Primary Culture Of Human Colorectal Carcinoma Associated Fibroblasts And Their Metabolic Characteristics

Objective： To screen and optimize the conditions for the primary culture ofcolorectal carcinoma associated fibroblasts (CCAFs) and to explore their metaboliccharacteristics so as to search for potential target for the treatment of colorectal cancer.Methods：1. Fresh specimens from rectal carcinoma were pretreated with highconcentration antibiotics in low temperature.2. The collagenase digestion method andtissue explantation method were compared with12-day cell counts.3. The effects ofdifferent culture conditions on6-day emigration ratios and12-day cell counts wereinvestigated to optimize the culture surface，medium，FBS levels and block size in primaryculture of CCAFs.4. CCAFs were identified by immunofluorescence and morphologyand growth curve.5.Use immunohistochemical methods, immunofluorescence ways,18F-FDG intake, lactate test and western blot to compare CCAFs with CNFs and cancerinduced CCAFs from Characteristics of glycolysis then find the differences.6. SPSS18.0is for the data statistics.Results:1. Tissue explatation method had more cell yields than collagenase digestionmethod. Higher FBS concentration, larger tissue blocks and PRMI-1640medium led tomore12-day cell counts, while the texture of culture bottle had no such effects.6-dayemigration ratios were related to medium and the culture texture.2.The harvested cellsintensively expressed SMA-α and MCT4presenting typical characteristics ofCAFs,while cancer cells expressed MCT1.High rate18F-FDG intake and lactate exclusionare showed in CCAFs and cancer induced CCAFs.Conclusion: Pretreatment of surgical specimen with high concentration antibiotics inlow temperature, tissue explantation method with large blocks and PRMI-1640mediumwith high concentration FBS are the optimized conditions for primary culture of CCAFs.Tumor trigger the fibroblasts to CCAFs,and improve the capability of glucose intake,Then by aerobic glycolysis, CCAFs expel actate,which make the surrounding “lethal acidicconditions”.In contrast,the cancer cells uptake the actate to satisfy their proliferation andmetastasis.