Mechanistic Studies Of Disseminated Tumor Cells (DTCs) Formation Promoted By Tumor Microenvironment

Objectives:1) To explore the mechanisms of DTCs formation promoted by tumor microenvironment; 2) To further test whether blocking this process may reduce the recurrence and metastasis of prostate cancer (PCa).Methods:We firstly established stable luciferase labeled PCa cells by lentivirus transfection (PC3-luc, C4-2B-luc, and RM-1-luc), and then these cells were subcutaneously injected into the nude and C57BL/6J mice respectively. When subcutaneous tumor grew to a certain size, we removed the tumor and cultured the tumor cells in vitro. We examined the EMT morphological changes and alterations of EMT hall markers by Western blot. At the same time, DTCs were tested from bone marrow of these mice. In addition, we established cardiac injection and tibia injection PCa metastic mouse model, and re-cultured their lung-specific metastasis PCa cells.Results:We successfully constructed stable luciferase labeled PCa cells, and established subcutaneously injected PCa mouse model. We found that subcutaneously-implanted PCa cells developed EMT after interaction with microenvironment in vivo and DTCs were successfully identified and confirmed in mouse bone marrow. These subcutaneous PCa cells obtained stronger ability of proliferation, migration, invasion and angiogenesis. We further observed that these alterations may be caused by regulating MCP-1 and AP-1 via Akt signaling pathways.Conclusion:Subcutaneously-implanted PCa cells developed EMT after interaction with the tumor microenvironment and promoted DTCs formation in mouse bone marrow. PCa cells with mesenchymal phenotype obtained stronger ability of proliferation, migration, invasion and angiogenesis, which may be caused by regulating MCP-1 by AP-1 via Akt signaling pathways.Section 1:The establishment of luciferase labeled PCa cellsObjective:To construct stable luciferase labeled PCa cellsMethods:Transformed, amplified and extracted pCDNA3.1, pGL3-control, pGL4.50 and packing plasmid VSV-G, pRRE, RSV-REV; Compared lipofectin transfected pCDNA3.1 and pGL3-control together, lipofectin transfected pGL4.50, electroporation transfected pGL4.50 and pGLV4-EF 1 a-Luclentivirus transfected PCa cell lines to construct stable luciferase labeled PCa cells. Collected strong luciferase expression PC3-luc and C4-2B-luc by flow cytometry cell sorting according to the expression of GFP; constructed luciferase expressed lentivirus vector, identified correct constructed one by bacteria liquid PCR, double-enzyme cleavage method and sequencing. We packaged constructed lentivirus vector by VSV-G, pRRE and RSV-REV in 293T cells. Transfected RM-1 and other kind of cell lines by our packaged lentivirus, Blasticidin was used to select positive cells.Results:After comparing several kinds of transfection methods, our study selected pGLV4-EF1a-Luc lentivirus to construct the luciferase labeled PC3 and C4-2B PCa cell lines and collected strong positive cells by flow cytometry cell sorting according to GFP expression, PC3-luc and C4-2B-luc. And also we constructed the luciferase lentivirus vector, selected correct constructed one, packaged, transfected RM-1 and some other kinds of cell lines, such as lung cancer, liver cancer, ovarian cancer and their drug resistant cell lines for other research use.Conclusion:Compared with lipofection and electroporation transfection, lentivirus transfection method is more suitable for the construction of stable luciferase labeled cell lines.Section 2:To establish the mouse model of PCa cells occuring EMTObjective:To construct PC3-luc, C4-2B-lu, RM-1-luc subcutaneously injected PCa mouse model and identify early metastatic events.Methods:PC3-luc cells were subcutaneously injected into nude mouse, C4-2B-luc cells were subcutaneously injected into SCID mouse and RM-1-luc cells were subcutaneously injected into C57BL/6J mouse respectively. Bioluminescence image (BLI) will be obtained every week, Tumor measurement will be performed by calipers twice weekly and weigh twice weekly, when the tumor grew to a certain size, the mice were sacrificed, isolated the subcutaneous tumor and re-cultured in vitro (PC3-luc s1, C4-2B-luc s1, RM-1-luc s1), observed the morphological changes between subcutaneous tumor cells and parental cells, detected the EMT hallmarker changes of subcutaneous tumor cells.Results:Constructed PC3-luc, C4-2B-luc, RM-1-luc subcutaneously injected mouse model and obtained tumor growth curve; Phenotype changed after in vivo passing of tumor cells; compare to parental tumor cells, Vimentin and snail were up regulated in PC3-luc s1 cells, while E-cadherin and claudin-1 were down regulated in PC3-luc s1 cells.Conclusion:Tumor cells occurred EMT after in vivo passageSection3:Detection of DTCs in the bone marrow of subcutaneously injected mouseObjectives:To detect whether there are DTCs cells in the bone marrow of subcutaneously injected tumor cells mouse model.Methods:Subcutaneously injected tumor cells mouse model was constructed and sacrificed when tumor grew to a certain size, took normal mouse as control. Isolated ilium, femur and tibia (ilium and femur were used for bone marrow cells collection while tibia was used to fix, decalcification and paraffin embedding). When collected, bone marrow cells were used to extract total RNA for PCR detection. PC3-TxR, DU145, CNE2 mouse bone marrow were from other research groups in our lab. Detected the expression of Luc and Human GAPDH in mouse bone marrow by RT-PCR, took mouse GAPDH as internal reference. Put tibia into 10% neutral formalin, after 24 hours, transferred to 10% EDTA to decalcify, when finished transfer them to 70% ethanol for paraffin embedding.Results:The results of PCR show that there were Luc and Human GAPDH expression in subcutaneously injected tumor cell mouse model bone marrow. While there was no Luc and Human GAPDH expression in control normal mouse bone marrow.Conclusions:In subcutaneously injected tumor cells mouse model, tumor cells appear in their bone marrow.Section 4:Tumor microenvironment promotes PCa cells occurring biological changesObjective:To detect the biological changes occurring in PC3-luc s1 cells which obtained mesenchymal phenotype after EMTMethods:PC3-luc and PC3-luc s1 cells were subcutaneously injected in nude mice again (2×106 cells/mouse), BLI weekly and caliper measure tumor size, weight mice twice a week, compared the changes between PC3-luc and PC3-luc s1 cells mouse subcutaneous tumors. When tumor grew to a certain size, sacrificed the mice and isolated subcutaneous tumors, compared their tumor tissue difference between PC3-luc and PC3-luc s1 cells injected mice. Compared cell proliferation of PC3, PC3-luc and PC3-luc s1 cells in vitro by MTS, and compared their migration and invasion ability by wound scratch healing assay and transwell experiment.Results:PC3-luc s1 mice subcutaneous tumor grew faster than PC3-luc mice subcutaneous tumor in vivo. Compare to PC3-luc, PC3-luc s1 mice tumor were more uniformity and with more abundant blood vessels on their surface. In vitro, experiment indicated that compared with PC3 and PC3-luc cells, PC3-luc s1 cells did not increase in the proliferation, but their migration and invasion ability increases.Conclusion:Compare to PC3 and PC3-luc, PC3-luc s1 obtain more ability of proliferation, migration, invasion and angiogenesis.Section 5:The mechanism of tumor microenvironment inducing EMT and DTCsObjective:To explore the molecular mechanisms of prostate cancer cells occurring EMT and promoting DTCs formationMethods:Detected difference protein expression and transcription factors of PC3-luc, PC3-luc s1, C4-2B, C4-2B-luc, C4-2B-luc s1 in their conditioned medium and nuclear protein by antibody array and Protein/DNA array; Verified difference expression proteins in PC3, PC3-luc and PC3-luc s1 by ELISA, QRT-PCR and Western blot; Detected important signaling pathway proteins in PC3, PC3-luc and PC3-luc s1 by Western blot.Results:Compared with parental cells, the expression of MCP-1 in PC3-luc s1 and C4-2B-luc s1 conditioned medium was increased; In PC3-luc s1 nuclear extract, the expression of transcription factors c-Myb, Sp-1 were down regulated while the expression of c-Jun were upregulated and no obvious change in CREB. Western blot showed:compared with parental cells, the expression of p-mTOR was down regulated, the expression of p-Akt p-AMPKa, p-p70S6K, p-4E-BP1 were up regulated, the expression of total proteins were unchanged.Conclusion:Akt regulated MCP-1 by AP-1 or/and Akt/AMPKa/mTOR signaling pathway may regulate EMT and promote tumor cell proliferation, metastasis and angiogenesis.Section 6:The establishment of organ-specific metastasis mouse modelObjective:To establish PC3-luc and RM-1-luc cardiac injection and tibia injection metastasis mouse model.Methods:2×105 PC3-luc and RM-1-luc cells were injected into SCID mouse and C57BL/6J mouse by cardiac injection and tibia injection. BLI weekly and weigh mice twice every week to monitor tumor metastasis and mice health condition. When the tumor or the metastasis tumor grew to a certain size, sacrificed and obtained the internal organs, and put them into BLI. Isolated the lung metastasis tumor cells of PC3-luc and RM-1-luc lung metastasis model, and cultured them in vivo.Results:PC3-luc cells cardiac injected mouse model started to appear metastasis tumor about 4 weeks after injection, including lung metastasis, liver metastasis and bone metastasis. In PC3-luc cells tibia injected mouse model, cancer cells grew full fill femur, tibia and feet, and also they metastasis to the other side of leg and feet about 8 weeks after injection. RM-1-luc cells cardiac injected mouse model metastasis to whole body quickly, after 1 week injection; we found mice began to die because of metastasis. Isolated internal organ and exposed in imagine in vivo, RM-1-luc cells metastasized to brain, heart, lung, liver and kidney. And also RM-1-luc tibia injected mouse occurred metastasis too, including lung metastasis (primary), brain metastasis, heart metastasis. At last, we isolated PC3-luc and RM-1-luc lung metastasis tumor cells and re-cultured in vitro.Conclusion:PCa cells could develope whole body metastasis by cardiac injection and tibia injection.