatic adenocarcinoma and 2 with other gastrointestinal malignancies) have been treated on the dose escalation as of June 30, 2023. Preliminary pharmacokinetic measurements demonstrate a plasma half-life of 17.35 hours, distribution volume of 17.62 L, and clearance of 0.522 L/hr. ProAgio has been exceptionally well tolerated with only transient grade 1 or 2 treatment-related adverse events observed in most participants. Two participants had grade 2 infusion related reaction but were able to continue treatment with increased dose of diphenhydramine pre-medication. A serious adverse event of grade 3 hyperglycemia was observed in 1 participant with insulin-dependent diabetes within 24 hours of ProAgio infusion. No other treatment-associated hyperglycemia has been observed. Two participants with pancreatic cancer receiving weekly dosing of ProAgio had stable disease for >4 months and interval stabilization in CA 19-9 tumor marker. Conclusions: ProAgio is safe and well-tolerated. Upon completion of the dose escalation, a dose expansion phase of 15 participants treated at the RP2D will begin enrolling. NCT05085548 Citation Format: Nebojsa Skorupan, Cody J. Peer, Eve Koehler, Seth Steinberg, Jane B Trepel, William D Figg, Zhi-Ren Liu, Christine Alewine. Phase I trial of αvβ3 integrin cytotoxin ProAgio in patients with previously treated advanced pancreatic cancer and other solid tumor malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B047.

Unravelling heterogeneous effects of cancer‑associated fibroblasts on poor prognosis markers in breast cancer EM‑G3 cell line: Invitro‑targeted treatment (anti‑IL-6, anti‑VEGF-A, anti‑MFGE8) based on transcriptomic profiling.

Breast cancer is the most frequently diagnosed cancer in women worldwide. Although dramatically increased survival rates of early diagnosed cases have been observed, late diagnosed patients and metastatic cancer may still be considered fatal. The present study's main focus was on cancer‑associated fibroblasts (CAFs) which is an active component of the tumor microenvironment (TME) regulating the breast cancer ecosystem. Transcriptomic profiling and analysis of CAFs isolated from breast cancer skin metastasis, cutaneous basal cell carcinoma, and squamous cell carcinoma unravelled major gene candidates such as IL6, VEGFA and MFGE8 that induced co‑expression of keratins‑8/‑14 in the EM‑G3 cell line derived from infiltrating ductal breast carcinoma. Western blot analysis of selected keratins (keratin‑8, ‑14, ‑18, ‑19) and epithelial‑mesenchymal transition‑associated markers (SLUG, SNAIL, ZEB1, E‑/N‑cadherin, vimentin) revealed specific responses pointing to certain heterogeneity of the studied CAF populations. Experimental invitro treatment using neutralizing antibodies against IL-6, VEGF‑A and MFGE8 attenuated the modulatory effect of CAFs on EM‑G3 cells. The present study provided novel data in characterizing and understanding the interactions between CAFs and EM‑G3 cells invitro. CAFs of different origins support the pro‑inflammatory microenvironment and influence the biology of breast cancer cells. This observation potentially holds significant interest for the development of novel, clinically relevant approaches targeting the TME in breast cancer. Furthermore, its implications extend beyond breast cancer and have the potential to impact a wide range of other cancer types.

Cancer-associated fibroblasts undergoing neoadjuvant chemotherapy suppress rectal cancer revealed by single-cell and spatial transcriptomics

Neoadjuvant chemotherapy (NAC) for rectal cancer (RC) shows promising clinical response. The modulation of the tumor microenvironment (TME) by NAC and its association with therapeutic response remain unclear. Here, we use single-cell RNA sequencing and spatial transcriptome sequencing to examine the cell dynamics in 29 patients with RC, who are sampled pairwise before and after treatment. We construct a high-resolution cellular dynamic landscape remodeled by NAC and their associations with therapeutic response. NAC markedly reshapes the populations of cancer-associated fibroblasts (CAFs), which is strongly associated with therapeutic response. The remodeled CAF subsets regulate the TME through spatial recruitment and crosstalk to activate immunity and suppress tumor progression through multiple cytokines, including CXCL12, SLIT2, and DCN. In contrast, the epithelial-mesenchymal transition of malignant cells is upregulated by CAF_FAP through MIR4435-2HG induction, resulting in worse outcomes. Our study demonstrates that NAC inhibits tumor progression and modulates the TME by remodeling CAFs.

Critical requirement of SOS1 for tumor development and microenvironment modulation in KRASG12D-driven lung adenocarcinoma

The impact of genetic ablation of SOS1 or SOS2 is evaluated in a murine model of KRASG12D-driven lung adenocarcinoma (LUAD). SOS2 ablation shows some protection during early stages but only SOS1 ablation causes significant, specific long term increase of survival/lifespan of the KRASG12D mice associated to markedly reduced tumor burden and reduced populations of cancer-associated fibroblasts, macrophages and T-lymphocytes in the lung tumor microenvironment (TME). SOS1 ablation also causes specific shrinkage and regression of LUAD tumoral masses and components of the TME in pre-established KRASG12D LUAD tumors. The critical requirement of SOS1 for KRASG12D-driven LUAD is further confirmed by means of intravenous tail injection of KRASG12D tumor cells into SOS1KO/KRASWT mice, or of SOS1-less, KRASG12D tumor cells into wildtype mice. In silico analyses of human lung cancer databases support also the dominant role of SOS1 regarding tumor development and survival in LUAD patients. Our data indicate that SOS1 is critically required for development of KRASG12D-driven LUAD and confirm the validity of this RAS-GEF activator as an actionable therapeutic target in KRAS mutant LUAD.

267. IDENTIFYING CANCER-ASSOCIATED FIBROBLAST POPULATIONS DRIVING THERAPY RESISTANCE IN GASTROESOPHAGEAL ADENOCARCINOMA

Abstract Background Peri-operative docetaxel-based triplet chemotherapy is the standard-of-care treatment for advanced gastroesophageal adenocarcinoma (GEA). However, most patients recur due to innate or acquired resistance. Although distinct populations of cancer-associated fibroblasts (CAFs) within the tumor microenvironment play important roles in conferring chemoresistance in other cancer types, this paradigm has not been extensively studied in GEA. We aim to characterize CAF heterogeneity in a well-annotated cohort of GEA patients, identifying potential biomarkers and targets to overcome chemoresistance. Methods Immunofluorescence and flow cytometry assays were performed to validate previously reported CAF markers from the literature in primary patient fibroblasts. To identify specific CAF markers for GEA, an atlas was developed using single-cell RNA sequencing (scRNA-seq) data obtained from 46 GEA patient samples, including 28 patients with longitudinal samples over the docetaxel-based triplet chemotherapy treatment trajectory. Differential and gene ontology analyses were performed to characterize distinct CAF subpopulations and identify dynamic CAF markers across treatment timepoints, while correlating with in-patient treatment response to chemotherapy. In parallel, tumor organoid-CAF co-cultures were established for subsequent in vitro drug testing with standard-of-care chemotherapy. Results CAF markers (VIM, FAP, PDPN, and S100A4) were found to be differentially expressed in fibroblasts isolated from good and poor pathological-response tumours. Analysis of scRNA-seq data reveals two main subpopulations of CAFs: myofibroblast CAFs (myCAFs) and inflammatory CAFs (iCAFs). Two markers, CCL20 and CHRDL1, were identified as differentially expressed between good and poor pathological responders in iCAFs, while nine markers, including ISG20 and C20orf27, were differentially expressed between clinical partial responders and poor responders. Dynamic CAF markers, including STAG2 and HAT1, were differentially expressed across treatment timepoints and were associated with pathological or clinical response. Conclusions GEA CAFs demonstrate extensive heterogeneity and comprise two major subpopulations. Several CAF markers were found to be associated with patient chemotherapy pathological or clinical response to chemotherapy, either in the chemonaïve setting or in the context of changes over the course of therapy. These putative biomarkers will be further validated and investigated, both in additional samples and in co-culture settings, to gain a better understanding of their clinical relevance and targeting potential.

Novel CAF-identifiers via transcriptomic and protein level analysis in HNSC patients

Cancer-associated fibroblasts (CAFs), a prominent component of the tumor microenvironment, play an important role in tumor development, invasion, and drug resistance. The expression of distinct “CAF-markers” which separates CAFs from normal fibroblasts and epithelial cells, have traditionally been used to identify them. These commonly used CAF-markers have been reported to differ greatly across different CAF subpopulations, even within a cancer type. Using an unbiased -omic approach from public data and in-house RNAseq data from patient derived novel CAF cells, TIMP-1, SPARC, COL1A2, COL3A1 and COL1A1 were identified as potential CAF-markers by differential gene expression analysis using publicly available single cell sequencing data and in-house RNAseq data to distinguish CAF populations from tumor epithelia and normal oral fibroblasts. Experimental validation using qPCR and immunofluorescence revealed CAF-specific higher expression of TIMP-1 and COL1A2 as compared to other markers in 5 novel CAF cells, derived from patients of diverse gender, habits and different locations of head and neck squamous cell carcinoma (HNSC). Upon immunohistochemical (IHC) analysis of FFPE blocks however, COL1A2 showed better differential staining between tumor epithelia and tumor stroma. Similar data science driven approach utilizing single cell sequencing and RNAseq data from stabilized CAFs can be employed to identify CAF-markers in various cancers.

High CD142 Level Marks Tumor-Promoting Fibroblasts with Targeting Potential in Colorectal Cancer.

Colorectal cancer (CRC) has a high incidence and is one of the leading causes of cancer-related death. The accumulation of cancer-associated fibroblasts (CAF) induces an aggressive, stem-like phenotype in tumor cells, and it indicates a poor prognosis. However, cellular heterogeneity among CAFs and the targeting of both stromal and CRC cells are not yet well resolved. Here, we identified CD142high fibroblasts with a higher stimulating effect on CRC cell proliferation via secreting more hepatocyte growth factor (HGF) compared to CD142low CAFs. We also found that combinations of inhibitors that had either a promising effect in other cancer types or are more active in CRC compared to normal colonic epithelium acted synergistically in CRC cells. Importantly, heat shock protein 90 (HSP90) inhibitor selected against CD142high fibroblasts, and both CRC cells and CAFs were sensitive to a BCL-xL inhibitor. However, targeting mitogen-activated protein kinase kinase (MEK) was ineffective in fibroblasts, and an epigenetic inhibitor selected for a tumor cell population with markers of aggressive behavior. Thus, we suggest BCL-xL and HSP90 inhibitors to eliminate cancer cells and decrease the tumor-promoting CD142high CAF population. This may be the basis of a strategy to target both CRC cells and stromal fibroblasts, resulting in the inhibition of tumor relapse.

Evaluation of THEO-260 as a novel oncolytic virus therapy for treating stromal rich tumours.

e14573 Background: Stromal rich tumours pose a significant challenge to the effectiveness of systemic anticancer therapies, including immunotherapies and oncolytic viruses. This is due to the abundance of cancer associated fibroblasts (CAFs) that suppress immune cells and limit drug penetration. THEO-260 is a potent, mechanistically novel oncolytic virus therapy intrinsically capable of lysing both cancer cells and CAFs, and is currently in development for late stage relapsed refractory ovarian cancer. Methods: The efficacy and safety of THEO-260 was assessed in multicellular spheroids, in patient-derived ex vivo fresh ovarian samples and in mouse models of human stromal tumours. Results: Unpassaged ascites and tumour samples collected from cytoreductive surgeries for advanced ovarian cancer (n=24 independent patients) were treated with a clinically-relevant dose of THEO-260. THEO-260 effectively killed the cancer cell (EpCAM+ EGFR+PDL1+ CA125+) and CAF (FAP+) populations, being efficacious in treatment-naive and platinum-resistant patient samples. Importantly, the T cell population in these patients, in addition to primary normal fibroblasts (including intestinal, pulmonary, dermal, cardiac, uterine fibroblasts) from healthy donors, were not damaged. Ex vivo studies show THEO-260 stimulates effector T cell responses upon tumour lysis as indicated by IFNγ release. Demonstrating suitability as an IV delivered agent, THEO-260 was active in pooled human serum and whole human blood (n = 10 donors). THEO-260 excels in low nutrient, low proliferative environments, typical of human solid tumours and has been developed with a view to persist in the tumour microenvironment without the need for repeated cycles of delivery. THEO-260 is well tolerated in vivo following a clinically relevant intravenous dosing regimen. In PARP inhibitor and platinum resistant ovarian cancer murine models containing human CAFs and cancer cells, THEO-260 shows antitumor efficacy, with a complete reduction in tumour volume in subcutaneous and intraperitoneal systems, and superiority to SOC. Virus activity is not restricted to ovarian cancer, with cell line data demonstrating killing in additional solid tumour types, including pancreatic, colorectal, lung, and breast cancer. THEO-260 exhibits genetic and temperature based stability, and GMP manufacturing to high yields is underway. Conclusions: Taken together, we present a preclinical data package for THEO-260 supporting progression to Phase I clinical trials.

Understanding Long-Term Survival of Patients with Ovarian Cancer—The Tumor Microenvironment Comes to the Forefront

High-grade serous ovarian cancer (HGSOC) is the deadliest subtype of ovarian cancer, and most patients do not survive more than 5 years after diagnosis. Yet, for reasons that are often elusive, approximately 15% of women with advanced-stage HGSOC will survive longer than 10 years. An understanding of the biological basis of long-term survival with HGSOC may elucidate novel prognostic factors and targets for treatment. Past analyses of the clinicopathologic features of these women and genetic profiles of their tumors have not revealed a unifying explanation for their increased longevity. In this issue of Cancer Research, Ferri-Borgogno and colleagues investigate the tumor microenvironment (TME) in samples from both long- and short-term survivors using spatial transcriptomics and single-cell RNA sequencing. They found that, in metastatic tumors, various populations of cancer-associated fibroblasts (CAF) in the TME play different roles in supporting the malignant phenotype of ovarian cancer cells. Higher density of CAFs, particularly αSMA+VIM+PDGFRβ+ CAFs, was associated with lower tumor immune infiltration and short-term survival. There was also marked expression of periostin and CD36 in spatially resolved CAFs, as well as a prevalence of the APOE-LRP5 ligand-receptor pair at the tumor-stromal interface in tissue from short-term survivors. These findings suggest that, in short-term survivors, CAFs are able to more effectively promote tumorigenicity, stemness, and chemoresistance in the nearby tumor. See related article by Ferri-Borgogno et al., p. 1503.

Investigating the interactions between regulatory T cells and cancer-associated fibroblasts and their role in lung cancer progression

Abstract The tumor microenvironment (TME) is a highly complex multicellular system and has been shown to play a fundamental role in dictating tumorigenesis. High expression of regulatory surface molecules, cytokines, and chemokines in fibroblast niches within lung tumor stroma, paired with their colocalization with tumor infiltrating lymphocytes (TILs), suggests that cancer-associated fibroblast (CAF) niches may alter the functional response of TILs and influence the anti-tumor immune response. Here, utilizing a transcriptomic approach, we have uncovered a distinct CAF population, which we term “iCAFs,” that is enriched for immunomodulatory gene signatures, T cell ligands, and chemoattractants. This population expresses a unique set of cell surface molecules that permit their identification and isolation from lung tumor tissue. Given the demonstrated importance of Tregs in suppressing anti-tumor immunity, we chose to evaluate how Tregs may be interacting with iCAFs within the TME of lung adenocarcinoma. We uncovered significant colocalization patterns of Foxp3 +Tregs with iCAF—a pattern which was found to be unique for Tregs when compared to conventional CD4 +Foxp3 −T cells. Moreover, the Treg to conventional CD4 +T cell ratio is significantly decreased in tumor tissue relative to normal adjacent tissue and healthy controls. Our analyses demonstrate that Tregs preferentially associate with iCAF populations within the tumor, leading us to hypothesize that interactions between Tregs and iCAFs may, in turn, influence Treg trafficking, phenotype and function within the TME. T32AI106711

Targeting the IL1β Pathway for Cancer Immunotherapy Remodels the Tumor Microenvironment and Enhances Antitumor Immune Responses.

High levels of IL1β can result in chronic inflammation, which in turn can promote tumor growth and metastasis. Inhibition of IL1β could therefore be a promising therapeutic option in the treatment of cancer. Here, the effects of IL1β blockade induced by the mAbs canakinumab and gevokizumab were evaluated alone or in combination with docetaxel, anti-programmed cell death protein 1 (anti-PD-1), anti-VEGFα, and anti-TGFβ treatment in syngeneic and humanized mouse models of cancers of different origin. Canakinumab and gevokizumab did not show notable efficacy as single-agent therapies; however, IL1β blockade enhanced the effectiveness of docetaxel and anti-PD-1. Accompanying these effects, blockade of IL1β alone or in combination induced significant remodeling of the tumor microenvironment (TME), with decreased numbers of immune suppressive cells and increased tumor infiltration by dendritic cells (DC) and effector T cells. Further investigation revealed that cancer-associated fibroblasts (CAF) were the cell type most affected by treatment with canakinumab or gevokizumab in terms of change in gene expression. IL1β inhibition drove phenotypic changes in CAF populations, particularly those with the ability to influence immune cell recruitment. These results suggest that the observed remodeling of the TME following IL1β blockade may stem from changes in CAF populations. Overall, the results presented here support the potential use of IL1β inhibition in cancer treatment. Further exploration in ongoing clinical studies will help identify the best combination partners for different cancer types, cancer stages, and lines of treatment.

Abstract 1255: Glucose dependent CD73+ CAFs enforce a tumor metabolic barrier that promotes T cell exclusion

Abstract Trafficking of T lymphocytes from the lymph nodes to the tumor microenvironment is a critical process of the tumor immunity cycle to elicit cytotoxic anti-tumor responses driven by CD8+ T cells. However, some tumors termed “immune excluded” recruit lymphocytes to the tumor site, but the lymphocytes are unable to penetrate the tumor parenchyma and localize primarily in the peritumoral region. In soft tissue sarcoma patients, most tumors are poorly infiltrated by T cells, which is associated with a poor response to immunotherapies. It has been described that cancer associated fibroblasts (CAFs) are enriched in immune excluded tumors and may directly block the migration of T cells via the production of dense extracellular matrix or by forging an immunosuppressive niche. We generated two models of undifferentiated pleomorphic sarcoma (UPS) that recapitulate the “immune excluded” and “inflamed” microenvironments observed in sarcoma patients. These syngeneic models rely on p53KO mesenchymal stem cells overexpressing either Ccne1 or Vgll3, which are frequently amplified in UPS patients. These models differ in their overall proportion of infiltrating TILs, and specifically T cells, making them ideal for comparative studies to investigate the mechanisms driving T cell exclusion in the TME. Using single-cell RNA-sequencing, we identified a population of CAFs expressing Nt5e, encoding CD73, which are spatially enriched in the peritumoral region of immune excluded Ccne1 tumors and closely associate with CD8+ T cells located at the tumor margin. Using transwell invasion assays, we show that CD73+ CAFs but not CD73- CAFs are able to block the migration of activated T cells towards tumor cells, even in the presence of CXCL10. Further, we show that Nt5e CAFs are enriched for signatures of glucose metabolism, and hypoxia, thus we hypothesized that CD73+ CAFs may block the migration of T cells into tumors by forging a nutrient poor metabolic barrier around the tumor. To test this, we treated Ccne1 tumors with BAY-876, a GLUT1 inhibitor and observed a significant accumulation of infiltrating CD8+ T cells compared to controls. GLUT1 treated CAFs expressed significantly less Nt5e, indicating that CD73 may play a role in the maintenance of glucose metabolism in CAFs. Furthermore, blockade of CD73 in CD73+ CAFs decreases the expression of the glucose transporter, Glut1. All together, these data suggest that CD73 may serve as a marker of glucose dependent CAFs that alter the metabolic niche to block T cell infiltration into tumors. Citation Format: Marina Broz, Emily Ko, Jinfen Xiao, Marco DeSimone, Roberta Piras, Kristin Ishaya, Xen Ping Hoi, Jlenia Guarnerio. Glucose dependent CD73+ CAFs enforce a tumor metabolic barrier that promotes T cell exclusion [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 1255.

Abstract 2618: IQGAP3 regulates intra-cellular Ras signaling and intra-tumoral malignant cycles via TGFβ signaling

Abstract Scaffold protein IQ motif-containing GTPase-activating protein 3 (IQGAP3; IQ3) is a member of the multifunctional IQGAP family. The interaction between IQ3 and Ras-ERK signaling has been reported, but how IQ3 regulates Ras-ERK signaling in malignancies is still unclear. The purpose of this study is to elucidate the role of IQ3 in coordinating diverse pathways, such as ERK-Ras and TGFβ in gastric cancer (GC).According to Stomach Adenocarcinoma of TCGA Pan-Cancer Atlas, the frequency of gene mutations is high in TP53 (60.1%), RTK-Ras (51.8%), and TGFβ pathways (36.4%). Gene Set Enrichment Analysis (GSEA) indicated that KRAS signaling was significantly downregulated after IQ3 knockdown (KD) in three GC cell lines (AGS, NUGC3, Hs746T). Ingenuity upstream regulator analysis (IPA) of RNA sequencing data revealed that TGFβ1, the top-ranked growth factor, is significantly inhibited after IQ3 KD. Immunoblot analysis showed that IQ3 KD led to suppression of TGFβ-induced phosphorylation of MEK/ERK in three GC cell lines, particularly NUGC3. While TGFβ treatment promoted invasion and migration in transwell migration assays, IQ3 KD significantly diminished TGFβ-mediated cell migration. In vivo, the size and volume of subcutaneous tumors from IQ3 KD NUGC3 cells were significantly smaller than that from Control (Ctrl) cells. According to RNA sequencing using bulk tumors, mouse stromal cells in Ctrl tumors showed significantly higher expression of acta2, fap, pdgfa, which are the markers of cancer-associated fibroblasts, than in IQ3 KD tumors. The TGFβ1 expression levels in the NUGC3 xenograft and mouse stromal cells harvested from Ctrl were significantly higher than that from IQ3 KD, respectively. Additionally, digital spatial profiler (DSP) GeoMx evaluation of the intra-tumoral expressions from the human xenograft and mouse stromal cells showed that the mouse stromal cells from Ctrl expressed significantly higher acta2 and tgfβ1 than that from IQ3 KD. Furthermore, metastasis assays via tail vein injection in vivo showed dramatically lesser lung metastases from IQ3 KD cells than that from Ctrl cells. This study shows that IQ3 KD suppressed Ras signaling and blocked upstream regulator TGFβ1 in vitro. The stromal cells in the IQ3 KD xenografts showed a reduced population of cancer-associated fibroblasts in vivo. Consequently, tumorigenesis and metastasis were strongly suppressed after IQ3 KD. IQ3 is thus a highly relevant therapy target in GC. Citation Format: Mitsuhiro Shimura, Pang SHUCHIN, Junichi Matsuo, Linda Shyue Chuang, Yoshiaki Ito. IQGAP3 regulates intra-cellular Ras signaling and intra-tumoral malignant cycles via TGFβ signaling [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 2618.

Abstract 1344: myCAFs and iCAFs have similar lineage

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense stroma primarily composed of cancer associated fibroblasts (CAFs). These fibroblasts are a diverse population of cells; some groups of fibroblasts have been shown to play tumor supporting roles while others have been shown to be tumor suppressing. Two prolific and well-characterized CAF subtypes—myofibroblasts (myCAFs) and inflammatory CAFs (iCAFs)—could potentially have different regulatory effects on tumor growth. Understanding the tissue origin of these two groups of CAFs could assist in understanding the complex and heterogeneous roles they might play in PDAC development. Previous studies in our group showed that the splanchnic mesenchyme, a layer of mesenchyme adjacent to the fetal pancreatic epithelium, gives rise to the majority of the total CAF population. Here, I aim to test the hypothesis that both iCAFs and myCAFs originate from the splanchnic mesenchyme. myCAFs have been previously observed to be proximal to tumor cells and express αSMA at a higher level, while iCAFs have been observed to be distal to tumor cells and express αSMA at a lower level. Here, we used a genetically engineered mouse PDAC model carrying KrasG12D/+;p53Frt/+;Pdx1FlpO/+;Isl1cre/+;R26Tomato/+ alleles. In this model, epithelial specific FlpO expression leads to Kras activation and p53 deletion, causing tumorigenesis. Simultaneously, splanchnic specific Cre expression leads to permanent tomato expression in the splanchnic descendants. The pancreata of these mice were harvested, fixed, and mounted onto slides. Co-immunostaining with αSMA and tomato was then done. The stained slides were scanned, and the Inform software was used to segment and phenotype the cells. More than 95% of αSMA high cells were tomato positive, and more than 95% of αSMA low cells were also tomato positive. We thus conclude that both myCAFs and iCAFs originate from the splanchnic mesenchyme. Consistent with previous in vitro studies, this lineage tracing study suggests that different CAF subtypes are likely due to patterning influenced by different factors within the tumor microenvironment. Citation Format: Thomas S. Walter, Lu Han, Michael Ostrowski. myCAFs and iCAFs have similar lineage [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 1344.

Single-Cell Analysis Reveals Transcriptomic Features of Drug-Tolerant Persisters and Stromal Adaptation in a Patient-Derived EGFR-Mutated Lung Adenocarcinoma Xenograft Model.

Targeted therapies require life-long treatment, as drug discontinuation invariably leads to tumor recurrence. Recurrence is mainly driven by minor subpopulations of drug-tolerant persister (DTP) cells that survive the cytotoxic drug effect. In lung cancer, DTP studies have mainly been conducted with cell line models. We conducted an invivo DTP study using a lung adenocarcinoma patient-derived xenograft tumor driven by an EGFR mutation. Daily treatment of tumor-bearing mice for 5 to 6 weeks with the EGFR inhibitor erlotinib markedly shrunk tumors and generated DTPs, which were analyzed by whole exome, bulk population transcriptome, and single-cell RNA sequencing. The DTP tumors maintained the genomic clonal architecture of untreated baseline (BL) tumors but had reduced proliferation. Single-cell RNA sequencing identified a rare (approximately 4%) subpopulation of BL cells (DTP-like) with transcriptomic similarity to DTP cells and intermediate activity of pathways that are up-regulated in DTPs. Furthermore, the predominant transforming growth factor-β activated cancer-associated fibroblast (CAF) population in BL tumors was replaced by a CAF population enriched for IL6 production. Invitro experiments indicate that these populations interconvert depending on the levels of transforming growth factor-β versus NF-κB signaling, which is modulated by tyrosine kinase inhibitor presence. The DTPs had signs of increased NF-κB and STAT3 signaling, which may promote their survival. The DTPs may arise from a specific preexisting subpopulation of cancer cells with partial activation of specific drug resistance pathways. Tyrosine kinase inhibitor treatment induces DTPs revealing greater activation of these pathways while converting the major preexisting CAF population into a new state that may further promote DTP survival.

Cancer-Associated Fibroblasts: Lessons from Pancreatic Cancer

Cancer-associated fibroblasts (CAFs) are present in all malignancies. Arguably, in none are they as prevalent as they are in pancreatic ductal adenocarcinoma (PDAC), where they often outnumber cancer cells. The origin and function of CAFs are still not completely understood, and attempts to target this cell population as a component of combination therapy have so far not succeeded. Our understanding of pancreatic CAFs is in rapid evolution. Heterogeneity of CAFs is the key concept to understand this cell population. We discuss heterogeneity of origin, with recent findings challenging the notion that CAFs uniformly derive from pancreatic stellate cells, and instead suggesting that multiple types of resident fibroblasts contribute to CAF expansion. Heterogeneity in gene expression divides CAFs in different subpopulations. Most importantly, heterogeneity in function underlies the complexity of CAFs. CAFs deposit components of the extracellular matrix, contributing to the high interstitial pressure in pancreatic cancer. CAFs serve as “feeder” cells for cancer cells by providing metabolites, thus mitigating the effect of the low-nutrient environment of PDAC. At the same time, CAFs regulate the function of the immune system, inhibiting antitumor immune responses. Understanding the functional role of different CAF populations and the drivers of each of their functional roles is key to devising new ways to target this cell population in PDAC.

Fibroblast Isolation from Mammary Gland Tissue and Syngeneic Murine Breast Cancer Models.

Cancer-associated fibroblasts (CAFs) are vital within the tumor ecosystem, regulating tumor growth, dissemination, and response to therapy through crosstalk with tumor cells, infiltrating immune and vascular cells, as well as components of the extracellular matrix (ECM). CAFs have thus emerged as potential therapeutic targets to complement cancer cell-targeted therapies. To study CAF-tumor cell crosstalk ex vivo, robust isolation methods of primary CAFs are required. Here, we present protocols to isolate, expand, and culture two types of fibroblasts: (1) healthy murine mammary gland fibroblasts, a key source of the CAF population in breast tumor models and (2) CAFs derived from syngeneic murine breast tumors. Isolated mammary fibroblasts and CAFs are suitable for use in a variety of downstream cellular and molecular experiments. We expect these methods to be useful to scientists studying the properties of fibroblasts and CAFs and the interaction between CAFs and the various components of the tumor microenvironment (TME).