Establishment And Characterization Of A Human Renal Clear Cell Carcinoma Cell Clones With Different Metastatic Potential

Renal cell carcinomar (RCC) occurs more commonly in urinary system, which is associated with more aggressive and extensive metastatic characteristics. One thirds patients have been in advanced stages when being detected for the first time, and forty percent patients reoccur or metastasis after operations with worse prognosis. Renal Clear cell carcinoma is first most common subtype of renal cell carcinomar. Therefore, developing a series of matastic associated biological marks will be of great significance in detecting RCC effectively with the potential to reduce the death rate. It's critical step for screening and identifying metastatic associated biological mark that renal clear cell carcinomar cell lines with different metastatic potential is established in the same genetic background. The present study was divided into three parts: First, establishment of human renal clear cell carcinomar orthotopictransplant metastatic models; Secondly, estanlishment of sublines with high metastatic potential isolated by selecting in vitro and vivo successsively. Thir -dly, establishment and characterization of human renal clear cell carcinoma cell clones with different meta static potential.Establish human clear cell carcinomar orthotopic-transplant metastatic models.Exponentially growing 786-0 cells were harvested from subconfluent cultures by overla -ying the monolayers with a solution of 0.25 % trypsin. Subcutaneous inoculation of 786-0 cells yielded tumors in nude mice. The tumor was resectioned when it reached 1.5cm in diameter and was passaged in the same way (2mice/one passage) in nude mice. Tumor in the subcuits was removed after 3-4 passages and transplanted orthotopicly into renal subcapsule of nude mice to construct SOI (Surgical Orthotopic Implantation) models. Themice were killed and autopsied when they became moribund. Lung matastic lesion was harvested and secondary culture in vitro. After 4-5 passages with a solution of 0.25 % trypsin, the subline 786-0LM1 was obtained. At the same time 786-0 cells suspensions were injected orthotopicly into renal subcapsule and constructed COI (Cellular Orthoto -pic Injection) models, but no lung mastastic lesion was detected. The tumorigenicity and lung metastatic rates were high in SOI comparing to COI. Hence SOI model is appropriate for isolating metastatic cells in vivo.Establishment of sublines with high metastatic potential by selecting in vitro and in vivo successively.According to tumor heterogeneity theory: neoplasms contain subpopulations of cells having different metastatic potential, only those tumor cells can establish a metastasis which are capable of completing the entire process: dissociating from the primary mass, migrating through the interstitial extracellular matrix, invading the basement membrane underlying the vascular endothelium and entering the circulation, then attaching to and invading though the endothelial cell layer and basement membrane to enter the interstitial matrix and establish a metastasis. Therefore, invasive and matastatic associated factors can be used as basis for isolating tumor cells with different invasive, matastatic potentials.Malignant cells must traverse basement membranes during their migration to sites distant from the primary tumor. Matrige is a solubulized basement membrane preparation extracted from the Englebreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in ECM proteins. Its major component is laminin, followed by collagen IV, heparan sulfate proteoglycans, and entactin. At room temperature, Matrigel polymerizes to produce boilo -gically active matrix material resembling the mammalian cellular basement membrane, which are thin continuous sheets applied over filters in the transwell insert, blocking non-invasive cells from migrating through the membrane. In contrast, invasive tumorcells are able to detach themselves from and invade through the Matrigel and the 8 micr -on membrane pores. Therefor, it is well suited for selecting invasive cell phenotypes from non-invasive cell phenotypes in response to a chemoattractant.In our study, exponentially growing 786-0LM1 cells ( NO.5 passage ) were used for isolation in transwell. In brief: the polycarbonate membranes (containing 8-/mi pores) of the 24-well transwell inserts were coated with diluted Matrigel, 3T3 fibroblast condition -ed medium was added to the lower compartment as a chemoattractant, and 105 tumor cells resuspended in serum-free 1640 were added to the upper compartment, After 72h incubation at 37℃ 5% CO₂, the inserts were removed. The cells that penetrate the Matrigel and attached to the lower-chamber compartments were harvested and expanded for the second-round in vivo selection: subcutaneous injection and then orthotopic trans -plantation into renal subcapsule of nude mice. The same procedure was repeated twice, a subline 786-0LM3 from the third round of lung metastasis was thus established.Establishment and characterization of human renal clear cell carcinoma Cell Clones with Different Metastatic PotentialCell clone culture was conducted on the 1 Oth passage of the source cell line 786-0LM3 using limited dilution methord . Taget clones were selected by in vivo screening in nu -de mice. Human renal clear cell carcinoma Cell Clones with high (786-OH) and low (786-OL) metastatic potential were established, when 786-OH was compared with 786-OL, the pulmonary metastatic rate was 100% versus 20%. 786-OH cell clone exhibited different characteristics in vitro invasion assay, Chromosomal analysis, colony formation in soft agarose, tumor formation in nude mice by a Xenotransplanta -tion of cells from 786-OL. Compared with 786-OL, 786-OH has the characteristics of shorter multiply time and high agar clone forming rate ( P < 0. 01). SP immunocytoch -emistry was applied in to detect protein expression of RCC metastatic associated...