Vesicles Derived From Tumor Cells Are Used As Sensitizers To Enhance The Chemotherapy Effect Of Bladder Cancer

Purpose:Nonmuscle-invasive bladder cancer(NMIBC)is usually treated by surgical resection following with chemotherapy in clinic.However,the residual tumor-repopulating cells(TRCs)could not be eliminated,resulting in the tumor recurrence and drugs resistance development.Tumor cells derived microparticles(T-MPs)are a type of vesicle with a diameter ranging from 100 to 1000 nm,which are composed of cell membranes encapsulating cytoplasmic contents.Those microparticles are released by tumor cells through changing the cytoskeletal structure,when tumor cells are stimulated by the extra-cellular signals or undergoing apoptosis.Previous studies have demonstrated that T-MPs could encapsulate chemotherapeutic agents and significantly enhance the anticancer effects in several tumors.Moreover,the specific lipid rafts structure enables the physical and chemical stability of microparticles,which reveals crucial potential application in clinical cancer therapy.T-MPs serve as a superior drugs carrier,however,the limited drug loading efficiency restricts the promotion of T-MPs application.Additionally,the metabolism and underlying mechanism of the T-MPs in tumor cells remain unclear.Here,we further investigated the T-MPs based drug delivery system and employed T-MPs as a chemo-sensitizer to combine with chemotherapeutic agents for NMIBC treatment.We also expounded the metabolism of T-MPs and the mechanism of T-MPs serving as a chemo-sensitizer,which provides an innovative approach for clinical NMIBC treatment.Methods:(1)To evaluate the sensitization effects induced by T-MPs to NMIBC,we established the orthotopic non-invasive superficial bladder cancer model of C57 mice.We isolated T-MPs from different tumor cells and used those T-MPs to treat the bladder tumors for chemotherapy sensitization analysis.We also performed H&E staining for tumor tissues to evaluate the anti cancer effects.(2)To detect whether the sensitization effect of T-MPs to chemotherapy was immune cells dependent,we used immunodeficient nude mice or macrophage deficient mice to establish the orthotopic bladder model.Then we treated those mice with T-MPs and evaluated the sensitization effect to chemotherapy.(3)To investigate whether T-MPs could enhance the cytotoxicity of chemotherapeutic agents to tumor cells,we used the flow cytometry to detect the Annexin-V and PI staining of tumor cells treated with T-MPs or chemotherapeutic agents.(4)To detect the influence of T-MPs to drugs uptake and efflux,we used the flow cytometry and HPLC to analyze the drugs uptake and efflux of tumor cells.(5)To detect the T-MPs influence to the P-pg expression of tumor cells,we used RT-PCR and western blotting to evaluate the expression of ABCB1 gene and P-gp protein of tumor cells treated with T-MPs.(6)To detect the T-MPs influence to tumor cells lysosomes,we used siRNA to knockdown the lysosomes associated gene TFEB.(7)To detect the co-location of Rab7,dynein,lysosomes and other proteins or organelles in tumor cells,we used immunofluorescence to mark those proteins and performed confocal analysis.(8)To detect the influence of tumor cells lysosomes pH value from T-MPs,we used lysosome sensor staining and lysosomal pH detection kit to calculate the pH value of tumor cells.(9)To evaluate the effects of chemotherapy to tumor repopulating cells treated with T-MPs,we used 3D soft gel culture system for tumor repopulating cells culture and detected the cytotoxicity of chemotherapeutic agents to tumor repopulating cells treated with T-MPs.Conclusion:T-MPs could be efficiently uptaken by tumor cells.T-MPs could down-regulate the expression of P-gp to enhance the drugs uptake and reduce the drugs efflux,resulting in elevated cytotoxicity of chemotherapeutic agents.Simultaneously,lysosome-entering T-MPs increase lysosomal pH value and changeits function;Next,Rab7 is recruited to the lysosomal membrane and dynein is then recruited to Rab7 complex.Then Rab7-dynein complex acts as a linker to bridge lysosome and microtubule.Next,dynein as motor protein pushes lysosome migration toward the nucleus along microtubule;Lastly,lysosome downloads drug moleculesaround the periphery of the nucleus;And drug molecules subsequently enter the nucleus via nuclear pores,resulting in the sensitization effects.T-MPs serve as lysosomes inhibitors,efficiently suppressing bladder tumor growth and providing innovative approach for clinical bladder cancer treatment.