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Renal cell carcinoma (RCC) is a common lethal urological cancer,the distant metastasis of which is the leading cause of death.Although targeted agents have remarkably improved the overall prognosis of RCC patients,nearly all the patients eventually acquire therapeutic resistance.With the advent of immune checkpoint inhibitors,immunotherapy based on tumor microenvironment (TME) has shown a broad scope in clinical application.The deepening understanding of TME leads to the changes of therapeutic strategies for advanced RCC,and the combination of targeted therapy and immunotherapy is exhibiting a promising prospect.Herein,we reviewed the TME characteristics,candidate predictive biomarkers,and possible targets for future development of drugs against RCC.
Subject(s)
Female , Humans , Male , Carcinoma, Renal Cell/therapy , Immune Checkpoint Inhibitors , Immunotherapy , Kidney Neoplasms/therapy , Tumor MicroenvironmentABSTRACT
@#AIM: To investigate the role and significance of carcinoma-associated fibroblasts(CAFs)in the occurrence and development of eyelid basal cell carcinoma(BCC), and study on the biological characteristics and the expression of fibroblast activation protein(FAP)of two kinds of fibroblasts associated with eyelid BCC and normal eyelid skin fibroblasts(normal fibroblasts, NFs).<p>METHODS: CAFs and NFs were obtained by tissue primary cultured. The cell morphology of the 3<sup>rd</sup> generation purified cells were observed under an inverted phase contrast microscope and identified them using their biomarker by immunohistochemistry for CK, VIM, α-SMA and FAP. Cell growth and proliferation were measured by MTT assay. The expression of FAP mRNA and protein in cells was detected by RT-qPCR and Western Blot.<p>RESULTS: The CAFs of the eyelid was long fusiform or spindle-shaped, with reduced cytoplasmic processes, more cytoplasmic granules, different cell sizes, disordered arrangement, overlapping growth, and loss of contact inhibition. NFs were in the form of extensive shuttle, radial arrangement, the cytoplasmic particles were rare, there was no overlapping growth phenomenon, and no contact inhibition. The proliferation rate of eyelid CAFs was faster than that of NFs. And the expression of FAP mRNA in CAFs was 3.672±0.221, which was significantly higher than that in NFs(1.034±0.024)(<i>P</i><0.05). In addition, the expression level of FAP protein in CAFs was high, while NFs were not expressed(<i>P</i>< 0.05).<p>CONCLUSION: There were significant differences in the biological characteristics of CAFs and NFs, such as morphological structure, growth and the proliferation, growth factor expression and so on. It was therefore suggested that the tumor microenvironment of eyelid basal cell carcinoma had changed, and further induced the biological characteristics and function of NFs, and finally transformed into CAFs. In addition, eyelid CAFs expressed higher expression of FAP than NFs, indicating that FAP may be involved in the occurrence and development of tumor cells in tumor microenvironment, which is associated with the invasive growth of CAFs in eyelid.
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Objective@#To observe the effect of transforming growth factor-β1 (TGF-β1) on the migration of oral carcinoma associated fibroblasts (CAFs) with two-dimensional culture model and three-dimensional model.@*Methods @# Under two-dimensional culture conditions, CAFs stimulated by TGF-β1 with the addition of 10 ng/mL medium were used as the experimental group, and untreated CAFs were used as the control group. The migration of CAFs with the stimulation of TGF-β1 was measured by cell scratch assay and transwell assay. CAFs positive for green fluorescent protein (GFP) were cultured by retrovirus transfection. Human tongue squamous cell carcinoma cells SCC25, GFP(+) CAFs and CAFs with three-dimensional cell co-culture models were established. The three-dimensional model cultured under the stimulation of TGF-β1 with 10 ng/mL medium was used as the experimental group, and the three-dimensional model without TGF-β1 was used as the control group. The migration of CAFs with the stimulation of TGF-β1 was also measured by the three-dimensional models.@*Results@# It was verified that 10 ng/mL TGF-β1 promoted the migration of CAFs in the two-dimensional culture model. The three-dimensional co-culture models of SCC25, GFP(+) CAFs and CAFs were successfully established. The migration of SCC25 and CAFs was detected in the three-dimensional model. However, 10 ng/mL TGF-β1 had little effect on their migration.@*Conclusion@#The effect of TGF-β1 in vitro on the migration of oral CAFs was associated with different culture models in two and three dimensions.
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Objective@#To research the expression levels of FEN1 and PCNA in carcinoma-associated fibroblasts (CAFs) and analyze their correlation. @*Methods@#Fresh specimens of oral squamous cell carcinoma tissues and normal oral mucosal tissues excised during oral and maxillofacial plastic surgery were collected. Primary oral CAFs and normal fibroblasts (NFs) were obtained by tissue culture, identified by immunocytochemistry and divided into the CAF and NF groups. Western blot and quantitative real-time PCR were used to detect the protein and mRNA expression levels of both FEN1 and PCNA in the oral CAFs and NFs. The correlation between FEN1 and PCNA expression in oral CAFs was analyzed. @*Results@#Oral CAFs and oral NFs were successfully cultured and identified from 12 samples. Both the protein and mRNA expression levels of FEN1 and PCNA were higher in the oral CAFs than NFs, but there were no significant differences (P > 0.05). In the oral CAFs, the linear correlation coefficient between FEN1 and PCNA was 0.677 (P = 0.016) at the mRNA level, indicating a strong positive correlation; however, at the protein level, no correlation was found (P > 0.05). @*Conclusion@# In primary cultured oral CAFs and NFs, there were no significant differences in the FEN1 and PCNA protein and mRNA expression levels. However, in the CAFs, the mRNA levels of FEN1 and PCNA had a strong positive correlation. The relationship and the regulatory mechanism of the two genes require further study.
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Objective To investigate the expression differences and significance of periostin (PN) in eyelid basal cell carcinoma associated fibroblasts (BCAFs) andnormal fibroblasts (NFs) after separation,culture,purification and identification.Methods The third generation of purified BCAFs and NFs was selected,and the concentrations of cell suspensions were modulated to 20 × 106 L-1 by trypsin,and then the cell suspension were seeded and cultured in 6-well plate by 2 mL per well.The cell culture supernatants were collected when BCAFs and NFs were cultured by serum-free medium for 48 h,then the content of PN in cell culture supernatants from BCAFs and NFs was detected by enzyme-linked immunosorbent assay (ELISA).The glass coverslips were placed at the bottom of the 6-well plate to make cell slides,and then the expression of PN in BCAFs and NFs cells were tested by immunofluorescence staining.Results ELISA showed that the content of PN in cell culture supernatants from BCAFs and NFs was (9.26 ± 2.35) μg · L-1 and (2.57 ± 0.41) μg · L-1.And the expression level of PN in BCAFs tested by immunofluorescence staining technology was higher than that in NFs cells,and the differences were statistically significant (all P ＜ 0.05).Conclusion The expression and secretion of PN in the eyelid BCAFs were highly enhanced when compared with NFs,suggesting that periostein may promote or inhibit the occurrence and development of the eyelid basal cell carcinoma in the microenvironment of the eyelid basal cell carcinoma.
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Objective:To investigate whether the omental-adipose stromal cells (O-ASCs) exposing to gastric cancer-conditioned medi-um (CM) could be inducted to differentiate into carcinoma-associated fibroblasts (CAFs) and the effect of ERK signaling pathway in the process. Methods: We identified O-ASCs by examining their ability to differentiate osteogenic and adipogenic lineages and through flow cytometry. O-ASCs were co-cultured with MGC803 and SGC7901CM. The expression of CAFs markers (α-SMA, FSP-1, and vimentin) and paracrine factors (VEGFA, TGF-β1, FAP, and SDF-1) were evaluated by RT-PCR and Western blot. In vitro cultures of O-ASCs were divided into three groups:the control, SGC7901-CM, and SGC7901-CM+U0126 groups. Cells were collected after 12 h. West-ern blot was performed to evaluate the expression ofα-SMA, FSP-1, ERK, and p-ERK1/2. Results:The primary cells were O-ASCs. The expression levels of CAFs markers (α-SMA, FSP-1, and vimentin) and O-ASC paracrine factors (VEGFA, TGF-β1, FAP, and SDF-1) clearly in-creased (P0.05), while the ex-pression of p-ERK1/2,α-SMA, and FSP-1 significantly improved (P0.05), while the expression levels of p-ERK1/2,α-SMA, and FSP-1 decreased (P<0.05). Conclusion:O-ASCs participate in the peritoneal metastasis of gastric cancer through differentiation by CAF and paracrine factors. The ERK signaling pathway is important in the differentiation of O-ASCs towards CAFs.
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Tumor microenvironment (TME) plays a key role in the development and progression of tumors, such as pro?moting local drug resistance, immune escape, and distal metastasis. According to the TME of different individuals, accurate evaluation and selection of clinical medication can effectively control the malignant transformation of carcinoma in situ and metastatic cancer. At present, the main method to treat cancer is chemotherapy, TME can regulate the reaction of the tumor cells to the standard chemotherapy and target drug therapy, so the combination of the targeted TME therapy and chemothera?py will achieve better clinical efficacy. In this review, we summarized the mechanisms of TME in breast cancer, including ex?tracellular matrix, carcinoma-associated fibroblasts, carcinoma-associated macrophages, regulatory T cells and bone marrow mesenchymal stem cells, which providing a theoretical basis for the development of TME targeted therapy.
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The biological characteristic of carcinomas is determined both by the stromal microenvironment and the oncogene or anti-oncogene.The tumor stroma is also known as the reactive stroma which is composed of base member,immunocell,capillary,fibroblasts and ECM.Fibroblasts are the majority of tumor stromal cells.The relationship between fibroblasts and the initiation,progression of carcinoma has being in the spot light.In this review,we summarized the advance in the study of carcinoma-associated fibroblasts.