Adipose tissue-derived stem cells differentiate into carcinoma-associated fibroblast-like cells under the influence of tumor-derived factors

Carcinoma-associated fibroblasts (CAF) are considered to contribute to tumor growth, invasion and metastasis. However, the cell type of origin remains unknown. Since human adipose tissue-derived stem cells (hASCs) are locally adjacent to breast cancer cells and might directly interact with tumor cells, we investigated whether CAFs may originate from hASCs. We demonstrated that a significant percentage of hASCs differentiated into a CAF-like myofibroblastic phenotype (e.g., expression of alpha smooth muscle actin and tenascin-C) when exposed to conditioned medium from the human breast cancer lines MDAMB231 and MCF7. The conditioned medium from MDAMB231 and MCF7 contains significant amounts of transforming growth factor-beta 1 (TGFβ1) and the differentiation of hASCs towards CAFs is dependent on TGFβ1 signaling via Smad3 in hASCs. The induction of CAFs can be abolished using a neutralizing antibody to TGFβ1 as well as by pretreatment of the hASCs with SB431542, a TGFβ1 receptor kinase inhibitor. Additionally, we found that these hASC-derived CAF-like cells exhibit functional properties of CAFs, including the ability to promote tumor cell invasion in an in vitro invasion assay, as well as increased expression of stromal-cell-derived factor 1 (SDF-1) and CCL5. Taken together, these data suggest that hASCs are a source of CAFs which play an important role in the tumor invasion.     

Reprogramming of cancer-associated fibroblasts combined with immune checkpoint inhibitors: A potential therapeutic strategy for cancers

Activated fibroblasts, namely cancer-associated fibroblasts (CAFs), are highly heterogeneous in phenotypes, functions, and origins. CAFs originated from varieties of cell types, including local resident fibroblasts, epithelial cells, mesenchymal stromal cells, or others. These cells participate in tumor angiogenesis, mechanics, drug access, and immune suppression, with the latter being particularly important. It was difficult to distinguish CAFs by subsets due to their complex origins until the use of scRNA-seq. Reprogramming CAFs with TGFβ-RI inhibitor, a CXCR4 blocker, or other methods increases T cells activation and infiltration, together with a decrease in CAFs recruitment, thus improving the prognosis. As depletion of CAFs can't bring clinical benefit, the combination of reprogramming CAFs and immune checkpoint inhibitors (ICIs) come into consideration. It has shown better outcomes compared with monotherapy respectively in basic/preclinical researches, and needs more data on clinical trials. Combination therapy may be a promising and expecting method for treatment of cancer.     

Aberrant hypermethylation of the FGFR2 gene in human gastric cancer cell lines

We have previously shown that fibroblast growth factor receptor 2 (FGFR2) plays an important role in gastric carcinogenesis. In this study, we assessed DNA methylation status in the promoter region of FGFR2 gene in gastric cancer cell lines, and indicated that this region was highly methylated, compared with FGFR2-expressing gastric cancer cell lines. Moreover, the restoration of FGFR2 expression by treating methylated cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine strongly suggests that the loss of FGFR2 expression may be due to the aberrant hypermethylation in the promoter region of the FGFR2 gene. Thus, our results suggest that the epigenetic silencing of FGFR2 through DNA methylation in gastric cancer may contribute to tumor progression.     

Downregulation of Microrna‐148a in Cancer‐Associated Fibroblasts from Oral Cancer Promotes Cancer Cell Migration and Invasion by Targeting Wnt10b

It is well established that crosstalk between cancer‐associated fibroblasts (CAFs) and cancer cells plays a critical role in the occurrence and development of oral squamous cell carcinoma (OSCC). The molecular mechanisms underlying such interaction, however, remain far from clear. Accumulating data have indicated that microRNAs involved in tumor microenvironment, particularly in CAFs, contribute to the activation of fibroblasts and metastasis of cancer cells. Here, we showed that miR‐148a was downregulated in CAFs compared with normal fibroblasts isolated from clinical OSCC tissue. Investigation of miR‐148a function in fibroblasts demonstrated that overexpression of miR‐148a in CAFs significantly impaired the migration and invasion of oral carcinoma cells (SCC‐25) by directly targeting WNT10B. Taken together, these data suggested that miR‐148a might be a novel candidate target for the treatment of OSCC.     

Breakdown of the endothelial barrier function in tumor cell transmigration

The ability of tumor cells to metastasize is associated with a poor prognosis for cancer. During the process of metastasis, tumor cells circulating in the blood or lymph vessels can adhere to, and potentially transmigrate through, the endothelium and invade the connective tissue. We studied the effectiveness of the endothelium as a barrier against the invasion of 51 tumor cell lines into a three-dimensional collagen matrix. Only nine tumor cell lines showed attenuated invasion in the presence of an endothelial cell monolayer, whereas 17 cell lines became invasive or showed a significantly increased invasion. Endothelial cells cocultured with invasive tumor cells increased chemokine gene expression of IL-8 and Gro-β. Expression of the IL-8 and Gro-β receptor, CXCR2, was upregulated in invasive tumor cells. Addition of IL-8 or Gro-β increased tumor cell invasiveness by more than twofold. Tumor cell variants selected for high CXCR2 expression were fourfold more invasive in the presence of an endothelial cell layer, whereas CXCR2 siRNA knock-down cells were fivefold less invasive. We demonstrate that Gro-β and IL-8 secreted by endothelial cells, together with CXCR2 receptor expression on invasive tumor cells, contribute to the breakdown of the endothelial barrier by enhancing tumor cell force generation and cytoskeletal remodeling dynamics.     

Tumor cell and carcinoma-associated fibroblast interaction regulates matrix metalloproteinases and their inhibitors in oral squamous cell carcinoma

Co-culture of periodontal ligament (PDL) fibroblasts and SCC-25 oral squamous carcinoma cells (OSCC), results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs). Paracrin circuits between CAFs and OSCC cells were hypothesized to regulate the gene expression of matrix remodeling enzymes in their co-culture, which was performed for 7days, followed by analysis of the mRNA/protein expression and activity of metalloproteinases (MMPs), their tissue inhibitors (TIMPs) and other relevant genes. Interleukin1-β, transforming growth factor-β1, fibronectin and αvβ6 integrin have shown to be involved in the regulation of the MMP and TIMP gene expression in co-culture of CAFs and tumor cells. In addition, these cells also cooperated in activation of MMP pro-enzymes. It is particularly interesting that the fibroblast-produced inactive MMP-2 has been activated by the tumor-cell-produced membrane-type 1 matrix metalloproteinase (MT1-MMP). The crosstalk between cancer- and the surrounding fibroblast stromal-cells is essential for the fine tuning of cancer cells invasivity.     

CXCL12 derived from CD248-expressing cancer-associated fibroblasts mediates M2-polarized macrophages to promote nonsmall cell lung cancer progression

Nonsmall cell lung cancer (NSCLC) is among the most prevalent malignant tumours threatening human health. In the tumour microenvironment (TME), cancer-associated fibroblasts (CAFs) induce M2-polarized macrophages, which strongly regulate tumour progression. However, little is known about the association between CAFs and M2 macrophages. CD248 is a transmembrane glycoprotein found in several cancer cells, tumour stromal cells, and pericytes. Here, we isolated CAFs from tumour tissues of NSCLC patients to detect the relationship between CD248 expression and patient prognosis. We knocked down the expression of CD248 on CAFs to detect CXCL12 secretion and macrophage polarization. We then examined the effects of CD248-expressing CAF-induced M2 macrophage polarization to promote NSCLC progression in vitro and in vivo. We found that CD248 is expressed mainly in NSCLC-derived CAFs and that the expression of CD248 correlates with poor patient prognosis. Blocking CXCL12 receptor (CXCR4) drastically decreased M2 macrophage chemotaxis. CD248 promotes CAFs secreting CXCL12 to mediate M2-polarized macrophages to promote NSCLC progression both in vitro and in vivo. Collectively, our data suggest that CD248-positive CAFs induce NSCLC progression by mediating M2-polarized macrophages.     

Candidate Markers That Associate with Chemotherapy Resistance in Breast Cancer through the Study on Taxotere-Induced Damage to Tumor Microenvironment and Gene Expression Profiling of Carcinoma-Associated Fibroblasts (CAFs)

Recently, emerging evidence has suggested that carcinoma-associated fibroblasts (CAFs) could contribute to chemotherapy resistances in breast cancer treatment. The aim of this study is to compare the gene expression profiling of CAFs before and after chemotherapy and pick up candidate genes that might associate with chemotherapy resistance and could be used as predictors of treatment response. CAFs were cultured from surgically resected primary breast cancers and identified with immunohistochemistry (IHC) and Flow cytometry (FCM). MDA-MB-231 cells were cultured as the breast cancer cell line. Cell adhesion assay, invasion assay, and proliferation assay (MTT) were performed to compare the function of MDA-MB-231 cells co-cultured with CAFs and MDA-MB-231 cells without co-culture, after chemotherapy. Totally 6 pairs of CAFs were prepared for microarray analysis. Each pair of CAFs were obtained from the same patient and classified into two groups. One group was treated with Taxotere (regarded as after chemotherapy) while the other group was not processed with Taxotere (regarded as before chemotherapy). According to our study, the primary-cultured CAFs exhibited characteristic phenotype. After chemotherapy, MDA-MB-231 cells co-cultured with CAFs displayed increasing adhesion, invasiveness and proliferation abilities, compared with MDA-MB-231 cells without CAFs. Moreover, 35 differentially expressed genes (absolute fold change >2) were identified between CAFs after chemotherapy and before chemotherapy, including 17 up-regulated genes and 18 down-regulated genes. CXCL2, MMP1, IL8, RARRES1, FGF1, and CXCR7 were picked up as the candidate markers, of which the differential expression in CAFs before and after chemotherapy was confirmed. The results indicate the changes of gene expression in CAFs induced by Taxotere treatment and propose the candidate markers that possibly associate with chemotherapy resistance in breast cancer.     

Cancer-Associated Fibroblasts in a Human HEp-2 Established Laryngeal Xenografted Tumor Are Not Derived from Cancer Cells through Epithelial-Mesenchymal Transition,Phenotypically Activated but Karyotypically Normal

Cancer-associated fibroblasts (CAFs) play a crucial role in cancer progression and even initiation. However, the origins of CAFs in various cancer types remain controversial, and one of the important hypothesized origins is through epithelial-mesenchymal transition (EMT) from cancer cells. In this study, we investigated whether the HEp-2 laryngeal cancer cells are able to generate CAFs via EMT during tumor formation, which is now still unknown. The laryngeal xenografted tumor model was established by inoculating the HEp-2 laryngeal cancer cell line in nude mice. Primary cultured CAFs from the tumor nodules and matched normal fibroblasts (NFs) from the adjacent connective tissues were subcultured, purified, and verified by immunofluorescence. Migration, invasion, and proliferation potentials were compared between the CAFs and NFs. A co-culture of CAFs with HEp-2 cells and a co-injection of CAFs with HEp-2 cells in nude mice were performed to examine the cancer-promoting potential of CAFs to further verify their identity. Karyotypic analyses of the CAFs, NFs, and HEp-2 cells were conducted. A co-culture of NFs with HEp-2 cells was also performed to examine the expression of activated markers of CAFs. A pathological examination confirmed that the laryngeal xenografted tumor model was successfully established, containing abundant CAFs. Immunocytochemical staining verified the purities and identities of the CAFs and NFs. Although the CAFs manifested higher migration, invasion, proliferation, and cancer-promoting capacities compared with the NFs, an analysis of chromosomes revealed that both the CAFs and NFs showed typical normal mouse karyotypes. In addition, the NFs co-cultured with HEp-2 cells did not show induced expressions of activated markers of CAFs. Our findings reveal that the CAFs in the HEp-2 established laryngeal xenografted tumor are not of laryngeal cancer origin but of mouse origin, indicating that the HEp-2 laryngeal cancer cells cannot generate their own CAFs via EMT in this model.     

Effect of insulin receptor down regulation on insulin-stimulated thymidine incorporation in cultured human fibroblasts and tumor cell lines.

Insulin receptors in transformed tissue are relatively resistant to down regulation by insulin, and although receptor downregulation reduces rapid onset biologic responses to insulin in normal tissue, this is not observed in tumor cells. The present study compares longterm insulin responses (thymidine incorporation and cell growth) in normal human fibroblasts with responses in human tumor cell lines (MCF-7, T-47D and HCT-8) to determine whether these responses are also resistant to the effects of receptor down regulation. Thymidine incorporation into fibroblasts was more responsive to insulin than was incorporation into tumor cells, although stimulation of uptake into fibroblasts was not paralleled by changes in cell replication. In contrast, physiological insulin concentrations inhibited, and high concentrations of insulin stimulated, thymidine incorporation and cell replication in MCF-7 and T-47D cells. All insulin concentrations inhibited thymidine incorporation in HCT-8 cells without affecting cell replication. The responsiveness of fibroblasts, MCF-7 and HCT-8 cells to insulin was unaltered by down regulation of insulin receptors prior to measuring thymidine incorporation, whereas receptor down regulation paradoxically increased the responsiveness of T-47D cells to insulin. Exposure of fibroblasts to 5 x 10(-8) M dexamethasone for 24h increased their responsiveness to insulin but did not influence the response of MCF-7 or HCT-8 cells, whereas insulin-stimulated incorporation of thymidine in T-47D cells was inhibited. Thus, receptor down regulation does not influence the longterm biologic response to insulin in normal cells, and paradoxically increases responsiveness in one of three tumor cell lines. These changes may contribute to the well-described stimulatory effects of insulin on tumor cell growth and inhibition of this response with dexamethasone may be relevant to cancer treatment programs.     

Human breast cancer associated fibroblasts exhibit subtype specific gene expression profiles

ABSTRACT: BACKGROUND: Breast cancer is a heterogeneous disease for which prognosis and treatment strategies are largely governed by the receptor status (estrogen, progesterone and Her2) of the tumor cells. Gene expression profiling of whole breast tumors further stratifies breast cancer into several molecular subtypes which also co-segregate with the receptor status of the tumor cells. We postulated that cancer associated fibroblasts (CAFs) within the tumor stroma may exhibit subtype specific gene expression profiles and thus contribute to the biology of the disease in a subtype specific manner. Several studies have reported gene expression profile differences between CAFs and normal breast fibroblasts but in none of these studies were the results stratified based on tumor subtypes. METHODS: To address whether gene expression in breast cancer associated fibroblasts varies between breast cancer subtypes, we compared the gene expression profiles of early passage primary CAFs isolated from twenty human breast cancer samples representing three main subtypes; seven ER+, seven triple negative (TNBC) and six Her2+. RESULTS: We observed significant expression differences between CAFs derived from Her2+ breast cancer and CAFs from TNBC and ER + cancers, particularly in pathways associated with cytoskeleton and integrin signaling. In the case of Her2+ breast cancer, the signaling pathways found to be selectively up regulated in CAFs likely contribute to the enhanced migration of breast cancer cells in transwell assays and may contribute to the unfavorable prognosis of Her2+ breast cancer. CONCLUSIONS: These data demonstrate that in addition to the distinct molecular profiles that characterize the neoplastic cells, CAF gene expression is also differentially regulated in distinct subtypes of breast cancer.     

Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion

Fibroblasts often constitute the majority of the stromal cells within a breast carcinoma, yet the functional contributions of these cells to tumorigenesis are poorly understood. Using a coimplantation tumor xenograft model, we demonstrate that carcinoma-associated fibroblasts (CAFs) extracted from human breast carcinomas promote the growth of admixed breast carcinoma cells significantly more than do normal mammary fibroblasts derived from the same patients. The CAFs, which exhibit the traits of myofibroblasts, play a central role in promoting the growth of tumor cells through their ability to secrete stromal cell-derived factor 1 (SDF-1); CAFs promote angiogenesis by recruiting endothelial progenitor cells (EPCs) into carcinomas, an effect mediated in part by SDF-1. CAF-secreted SDF-1 also stimulates tumor growth directly, acting through the cognate receptor, CXCR4, which is expressed by carcinoma cells. Our findings indicate that fibroblasts within invasive breast carcinomas contribute to tumor promotion in large part through the secretion of SDF-1.     

Autophagic secretion of HMGB1 from cancer-associated fibroblasts promotes metastatic potential of non-small cell lung cancer cells via NFκB signaling

Tumor progression requires the communication between tumor cells and tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are major components of stromal cells. CAFs contribute to metastasis process through direct or indirect interaction with tumor cells; however, the underlying mechanism is largely unknown. Here, we reported that autophagy was upregulated in lung cancer-associated CAFs compared to normal fibroblasts (NFs), and autophagy was responsible for the promoting effect of CAFs on non-small cell lung cancer (NSCLC) cell migration and invasion. Inhibition of CAFs autophagy attenuated their regulation on epithelial–mesenchymal transition (EMT) and metastasis-related genes of NSCLC cells. High mobility group box 1 (HMGB1) secreted by CAFs mediated CAFs’ effect on lung cancer cell invasion, demonstrated by using recombinant HMGB1, HMGB1 neutralizing antibody, and HMGB1 inhibitor glycyrrhizin (GA). Importantly, the autophagy blockade of CAFs revealed that HMGB1 release was dependent on autophagy. We also found HMGB1 was responsible, at least in part, for autophagy activation of CAFs, suggesting CAFs remain active through an autocrine HMGB1 loop. Further study demonstrated that HMGB1 facilitated lung cancer cell invasion by activating the NFκB pathway. In a mouse xenograft model, the autophagy specific inhibitor chloroquine abolished the stimulating effect of CAFs on tumor growth. These results elucidated an oncogenic function for secretory autophagy in lung cancer-associated CAFs that promotes metastasis potential, and suggested HMGB1 as a novel therapeutic target.Subject terms: Cancer microenvironment, Non-small-cell lung cancer, Metastasis, Translational research     

Cancer-associated fibroblasts–derived exosomes-mediated transfer of LINC00355 regulates bladder cancer cell proliferation and invasion

The aim of this study was to investigate the regulatory mechanism of cancer-associated fibroblasts (CAFs) exosome in bladder cancer (BC) cell proliferation and invasion. CAFs and normal fibroblasts (NFs) were isolated from tumor tissues and adjacent normal tissues of BC patients, and examined by immunocytochemistry for the expression of fibroblast activation protein alpha (FAP) and α-smooth muscle actin (α-SMA). Exosomes were extracted from CAFs and NFs and observed under a transmission electron microscope, and expression of the exosome markers CD9 and CD63 was confirmed by western blotting. The distribution and intensity of fluorescence were observed by confocal laser microscopy to analyze exosomes uptake by BC cell lines T24 or 5367. BC cell proliferation and invasion were detected by MTT and Transwell assays, respectively. LINC00355 levels in CAFs, NFs, CAFs exosome, NFs exosome, and BC cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Results showed that CAFs exosome significantly promoted BC cell proliferation and invasion relative to NFs exosome. LINC00355 expression was significantly elevated in CAFs exosome when compared with that in NFs-exosome. Up-regulated LINC00355 expression was observed both in T24 and 5367 cells co-incubated with CAFs exosome. Exosomes derived from LINC00355 siRNA-transfected CAFs observably repressed BC cell proliferation and invasion when compared with control siRNA-CAFs exosome. In conclusion, CAFs exosome–mediated transfer of LINC00355 regulates BC cell proliferation and invasion. Significance of the study. In this study, our data suggest that the exosomes released from CAFs promote BC cell proliferation and invasion. The mechanism of this effect is, at least in part, related to the increased LINC00355. Regulation of LINC00355 expression in exosomes released from CAFs might be a putative therapeutic strategy against the pathogenesis of BC.     

Carcinoma-Associated Fibroblasts Lead the Invasion of Salivary Gland Adenoid Cystic Carcinoma Cells by Creating an Invasive Track

Carcinoma-associated fibroblasts (CAFs) are critical in determining tumor invasion and metastasis. However the role of CAFs in the invasion of salivary gland adenoid cystic carcinoma (ACC) is poorly understood. In this study, we isolated primary CAFs from two ACC patients. ACC-derived CAFs expressed typical CAF biomarkers and showed increased migration and invasion activity. Conditioned medium collected from CAFs significantly promoted ACC cell migration and invasion. Co-culture of CAFs with ACC cells in a microfluidic device further revealed that CAFs localized at the invasion front and ACC cells followed the track behind the CAFs. Interfering of both matrix metalloproteinase and CXCL12/CXCR4 pathway inhibited ACC invasion promoted by CAFs. Overall, our study demonstrates that ACC-derived CAFs exhibit the most important defining feature of CAFs by promoting cancer invasion. In addition to secretion of soluble factors, CAFs also lead ACC invasion by creating an invasive track in the ECM.