Parallel Screening And Validation Of Metastatic Human Pancreatic Cancer Cells And Related Genes From Mixed Subpopulations

Objective To identify and validate metastatic potential of mixed pool of candidate human pancreatic cell subpopulations. To study the molecular mechanism of malignant tumor metastasis, and lay the foundation for pancreatic cancer clinical diagnosis, treatment as well as prevention.Methods A pool of mixed subpopulations of metastatic human pancreatic cancer cells were cultured and, in the first round of nude mice in vivo screening, injected into two groups (Doxycycline treated and untreated) intravenously. Metastatic tumor cells were harvested from lungs tissue of the injected nude mice through primary tissue culture; genomic DNAs were extracted from metastatic tumor cells from each mouse, and characterized with genomic PCR analysis with specific gene probes.In the second round of nude mice in vivo screening, the pool of mixed subpopulations of metastatic human pancreatic cancer cells were injected into two groups (Doxycycline treated and untreated) intravenously as well as intraperitoneally. The other procedures (primary tissue culture、genomic PCR) are the same as the first round.Results Both rounds of in vivo screening of high lung metastatic potential have successfully obtained pancreatic cell subgroups of highly pulmonary specific metastastasis. The results of the first round of in vivo screening and genomic PCR show that62.5%(5/8) nude mice without Doxycycline treatment have been detected the expresstion of SQSTM1gene, while only37.5%(3/8) nude mice detected SQSTM1gene expression.The results of the second round of in vivo screening (intravenously) and genomic PCR show that75%(6/8) nude mice without Doxycycline treatment have been detected the expresstion of SQSTM1gene, while only37.5%(3/8) nude mice detected SQSTM1gene expression. Nude mice without Doxycycline treatment developed signs of lung metastasis much earlier and have shorter survival time than ones with doxycycline treatment.The results of the second round of in vivo screening (intraperitoneally) and genomic PCR show that62.5%(5/8) nude mice without Doxycycline treatment have been detected the expresstion of SQSTM1gene, while only33.34%(2/6)nude mice detected SQSTM1gene expression.The two rounds of in vivo screening of high lung metastatic potential showed that nude mice without Doxycycline treatment developed signs of lung metastasis much earlier and have shorter survival time than ones with doxycycline treatment. Metastatic tumor cells were successfully cultured from lungs of both experimental groups. Genomic DNA PCR analyses of other candidate genes, including SEMA4B, UBE2K and GNA13, did not show positive association with metastatic potential. This further validated that Doxycycline mediated expression of SQSTM1gene played an important role in lung metastasis of human pancreatic tumor cells in nude mice. What is more, the results of Western-Blot and q-PCR vindicate that the expression the SQSTM1gene can be rheostatically manipulated by Doxycyclin in our experiment.Conclusion Parallel screening and analysis identified a subpopulation of human pancreatic cancer cells, which had high lung metastatic potential and carried a regulated expression of SQSTM1gene. SQSTM1gene plays an important role in the lung metastasis of pancreatic cancer cells. On the basis of the Genome-wide Random Homozygous Knockout (RHKO) technology, SQSTM1gene expression can be rheostatically manipulated with and without Doxycyclin.