Alterations In Mechanical Properties With Prostate Cancer Progression And Interferes The Apoptosis And Invasion In Prostate Cancer Cells

Prostate cancer (Pca) is the most frequently diagnosed malignant tumor amongmen in the United States and European countries, and it is often palpable during adigital rectal examination (DRE). Physicians have noted that tumors are typically harderthan the surrounding tissue. However, after splitting prostate cancer specimens in half,we often find the perceived hardness of part of a tumor tissue is different from that ofthe whole. The connections between tissue mechanics and cancer progression have beenstudied in many malignancies, but the relationship between mechanical properties andmalignant behavior in Pca has yet to be determined.Collagen is the most abundant extracellular matrix (ECM) scaffolding protein andcontributes significantly to the tensile strength of tissue. Furthermore, there have beenreports that alterations in ECM stiffness are correlated with tumor progression andmetastasis. As a “bridge” located between the ECM and cell membranes, fibronectinmolecules contain binding sites for collagens and cell surface receptors (integrins).Moreover, integrins and fibronectin are necessary elements for most mechano-sensingmodels and lie at the beginning of the sensing pathway that correlates with tumorbehavio. In previous studies, several methods such as atomic force microscopy (AFM),unconfined compression, transrectal real-time tissue elastography (TRTE), andindentation have been used to measure the mechanical properties of tissues. However,few of these studies measured the mechanical properties of tissues on both the macro- and micro-scales in the same study.Objective1. Measure the differences in mechanical properties between cancerous and BPHtissues by TRTE and AFM on both the macro-and micro-scales, and study therelationship between the mechanical properties and pathological grade and metastasis;2. Evaluate the probable cause of the difference on mechanical properties, and toidentify the key factors of the mechanotransduction pathway usingimmunohistochemistry (IHC); and try to explain the relationships between themechanical properties and malignant behavior.3. Investigate the effect of different concentration of collagen I on cell adherent,apoptosis，Young’s modulus and invasion.Methods1. A total of43patients participated in this study. Twenty patients underwentsurgery for BPH, and twenty-three patients underwent surgery for Pca. None of thepatients included in this study had had chemotherapy or radiotherapy before surgery.Pca tissues were collected from the foci of23patients who underwent transurethralresection of the prostate cancer (TURP) and suprapubic prostatectomy. The BPH tissueswere collected from20patients who underwent TURP. The pathological biopsydiagnosis was performed and confirmed both preoperatively and postoperatively.Gleason scores were examined by two senior pathologists experienced in Pca diagnosis.The20BPH patients composed the control group, and the Pca group was divided intotwo sub-groups based on the Gleason score (11patients for Gleason2-7and12patientsfor Gleason8-10) and the presence of metastasis (16patients for non-metastasis and7patients for metastasis). All the patients was tested by TRTE andAFM.2. For immunohistochemical analysis, the extent of target proteins expressionwere calculated by two independent pathologists who were blinded to theclinicopathological parameters of the patients. The percentage of positive cells whichshow immunorative staining on specific location were counted by10representativemicroscopic fields. And then, the specimens were scored in a semiquantitative manner based on the percentage of tumor cells showing immunoreactivity. The criteria used forthe assessment of staining for Collagen I, III, IV, MMP-2, FN, and Integrin α5were:0(negative),1(weak),2(medium), and3(strong).3. With different concentration of collagen I, culture the PC-3cell line and LNCaPcell line in3-dimension, after that test adherent ability and invasion ability and sensitiveof apoptosis on prostate cancer cells.Results1.Mechanical parameters of prostate tissues measured by TRTE andAFMTo investigate the mechanical properties of BPH and Pca, a total of43patients,including20BPH patients and23Pca patients, were subjected to TRTE. The mean SI ofPca tissues were significant higher than that of BPH tissue, especially in high Gleasonscore (8-10)(P<0.05). To further investigate the differences in the mechanical propertiesof BPH and Pca tissues, AFM testing was conducted. A total of43tissue sections (20BPH and23Pca) were obtained from patients as described previously, and for eachtissue section, three random sites were probed by AFM. The Young’s modulus of thePca tissues was significant lower than that of BPH tissues (P<0.05); and the meanYoung’s modulus of low-to-medium Gleason score (2-7) Pca tissues was lower than thatof high Gleason score (8-10) Pca tissues; there were significant difference between theYoung’s moduli of non-metastatic and metastatic Pca tissues (P<0.05).2. The expression of proteins in ECM and the expression level of importantmolecules on mechanotransduction signaling pathwayThe expression levels of Collagen I, III, IV in Pca tissues were significant lowerthan that of BPH tissues (P<0.05). Further more, in advance cancer, their expressionwere decrease further. The expression of MMP-2in Pca tissues were significant higherthan that of BPH tissues, but the expression in low-meditate Gleason score (2-7) tissue were higher than that of high Gleason score, and there was significant differencebetween metastasis and non-metastasis tissues (P<0.05). There was no differencebetween the expression of fibronectin in BPH and Pca tissues, but the location wasdifferernce. The expression of integrin α5was higher in Pca tissues than in BPHtissues (P<0.05).3. Experimental study of miRNA-198mimic inhibits the Livin geneexpression and interferes the apoptosis and invasion in prostate cancer cellsDilute the collagen I into1mg/ml and5mg/ml, and culture the PC-3cell line andLNCaP cell line in3-dimension. Use computer software to calculate the number ofadherent cells and cell areas, and then number the cell witch invasive through theTranswell insert, and finally, use TUNEL kit to measure the sensitive of prostate cancercells apoptosis. We found that PC-3cells were better adhere to collagen I than that ofLNCaP cells. And in lower concentration of collagen I, the ability of invasion washigher than that of higher concentration in PC-3cells, and PC-3cells were moresensitive to higher concentration collagen I on aoptosis.Conclusions1. On macro-scale, higher mechanical properties are associate with high Gleasonscore. And on micro-scale, lower mechanical properties are associate with high Gleasonscore.2. The prostate cancer cells proliferation were our of control, and the cells squeezewith each other and generate tension to made the mechanical properties higher onmacro-scale. The degradation collagens by MMP-2made the mechanical propertieslower on micro-scale, and fibronecin, MMP-2and integrin α5were importantmolecules on mechanotransductin signaling pathway.3. Collagen I was a kind of barrier structure of cell invasion in ECM, at the sametime, PC-3cell were tend to adhere on collagen I. And high concentration of collagen Icue to the higher sensation of apoptosis in prostate cancer cells.