ABSTRACT
This study introduces a new imaging, spatial transcriptomics (ST), and single-cell RNA-sequencing integration pipeline to characterize neoplastic cell state transitions during tumorigenesis. We applied a semi-supervised analysis pipeline to examine premalignant pancreatic intraepithelial neoplasias (PanINs) that can develop into pancreatic ductal adenocarcinoma (PDAC). Their strict diagnosis on formalin-fixed and paraffin-embedded (FFPE) samples limited the single-cell characterization of human PanINs within their microenvironment. We leverage whole transcriptome FFPE ST to enable the study of a rare cohort of matched low-grade (LG) and high-grade (HG) PanIN lesions to track progression and map cellular phenotypes relative to single-cell PDAC datasets. We demonstrate that cancer-associated fibroblasts (CAFs), including antigen-presenting CAFs, are located close to PanINs. We further observed a transition from CAF-related inflammatory signaling to cellular proliferation during PanIN progression. We validate these findings with single-cell high-dimensional imaging proteomics and transcriptomics technologies. Altogether, our semi-supervised learning framework for spatial multi-omics has broad applicability across cancer types to decipher the spatiotemporal dynamics of carcinogenesis.
Subject(s)
Cancer-Associated Fibroblasts , Carcinogenesis , Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Humans , Pancreatic Neoplasms/genetics , Carcinogenesis/genetics , Cancer-Associated Fibroblasts/metabolism , Carcinoma, Pancreatic Ductal/genetics , Tumor Microenvironment/genetics , Single-Cell Analysis/methods , Transcriptome/genetics , Gene Expression Regulation, Neoplastic/genetics , Carcinoma in Situ/genetics , Carcinoma in Situ/pathologyABSTRACT
Colorectal cancer (CRC) is a leading cause of cancer mortality worldwide, and cancer-associated fibroblasts (CAFs) play a major role in the tumor microenvironment (TME), which facilitates the progression of CRC. It is critical to understand how CAFs promote the progression of CRC for the development of novel therapeutic approaches. The purpose of this study was to understand how CAF-derived stromal-derived factor-1 (SDF-1) and its interactions with the corresponding C-X-C motif chemokine receptor 4 (CXCR4) promote CRC progression. Our study focused on their roles in promoting tumor cell migration and invasion and their effects on the characteristics of cancer stem cells (CSCs), which ultimately impact patient outcomes. Here, using in vivo approaches and clinical histological samples, we analyzed the influence of secreted SDF-1 on CRC progression, especially in terms of tumor cell behavior and stemness. We demonstrated that CAF-secreted SDF-1 significantly enhanced CRC cell migration and invasion through paracrine signaling. In addition, the overexpression of SDF-1 in CRC cell lines HT29 and HCT-116 triggered these cells to generate autocrine SDF-1 signaling, which further enhanced their CSC characteristics, including those of migration, invasion, and spheroid formation. An immunohistochemical study showed a close relationship between SDF-1 and CXCR4 expression in CRC tissue, and this significantly affected patient outcomes. The administration of AMD3100, an inhibitor of CXCR4, reversed the entire phenomenon. Our results strongly suggest that targeting this signaling axis in CRC is a feasible approach to attenuating tumor progression, and it may, therefore, serve as an alternative treatment method to improve the prognosis of patients with CRC, especially those with advanced, recurrent, or metastatic CRC following standard therapy.
Subject(s)
Autocrine Communication , Cancer-Associated Fibroblasts , Cell Movement , Chemokine CXCL12 , Colorectal Neoplasms , Neoplastic Stem Cells , Paracrine Communication , Receptors, CXCR4 , Signal Transduction , Humans , Colorectal Neoplasms/pathology , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/genetics , Chemokine CXCL12/metabolism , Receptors, CXCR4/metabolism , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/pathology , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Animals , Neoplasm Invasiveness , Mice , Tumor Microenvironment , Cell Line, Tumor , HCT116 Cells , Male , Female , HT29 CellsABSTRACT
Metastasis is a crucial stage in tumour progression, and cancer-associated fibroblasts (CAFs) support metastasis through their participation in extracellular matrix (ECM) stiffness. CD248 is a possible biomarker for non-small cell lung cancer (NSCLC)-derived CAFs, but its role in mediating ECM stiffness to promote NSCLC metastasis is unknown. We investigated the significance of CD248+ CAFs in activating the Hippo axis and promoting connective tissue growth factor (CTGF) expression, which affects the stromal collagen I environment and improves ECM stiffness, thereby facilitating NSCLC metastasis. In this study, we found that higher levels of CD248 in CAFs induced the formation of collagen I, which in turn increased extracellular matrix stiffness, thereby enabling NSCLC cell infiltration and migration. Hippo axis activation by CD248+ CAFs induces CTGF expression, which facilitates the formation of the collagen I milieu in the stromal matrix. In a tumour lung metastasis model utilizing fibroblast-specific CD248 gene knockout mice, CD248 gene knockout mice showed a significantly reduced ability to develop tumour lung metastasis compared to that of WT mice. Our findings demonstrate that CD248+ CAFs activate the Hippo pathway, thereby inducing CTGF expression, which in turn facilitates the collagen I milieu of the stromal matrix, which promotes NSCLC metastasis.
Subject(s)
Cancer-Associated Fibroblasts , Carcinoma, Non-Small-Cell Lung , Connective Tissue Growth Factor , Extracellular Matrix , Hippo Signaling Pathway , Lung Neoplasms , Mice, Knockout , Protein Serine-Threonine Kinases , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/genetics , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/pathology , Animals , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Lung Neoplasms/genetics , Lung Neoplasms/secondary , Extracellular Matrix/metabolism , Mice , Connective Tissue Growth Factor/metabolism , Connective Tissue Growth Factor/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Cell Line, Tumor , Antigens, CD/metabolism , Antigens, CD/genetics , Neoplasm Metastasis , Signal Transduction , Gene Expression Regulation, Neoplastic , Cell Movement , Tumor MicroenvironmentABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) still has a poor response to therapies, partly due to their cancer-associated fibroblasts (CAFs). Here, we investigate the synergistic impact of a combinatory approach between a known chemotherapy agent, such as gemcitabine (GEM), and gene-modified human mesenchymal stromal/stem cells (MSCs) secreting the pro-apoptotic soluble (s)TRAIL (sTRAIL MSCs) on both PDAC cells and CAFs. The combo significantly impacts on PDAC survival in 2D and 3D models. In orthotopic xenograft models, GEM and sTRAIL MSCs induce tumor architecture shredding with a reduction of CK7- and CK8/18-positive cancer cells and the abrogation of spleen metastases. A cytotoxic effect on primary human CAFs is also observed along with an alteration of their transcriptome and a reduction of the related desmoplasia. Collectively, we demonstrate a promising therapeutic profile of combining GEM and sTRAIL MSCs to target both tumoral and stromal compartments in PDAC.
Subject(s)
Carcinoma, Pancreatic Ductal , Deoxycytidine , Gemcitabine , Mesenchymal Stem Cells , Pancreatic Neoplasms , TNF-Related Apoptosis-Inducing Ligand , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Deoxycytidine/therapeutic use , Humans , TNF-Related Apoptosis-Inducing Ligand/pharmacology , TNF-Related Apoptosis-Inducing Ligand/metabolism , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/metabolism , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/drug effects , Animals , Cell Line, Tumor , Mice , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/therapy , Xenograft Model Antitumor Assays , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/drug effects , Cancer-Associated Fibroblasts/pathology , Adenocarcinoma/pathology , Adenocarcinoma/drug therapy , Adenocarcinoma/metabolismABSTRACT
BACKGROUND: Oral cancer poses a significant health challenge due to limited treatment protocols and therapeutic targets. We aimed to investigate the invasive margins of gingivo-buccal oral squamous cell carcinoma (GB-OSCC) tumors in terms of the localization of genes and cell types within the margins at various distances that could lead to nodal metastasis. METHODS: We collected tumor tissues from 23 resected GB-OSCC samples for gene expression profiling using digital spatial transcriptomics. We monitored differential gene expression at varying distances between the tumor and its microenvironvent (TME), and performed a deconvulation study and immunohistochemistry to identify the cells and genes regulating the TME. RESULTS: We found that the tumor-stromal interface (a distance up to 200 µm between tumor and immune cells) is the most active region for disease progression in GB-OSCC. The most differentially expressed apex genes, such as FN1 and COL5A1, were located at the stromal ends of the margins, and together with enrichment of the extracellular matrix (ECM) and an immune-suppressed microenvironment, were associated with lymph node metastasis. Intermediate fibroblasts, myocytes, and neutrophils were enriched at the tumor ends, while cancer-associated fibroblasts (CAFs) were enriched at the stromal ends. The intermediate fibroblasts transformed into CAFs and relocated to the adjacent stromal ends where they participated in FN1-mediated ECM modulation. CONCLUSION: We have generated a functional organization of the tumor-stromal interface in GB-OSCC and identified spatially located genes that contribute to nodal metastasis and disease progression. Our dataset might now be mined to discover suitable molecular targets in oral cancer.
Subject(s)
Fibroblasts , Gene Expression Regulation, Neoplastic , Lymphatic Metastasis , Mouth Neoplasms , Myeloid Cells , Tumor Microenvironment , Humans , Mouth Neoplasms/pathology , Mouth Neoplasms/genetics , Lymphatic Metastasis/pathology , Fibroblasts/pathology , Fibroblasts/metabolism , Myeloid Cells/pathology , Myeloid Cells/metabolism , Cancer-Associated Fibroblasts/pathology , Cancer-Associated Fibroblasts/metabolism , Gene Expression Profiling , Female , Male , Middle Aged , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/geneticsABSTRACT
Cancer-associated fibroblasts (CAFs) are an important component of the tumor microenvironment (TME) and can induce functional polarization of tumor macrophages. This study aimed to explore the effect of CAFs-derived exosome LINC01833 on the malignant biological behavior of non-small cell lung cancer (NSCLC) cells and its mechanism. Tumor tissues (n = 3) and adjacent noncancerous tissues (n = 3) were collected from patients with NSCLC, and fibroblasts (CAF, NF) were isolated from the two tissues. Expression of LINC01833/miR-335-5p/VAPA in NSCLC clinical tissues and cell lines was detected by RT-qPCR. Exosomes of CAFs and NFs were isolated by ultracentrifugation. Cell proliferation, migration, invasion, and M2 macrophage polarization were detected by MTT, transwell, wound-healing assay, and flow cytometry assay, while western blot was used to verify the expression of M2 macrophage polarization-related proteins. Tumor volume weight and M2 macrophage polarization were detected by tumor xenografts in nude mice. LINC01833 was highly expressed in NSCLC tumor tissues and cells. Knockdown of LINC01833 exosomes could significantly inhibit proliferation, migration, invasion of NSCLC cells, and M2 macrophage polarization of THP-1 cells, while simultaneous knockdown of miR-335-5p on the above basis could reverse the effect of knockdown of LINC01833. In vivo experiments also indicated that knockdown of LINC01833 exosomes suppressed tumor growth and M2 macrophage polarization. CAF-derived LINC01833 exosomes can promote the proliferation, migration and invasion of NSCLC cells and M2 macrophage polarization by inhibiting miR-335-5p and regulating VAPA activity.
Subject(s)
Cancer-Associated Fibroblasts , Carcinoma, Non-Small-Cell Lung , Exosomes , Lung Neoplasms , Mice, Nude , MicroRNAs , RNA, Long Noncoding , MicroRNAs/genetics , MicroRNAs/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/genetics , Humans , Exosomes/metabolism , Exosomes/genetics , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Lung Neoplasms/genetics , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/pathology , Animals , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Mice , Cell Proliferation , Male , Female , Cell Line, Tumor , Cell Movement , A549 Cells , Mice, Inbred BALB CABSTRACT
The pleiotropic effect of cancer-associated fibroblasts (CAFs) on tumour progression depends on the environment. circFARP1 is critical for CAFs-induced gemcitabine (GEM) resistance in pancreatic cancer. Its specific role and mechanism in non-small cell lung cancer (NSCLC) have not been reported yet. We prepared a cancer-associated fibroblasts-conditioned medium (CAF-CM) to incubate the A549 cells. Quantitative real-time polymerase chain reaction was used to detect RNA levels. We detected protein expression by immunohistochemistry, immunocytochemistry, western blot and immunofluorescence. We also detected the targeting impact between circFARP1, miR-338-3p and SRY-box transcription factor 4 (SOX4) by using dual-luciferase reporter and RNA pull-down assays. We determined cell proliferation, migration and invasion capabilities through Cell Counting Kit-8 and transwell assays. In addition, we measured tumour volume and weight in vivo by establishing a xenograft tumour model. CircFARP1 levels were remarkably high in the CAFs. The transfection experiments found that circFARP1 downregulation in CAFs caused migration, proliferation and invasion inhibition of CAFs and A549 cells, whereas inhibiting miR-38-3p or overexpressing SOX4 in CAFs could significantly reverse the inhibition. In vivo study in nude mice confirmed that CAFs could promote NSCLC tumour growth and knockdown of circFARP1 could inhibit tumour growth of NSCLC, whereas miR-38-3p downregulation or SOX4 overexpression could significantly reverse the inhibition. circFARP1 promotes NSCLC development by stimulating miR-338-3p/SOX4 signalling axis to regulate CAFs.
Subject(s)
Cancer-Associated Fibroblasts , Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , MicroRNAs , Neoplasm Invasiveness , RNA, Circular , SOXC Transcription Factors , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , SOXC Transcription Factors/genetics , SOXC Transcription Factors/metabolism , Lung Neoplasms/pathology , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Animals , RNA, Circular/genetics , RNA, Circular/metabolism , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/pathology , Cell Proliferation/genetics , Mice , A549 Cells , Neoplasm Metastasis , Cell Movement , Gene Expression Regulation, Neoplastic , Mice, Nude , MaleABSTRACT
Senescent cells are known to secrete proteins, including inflammatory cytokines and damageassociated molecular patterns. This phenomenon is known as the senescenceassociated secretory phenotype (SASP). SASP in cancer stromal fibroblasts is involved in cancer growth and progression. Conversely, metformin, an antidiabetic drug, has been reported to inhibit SASP induction by inhibiting the activation of NFκB, a regulator of SASP. To date, at least to the best of our knowledge, there have been no reports regarding cellular senescence in fibroblasts and tumor progression via the SASPmediated paracrine pathway. The present study thus aimed to elucidate the induction mechanisms of SASP in radiationinduced fibroblasts and to determine its effects on cancer progression via the paracrine pathway. Furthermore, the present study aimed to determine whether controlling SASP using metformin suppresses cancer progression. A welldifferentiated esophageal cancer cell line established by the authors' department and fibroblasts isolated and cultured from the noncancerous esophageal mucosa of resected esophageal cancer cases were used for the experiments. Fibroblasts were irradiated with 8 Gy radiation, and the changes in the expression of the senescence markers, SAßgal, p21, p16 and NFκB were evaluated using immunofluorescent staining and western blot analysis in the presence or absence of metformin treatment. The culture supernatants of irradiated fibroblasts treated with metformin and those treated without metformin were collected and added to the cancer cells to evaluate their proliferative, invasive and migratory abilities. Vimentin and Ecadherin expression levels were also evaluated using immunofluorescent staining and western blot analysis. The expression levels of p16, p21 and NFκB in irradiated fibroblasts were attenuated by treatment with metformin. Supernatants collected from irradiated fibroblasts exhibited the proliferative activity of esophageal cancer cells, and the promotion of migratory and invasion abilities, which may be due to epithelialmesenchymal transition and changes in cell morphology. These reactions were confirmed to be suppressed by the addition of the supernatant of cultured fibroblasts pretreated with metformin. On the whole, the present study demonstrates that fibroblasts in the cancer stroma may be involved in tumor progression through cellular senescence.
Subject(s)
Cancer-Associated Fibroblasts , Cell Proliferation , Cellular Senescence , Esophageal Neoplasms , Metformin , Metformin/pharmacology , Humans , Cellular Senescence/drug effects , Cellular Senescence/radiation effects , Esophageal Neoplasms/pathology , Esophageal Neoplasms/metabolism , Esophageal Neoplasms/drug therapy , Cancer-Associated Fibroblasts/drug effects , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/radiation effects , Cancer-Associated Fibroblasts/pathology , Cell Proliferation/drug effects , Disease Progression , NF-kappa B/metabolism , Cell Line, Tumor , Senescence-Associated Secretory Phenotype , Cell Movement/drug effects , Cell Movement/radiation effects , Epithelial-Mesenchymal Transition/drug effects , Epithelial-Mesenchymal Transition/radiation effects , Hypoglycemic Agents/pharmacology , Fibroblasts/metabolism , Fibroblasts/radiation effects , Fibroblasts/drug effectsABSTRACT
Cell-type-specific domains are the anatomical domains in spatially resolved transcriptome (SRT) tissues where particular cell types are enriched coincidentally. It is challenging to use existing computational methods to detect specific domains with low-proportion cell types, which are partly overlapped with or even inside other cell-type-specific domains. Here, we propose De-spot, which synthesizes segmentation and deconvolution as an ensemble to generate cell-type patterns, detect low-proportion cell-type-specific domains, and display these domains intuitively. Experimental evaluation showed that De-spot enabled us to discover the co-localizations between cancer-associated fibroblasts and immune-related cells that indicate potential tumor microenvironment (TME) domains in given slices, which were obscured by previous computational methods. We further elucidated the identified domains and found that Srgn may be a critical TME marker in SRT slices. By deciphering T cell-specific domains in breast cancer tissues, De-spot also revealed that the proportions of exhausted T cells were significantly increased in invasive vs. ductal carcinoma.
Subject(s)
Breast Neoplasms , Transcriptome , Tumor Microenvironment , Tumor Microenvironment/immunology , Humans , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Breast Neoplasms/immunology , Female , Gene Expression Profiling/methods , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Mice , Animals , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/pathologyABSTRACT
BACKGROUND: Cancer-associated fibroblasts (CAFs) are the prominent cell type in the tumor microenvironment (TME), and CAF subsets have been identified in various tumors. However, how CAFs spatially coordinate other cell populations within the liver TME to promote cancer progression remains unclear. METHODS: We combined multi-region proteomics (6 patients, 24 samples), 10X Genomics Visium spatial transcriptomics (11 patients, 25 samples), and multiplexed imaging (92 patients, 264 samples) technologies to decipher the expression heterogeneity, functional diversity, spatial distribution, colocalization, and interaction of fibroblasts. The newly identified CAF subpopulation was validated by cells isolated from 5 liver cancer patients and in vitro functional assays. RESULTS: We identified a liver CAF subpopulation, marked by the expression of COL1A2, COL4A1, COL4A2, CTGF, and FSTL1, and named F5-CAF. F5-CAF is preferentially located within and around tumor nests and colocalizes with cancer cells with higher stemness in hepatocellular carcinoma (HCC). Multiplexed staining of 92 patients and the bulk transcriptome of 371 patients demonstrated that the abundance of F5-CAFs in HCC was associated with a worse prognosis. Further in vitro experiments showed that F5-CAFs isolated from liver cancer patients can promote the proliferation and stemness of HCC cells. CONCLUSIONS: We identified a CAF subpopulation F5-CAF in liver cancer, which is associated with cancer stemness and unfavorable prognosis. Our results provide potential mechanisms by which the CAF subset in the TME promotes the development of liver cancer by supporting the survival of cancer stem cells.
Subject(s)
Cancer-Associated Fibroblasts , Carcinoma, Hepatocellular , Liver Neoplasms , Neoplastic Stem Cells , Tumor Microenvironment , Humans , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/pathology , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/pathology , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Tumor Microenvironment/genetics , Proteomics/methods , Transcriptome , Gene Expression Regulation, Neoplastic , Genomics/methods , Cell Proliferation , Gene Expression Profiling , Cell Line, Tumor , Prognosis , MultiomicsABSTRACT
Pancreatic stellate cells (PSC) are one source of cancer-associated fibroblasts (CAF) and play, therefore, an essential role in pancreatic ductal adenocarcinoma (PDA). Paracrine signalling between PDA cells and CAF has been widely studied, yet external influences on paracrine crosstalk are poorly understood. This study aimed to gain a deeper insight into the communication of PSC and cancer cells under different co-culture conditions via analysis of PSC gene expression profiles. Two contactless co-culture models with tumor cells from the p48-Cre; lox-stop-lox-KrasG12D/+; lox-stop-lox-Trp53R172H/+ mouse model (KPC) and murine PSC separated through a microporous membrane and grown in different compartments (standard co-culture) or on different sides of the same membrane (inverse co-culture), were established. RNA-Sequencing analysis of PSC mRNA was performed 24 h and 72 h after co-culture with KPC cells. For selected genes, results were confirmed by quantitative RT-PCR and immunocytochemistry. Standard co-culture displayed 19 differentially expressed genes (DEG) at 24 h and 52 DEG at 72 h. In inverse co-culture, 800 DEG at 24 h and 2213 DEG at 72 h were enriched. PSC showed great heterogeneity in their gene expression profiles; however, mutually regulated genes of both co-cultures, such as VCAN and CHST11, could be identified. VCAN-protein-protein interaction-network analysis revealed several shared genes between co-culture models, such as SDC4 and FN1. In conclusion, PSC show a varying susceptibility to cancer cell signals depending on the co-culture method, with intensified transcriptome changes with closer proximity.
Subject(s)
Carcinoma, Pancreatic Ductal , Coculture Techniques , Pancreatic Neoplasms , Pancreatic Stellate Cells , Paracrine Communication , Transcriptome , Pancreatic Stellate Cells/metabolism , Pancreatic Stellate Cells/pathology , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/metabolism , Animals , Mice , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Cell Line, Tumor , Humans , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/pathology , Gene Expression ProfilingABSTRACT
Introduction: Fibroblast activation protein (FAP) overexpression on cancer-associated fibroblasts (CAFs) is associated with poor prognosis and worse clinical outcomes. Selective ablation of pro-tumorgenic FAP+ stromal cells with CAR-T cells may be a new therapeutic strategy. However, the clinical use of FAP-CAR T cells is suggested to proceed with caution for occasional poor efficacy and induction of on-target off-tumor toxicity (OTOT), including lethal osteotoxicity and cachexia. Hence, more investigations and preclinical trials are required to optimize the FAP-CAR T cells and to approve their safety and efficacy. Methods: In this study, we designed second-generation CAR T cells targeting FAP with 4-1BB as a co-stimulatory molecule, and tested their cytotoxicity against FAP-positive cells (hFAP-HT1080 cells and a variety of primary CAFs) in vitro and in Cell line-derived xenograft (CDX) and a patient-derived xenograft (PDX) model. Results: Results showed that our FAP-CAR T cells were powerfully potent in killing human and murine FAP-positive tumor cells and CAFs in multiple types of tumors in BALB/c and C57BL/6 mice and in patient-derived xenografts (PDX) model. And they were proved to be biologically safe and exhibit low-level OTOT. Discussion: Taken together, the human/murine cross-reactive FAP-CAR T cells were powerfully potent in killing human and murine FAP positive tumor cells and CAFs. They were biologically safe and exhibit low-level OTOT, warranting further clinical investigation into our FAP-CAR T cells.
Subject(s)
Immunotherapy, Adoptive , Receptors, Chimeric Antigen , Animals , Female , Humans , Mice , Cancer-Associated Fibroblasts/immunology , Cancer-Associated Fibroblasts/metabolism , Cell Line, Tumor , Cross Reactions/immunology , Endopeptidases , Gelatinases/immunology , Gelatinases/metabolism , Immunotherapy, Adoptive/methods , Immunotherapy, Adoptive/adverse effects , Membrane Proteins/immunology , Membrane Proteins/genetics , Mice, Inbred BALB C , Mice, Inbred C57BL , Neoplasms/immunology , Neoplasms/therapy , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/genetics , Serine Endopeptidases/immunology , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , T-Lymphocytes/immunology , Xenograft Model Antitumor AssaysABSTRACT
Helicobacter pylori (Hp) is a Gram-negative bacterium that colonizes in the gastric mucosa. Hp induces the production of cancer-associated fibroblasts (CAF) in the stomach. The virulence factors of Hp and CAF trigger epithelial-mesenchymal transition (EMT), leading to local inflammation, damage to the gastric mucosa, and the occurrence of chronic gastritis. Here, we summarize the molecular mechanisms of CAF mediated gastric EMT after Hp infection, providing new insights into potential molecular targets and strategies for the future treatment of Hp infection associated gastric cancer.
Subject(s)
Epithelial-Mesenchymal Transition , Gastric Mucosa , Helicobacter Infections , Helicobacter pylori , Stomach Neoplasms , Humans , Helicobacter Infections/complications , Helicobacter pylori/physiology , Stomach Neoplasms/microbiology , Stomach Neoplasms/pathology , Stomach Neoplasms/etiology , Gastric Mucosa/microbiology , Gastric Mucosa/pathology , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/physiology , AnimalsABSTRACT
BACKGROUND: Cancer-associated fibroblasts (CAFs) are one of the major components of prostate stromal cells, which play a crucial part in tumor development and treatment resistance. This study aimed to establish a model of CAFs-related microRNAs (miRNAs) to assess prognostic differences, tumor microenvironments, and screening of anticancer drugs by integrating data from single-cell RNA sequencing (scRNA-seq) and bulk RNA sequencing (buRNA-seq). METHODS: scRNA-seq and buRNA-seq data of primary prostate cancer (PCa) were downloaded from Gene Expression Omnibus and The Cancer Genome Atlas databases. Statistical methods including Least absolute shrinkage and selection operator (Lasso), Lasso penalized, Random Forest, Random Forest Combination, and Support Vector Machine (SVM) were performed to select hub miRNAs. Pathway analyses and assessment of infiltrating immune cells were conducted using Gene Set Enrichment Analysis and the CIBERSORT algorithm. The expression of CAFs-related miRNAs in fibroblast cell lines were validated through quantitative real-time PCR. Cell Counting Kit 8 (CCK8), wound-healing, clone formation, and cell migration assays were used to explore cell proliferation, growth, and migration in vitro. A mouse xenograft model was established to investigate the effect of CAFs on tumor growth in vivo. RESULTS: Through single-cell transcriptomics analysis in 34 PCa patients, 89 CAFs-related mRNAs were identified. A prognostic model based on 9 CAFs-related miRNAs (hsa-miR-1258, hsa-miR-133b, hsa-miR-222-3p, hsa-miR-145-3p, hsa-miR-493-5p, hsa-miR-96-5p, hsa-miR-15b-5p, hsa-miR-106b-5p, and hsa-miR-191-5p) was established to predict biochemical recurrence (BCR). We have determined through two prediction methods that NVP-TAE684 may be the optimal targeted therapy drug for treating CAFs. Downregulation of hsa-miR-106b-5p in CAFs significantly suppressed cell proliferation, migration, and colony formation in vitro. In vivo studies using a xenograft model further confirmed that hsa-miR-106b-5p downregulation significantly reduced tumor growth. CONCLUSION: Our findings conducted an integrated bioinformatic analysis to develop a CAFs-related miRNAs model that provides prognostic insights into individualized and precise treatment for prostate adenocarcinoma patients. Downregulation of miR-106b-5p in CAFs significantly suppressed tumor growth, suggesting a potential therapeutic target for cancer treatment.
Subject(s)
Cancer-Associated Fibroblasts , MicroRNAs , Prostatic Neoplasms , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Prostatic Neoplasms/metabolism , Male , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Cancer-Associated Fibroblasts/metabolism , Mice , Animals , Neoplasm Recurrence, Local/genetics , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Mice, NudeABSTRACT
BACKGROUND: Cancer-associated fibroblasts (CAFs) have been reported that can affect cancer cell proliferation, metastasis, ferroptosis, and immune escape. METTL3-mediated N6-methyladenine (m6A) modification is involved in the tumorigenesis of colorectal cancer (CRC). Herein, we investigated whether METTL3-dependent m6A in CAFs-derived exosomes (exo) affected CRC progression. METHODS: qRT-PCR and western blotting analyses detected levels of mRNAs and proteins. Cell proliferation and metastasis were evaluated using MTT, colony formation, transwell, and wound healing assays, respectively. Cell ferroptosis was assessed by detecting cell viability and the levels of Fe+, reactive oxygen species, and glutathione after erastin treatment. Exosomes were isolated from CAFs by ultracentrifugation. The m6A modification profile was determined by methylated RNA immunoprecipitation assay and the interaction between METTL3 and ACSL3 (acyl-CoA synthetase 3) was verified using dual-luciferase reporter assay. Animal models were established for in vivo analysis. RESULTS: CAFs promoted CRC cell proliferation and metastasis, and suppressed cell ferroptosis. METTL3 was enriched in CAFs and was packaged into exosomes. The m6A modification and METTL3 expression were increased in CRC samples. Knockdown of METTL3 in CAFs-exo suppressed CRC cell proliferation and metastasis, and induced cell ferroptosis. Mechanistically, METTL3 induced ACSL3 m6A modification and stabilized its expression. The anticancer effects mediated by METTL3-silenced CAFs-exo could be rescued by ACSL3 overexpression. Moreover, in vivo assay also showed that CAFs-exo with decreased METTL3 could hinder CRC growth and metastasis in mice models. CONCLUSION: CAFs promoted the proliferation and metastasis, and restrained the ferroptosis in CRC by exosomal METTL3-elicited ACSL3 m6A modification.
Subject(s)
Cancer-Associated Fibroblasts , Cell Proliferation , Coenzyme A Ligases , Colorectal Neoplasms , Exosomes , Ferroptosis , Methyltransferases , Ferroptosis/genetics , Methyltransferases/metabolism , Methyltransferases/genetics , Exosomes/metabolism , Exosomes/genetics , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Humans , Mice , Cancer-Associated Fibroblasts/metabolism , Animals , Coenzyme A Ligases/genetics , Coenzyme A Ligases/metabolism , Neoplasm Metastasis , Cell Line, Tumor , Mice, Nude , MaleABSTRACT
BACKGROUND: Cancer-associated fibroblasts (CAFs) play a crucial role in the progression of colorectal cancer (CRC). However, the impact of CAF subpopulation trajectory differentiation on CRC remains unclear. METHODS: In this study, we first explored the trajectory differences of CAFs subpopulations using bulk and integrated single-cell sequencing data, and then performed consensus clustering of CRC samples based on the trajectory differential genes of CAFs subpopulations. Subsequently, we analyzed the heterogeneity of CRC subtypes using bioinformatics. Finally, we constructed relevant prognostic signature using machine learning and validated them using spatial transcriptomic data. RESULTS: Based on the differential genes of CAFs subpopulation trajectory differentiation, we identified two CRC subtypes (C1 and C2) in this study. Compared to C1, C2 exhibited worse prognosis, higher immune evasion microenvironment and high CAF characteristics. C1 was primarily associated with metabolism, while C2 was primarily associated with cell metastasis and immune regulation. By combining 101 combinations of 10 machine learning algorithms, we developed a High-CAF risk signatures (HCAFRS) based on the C2 characteristic gene. HCAFRS was an independent prognostic factor for CRC and, when combined with clinical parameters, significantly predicted the overall survival of CRC patients. HCAFRS was closely associated with epithelial-mesenchymal transition, angiogenesis, and hypoxia. Furthermore, the risk score of HCAFRS was mainly derived from CAFs and was validated in the spatial transcriptomic data. CONCLUSION: In conclusion, HCAFRS has the potential to serve as a promising prognostic indicator for CRC, improving the quality of life for CRC patients.
Subject(s)
Cancer-Associated Fibroblasts , Colorectal Neoplasms , Tumor Microenvironment , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Humans , Cancer-Associated Fibroblasts/pathology , Cancer-Associated Fibroblasts/metabolism , Prognosis , Tumor Microenvironment/genetics , Cluster Analysis , Machine Learning , Biomarkers, Tumor/genetics , Transcriptome , Gene Expression Regulation, Neoplastic , Gene Expression Profiling/methods , Female , MaleABSTRACT
Pancreatic cancer is a very aggressive disease with a dismal prognosis. The tumor microenvironment exerts immunosuppressive activities through the secretion of several cytokines, including interleukin (IL)-1. The IL-1/IL-1 receptor (IL-1R) axis is a key regulator in tumor-promoting T helper (Th)2- and Th17-type inflammation. Th2 cells are differentiated by dendritic cells endowed with Th2-polarizing capability by the thymic stromal lymphopoietin (TSLP) that is secreted by IL-1-activated cancer-associated fibroblasts (CAFs). Th17 cells are differentiated in the presence of IL-1 and other IL-1-regulated cytokines. In pancreatic cancer, the use of a recombinant IL-1R antagonist (IL1RA, anakinra, ANK) in in vitro and in vivo models has shown efficacy in targeting the IL-1/IL-1R pathway. In this study, we have developed sphingomyelin nanosystems (SNs) loaded with ANK (ANK-SNs) to compare their ability to inhibit Th2- and Th17-type inflammation with that of the free drug in vitro. We found that ANK-SNs inhibited TSLP and other pro-tumor cytokines released by CAFs at levels similar to ANK. Importantly, inhibition of IL-17 secretion by Th17 cells, but not of interferon-γ, was significantly higher, and at lower concentrations, with ANK-SNs compared to ANK. Collectively, the use of ANK-SNs might be beneficial in reducing the effective dose of the drug and its toxic effects.
Subject(s)
Interleukin 1 Receptor Antagonist Protein , Interleukin-1 , Pancreatic Neoplasms , Sphingomyelins , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Humans , Interleukin 1 Receptor Antagonist Protein/pharmacology , Interleukin-1/metabolism , Sphingomyelins/metabolism , Cytokines/metabolism , Cell Line, Tumor , Inflammation/drug therapy , Inflammation/metabolism , Inflammation/pathology , Th17 Cells/immunology , Th17 Cells/drug effects , Th17 Cells/metabolism , Th2 Cells/immunology , Th2 Cells/drug effects , Th2 Cells/metabolism , Tumor Microenvironment/drug effects , Nanoparticles/chemistry , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/drug effectsABSTRACT
Understanding the tumor microenvironment (TME) and extracellular matrix (ECM) is crucial in cancer research due to their impact on tumor progression. Collagens, major ECM components, regulate cell signaling and behavior. Of the 28 reported collagens, type XII collagen is known to be vital for ECM organization. Over-produced by cancer-associated fibroblasts (CAFs), its upregulation correlates with poor survival in various cancers. This study aimed to develop an ELISA for quantifying circulating type XII collagen as a cancer biomarker. A specific ELISA targeting the C-terminal of type XII collagen was developed and used to analyze serum samples from cancer patients (n = 203) and healthy controls (n = 33). Additionally, type XII collagen expression was assessed in CAFs and normal fibroblasts (NFs) from different tissues, both under TGF-ß stimulated and non-stimulated conditions. The nordicPRO-C12 ELISA demonstrated robustness and specificity for type XII collagen. PRO-C12 levels were significantly elevated in patients with various cancers compared to healthy controls and effectively distinguished between cancer patients and controls. Findings were validated using gene expression data. Furthermore, Western blot analysis revealed increased type XII collagen expression in both CAFs and NFs upon TGF-ß1 stimulation, suggesting a potential role of TGF-ß1 in modulating the expression of type XII collagen in cancerous and normal tissue microenvironments. This study unveils a promising avenue for harnessing PRO-C12 as a non-invasive serum biomarker, enabling the quantification of type XII collagen fragments in cancer patients. Further investigations are warranted to explore the potential of PRO-C12 across different cancer types and disease stages, shedding light on its multifaceted role in cancer development.
Subject(s)
Biomarkers, Tumor , Collagen Type XII , Enzyme-Linked Immunosorbent Assay , Neoplasms , Humans , Neoplasms/blood , Neoplasms/pathology , Biomarkers, Tumor/blood , Collagen Type XII/blood , Female , Male , Tumor Microenvironment , Middle Aged , Fibroblasts/metabolism , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/pathology , Transforming Growth Factor beta1/blood , Adult , AgedABSTRACT
Resistance to immune checkpoint therapy (ICT) presents a growing clinical challenge. The tumor microenvironment (TME) and its components, namely tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs), play a pivotal role in ICT resistance; however, the underlying mechanisms remain under investigation. In this study, we identify expression of TNF-Stimulated Factor 6 (TSG-6) in ICT-resistant pancreatic tumors, compared to ICT-sensitive melanoma tumors, both in mouse and human. TSG-6 is expressed by CAFs within the TME, where suppressive macrophages expressing Arg1, Mafb, and Mrc1, along with TSG-6 ligand Cd44, predominate. Furthermore, TSG-6 expressing CAFs co-localize with the CD44 expressing macrophages in the TME. TSG-6 inhibition in combination with ICT improves therapy response and survival in pancreatic tumor-bearing mice by reducing macrophages expressing immunosuppressive phenotypes and increasing CD8 T cells. Overall, our findings propose TSG-6 as a therapeutic target to enhance ICT response in non-responsive tumors.
Subject(s)
Cancer-Associated Fibroblasts , Cell Adhesion Molecules , Immune Checkpoint Inhibitors , Pancreatic Neoplasms , Tumor Microenvironment , Animals , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/metabolism , Humans , Tumor Microenvironment/immunology , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/drug effects , Mice , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic use , Cell Line, Tumor , Cell Adhesion Molecules/metabolism , Cell Adhesion Molecules/genetics , Myeloid Cells/metabolism , Myeloid Cells/immunology , Tumor-Associated Macrophages/metabolism , Tumor-Associated Macrophages/immunology , Tumor-Associated Macrophages/drug effects , Mice, Inbred C57BL , Female , Drug Resistance, Neoplasm , Macrophages/immunology , Macrophages/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolismABSTRACT
Tumor-stromal interactions and stromal heterogeneity in the tumor microenvironment are critical factors that influence the progression, metastasis, and chemoresistance of pancreatic ductal adenocarcinoma (PDAC). Here, we used spatial transcriptome technology to profile the gene expression landscape of primary PDAC and liver metastatic PDAC after bioactive black phosphorus nanomaterial (bioactive BP) treatment using a murine model of PDAC (LSL-KrasG12D/+; LSL-Trp53R172H/+; and Pdx-1-Cre mice). Bioinformatic and biochemical analyses showed that bioactive BP contributes to the tumor-stromal interplay by suppressing cancer-associated fibroblast (CAF) activation. Our results showed that bioactive BP contributes to CAF heterogeneity by decreasing the amount of inflammatory CAFs and myofibroblastic CAFs, two CAF subpopulations. Our study demonstrates the influence of bioactive BP on tumor-stromal interactions and CAF heterogeneity and suggests bioactive BP as a potential PDAC treatment.