Study On The Characteristics Of Cancer-associated Fibroblasts And The Detection By Multi-voxel ~1H-MRS

Objective:In this study,we separated cancer-associated fibroblasts(CAF)cells and non-activated fibroblasts(NAF)cells from clinical tissue samples.The phenotype and biological function change between CAF and NAF were explored and it turned out that the proliferation rate and cycle of CAF was slower than NAF.The lactic acid distribution of head and neck tumors was detected by multi-voxel ~1H-MRS to form multidum polylactic acid 2D~1H-MRSI/anatomically superimposed pseudo-color image,and the results were consistent with immunohistochemical assay.The pseudo-color image can reflect the distribution range and even the density of CAF cells in the head and neck tumor tissue,and provide the basis for the further study of the effective range of tumor evaluation.Methods: 1.The expression and distribution of fibroblast-specific protein(FSP1)and tumor proliferating cell nuclear antigen(Ki67)in tumor tissue were detected by immunofluorescence.2.The CAF cells and NAF cells were isolated by enzymatic digestion.All cells were purified by adherent method.The morphology and characteristics of the cells were observed under microscope.Frozen spare part of the cells for about 3 generations.3.The fibroblast activation protein(FAP)of CAF cells was detected by Western Blot,and the effect of CAF cells on the growth of tumor cells was detected by clonal formation assay and nude mice tumor growth experiment.4.Cell Count and Brdu experiments were used to compare the difference of CAF cells and NAF cells,and the distribution of CAF cells and NAF cells was detected and analyzed by flow cytometry.5.The expression profile of total RNA of CAF cells and NAF cells was detected by RNA-sequencing technique.The differential expression of related cyclin was analyzed by IPA(Ingenuity Pathway Analysis)software,and the related pathways were screened and verified by Western Blot and immunofluorescence.6.The content and distribution of lactic acid in tumor tissue and its surrounding tissues were detected by multi-voxel ~1H-MRS,and 2D~1H-MRSI/anatomically complex pseudo-color images were reconstructed.7.FSP1 immunohistochemistry was used to detect the distribution and density of CAF cells in tumor tissues,and the results were compared with the 2D~1H-MRSI/anatomically superimposed pseudo-color images.Results: 1.Immunofluorescence revealed that CAF cells were distributed around tumor cells,especially surrounding fast-proliferating tumor cells.2.CAF cells,which were isolated from fresh tumor tissues,were spindle like in NAF cells,but CAF cells were larger in size and more branched synapses.3.The expression of FAP protein in CAF cells was significantly higher than that in NAF cells.Cloning formation experiment,nude mice tumor growth experiments found that colon cancer and liver cancer CAF cells on the corresponding cancer cells have significant role in promoting growth.4.Cell count and Brdu experiments showed that CAF cells was significantly slower than that of NAF cells.Further analysis of flow cytometry showed that CAF cell cycle was significantly blocked in G1/S phase.5.The related pathway proteins after screening,immunofluorescence analysis showed that the tumor cells secrete large amounts of TGF-?2 and act on CAF cells,the cell cycle of its downstream pathway changes,including:TGF-?2/p15/CDK4,TGF-?2/p21/CDK2 and TGF-?2/CDC25A/Cyclin E2 or Cyclin D3 pathway is activated,and the cell cycle in the G1/S phase was blocked,resulting in the proliferation of CAF cells decreased.6.Multi-voxel ~1H-MRS imaging showed that the content of lactic acid in the head and neck tumor tissues and its surroundings increased significantly,and the edge of the tumor was the most obvious.7.Immunohistochemistry revealed that the tumor stroma and its surroundings contained a large number of CAF cells,and the localized CAF cells were the most rapidly proliferated on the edge of the tumor,which was consistent with the distribution of lactic acid in the multi-voxel ~1H-MRS.Conclusion: 1.CAF cells are closely related to the rapid proliferation of tumor cells and can significantly promote the growth of tumor cells.2.Tumor cells secrete large amounts of TGF-?2 in CAF cells,and their downstream cell cycle-related pathways change,resulting in cell cycle arrest in G1/S phase,leading to CAF cells significantly slower than NAF cells.3.Multi-voxel ~1H-MRS lactate imaging can showed the concentration of lactate in the tumor and its edge,which was consistent with the distribution of CAF cells found in immunohistochemistry.