Carcinoma-associated Fibroblasts Research Articles

FAP and PSMA Expression by Immunohistochemistry and PET Imaging in Castration-Resistant Prostate Cancer: A Translational Pilot Study.

Prostate-specific membrane antigen (PSMA) is a theranostic target for metastatic prostate cancer (PCa). However, castration-resistant PCa (CRPC) may lose PSMA expression after systemic therapy. Fibroblast activation protein (FAP), expressed by carcinoma-associated fibroblasts in various cancer types, including PCa, has the potential to be an alternative target. In this study, we evaluated FAP expression in CRPC to assess its potential, using PSMA as a comparison. Methods: FAP expression was assessed using immunohistochemistry in 116 CRPC tumors: 78 adenocarcinomas, 11 small cell carcinomas, and 27 anaplastic carcinomas. Correlation analysis between manual scoring and automated scoring was performed on 54 whole-slide sections of metastatic CRPC. Paired FAP and PSMA stains were assessed in tissue microarray cores of CRPC (n = 62), consisting of locally advanced CRPC (n = 9) and metastatic CRPC (n = 53). FAP and PSMA positivity was defined by an immunohistochemistry score of at least 10. To explore the correlation of PSMA and FAP inhibitor (FAPi) PET imaging and immunohistochemistry, a preliminary analysis of 4 patients included in a [68Ga]-FAPi-46 imaging trial (NCT04457232) was conducted. Results: Manual and automated scoring of FAP yielded results with strong correlations. Overall, FAP expression in CRPC was notably lower than PSMA expression (median immunoscores, 14 vs. 72; P < 0.001). Different histologic subtypes of CRPC demonstrated distinct levels of PSMA expression, whereas their FAP expression levels were comparable. Among the 19 PSMA-negative tumors, 11 (58%) exhibited FAP positivity. FAP expression levels in lymph node metastases were significantly lower than those in nonnodal metastases (P = 0.021). Liver metastases showed significant enrichment of tumors with strong FAP expression compared with nonliver lesions (P = 0.016). In the 4 clinical trial patients, the biopsied metastatic lesions showed lower uptake on FAPi PET than on PSMA PET (median SUVmax, 9.6 vs. 14.5), consistent with FAP expression that was lower than PSMA expression in the corresponding tumor biopsy samples (median immunoscores, 30 vs. 160). Conclusion: Because of the low FAP expression levels in CRPC, the utility of FAPi PET imaging may be limited. Although FAPi PET imaging may be further tested in PSMA-negative CRPC, such as small cell carcinoma, other molecular imaging modalities should be evaluated as alternative choices.

Extracellular vesicles derived from melanoma cells induce carcinoma-associated fibroblasts via miR-92b-3p mediated downregulation of PTEN.

In melanoma, carcinoma-associated fibroblasts (CAFs) are important cellular components in the tumour microenvironment due to their potential to promote tumour growth and metastatic spread of malignant cells. Melanoma cells have the ability to affect non-tumour cells in the microenvironment by releasing extracellular vesicles (EVs). The mechanisms responsible for reprogramming normal dermal fibroblasts (NHDFs) into CAFs remain incompletely understood. However, it is likely thought to be mediated by melanoma-specific miRNAs, which are transported by EVs derived from melanoma cells. Therefore, we wondered if one of the most enriched miRNAs in EVs secreted by melanoma cells, miR-92b-3p, is involved in the conversion of normal fibroblasts into CAFs. We observed that melanoma cell-derived EVs indeed delivered miR-92b-3p into NHDFs and that its accumulation correlated with CAF formation, as demonstrated by enhanced expression of CAF marker genes and increased proliferation and migration. Overexpression of miR-92b-3p in NHDFs revealed similar results, while EVs deficient of miR-92b-3p did not induce a CAF phenotype. As a target we identified PTEN, whose repression led to increased expression of CAF markers. We thus provide a novel pathway of intercellular communication by which melanoma cells control the transformation of CAFs by virtue of EV-transported miRNAs.

Paracrine Activation of STAT3 Drives GM-CSF Expression in Breast Carcinoma Cells, Generating a Symbiotic Signaling Network with Breast Carcinoma-Associated Fibroblasts.

This study evaluated the paracrine signaling between breast carcinoma-associated fibroblasts (CAFs) and breast cancer (BCa) cells. Resolving cell-cell communication in the BCa tumor microenvironment (TME) will aid the development of new therapeutics. Here, we utilized our patented TAME (tissue architecture and microenvironment engineering) 3D culture microphysiological system, which is a suitable pathomimetic avatar for the study of the BCa TME. We cultured in 3D BCa cells and CAFs either alone or together in cocultures and found that when cocultured, CAFs enhanced the invasive characteristics of tumor cells, as shown by increased proliferation and spread of tumor cells into the surrounding matrix. Secretome analysis from 3D cultures revealed a relatively high secretion of IL-6 by CAFs. A marked increase in the secretion of granulocyte macrophage-colony stimulating factor (GM-CSF) when carcinoma cells and CAFs were in coculture was also observed. We theorized that the CAF-secreted IL-6 functions in a paracrine manner to induce GM-CSF expression and secretion from carcinoma cells. This was confirmed by evaluating the activation of STAT3 and gene expression of GM-CSF in carcinoma cells exposed to CAF-conditioned media (CAF-CM). In addition, the treatment of CAFs with BCa cell-CM yielded a brief upregulation of GM-CSF followed by a marked decrease, indicating a tightly regulated control of GM-CSF in CAFs. Secretion of IL-6 from CAFs drives the activation of STAT3 in BCa cells, which in turn drives the expression and secretion of GM-CSF. As a result, CAFs exposed to BCa cell-secreted GM-CSF upregulate inflammation-associated genes such as IL-6, IL-6R and IL-8, thereby forming a positive feedback loop. We propose that the tight regulation of GM-CSF in CAFs may be a novel regulatory pathway to target for disrupting the CAF:BCa cell symbiotic relationship. These data provide yet another piece of the cell-cell communication network governing the BCa TME.

Detection of Carcinoma-Associated Fibroblasts Derived from Mesothelial Cells via Mesothelial-to-Mesenchymal Transition in Primary Ovarian Carcinomas.

Carcinoma-associated fibroblasts (CAFs) are highly accumulated in the tumor-surrounding stroma of primary epithelial ovarian cancer (OC). CAFs exert important functions for the vascularization, growth, and progression of OC cells. However, the origin of CAFs in primary OC had not yet been studied, and they were assumed to arise from the activation of resident fibroblasts. Here, we compared CAFs in the ovary to CAFs found in peritoneal metastases from patients with advanced OC. Our findings show that CAFs from primary tumors and peritoneal metastases share the expression of mesothelial markers. Therefore, similar to peritoneal carcinomatosis, CAFs in primary ovarian carcinomas may originate from mesothelial cells via a mesothelial-to-mesenchymal transition. The detection of mesothelial-derived CAFs in tumors confined to the ovary and identification of biomarkers could be the key to the early detection of OC and peritoneal spread.

Retraction: Tumor-derived extracellular vesicles promote activation of carcinoma-associated fibroblasts and facilitate invasion and metastasis of ovarian cancer by carrying miR-630

Retraction: Tumor-derived extracellular vesicles promote activation of carcinoma-associated fibroblasts and facilitate invasion and metastasis of ovarian cancer by carrying miR-630

Association analysis of FXYD5 with prognosis and immunological characteristics across pan-cancer

BackgroundThe FXYD domain-containing ion transport regulator 5 (FXYD5) gene is a cancer promoter. However, evidence for an association between FXYD5 and various types of cancer is still lacking. Using multi-omics bioinformatics, our study aimed to reveal the expression distribution, prognostic value, immune infiltration correlation, and molecular functions of FXYD5. MethodsUsing pan-cancer multi-omics data (including The Cancer Genome Atlas, PrognoScan, Gene Expression Profiling Interactive Analysis, cBioPortal, Gene Expression Omnibus, TIMER and scTIME Portal), we assessed the differences in the expression and prognostic value of FXYD5 in malignant tumors. Furthermore, at the single-cell level, we analyze the expression distribution of FXYD5 across different cell types within the tumor microenvironment, and its relationship with the immune microenvironment. Finally, focusing on ovarian cancer, we conducted preliminary validation of the above findings using cell and molecular biology techniques. ResultsOur results indicated that FXYD5 was up-regulated in various tumor types and was positively associated with tumor progression. We also revealed that FXYD5 was ubiquitously expressed in microenvironmental cells at the single-cell level, and its upregulation was associated with enhanced immune infiltration, cancer-associated fibroblast infiltration, and dysfunction of tumor-infiltrating cytotoxic T lymphocyte. Additionally, its expression was positively correlated with immune checkpoint genes, DNA mismatch repair genes, MSI (microsatellite instability) and TMB (tumor mutational burden) across various cancers. Its higher expression in cytotoxic T lymphocytes attenuated its ability to predict patient survival with PD-L1 (programmed death-ligand 1) blockade therapy, and FXYD5 was found to be a potential regulator of tumor immune escape and resistance to cancer immunotherapies. Based on GSEA (gene set enrichment analysis) and experimental verification, FXYD5 activated TGF‐β/SMAD signaling and drove EMT (epithelial-mesenchymal transition) to promote ovarian cancer progression. ConclusionIn summary, our study revealed that FXYD5-TGFβ axis may coregulate the interaction between tumors, CAFs (carcinoma-associated fibroblasts) and immune cells to reshape the tumor immune microenvironment and promote tumorigenesis and tumor progression. Thus, FXYD5 could be used as an immune-related biomarker for diagnosing and predicting the prognosis of multiple cancer types. Therefore, our findings suggest that targeting FXYD5 in TME (tumor microenvironment) may be a promising therapeutic strategy.

Stromal cartilage oligomeric matrix protein as a tumorigenic driver in ovarian cancer via Notch3 signaling and epithelial-to-mesenchymal transition

BackgroundCartilage oligomeric matrix protein (COMP), an extracellular matrix glycoprotein, is vital in preserving cartilage integrity. Further, its overexpression is associated with the aggressiveness of several types of solid cancers. This study investigated COMP’s role in ovarian cancer, exploring clinicopathological links and mechanistic insights.MethodsTo study the association of COMP expression in cancer cells and stroma with clinicopathological features of ovarian tumor patients, we analyzed an epithelial ovarian tumor cohort by immunohistochemical analysis. Subsequently, to study the functional mechanisms played by COMP, an in vivo xenograft mouse model and several molecular biology techniques such as transwell migration and invasion assay, tumorsphere formation assay, proximity ligation assay, and RT-qPCR array were performed.ResultsBased on immunohistochemical analysis of epithelial ovarian tumor tissues, COMP expression in the stroma, but not in cancer cells, was linked to worse overall survival (OS) of ovarian cancer patients. A xenograft mouse model showed that carcinoma-associated fibroblasts (CAFs) expressing COMP stimulate the growth and metastasis of ovarian tumors through the secretion of COMP. The expression of COMP was upregulated in CAFs stimulated with TGF-β. Functionally, secreted COMP by CAFs enhanced the migratory capacity of ovarian cancer cells. Mechanistically, COMP activated the Notch3 receptor by enhancing the Notch3-Jagged1 interaction. The dependency of the COMP effect on Notch was confirmed when the migration and tumorsphere formation of COMP-treated ovarian cancer cells were inhibited upon incubation with Notch inhibitors. Moreover, COMP treatment induced epithelial-to-mesenchymal transition and upregulation of active β-catenin in ovarian cancer cells.ConclusionThis study suggests that COMP secretion by CAFs drives ovarian cancer progression through the induction of the Notch pathway and epithelial-to-mesenchymal transition.

Abstract LB446: Decoding the mechanism of metastasis: A novel CAF-therapy that blocks the tumor microenvironment and reverses metastatic traits in all solid tumors

Abstract Progression and metastatic spreading of cancer cells are modulated by the carcinoma-associated fibroblasts (CAFs) within the primary tumor microenvironment in solid tumors. However, the mechanisms through which CAFs exert their pro-metastatic effects on cancer cells remain unknown. In this study, we have developed a small molecule that blocks Neuropilin-1 signaling in metastatic CAFs (mCAFs), reversing the mechanisms that promote metastasis in prostate, breast, colorectal and pancreatic cancer. To address these critical needs, in this study, we sought to assess the potential role of proteoglican neuropilin 1 (PG-NRP1) signaling in the maintenance of the activated CAF phenotype. Our results by gene expression profile, qPCR, WB, and ELISA indicate that CAFs isolated from primary tumors of metastatic patients (mCAFs) are functionally different from CAFs isolated from primary tumors of non-metastatic patients, promoting metastasis traits of cancer cells. Among the most prominent differences is an increased PG-NRP1 expression. siRNA studies demonstrated that the knockdown of NRP-1 in mCAFs showed the reversion of the activated phenotype, evaluated by the activation markers a-SMA, Tenascin C, and Fibronectin. Furthermore, conditioned media from PG-NRP1-siRNA-mCAFs failed to increase cancer cells' metastasis traits (migration and invasion) in vitro and tumor progression in vivo. Furthermore, we engineered a small molecule designed to bind PG-NRP1 in mCAFs. Our findings, demonstrate in vitro and in vivo models that the inhibition of PG-NRP1 impedes tumor growth and metastatic dissemination of cancer cells in spontaneous metastasis assay. We designed and validated, using in vivo models, an inhibitor that blocks NRP1-mCAF. Our research demonstrates that blocking PG-NRP1 in mCAF, with a small molecule, is a transversal therapeutic strategy to prevent progression and metastatic spread in solid cancer tumors. Citation Format: Benjamin Prieto, Muriel Núñez, Valentina I. Cerdfa, Javier Cerda-Infante. Decoding the mechanism of metastasis: A novel CAF-therapy that blocks the tumor microenvironment and reverses metastatic traits in all solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB446.

Abstract 2148: Tumor-microenvironment from African-American prostate cancer exhibit methylation of multiple immune modulators

Abstract Background: Non-Hispanic Black American patients (BA) with prostate cancer (PCa) tend to fare worse than their Non-Hispanic White American counterparts (WA), even with consideration of socioeconomic disparities. Clinically, BA face diagnosis at younger age with worse clinical outcomes. Stromal cells adjacent to the tumor, including carcinoma-associated fibroblasts (CAFs), play a critical role in tumorigenesis of PCa. Dysfunction in the tumor microenvironment play a crucial role in mediating prostate cancer tumorigenesis and is emerging as a key target for cancer therapy in solid tumors. Methods: To investigate whether DNA methylation varies in tumor adjacent stroma (TAS) among PCa patients with different geographical ancestries, areas immediate adjacent to tumor (i.e., less than 1mm) removed for methylation analysis. A complete genome-wide DNA methylation of PCa stroma using 17 BA and 15 WA patients who had radical prostatectomy was carried out. Methyl-Captured (mCap) sequencing data was generated for TAS of prostate cancer FFPE tissues. Methylated sites were identified with the MACS2 callpeak function using a band width of 300 bp and a false discovery rate of q &amp;lt; 0.05. A consensus peak set was derived from the aligned reads of BA and WA prostate cancer stroma samples that were sequenced. Pathway analysis was performed using Ingenuity Pathway Analysis (IPA) tools. CAFs from primary tumors of BA and WA was prepared by tissue culture and treated with demethylating agent (i.e., 5-Azacytidine) for 24 hours followed by maintenance of the cells in drug-free medium for 7-10 days and subjected to mCap sequencing. Results: We found racial disparities in genome-wide DNA methylation in TAS between the two populations in which BA PCa patients had significantly increased in global DNA methylation as compared to their WA counterpart (p value &amp;lt; 0.001). Hypomethylation occurred in CAFs from BA patients treated with 5-Azacytidine. We identified 1041 methylated sites corresponding to 409 unique genes that were differentially methylated in BA vs WA (p value &amp;lt; 0.05). Pathway analysis identified significant association between methylated genes in BA TAS and immune response pathways including IL-13, JACK/STAT, T cell receptors, NF-KB, and PD-1, PDL1 cancer immunotherapy. Evaluation of the methylome profile between patients based on the post-prostatectomy resection margins, negative (R0) vs positive (R1), showed that R0 resections had significantly greater (p value &amp;lt; 0.001) methylation than R1 resections. Further analysis identified 984 sites corresponding to 373 differentially methylated, unique genes (p value &amp;lt; 0.05). Conclusion: Tumor-adjacent stroma of BA prostate cancer patients has a distinct methylome as compared to WA patients. Differences in immune response pathways suggests a distinct immune response in TAS in men of different races. Citation Format: Santosh Sankaran, James Nguyen, Tara Jennings, Vinay Kumar, Michael B. Lilly, Michael M. Ittmann, Patricia Castro, Liankun Song, Thomas Keane, Weiping Chu, Xiaolin Zi, Omid Yazdanpanah, Arash Rezazadeh Kalebasty, Farah Rahmatpanah. Tumor-microenvironment from African-American prostate cancer exhibit methylation of multiple immune modulators [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 2148.

Abstract 773: Overexpression of phosphoserine phosphatase (PSPH) in prostate cancer and in tumor microenvironment promotes tumor cell proliferation

Abstract Reprogrammed cellular metabolism is one of the key features of cancer and tumor microenvironment. L-3-phosphoserine phosphatase (PSPH) is one of the five rate-limiting enzymes in the biosynthesis of serine from glucose that supports cell proliferation. Here, we aim to study the role of PSPH in prostate cancer (PCa) and its tumor microenvironment. We find that PSPH not only is overexpressed in prostate cancer cell lines compared to normal prostate epithelial cells (PrEC) and benign prostate epithelial cells (BPH-1), but also exhibits a higher level in patient-derived carcinoma-associated fibroblasts (CAFs) compared to the matched benign associated fibroblasts (BAFs), which are derived from fresh radical prostatectomy specimens of African American (AA) and European American (EA) men. Notably, MDAPCa2b cells derived from an AA prostate cancer patient and AA CAFs have even higher expression of PSPH protein. The mRNA levels of PSPH are also significantly elevated in prostate cancer tissues in AA men compared to EA men and predict poor survival in PCa patients. Knock-down of PSPH expression significantly inhibits the proliferation and colony formation of prostate cancer cells. Growth of xenograft tumors derived from PSPH knockdown MDAPCa2b cells has also been suppressed markedly. Gene expression profiling revealed that suppression of PSPH expression by shRNAs in LNCaP and MDAPCa2b cells is associated with the regulation of gene expression related to metabolism, immunity, extracellular matrix remodeling, and Ion channels. Similarly, analysis of publicly available PCa RNA seq databases also demonstrates that PSPH amplification or over-expression is associated with alterations of gene expression related to metabolism and immunity. Our results suggest that PSPH is upregulated in prostate cancer cells and their surrounding fibroblasts to support tumor growth via reprogramming metabolic pathways and transcriptomes. Citation Format: Liankun Song, Noriko Yokoyama, Jun Xie, Yunjie Hu, Zhenyu (Arthur) Jia, Beverly Wang, Dan Mercola, Edward Uchio, Thomas Ahlering, Michael Lilly, Xiaolin Zi. Overexpression of phosphoserine phosphatase (PSPH) in prostate cancer and in tumor microenvironment promotes tumor cell proliferation [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 773.

Single-Cell RNA Sequencing: A Deep Dive into The Cellular Landscape and Interactions in Osteosarcoma

OSTEOSARCOMA is a leading cause of cancer-related deaths worldwide, with its progression highly influenced by the cellular interplay within the tumor microenvironment that is underexplored. Aiming to bridge this gap, our study utilizes single-cell RNA sequencing (scRNA-seq) to examine the cellular heterogeneity of OSTEOSARCOMA and investigate the roles of distinct cell populations. scRNA-seq was performed on eight DEN mice OSTEOSARCOMA samples, followed by bioinformatic analysis with Seurat package. 12 distinct cell populations were identified, with three unique macrophage populations suggestive of their role as tumor-associated macrophages (TAMs). The detected endothelial cells and pericytes hint at ongoing neoangiogenesis, with implications that endothelial cells might function as tumor-associated endothelial cells (TECs) and pericytes as carcinoma-associated fibroblasts (CAFs). Our findings provide insights into the potential roles of various cell populations in the OSTEOSARCOMA tumor microenvironment, which paves the way for developing novel therapies. These postulations, while offering a deeper understanding of OSTEOSARCOMA's cellular landscape, necessitate experimental validation for confirmation.

Single-Cell RNA Sequencing: A Deep Dive into The Cellular Landscape and Interactions in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide, with its progression highly influenced by the cellular interplay within the tumor microenvironment that is underexplored. Aiming to bridge this gap, our study utilizes single-cell RNA sequencing (scRNA-seq) to examine the cellular heterogeneity of HCC and investigate the roles of distinct cell populations. scRNA-seq was performed on eight DEN mice HCC samples, followed by bioinformatic analysis with Seurat package. Nine distinct cell populations were identified, with three unique macrophage populations suggestive of their role as tumor-associated macrophages (TAMs). The detected endothelial cells and pericytes hint at ongoing neoangiogenesis, with implications that endothelial cells might function as tumor-associated endothelial cells (TECs) and pericytes as carcinoma-associated fibroblasts (CAFs). Our findings provide insights into the potential roles of various cell populations in the HCC tumor microenvironment, which paves the way for developing novel therapies. These postulations, while offering a deeper understanding of HCC's cellular landscape, necessitate experimental validation for confirmation.

Glutamine deficiency drives transforming growth factor-β signaling activation that gives rise to myofibroblastic carcinoma-associated fibroblasts.

Tumor-promoting carcinoma-associated fibroblasts (CAFs), abundant in the mammary tumor microenvironment (TME), maintain transforming growth factor-β (TGF-β)-Smad2/3 signaling activation and the myofibroblastic state, the hallmark of activated fibroblasts. How myofibroblastic CAFs (myCAFs) arise in the TME and which epigenetic and metabolic alterations underlie activated fibroblastic phenotypes remain, however, poorly understood. We herein show global histone deacetylation in myCAFs present in tumors to be significantly associated with poorer outcomes in breast cancer patients. As the TME is subject to glutamine (Gln) deficiency, human mammary fibroblasts (HMFs) were cultured in Gln-starved medium. Global histone deacetylation and TGF-β-Smad2/3 signaling activation are induced in these cells, largely mediated by class I histone deacetylase (HDAC) activity. Additionally, mechanistic/mammalian target of rapamycin complex 1 (mTORC1) signaling is attenuated in Gln-starved HMFs, and mTORC1 inhibition in Gln-supplemented HMFs with rapamycin treatment boosts TGF-β-Smad2/3 signaling activation. These data indicate that mTORC1 suppression mediates TGF-β-Smad2/3 signaling activation in Gln-starved HMFs. Global histone deacetylation, class I HDAC activation, and mTORC1 suppression are also observed in cultured human breast CAFs. Class I HDAC inhibition or mTORC1 activation by high-dose Gln supplementation significantly attenuates TGF-β-Smad2/3 signaling and the myofibroblastic state in these cells. These data indicate class I HDAC activation and mTORC1 suppression to be required for maintenance of myCAF traits. Taken together, these findings indicate that Gln starvation triggers TGF-β signaling activation in HMFs through class I HDAC activity and mTORC1 suppression, presumably inducing myCAF conversion.

Carcinoma-associated fibroblast-derived lysyl oxidase-rich extracellular vesicles mediate collagen crosslinking and promote epithelial-mesenchymal transition via p-FAK/p-paxillin/YAP signaling

Carcinoma-associated fibroblasts (CAFs) are the main cellular components of the tumor microenvironment and promote cancer progression by modifying the extracellular matrix (ECM). The tumor-associated ECM is characterized by collagen crosslinking catalyzed by lysyl oxidase (LOX). Small extracellular vesicles (sEVs) mediate cell-cell communication. However, the interactions between sEVs and the ECM remain unclear. Here, we demonstrated that sEVs released from oral squamous cell carcinoma (OSCC)-derived CAFs induce collagen crosslinking, thereby promoting epithelial-mesenchymal transition (EMT). CAF sEVs preferably bound to the ECM rather than being taken up by fibroblasts and induced collagen crosslinking, and a LOX inhibitor or blocking antibody suppressed this effect. Active LOX (αLOX), but not the LOX precursor, was enriched in CAF sEVs and interacted with periostin, fibronectin, and bone morphogenetic protein-1 on the surface of sEVs. CAF sEV-associated integrin α2β1 mediated the binding of CAF sEVs to collagen I, and blocking integrin α2β1 inhibited collagen crosslinking by interfering with CAF sEV binding to collagen I. CAF sEV-induced collagen crosslinking promoted the EMT of OSCC through FAK/paxillin/YAP pathway. Taken together, these findings reveal a novel role of CAF sEVs in tumor ECM remodeling, suggesting a critical mechanism for CAF-induced EMT of cancer cells.

Renal clear cancer cells regulate the differentiation of urine-derived stem cells to carcinoma-associated fibroblasts via RUNX3/TGF-β1 signaling axis.

Clear cell renal cell carcinoma (ccRCC), the most common pathological subtype of renal cancer, is one of the significant health concerns due to limited clinically effective treatments. Nevertheless, targeting carcinoma-associated fibroblasts in the tumor microenvironment has emerged as a promising innovative strategy for renal cancer therapy. Thus, this study is aimed to explore the role and molecular mechanism of urine-derived stem cells (USCs) in the progression and metastasis of ccRCC. Initially, wound-healing and transwell experiments were used to assess the migration and invasion abilities of the cells. Then, western blot analysis (WB) and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analyses were used to demonstrate the relevant protein and messenger RNA expression levels. Finally, hematoxylin-eosin and immunohistochemical stainings were performed to evaluate metastasis and protein expression in lung tumors. The coculture of USCs with the ccRCC cell lines significantly enhanced their migratory and invasive abilities. WB and qRT-PCR analyses exhibited that ccRCC cell lines significantly increased cell mobility markers transcriptional and protein levels in USCs. Finally, the in vivo investigations in nude mice showed that USCs promoted the proliferation and migration of ccRCC-based xenograft tumors. In summary, these findings demonstrated that USCs promoted ccRCC tumorigenesis and development in vivo and in vitro by regulating the Runt-related transcription factor 3/transforming growth factor-β1 signaling axis.

Spatial analysis of stromal signatures identifies invasive front carcinoma-associated fibroblasts as suppressors of anti-tumor immune response in esophageal cancer

BackgroundIncreasing evidence indicates that the tumor microenvironment (TME) is a crucial determinant of cancer progression. However, the clinical and pathobiological significance of stromal signatures in the TME, as a complex dynamic entity, is still unclear in esophageal squamous cell carcinoma (ESCC).MethodsHerein, we used single-cell transcriptome sequencing data, imaging mass cytometry (IMC) and multiplex immunofluorescence staining to characterize the stromal signatures in ESCC and evaluate their prognostic values in this aggressive disease. An automated quantitative pathology imaging system determined the locations of the lamina propria, stroma, and invasive front. Subsequently, IMC spatial analyses further uncovered spatial interaction and distribution. Additionally, bioinformatics analysis was performed to explore the TME remodeling mechanism in ESCC. To define a new molecular prognostic model, we calculated the risk score of each patient based on their TME signatures and pTNM stages.ResultsWe demonstrate that the presence of fibroblasts at the tumor invasive front was associated with the invasive depth and poor prognosis. Furthermore, the amount of α-smooth muscle actin (α-SMA)+ fibroblasts at the tumor invasive front positively correlated with the number of macrophages (MØs), but negatively correlated with that of tumor-infiltrating granzyme B+ immune cells, and CD4+ and CD8+ T cells. Spatial analyses uncovered a significant spatial interaction between α-SMA+ fibroblasts and CD163+ MØs in the TME, which resulted in spatially exclusive interactions to anti-tumor immune cells. We further validated the laminin and collagen signaling network contributions to TME remodeling. Moreover, compared with pTNM staging, a molecular prognostic model, based on expression of α-SMA+ fibroblasts at the invasive front, and CD163+ MØs, showed higher accuracy in predicting survival or recurrence in ESCC patients. Regression analysis confirmed this model is an independent predictor for survival, which also identifies a high-risk group of ESCC patients that can benefit from adjuvant therapy.ConclusionsOur newly defined biomarker signature may serve as a complement for current clinical risk stratification approaches and provide potential therapeutic targets for reversing the fibroblast-mediated immunosuppressive microenvironment.

Brevilin A is a potent anti-metastatic CRC agent that targets the VEGF-IL6-STAT3 axis in the HSCs-CRC interplay

BackgroundMore than half of the colorectal cancer (CRC) patients will develop liver metastasis that underlies the cancer mortality. In the hepatic tumor microenvironment, the interplay between CRC cells and hepatic stellate cells (HSCs), and the activation of HSCs to become carcinoma-associated fibroblasts (CAFs) will further promote the cancer development. Nevertheless, the critical signaling molecule that involved in these processes remains unknown, which hinders the development of effective therapeutic agents for the treatment of metastatic CRC (mCRC).MethodsConditioned medium system and co-cultured system were used to examine the interplay between CRC cells and HSCs. Luminex liquid suspension chip detection and enzyme-linked immunosorbent assay were used to screen for the mediators in the conditioned medium that facilitated the CRC-HSCs interplay and HSCs-to-CAFs differentiation. Cell and animal models were used to examine whether brevilin A inhibited CRC liver metastasis via the VEGF-IL6-STAT3 axis.ResultsIn the CRC-HSCs interplay, CRC promoted HSCs-to-CAFs differentiation by releasing vascular endothelial growth factor (VEGF); and HSCs released interleukin 6 (IL6) that activated signal transducer and activator of transcription 3 (STAT3) in the CRC and hence increased the cancer metastatic potential. The functions of the VEGF-IL6-STAT3 axis in the HSCs-CRC interplay were further validated by VEGF recombinant protein and IL6 neutralizing antibody. More importantly, brevilin A, an active compound isolated from Centipeda minima (L.) A. Br. et Aschers, targeted the VEGF-IL6-STAT3 axis in the CRC-HSCs interplay, hence significantly inhibited colorectal liver metastasis and cancer growth both in vitro and in vivo.ConclusionsWe are the first to demonstrate brevilin A possesses potent anti-mCRC effect by targeting the VEGF-IL6-STAT3 axis in the CRC-HSCs interplay. Our findings not only support the development of brevilin A as a novel therapeutic agent for mCRC treatment, but also pave the path for the development of other VEGF-IL6-STAT3 targeting therapeutic strategies.

Targeting inhibition of prognosis-related lipid metabolism genes including CYP19A1 enhances immunotherapeutic response in colon cancer

BackgroundLipid metabolic reprogramming in colon cancer shows a potential impact on tumor immune microenvironment and is associated with response to immunotherapy. Therefore, this study aimed to develop a lipid metabolism-related prognostic risk score (LMrisk) to provide new biomarkers and combination therapy strategies for colon cancer immunotherapy.MethodsDifferentially expressed lipid metabolism-related genes (LMGs) including cytochrome P450 (CYP) 19A1 were screened to construct LMrisk in TCGA colon cancer cohort. The LMrisk was then validated in three GEO datasets. The differences of immune cell infiltration and immunotherapy response between LMrisk subgroups were investigated via bioinformatic analysis. These results were comfirmed by in vitro coculture of colon cancer cells with peripheral blood mononuclear cells, human colon cancer tissue microarray analysis, multiplex immunofluorescence staining and mouse xenograft models of colon cancer.ResultsSix LMGs including CYP19A1, ALOXE3, FABP4, LRP2, SLCO1A2 and PPARGC1A were selected to establish the LMrisk. The LMrisk was positively correlated with the abundance of macrophages, carcinoma-associated fibroblasts (CAFs), endothelial cells and the levels of biomarkers for immunotherapeutic response including programmed cell death ligand 1 (PD-L1) expression, tumor mutation burden and microsatellite instability, but negatively correlated with CD8+ T cell infiltration levels. CYP19A1 protein expression was an independent prognostic factor, and positively correlated with PD-L1 expression in human colon cancer tissues. Multiplex immunofluorescence analyses revealed that CYP19A1 protein expression was negatively correlated with CD8+ T cell infiltration, but positively correlated with the levels of tumor-associated macrophages, CAFs and endothelial cells. Importantly, CYP19A1 inhibition downregulated PD-L1, IL-6 and TGF-β levels through GPR30-AKT signaling, thereby enhancing CD8+ T cell-mediated antitumor immune response in vitro co-culture studies. CYP19A1 inhibition by letrozole or siRNA strengthened the anti-tumor immune response of CD8+ T cells, induced normalization of tumor blood vessels, and enhanced the efficacy of anti-PD-1 therapy in orthotopic and subcutaneous mouse colon cancer models.ConclusionA risk model based on lipid metabolism-related genes may predict prognosis and immunotherapeutic response in colon cancer. CYP19A1-catalyzed estrogen biosynthesis promotes vascular abnormality and inhibits CD8+ T cell function through the upregulation of PD-L1, IL-6 and TGF-β via GPR30-AKT signaling. CYP19A1 inhibition combined with PD-1 blockade represents a promising therapeutic strategy for colon cancer immunotherapy.

Bone mesenchymal stem cells promote gastric cancer progression through TGF-β1/Smad2 positive feedback loop

AimsBone marrow-derived mesenchymal stem cells (BMSCs) have been proven to be recruited into the tumor microenvironment and contribute to gastric cancer (GC) progression, but the underlying mechanism is still unclear. The purpose of this study is to explore the exact role and potential mechanism of BMSCs in the progression of GC. Materials and methodsBioinformatics analyzed were used to clarify the correlation between TGF-β1 and prognosis of gastric cancer. Cell co-culture were used to explore the interaction between gastric cancer cells (GCs) and BMSCs. Quantitative real time-PCR and Western blot assay were used to detect gene and protein expression, respectively. The biological characteristics of GCs and BMSCs were detected by immunofluorescence, Transwell migration, Elisa and invasion assay. Xenograft models in nude mice were constructed to evaluate GC development in vivo. Key findingsTGF-β1 was overexpressed in GC cells and tissues, and is positively related to the poor prognosis of patients. TGF-β1 from GCs activated the Smad2 pathway in BMSCs, promoting their differentiation into carcinoma-associated fibroblasts (CAFs) and TGF-β1 expression. Concomitantly, TGF-β1 secreted by CAFs activate Smad2 signaling in GC cells, thus inducing their epithelial-mesenchymal transition (EMT) and TGF-β1 secretion. BMSCs can dramatically promote the proliferation, migration, and invasion of GCs while blocking TGF-β1/Smad2 positive feedback loop can reverse these effects. SignificanceThe TGF-β1/Smad2 positive feedback loop between GCs and BMSCs, promotes the CAFs differentiation of BMSCs and the EMT of GCs, resulting in the progression of GC.

Modulating tumor-stromal crosstalk via a redox-responsive nanomedicine for combination tumor therapy.

Interaction between carcinoma-associated fibroblasts (CAFs) and tumor cells leads to the invasion and metastasis of breast cancer. Herein, we prepared a redox-responsive chondroitin sulfate (CS)-based nanomedicine, in which hydrophobic cabazitaxel (CTX) was conjugated to the backbone of CS via glutathione (GSH)-sensitive dithiomaleimide (DTM) to form an amphipathic CS-DTM-CTX (CDC) conjugate, and dasatinib (DAS) co-assembled with the CDC conjugate to obtain DAS@CDC. After CD44 receptor-mediated internalization by CAFs, the nanomedicine could reverse CAFs to normal fibroblasts, blocking their crosstalk with tumor cells and reducing synthesis of major tumor extracellular matrix proteins, including collagen and fibronectin. Meanwhile, the nanomedicine internalized by tumor cells could effectively inhibit tumor proliferation and metastasis, leading to shrinkage of the tumor volume and inhibition of lung metastasis in a subcutaneous 4 T1 tumor model with low side effects. Collectively, the nanomedicine showed a remarkably synergistic therapy effect against breast cancer by modulating tumor-stromal crosstalk.