Carcinoma-associated Fibroblasts Research Articles

Phenotypic heterogeneity, stability and plasticity in tumor-promoting carcinoma-associated fibroblasts.

Reciprocal interactions between cancer cells and stromal cells in the tumor microenvironment (TME) are essential for full-blown tumor development. Carcinoma-associated fibroblasts (CAFs) are a key component of the TME together with a wide variety of stromal cell types including vascular, inflammatory, and immune cells in the extracellular matrix. CAFs not only promote tumor growth, invasion, and metastasis, but also dampen the efficacy of various therapies including immune checkpoint inhibitors. CAFs are composed of distinct fibroblast populations presumably with diverse activated fibroblastic states and tumor-promoting phenotypes in a tumor, indicating intratumor heterogeneity in these fibroblasts. Given that CAFs have been implicated in both disease progression and therapeutic responses, elucidating the functional roles of each fibroblast population in CAFs and the molecular mechanisms mediating their phenotypic stability and plasticity in the TME would be crucial for understanding tumor biology. We herein discuss how distinct fibroblast populations comprising CAFs establish their cell identities, in terms of cells-of-origin, stimuli from the TME, and the phenotypes characteristic of activated states.

Sulfatase 2-Induced Cancer-Associated Fibroblasts Promote Hepatocellular Carcinoma Progression via Inhibition of Apoptosis and Induction of Epithelial-to-Mesenchymal Transition

BackgroundSulfatase 2 (SULF2) removes the 6-O-sulfate groups from heparan sulfate proteoglycans (HSPG) and consequently alters the binding sites for various signaling molecules. Here, we elucidated the role of SULF2 in the differentiation of hepatic stellate cells (HSCs) into carcinoma-associated fibroblasts (CAFs) in the hepatocellular carcinoma (HCC) microenvironment and the mechanism underlying CAF-mediated HCC growth.MethodsThe clinical relevance of SULF2 and CAFs was examined using in silico and immunohistochemical (IHC) analyses. Functional studies were performed to evaluate the role of SULF2 in the differentiation of HSCs into CAFs and elucidate the mechanism underlying CAF-mediated HCC growth. Mechanistic studies were performed using the chromatin immunoprecipitation, luciferase reporter, and RNA immunoprecipitation assays. The in vitro findings were verified using the nude HCC xenograft mouse model.ResultsThe Cancer Genome Atlas (TCGA) database and IHC analyses revealed that the expression of CAF markers, which was positively correlated with that of SULF2 in the HCC tissues, predicted unfavorable postsurgical outcomes. Co-culturing HSCs with HCC cells expressing SULF2 promoted CAF differentiation. Additionally, CAFs repressed HCC cell apoptosis by activating the SDF-1/CXCR4/PI3K/AKT signaling pathway. Meanwhile, SULF2-induced CAFs promoted epithelial-to-mesenchymal transition (EMT) of HCC cells by modulating the SDF-1/CXCR4/OIP5-AS1/miR-153-3p/SNAI1 axis. Studies using HCC xenograft mouse models demonstrated that OIP5-AS1 induced EMT by upregulating SNAI1 and promoted HCC growth in vivo.ConclusionThese data indicated that SULF2 secreted by the HCC cells induced the differentiation of HSCs into CAFs through the TGFβ1/SMAD3 signaling pathway. SULF2-induced CAFs attenuated HCC apoptosis by activating the SDF-1/CXCR4/PI3K/AKT signaling pathway and induced EMT through the SDF-1/CXCR4/OIP5-AS1/miR-153-3p/SNAI1 axis. This study revealed a novel mechanism involved in the crosstalk between HCC cells and CAFs in the tumor microenvironment, which can aid in the development of novel and efficient therapeutic strategies for primary liver cancer.

Pancreatic cancer is suppressed by fibroblast-derived collagen I.

Clinical implementation of anti-stromal therapies in pancreatic cancer has been delayed by unanticipated tumor-restraining properties of the desmoplastic stroma. In confronting these challenges, Chen etal. demonstrate in this issue of Cancer Cell that fibroblast-specific deletion of collagen I, in the background of oncogenic Kras-induced spontaneous murine pancreatic ductal adenocarcinoma, enhances immune suppression and accelerates progression of disease.

Tumor-penetrating therapy for \u03b25 integrin-rich pancreas cancer

Pancreatic ductal adenocarcinoma (PDAC) is characterized by marked desmoplasia and drug resistance due, in part, to poor drug delivery to extravascular tumor tissue. Here, we report that carcinoma-associated fibroblasts (CAFs) induce β5 integrin expression in tumor cells in a TGF-β dependent manner, making them an efficient drug delivery target for the tumor-penetrating peptide iRGD. The capacity of iRGD to deliver conjugated and co-injected payloads is markedly suppressed when β5 integrins are knocked out in the tumor cells. Of note, β5 integrin knock-out in tumor cells leads to reduced disease burden and prolonged survival of the mice, demonstrating its contribution to PDAC progression. iRGD significantly potentiates co-injected chemotherapy in KPC mice with high β5 integrin expression and may be a powerful strategy to target an aggressive PDAC subpopulation.

Tumour microenvironment-based molecular profiling reveals ideal candidates for high-grade serous ovarian cancer immunotherapy.

ObjectiveDue to limited immunological profiles of high‐grade serous ovarian cancer (HGSOC), we aimed to characterize its molecular features to determine whether a specific subset that can respond to immunotherapy exists.Materials and MethodsA training cohort of 418 HGSOC samples from TCGA was analysed by consensus non‐negative matrix factorization. We correlated the expression patterns with the presence of immune cell infiltrates, immune regulatory molecules and other genomic or epigenetic features. Two independent cohorts containing 482 HGSOCs and in vitro experiments were used for validation.ResultsWe identified immune and non‐immune groups where the former was enriched in signatures that reflect immune cells, infiltration and PD‐1 signalling (all, P < 0.001), and presented with a lower chromosomal aberrations but increased neoantigens, tumour mutation burden, and microsatellite instability (all, P < 0.05); this group was further refined into two microenvironment‐based subtypes characterized by either immunoactivation or carcinoma‐associated fibroblasts (CAFs) and distinct prognosis. CAFs‐immune subtype was enriched for factors that mediate immunosuppression and promote tumour progression, including highly expressed stromal signature, TGF‐β signalling, epithelial‐mesenchymal transition and tumour‐associated M2‐polarized macrophages (all, P < 0.001). Robustness of these immune‐specific subtypes was verified in validation cohorts, and in vitro experiments indicated that activated‐immune subtype may benefit from anti‐PD1 antibody therapy (P < 0.05).ConclusionOur findings revealed two immune subtypes with different responses to immunotherapy and indicated that some HGSOCs may be susceptible to immunotherapies or combination therapies.

Stromal WNTer Keeps the Tumor Cold and Drives Metastasis

Carcinoma-associated fibroblast (CAF) infiltration confers poor clinical outcomes in colorectal cancer (CRC) through mechanisms that are still unclear. In this issue of Developmental Cell, Kasashima etal. report that loss of PKCζ engenders a SOX2/SFRP2-positive CAF subpopulation that increases CRC aggressiveness by creating an immunosuppressed environment.

Carcinoma-Associated Fibroblasts Promote Growth of Sox2-Expressing Breast Cancer Cells.

Simple SummaryThe tumor microenvironment has a strong impact on the behavior of tumor cells. One major cell type residing in the tumor microenvironment is the carcinoma-associated fibroblast (CAF). We were interested in the effect of CAFs on Sox2 (sex determining region Y (SRY)-box 2), which not only is an essential embryonal stem cell transcription factor, but also plays a role in cancer stem cell activity. We found that long-term exposure of ERα-positive breast cancer cells to the cocktail of CAF-secreted factors strongly increased Sox2 expression involving tumor-related proteins and signaling pathways. However, Sox2 was not only present in those tumor cells that express stem cell markers, but was equally abundant in other tumor cells. By being widely expressed, Sox2 may have functions in non-stem cells. In fact, Sox2 was found to regulate ERα expression, to act anti-apoptotically, to promote cellular growth and to protect cells against the anti-estrogen fulvestrant.CAFs (Carcinoma-associated fibroblasts) play an important role in cancer progression. For instance, they promote resistance to anti-estrogens, such as fulvestrant. Here, we show that, in ERα-positive breast cancer cell lines, the cocktail of factors secreted by CAFs (CAF-CM) induce the expression of the embryonal stem cell transcription factor Sox2 (sex determining region Y (SRY)-box 2). Long-term exposure to CAF-CM was able to give rise to very high Sox2 levels both in the absence and presence of fulvestrant. IL-6 (interleukin-6), a major component of CAF-CM, failed to raise Sox2 expression. In MCF-7 sublines established in the presence of CAF-CM, almost all cells showed Sox2 expression, whereas long-term treatment of T47D cells with CAF-CM resulted in a ~60-fold increase in the proportions of two distinct populations of Sox2 high and low expresser cells. Exposure of BT474 cells to CAF-CM raised the fraction of Sox2 high expresser cells by ~3-fold. Cell sorting based on CD44 and CD24 expression or ALDH (aldehyde dehydrogenase) activity revealed that most Sox2 high expresser cells were not CD44hi/CD24lo- or ALDH-positive cells suggesting that they were not CSCs (cancer stem cells), though CD44 played a role in Sox2 expression. Functionally, Sox2 was found to protect CAF-CM-treated cells against apoptosis and to allow higher growth activity in the presence of fulvestrant. Mechanistically, the key drivers of Sox2 expression was found to be STAT3 (Signal transducer and activator of transcription 3), Bcl-3 (B-cell lymphoma 3) and the PI3K (Phosphoinositide 3-kinase)/AKT pathway, whose activities/expression can all be upregulated by CAF-CM. These data suggest that CAF-CM induces Sox2 expression in non-CSCs by activating proteins involved in growth control and drug resistance, leading to higher protection against apoptosis.

The Crosstalk Between Tumor-Associated Macrophages (TAMs) and Tumor Cells and the Corresponding Targeted Therapy.

Tumor microenvironment (TME) is composed of tumor cells and surrounding non-tumor stromal cells, mainly including tumor associated macrophages (TAMs), endothelial cells, and carcinoma-associated fibroblasts (CAFs). The TAMs are the major components of non-tumor stromal cells, and play an important role in promoting the occurrence and development of tumors. Macrophages originate from bone marrow hematopoietic stem cells and embryonic yolk sacs. There is close crosstalk between TAMs and tumor cells. With the occurrence of tumors, tumor cells secrete various chemokines to recruit monocytes to infiltrate tumor tissues and further promote their M2-type polarization. Importantly, M2-like TAMs can in turn accelerate tumor growth, promote tumor cell invasion and metastasis, and inhibit immune killing to promote tumor progression. Therefore, targeting TAMs in tumor tissues has become one of the principal strategies in current tumor immunotherapy. Current treatment strategies focus on reducing macrophage infiltration in tumor tissues and reprogramming TAMs to M1-like to kill tumors. Although these treatments have had some success, their effects are still limited. This paper mainly summarized the recruitment and polarization of macrophages by tumors, the support of TAMs for the growth of tumors, and the research progress of TAMs targeting tumors, to provide new treatment strategies for tumor immunotherapy.

Stromal microenvironment promoted infiltration in esophageal adenocarcinoma and squamous cell carcinoma: a multi-cohort gene-based analysis

The stromal microenvironment has been shown to affect the infiltration of esophageal carcinoma (ESCA), which is linked to prognosis. However, the complicated mechanism of how infiltration is influenced by the stromal microenvironment is not well-defined. In this study, a stromal activation classifier was established with ridge cox regression to calculate stroma scores for training (n = 182) and validation cohorts (n = 227) based on the stroma-related 32 hub genes identified by sequential bioinformatics algorithms. Patients with high stromal activation were associated with high T stage and poor prognosis in both esophagus adenocarcinoma and esophagus squamous cell carcinoma. Besides, comprehensive multi-omics analysis was used to outline stromal characterizations of 2 distinct stromal groups. Patients with activated tumor stoma showed high stromal cell infiltration (fibroblasts, endothelial cells, and monocyte macrophages), epithelial-mesenchymal transition, tumor angiogenesis and M2 macrophage polarization (CD163 and CD206). Tumor mutation burden of differential stromal groups was also depicted. In addition, a total of 6 stromal activation markers in ESCA were defined and involved in the function of carcinoma-associated fibroblasts that were crucial in the differentiation of distinct stromal characterizations. Based on these studies, a practical classifier for the stromal microenvironment was successfully proposed to predict the prognosis of ESCA patients.

Small extracellular vesicle-bound vascular endothelial growth factor secreted by carcinoma-associated fibroblasts promotes angiogenesis in a bevacizumab-resistant manner.

The blood vessel growth inhibitor bevacizumab targets vascular endothelial growth factor (VEGF), a crucial regulator of angiogenesis. Recently, small extracellular vesicles (sEVs) have been demonstrated to be important vehicles in the transport of growth factors to target cells. In this study, we isolated primary carcinoma-associated fibroblasts (CAFs) from four human oral squamous cell carcinoma (OSCC) specimens. Compared with other non-extracellular vesicle components, CAF-derived sEVs were found to be the main regulators of angiogenesis. The ability of CAF sEVs to activate VEGF receptor 2 (VEGFR2) signaling in human umbilical vein endothelial cells (HUVEC) was dependent on the association between sEVs and VEGF. In addition, sEV-bound VEGF secreted by CAFs further activated VEGFR2 signaling in HUVEC in a bevacizumab-resistant manner. VEGF was found to interact with heparan sulfate proteoglycans on the CAF sEV surface and could be released by heparinase I/III. The bioactivity of the dissociated VEGF was retained in vitro and in vivo and could be neutralized by bevacizumab. These findings suggest that the combined use of heparinase and bevacizumab might inhibit angiogenesis in patients with high levels of sEV-bound VEGF.

Modulating tumor reactive stroma by extracorporeal shock wavesto control prostate cancer progression.

Prostate cancer is the second most common cancer worldwide. Tumor microenvironment is composed of activated fibroblasts, the so called carcinoma-associated fibroblasts (CAFs). They express high levels of α-smooth muscle actin (α-SMA) and type I collagen (COL1), and support proliferation and migration of tumor epithelial cells. Extracorporeal shock waves (ESWs), acoustic waves, are effective in the treatment of hypertrophic scars, due to their ability to modulate fibrosis. Based on this rationale, the study evaluated the effects of ESWs on CAF activation and the influence of ESW-treated CAFs on the growth and migration of epithelial prostatic carcinoma cells. Primary cultures of CAFs (n = 10) were prepared from tumors of patients undergoing surgery for high-risk prostate carcinoma. CAFs were treated with ESWs (energy levels: 0.32 mJ/mm2 , 1000 pulses; 0.59 mJ/mm2 , 250 pulses). After treatment, the messenger RNA and protein levels of the stromal activation markers α-SMA and COL1 were determined. Subsequently, two different stabilized cell lines (PC3 and DU145) of androgen-resistant prostate cancer were treated with the conditioned media produced by ESW-treated CAFs. At different times, viability and migration of PC3 and DU145 cells were evaluated. Viability was also assessed by coculture system using CAFs and PC3 or DU145 cells. ESWs reduced gene expression and protein level of α-SMA and COL1 in CAFs. The treatment of PC3 and DU145 with conditioned media of ESW-treated CAFs determined a reduction of their growth and invasive potential. Coculture systems between ESW-treated CAFs and PC3 or DU145 cells confirmed the epithelial cell number reduction. This in vitro study demonstrates for the first time that ESWs are able to modulate the activation of prostate CAFs in favor of a less "reactive" stroma, with consequent slowing of the growth and migration of prostate cancer epithelial cells. However, only further studies to be performed in vivo will confirm the possibility of using this new therapy in patients with prostate cancer.

Conditioned medium of the osteosarcoma cell line U2OS induces hBMSCs to exhibit characteristics of carcinoma-associated fibroblasts via activation of IL-6/STAT3 signalling

As a research hotspot in recent years, bone mesenchymal stem cells (BMSCs) play an important role in the process of a variety of human diseases, including cancers. However, in osteosarcoma, the role of BMSCs and their communication with tumour cells are not clear. In this study, we validated the communication of osteosarcoma (OS) cells with BMSCs. The results showed that the conditioned medium of osteosarcoma cell line U2OS (U2OS-CM) induces the carcinoma-associated fibroblasts (CAFs)-like transformation of BMSCs and promotes the proliferation, migration and invasion of BMSCs. Mechanistically, treatment of human bone mesenchymal stem cells (hBMSCs) with U2OS-CM results in a significant increase in the IL-6 expression and phosphorylation of STAT3. Furthermore, blockade of the IL-6/STAT3 signalling in hBMSCs rescues the transformation of CAF phenotype induced by U2OS-CM. And, human IL-6 can directly increase the expression of the CAF marker genes in hMSCs. Meanwhile, IL-6/STAT3 signalling involves in promoting effects of U2OS-CM on the proliferation, migration and invasion of BMSCs. In summary, our results suggest that BMSCs communicate with OS cells through IL-6/STAT3 signalling and play an important role in the progress of osteosarcoma.

4078 Identification of distinct fibroblast populations with unique roles in pancreatic cancer progression and tumor immunity

OBJECTIVES/GOALS: The desmoplastic reaction in PDAC involves a significant accumulation of immune cells and fibroblasts.The functional diversity of carcinoma associated fibroblasts (CAFs) remains largely unknown, and identification of immune regulating subsets would have a substantial impact in augmentation of immunotherapy efficacy. METHODS/STUDY POPULATION: Employing histology, FACs, multiplex immunohistochemistry, single cell RNA sequencing (sc-RNA-seq) and genetically engineered mouse models, we demonstrate that aSMA+ cells are a dominant CAF population in PDAC with tumor restraining properties (TS-CAFs), as opposed those of the FAP+ CAFs, which demonstrate tumor promoting activity (TP-CAFs). RESULTS/ANTICIPATED RESULTS: Analysis of bulk tumor depleted of either TS-CAFs or TP-CAFs showed that TS-CAFs predominantly modulate extracellular matrix (ECM) production, facilitate cell-ECM adhesion and regulate adaptive immunity, while TP-CAFs exhibit a lineage that is skewed towards a pro-inflammatory, chemokine secreting phenotype. Further, scRNA-Seq analyses demonstrate that CAFs share distinct gene expression profiles characteristic of lymphocytic and myeloid lineages. Together our data distinguish two populations of CAFs, one which is tumor suppressing with roles in ECM remodeling and another which is tumor promoting with roles in cytokine production, both with immune modulating capabilities. DISCUSSION/SIGNIFICANCE OF IMPACT: Our study identifies a complex network of functionally heterogeneous fibroblasts during PDAC progression with significant immunotherapeutic implication. The identification of distinct fibroblast subsets will allow us to discriminately target fibroblast populations to augment immunotherapy efficacy in pancreatic cancer.

Immunoscore Predicts Survival in Early-Stage Lung Adenocarcinoma Patients.

Background: The lung cancer staging system is insufficient for a comprehensive evaluation of patient prognosis. We constructed a novel immunoscore model to predict patients with high risk and poor survival.Method: Immunoscore was developed based on z-score transformed enrichment score of 11 immune-related gene sets of 109 immune risk genes. The immunoscore model was trained in lung adenocarcinoma cohort from The Cancer Genome Atlas (TCGA-LUAD) (n = 400), and validated in other two independent cohorts from Gene Expression Omnibus (GEO), GSE31210 (n = 219) and GSE68465 (n = 356). Meta-set (n = 975) was formed by combining all training and testing sets.Result: High immunoscore conferred worse prognosis in all sets. It was an independent prognostic factors in multivariate Cox analysis in training, testing and meta-set [hazard ratio (HR) = 2.96 (2.24–3.9), P < 0.001 in training set; HR = 1.99 (1.21–3.26), P = 0.006 in testing set 1; HR = 1.48 (1.69–2.39), P = 0.005 in testing set 2; HR = 2.01 (1.69–2.39), P < 0.001 in meta-set]. Immunoscore-clinical prognostic signature (ICPS) was developed by integrating immunoscore and clinical characteristic, and had higher C-index than immunoscore or stage alone in all sets [0.72 (ICPS) vs. 0.7 (immunoscore) or 0.59 (stage) in training set; 0.75 vs. 0.72 or 0.7 in testing set 1; 0.65 vs. 0.61 or 0.62 in testing set 2; 0.7 vs. 0.66 or 0.64 in meta-set]. Genome analysis revealed that immunoscore was positively correlated with tumor mutation burden (R = 0.22, P < 0.001). Besides, high immunoscore was correlated with high proportion of carcinoma-associated fibroblasts (R = 0.32, P < 0.001) in tumor microenvironment but fewer CD8+ cells infiltration (R = −0.28, P < 0.001).Conclusion: The immunoscore and ICPS are potential biomarkers for evaluating patient survival. Further investigations are required to validate and improve their prediction accuracy.

Novel module and hub genes of distinctive breast cancer associated fibroblasts identified by weighted gene co-expression network analysis.

As abundant and heterogeneous stromal cells in tumor microenvironment, carcinoma-associated fibroblasts (CAFs) are critically involved in cancer progression. To identify co-expression module and hub genes of distinctive breast CAFs, weighted gene co-expression network analysis (WGCNA) was conducted based on the expression array results of CAFs from seven chemo-sensitive breast cancer (BC) patients and seven chemo-resistant ones before neo-adjuvant chemotherapy. A total of 4916 genes were included in WGCNA, and 12 modules were determined. Module-trait assay showed that the blue module (cor = 0.97, P < 0.001) was associated with CAF-related chemo-resistance, which was enriched mainly as "inflammatory response", "interferon-gamma-mediated signaling" and "NIK/NF-kappaB signaling" pathways. Moreover, CXCL8, CXCL10, CXCL11, PLSCR1, RIPK2 and USP18 were found to be potentially associated with chemo-resistance related to CAFs and prognosis of BC. Our current data offered valuable insights into the molecular mechanisms of distinctive breast CAFs, which was beneficial for revealing how chemo-resistance of BC was initiated.

MiR-3613-3p from carcinoma-associated fibroblasts exosomes promoted breast cancer cell proliferation and metastasis by regulating SOCS2 expression.

Exosomes carrying microRNAs (miRNAs) mediate cell-to-cell communication, which play important roles in cancer growth and progression. However, the roles and molecular mechanisms of the miRNAs in the exosomes from carcinoma-associated fibroblasts (CAFs) are still not clear. The miRNA array showed that miR-3613-3p was an upregulated miRNA in CAFs exosomes. It was verified that miR-3613-3p was upregulated in exosomes from fibroblasts educated by TGF-β1 and the fibroblasts from breast cancer tissues. Exosomal miR-3613-3p promoted breast cancer cell proliferation and metastasis. The cellular functions showed that miR-3613-3p downregulation in the CAFs exosomes suppressed cell proliferation and metastasis in breast cancer by targeting SOCS2 expression. The clinical data showed that miR-3613-3p levels were negatively related to SOCS2 expression in breast cancer tissues. In a conclusion, the study demonstrated that activated fibroblasts exosomes with high levels of miR-3613-3p played an oncogenic role in breast cancer cell survival and metastasis, which suggested that miR-3613-3p function as a therapeutic target.

Abstract A19: Metronomic chemotherapy enhances immunotherapy by preventing stroma-induced immunosuppression

Abstract Chemotherapy (C/T) drugs are usually administered to cancer patients every few weeks at a high, “maximum tolerated” dose (MTD). Though this approach kills the majority of tumor cells, it often spares a small number of stem-like cancer cells (CSCs) that subsequently give rise to new tumors. Moreover, these recurring tumors are often more aggressive and able to metastasize to other tissues, in part because high doses of C/T drugs also affect cells in the stromal tissue that surrounds tumors, including carcinoma-associated fibroblasts (CAFs) and immune cells. Many human solid tumors are characterized by a pronounced stromal reaction characterized by a desmoplastic response produced by CAFs as well as an extensive infiltration by immunosuppressive cell populations such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). Importantly, both the tumor epithelium and adjacent stroma respond to systemic C/T and a therapy-modified stroma can deleteriously influence treatment efficacy. Recently, we have demonstrated in assorted mouse tumor models that traditional C/T using the MTD regimens of C/T agents such as cyclophosphamide, doxorubicin, gemcitabine, and paclitaxel could paradoxically render CAFs oncogenic through the stromal-epithelial ELR+-chemokine/CXCR-2 signaling axis, which expands CSCs, enhances tumor neovascularization, and induces immunosuppressive M2 subtype TAM infiltration, together leading to tumor recurrence and treatment refractoriness (Chan et al., J. Exp. Med 2016). By contrast, the same total accumulated dose of the C/T agents administered as a low-dose metronomic (LDM) regimen could largely prevent the therapy-induced stromal alterations and CSC expansion and thereby substantially enhance the treatment response. We subsequently showed that LDM C/T not only obviated the MTD-C/T-elicited CSC expansion but also attenuated the influx of the highly immunosuppressive granulocytic MDSCs, thereby potentiating immunotherapy such as anti-PD-1. We thus anticipate that, while pharmacologic inhibition of CXCR-2 has enhanced the antitumor efficacy of immune checkpoint blockade, LDM C/T may provide a clinically applicable alternative of enhancing immune surveillance in stroma-rich and T-cell inflamed tumors. Collectively, our studies provide compelling evidence and novel mechanistic insights into the unique benefit of LDM C/T on the tumor stroma. Given that MTD C/T exerts favorable influences on tumor stemness and immunity, it may better synergize with emerging CSC-targeted or immunotherapeutics than traditional MTD C/T to further enhance the therapeutic outcome of human solid tumors. Citation Format: Kelvin K. Tsai, Tze-sian Chan. Metronomic chemotherapy enhances immunotherapy by preventing stroma-induced immunosuppression [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A19.

Abstract A108: Identification of distinct fibroblast populations with unique roles in PDAC progression and tumor immunity

Abstract The desmoplastic reaction in pancreatic ductal adenocarcinomas (PDAC) involves a significant accumulation of immune cells and fibroblasts. Although carcinoma-associated fibroblasts (CAFs) have been associated with immune suppression and immunotherapy resistance in PDAC mouse models, some studies have shown immune-promoting and tumor-suppressing roles of CAFs. The functional diversity of carcinoma-associated fibroblasts (CAFs) remains largely unknown, and identification of immune-regulating subsets would have a substantial impact in augmentation of immunotherapy efficacy. Employing histology, FACs, multiplex immunohistochemistry, single-cell RNA sequencing (sc-RNA-seq), and genetically engineered mouse models, we demonstrate that SMA+ cells are a dominant CAF population in PDAC with tumor-restraining properties (TS-CAFs), as opposed those of the FAP+ CAFs, which demonstrate tumor-promoting activity (TP-CAFs). Analysis of bulk tumor depleted of either TS-CAFs or TP-CAFs showed that TS-CAFs predominantly modulate extracellular matrix (ECM) production, facilitate cell-ECM adhesion, and regulate adaptive immunity, while TP-CAFs exhibit a lineage that is skewed towards a proinflammatory, chemokine-secreting phenotype. Further, scRNA-Seq analyses demonstrate that CAFs share distinct gene expression profiles characteristic of lymphocytic and myeloid lineages. Together our data distinguish two populations of CAFs, one that is tumor suppressing with roles in ECM remodeling and another that is tumor promoting with roles in cytokine production, both with immune-modulating capabilities. Collectively, our study identifies a complex network of functionally heterogeneous fibroblasts during PDAC progression with significant immunotherapeutic implication. Citation Format: J. Kebbeh Darpolor, Xiaofeng Zheng, Sujuan Yang, Kathleen M. McAndrews, Julienne L. Carstens, Patricia E. Phillips, Hikaru Sugimoto, Pedro Correa De Sampaio, Chia-Chin Wu, Valerie S. LeBleu, Raghu Kalluri. Identification of distinct fibroblast populations with unique roles in PDAC progression and tumor immunity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A108.

Mesenchymal stem cells maintain the stemness of colon cancer stem cells via interleukin-8/mitogen-activated protein kinase signaling pathway.

Mesenchymal stem cells (MSCs) can act as a carrier in tumor therapy, and tumor suppressor gene-modified MSCs can reach and suppress the tumor. However, in the colon cancer microenvironment, MSCs could promote tumor growth and create the environment that is conducive to the survival of cancer stem cells (CSCs). This study discovered MSCs from three sources (bone marrow, adipose, placenta) could induce the stemness and epithelial–mesenchymal transition (EMT) of HCT116 in vitro, meanwhile adipose- and placenta-derived MSCs increase the proportion of CD133+/CD44+ HCT116. Then, we explored the interaction mechanism between CD133+/CD44+ HCT116 and MSCs by the bioinformatics and in vitro assays. After CD133+/CD44+ HCT116 were co-cultured with MSCs, many cytokines in MSCs were stimulated, including interleukin-8 (IL-8). The binding of IL-8/CXCR2 activates the downstream mitogen-activated protein kinase (MAPK) signaling pathway in colon CSCs, thereby promoting the stemness and EMT. However, inhibition of IL-8/CXCR2/Erk1/2 could reverse the effect of MSCs on CSC stemness. In addition, MSCs co-cultured with CD133+/CD44+ HCT116 produce a carcinoma-associated fibroblast phenotype via intracellular FGF10–PKA–Akt–β-catenin signaling, which can be attenuated by IL-8 peptide inhibitor. To conclude, IL-8 promotes the interaction between colon CSCs and MSCs, and activates the MAPK signaling pathway in colon CSCs, which provides a theoretical basis for the application of MSCs in clinical practice. Impact statement MSCs have the property of chemotaxis and they can migrate to the tumor site by paracrine pathway in the tumor environment, and then interact with tumor cells. Although a mass of studies have been conducted about the impact of MSCs on tumors, it is still controversial whether the exogenous MSCs promote or inhibit tumor growth. In this work, we evaluated the effects of MSCs from three sources (bone marrow, adipose, placenta) on the proliferation, stemness, and metastasis of the colon cancer cells both in vitro and in vivo. Then, we proved the IL-8/CXCR2/MAPK and FGF10–PKA–Akt–β-catenin signaling pathway which mediate the interplay between MSC and CD133+/CD44+ colon cancer cell. This research aims to provide a theoretical basis for the safe application of MSCs in the clinical treatment of colon cancer.

Mesothelial-to-Mesenchymal Transition Contributes to the Generation of Carcinoma-Associated Fibroblasts in Locally Advanced Primary Colorectal Carcinomas.

During peritoneal metastasis, cancer cells spread from abdominal solid tumors, disseminate through the peritoneal fluid and attach to and invade through mesothelial cells (MCs) that line the peritoneum. Intestinal adenocarcinomas originating in the mucosa infiltrate the submucosa, muscle layer, and serosa in order to finally colonize the peritoneal cavity. However, the mechanism by which metastatic cells leave the primary tumor and reach the peritoneal cavity has not been previously described. Hence, we investigate whether MCs lining visceral peritoneum, through a mesothelial-to-mesenchymal transition (MMT), are a source of carcinoma-associated fibroblasts (CAFs), which could contribute to cancer progression toward the peritoneal cavity. CAFs detected in biopsies from patients with superficially invasive colorectal cancer differed from locally advanced tumors. An aberrant accumulation of myofibroblasts expressing mesothelial markers was found in the stroma of deeply infiltrative tumors located in the neighborhood of a frequently activated mesothelium. We suggest that MMT is a key event in the early stages of peritoneal dissemination.