| [Objective] Within the tumor microenvironment, in addition to tumor cells themselves, there are surrounding matrix, which are mainly composed of interstitial cells, immune inflammatory cells, cytokines, chemokines, microvascular and other components; this is the local pathology environment that provides tumors with growth environment and also interaction space between cancer cells and microenvironment. And interstitial cells is an important part of the tumor microenvironment. Recent studies found that partial tumors result from genetic changes in the tumor cells themselves, but a considerable part of them result from changes in the tumor microenvironment. Studies have confirmed that interstitial cells within the tumor microenvironment play an important role in the progression of other types of tumors by participating in abnormal expression of certain genes and specific cytokines as well as regulating many processes during tumor development; in addition, cytokines from endometrial interstitial cells can induce endometrial cancer differentiation and inhibit its growth and proliferation. However, the abnormal expression in endometrial cancer, its potential biological effects, and the involved signaling pathways remain to be further explored and determined. PTEN (Phosphatase and tensin homology on chromosome ten) gene is the first tumor suppressor gene with dual-specific phosphatase activity which can prevent cell growth, promote apoptosis, inhibit precancerous lesions and cancer incidence by dephosphorylation of phosphatidylinositol3-phosphate (phosphatidylinositol-3-phosphate, PIP3). It plays an important role in the development of tumors, including a low expression widely in endometrial cancer. Ras gene is one of the most common proto-oncogenes, which is closely related with tumor formation, proliferation, migration, diffusion, and angiogenesis. It has a wide selective overexpression in a variety of tumors in humans, including endometrial carcinoma. HGF (hepatocyte growth factor, HGF), the first named hepatocyte mitotic agent, which is mainly produced by interstitial cells, has a heterodimeric structure and is an important cytokine having a variety of biological functions. Its biological activity is mediated by a single receptor c-Met (c-met oncogene product). c-Met, which is also called MET and hepatocyte growth factor receptor, is a membrane receptor with tyrosine kinase activity, typically expresses in epithelial-origin cells. HGF and c-Met activate receptors by binding together, and their phosphorylation can cause a series of intracellular signal transduction cascade pathway activation, leading to different responses in various cells and tissues. Studies have shown that HGF/c-Met/Akt signaling pathway can regulate the proliferation and differentiation of tumor cells through activation of Akt in a series of tumors; and in many tumors including endometrial cancer, abnormalities in signal transduction are prevalent. This study explores the interaction between endometrial carcinoma interstitial cells (CIC) and normal endometrial epithelial cells (NEC) by establishing in vitro co-culture systems as well as in vivo animal experiment models between cancer cells and normal epithelial cells; at the same time, we will discuss about the abnormal expression of PTEN and Ras genes within the co-culture microenvironment between cancer interstitial cells and normal epithelial cells; we will further investigate the mediating role of HGF within the tumor microenvironment and study signaling pathways involved in the interaction, to make clear the key role of interstitial cells of endometrial carcinomer (EC) and paracrine factors in tumor progression. We are aimed to select appropriate therapeutic targets to provide useful information for improving patient survival.[Method]This study established the co-culture system of primary cells between endometrial interstitial cells and normal endometrial epithelial cells, and constructed a paracrine model to mimic the interaction between endometrial interstitial cells and normal epithelial cells, which can be used to detect the mediation role of endometrial carcinoma interstitial cells on normal endometrial epithelial cell proliferation and paracrine factor HGF within the tumor microenvironment; in addition, we investigated the possibly involved signaling pathways in their interaction.1. Collecting samples, culture and isolation of primary cells1) To obtain clinical tissue samples Clinical tissue samples were taken from Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, and Gynecologic Oncology Center, Isar right hospital, Technical University of Munich, including32cases of endometrial cancer surgical patients and48cases vaginal bleeding patients of childbearing age, among which21cases of vaginal bleeding patients were finally histopathologically diagnosed as secretory endometrium while27cases of vaginal bleeding patients were finally histopathologically diagnosed as proliferative endometrium. None patients received any hormone therapy within the previous six months of the surgery. All patients and their families were informed consent of the acquisition of samples and experimental purposes. All operations have been approved by the ethics committee from both countries.2) Primary cell isolation and culture Collagenase IV was used to digest and separate endometrial adenocarcinoma and normal endometrial tissue to get endometrial adenocarcinoma interstitial cells and normal endometrial epithelial cells as well as interstitial cells, which were identified with vimentin antibody respectively by immunocytochemistry.3) Immunocytochemistry The obtained three groups of carcinoma interstitial cells (CIC),normal endometrial epithelial cells (NEC), and normal interstitial cells (NIC) within petri dishes were seeded with cover slips for immunochemical detection of cells. Then4%paraformaldehyde was added for10minutes for rupture and incubated with5%NGS (normal goat serum) solution for30min in order to seal. The rabbit anti-human vimentin was added as the first antibody, and after4℃incubation overnight, HRP-conjugated secondary antibodies were added. DAB was used for color reaction, which was terminated timely according to the reaction. Then, the samples were counterstained with hematoxylin for30sec, followed by gradient dehydrationof alcohol (70%,80%,90%,95%,100%) with5min for each level. After that, the slides were mounted with neutral gum and sent to microscopy for imaging. Each sample was evaluated by two independent observers. With PBS as alternative primary antibody, these samples were treated as a negative control group. Immunohistochemical staining score was based on the strength of the sample and the number of positive cells. Immunostaining score=percentage of positive staining cells×staining intensity score.2. The effect of endometrial adenocarcinoma interstitial cells on normal epithelial cells1) Indirect cell co-culture Using indirect co-culture, the endometrial adenocarcinoma interstitial cells were seeded in the lower chamber of a6-well co-culture plate to provide paracrine factor, while the normal endometrial epithelial cells were seeded in the upper chamber filter of the6-well co-culture plate. They were cultured for1to5days. And the endometrial adenocarcinoma interstitial cells which were cultured alone were treated as a control group.2) Determination of cell growth curve The growth curve was used to measure cell proliferation. The endometrial adenocarcinoma interstitial cells and normal epithelial cells were co-cultured in96-well plates, with each group repeating three wells per day and seven days according to cell inoculation. Wait4h until cells adhere to the wells, CCK-8reagent was added and was placed at37℃in5%CO2incubator for1h. Microplate was used to measure absorbance at450nm to determine the actual starting cell density, which can be used as the relative growth baseline.The seven days’ detection data was processed using Excel to obtain cell proliferation plotted curves.3) Incorporation analysis of Brdu BrdU incorporation detection can be used to characterize the proportion of cells in S phase and proliferation. Immunofluorescence method was used to analyze BrdU incorporation in cells.The normal epithelial cells were seeded in well-prepared6-well plates, within which chambers seeded with adenocarcinoma interstitial cells were placed. At selected time points, culture fluid was aspirated and BrdU culture mediumwas added; then wash with PBS, add hydrochloric acid (HCl) for degeneration, add sodium borate solution for neutralization, and add BSA/PBS at room temperature for blocking; draw boundaries with impermeable pen around the four boundaries of covers lips with a middle line dividing the plate into two halves. One bigger half was diluted with BrdU antibody, whilePBS/BSA was added into the smaller half. They were incubated in a humid chamber at4℃overnight. Remove covers lips from the six-well plates, and turned over with cells face down affixed to glass slides. The samples were observed and imaged with microscope.4) Cell RNA extraction TRIzol (Invitrogen) reagent was used for RNA extraction. TRIzol reagent was added into each well to lyse the cells within the six-well plates, which were placed at room temperature. After adding chloroform and centrifuged, the supernatant was transferred into RNA FREE tubes with a pipette and added different treatment factors. A microplate reader was used to detect RNA concentration and purity, afterwards, the samples were stored in-80℃refrigerator.5) Reverse transcription of RNA into cDNA M-MLV reverse transcriptase (Promega) was used for cDNA synthesis. In the nuclease-free PCR tubes, RNA, Random Primer, Nuclease-Free H2O and other reagents were added in order. The PCR tubes were placed in PCR machine for heating and cooling. In the reaction system, different reagents were then added, which were placed inPCR instrument for incubation and inactivation.6) Real-time PCR Real-time PCR can determine the concentration of the PCR products by detecting fluorescence signal intensityin the PCR reaction which was emitted from the reaction system. SYBR(?) Premix Ex Taq TM (Perfect Real Time) kit (TaKaRa Biotechnology Co., Ltd. Dalian, China) was used for real-time quantitative PCR reaction, while operating instrument was Thermal Cycler Dice TM Real Time System (TP800real-time PCR instrument, TaKaRa). The product length met specification requirements of quantitative PCR amplification reference, with80bp-150bp as most appropriate (can be extended to300bp). Fluorescence threshold and background signal were as set as default value of the experimental apparatus. The number of cycles when the collected fluorescence signal intensity of each reaction tubereached thethreshold value(10times the baseline fluorescence intensity)is defined asCt value; in this study, each sample had three triplicates and the expression level of the target gene was expressed by2-△△Ct.The reaction product was analyzed with1.5%agarose gel electrophoresis and stained with ethidium bromide. Each product’s sequence was confirmed by automated sequencing.7) Western blot analysis Proteins were degraded using the protein lysate and cells were collected by low-temperature centrifugation. The protein extracts were treated with protein electrophoresis, transferred to a membrane, blocked and incubated with the first antibody overnight at a low temperature, followed by secondary antibodies incubation for30minutes. ODYSSEY infrared imaging system was used for membrane scanning and saving the image.8) Flow cytometry (FCM) for cell cycle Flow cytometry is a quantitative analysis technique for single cells.The cultured cells were trypsinized and transferred to a dedicated streaming tube, then centrifuged at room temperature; the supernatant was discarded and the cells were collected. Prechilled ethanol was added to fix cells, which were placed in-20℃for cryopreservation. The sample buffer was prepared to resuspend cells and then stored in the dark at room temperature for10min. With the excitation light of the appropriate wavelength, the fluorescence intensity emitted by PI was proportional to the DNA concentration. With fluorescence intensity as the horizontal and the number of cells as the vertical axis, the number of cells within each cycle can be obtained using flow cytometry analysis software.9) Nude mouse transplantation tumor experiment Cultured cells were inoculated subcutaneously in the4-week-old male BLAB/c nu nude mice shoulders, with each side of nude mice seeding2x106cells. For the right side, co-cultured normal endometrial interstitial cells and epithelial cells were seeded, while for the left side, co-cultured endometrial adenocarcinoma interstitial cells and normal epithelial cells were seeded, which can be used as controls. After the tumor was visible, the tumor size was measured with a vernier caliper periodically. The reference formula of tumor volume: Volume=longer diameter×shorter diameter×shorter diameter/2.3. Paracrine role of interstitial cells of endometrial adenocarcinoma1) Enzyme-linked immunosorbent assay(ELISA):The cell culture supernatant that the co-cultured adenocarcinoma interstitial cells and normal epithelial cells were seeded after72hours. Different interventions were given to cells. Then, the ELISA kit was used to detect levels of HGF and PGF2a within supernatants. And the absorbance at450nm wavelength was recorded.2) Immunohistochemistry(IHC):the samples will be detected by immunohistochemistry included endometrial adenocarcinoma (32cases) and normal endometrium (48cases), which were then treated with paraffin embedding, sectioning, dewaxing, and hydrationaccording to the manufacturer instructions on operation. The double-blindmethod was used for random selection and analysis of results. Each specimen was evaluated by two independent observers. With PBS as alternative primary antibody, these samples were treated as a negative control group. Immunohistochemical staining score was based on the strength of the sample and the number of positive cells. Immunostaining score=percentage of positive staining cells×staining intensity score.4%HGF mechanism in epithelial cell proliferation1) Western blot (WB): proteins were degraded using the protein lysate and cells were collected by low-temperature centrifugation.The protein extracts were treated with protein electrophoresis, transferred to a membrane, blocked and incubated with the first antibody (p-Akt, β-catenin, p-ERK1/2, p-NF-kB, P-actin) overnight at a low temperature, followed by secondary antibodies incubation for30minutes. ODYSSEY infrared imaging system was used for membrane scanning and saving the image.2) Determination of cell growth curve:the growth curve was used to measure cell proliferation. The endometrial adenocarcinoma interstitial cells and normal epithelial cells were co-cultured in96-well plates, with each group repeating three wells per day and seven days according to cell inoculation. Wait4h until cells adhere to the wells, CCK-8reagent was added and was placed at37℃in5%CO2incubator for1h. Microplate was used to measure absorbance at450nm to determine the actual starting cell density, which can be used as the relative growth baseline.The seven days’detection data was processed using Excel to obtain cell proliferation plotted curves.3) Real-time PCR:SYBR(?) Premix Ex Taq TM (Perfect Real Time) kit (TaKaRa Biotechnology Co., Ltd. Dalian, China) was used for real-time quantitative PCR reaction, while operating instrument was Thermal Cycler Dice TM Real Time System (TP800real-time PCR instrument, TaKaRa).The product length met specification requirements of quantitative PCR amplification reference, with80bp-150bp as most appropriate (can be extended to300bp). Fluorescence threshold and background signal were as set as default value of the experimental apparatus. The number of cycles when the collected fluorescence signal intensity of each reaction tube reached the threshold value(10times the baseline fluorescence intensity)is defined asCt value; in this study, each sample had three triplicates and the expression level of the target gene was expressed by2-△△Ct.The reaction product was analyzed with1.5%agarose gel electrophoresis and stained with ethidium bromide. Each product’s sequence was confirmed by automated sequencing.5. Statistical analysis: The resulting data was analyzed using SPSS16.0statistical software for variance analysis(Chicago, IL). Results were expressed in the form of mean±standard deviation, with at least three independent experiments. The comparison between the two groups or between multiple groups was conducted with variance analysis or t-test, and P<0.05was considered as statistically significant.[Results]1. Primary cell isolation, culture and immunocytochemistry1.1Interstitial cells derived from adenocarcinoma were elongated and disorganized, while interstitial cells from normal tissue were spindle-shaped and well arranged.1.2Cytokeratin and vimentin were characteristic proteins of endometrial interstitial cells and epithelial cells, respectively. Epithelial cells and interstitial cells were isolated from endometrial tissue and identified; it showed that epithelial cells were cytokeratin-positive and vimentin-negative while interstitial cells were cytokeratin-negative and vimentin-positive with the purity of more than95%; interstitial cells were isolated from endometrial adenocarcinoma and identified with immunocytochemistry, similar results were obtained as the normal interstitial cells, which were cytokeratin-negative and vimentin-positive with puritymore than95%.2. Paracrine role of endometrial adenocarcinoma interstitial cells can promote epithelial cell proliferation2.1It was shown by the indirect co-culture experiment which can mimic in vivo paracrine role, epithelial cells co-cultured with adenocarcinoma interstitial cells grew faster than those co-cultured with normal interstitial cells (P<0.05), indicating some specific factors secreted by adenocarcinoma interstitial cells can promote proliferation of epithelial cells.2.2BrdU incorporation experiment showed that the ratio of proliferative phase epithelial cellsco-cultured with adenocarcinoma interstitial cells was higher than those co-cultured with normal interstitial cells (P<0.05).2.3Quantitative PCR results showed that the tumor suppressor gene PTEN, oncogene K-Ras and the reference gene β-actin all have sharp and single melting curves, indicating that these primers were designed with good specificity. In terms of gene expression levels, PTEN mRNA expression levels of both normal endometrial interstitial cells and adenocarcinoma interstitial cells decreased while Ras mRNA expression levels gradually rose, indicating paracrine factors released by endometrial adenocarcinoma interstitial cells can decrease expression level of tumor suppressor gene PTEN while increase expression level of oncogene K-Ras, which can induce the malignant transformation of normal endometrial epithelial cells.2.4Western blot (WB) data analysis showed that the results indicated that PTEN expression levels of the two groups (normal endometrial interstitial cells and adenocarcinoma interstitial cells) decreased while Ras expression levels gradually increased, which indicated the same trend with mRNA expression levels.2.5Immunocytochemistry (ICC) data analysis showed that by immunocytochemistry approach, co-cultured epithelial cells were stained to observe the expression levels of the two proteins. The results showed that PTEN protein expression levels of the two groups (normal endometrial interstitial cells and adenocarcinoma interstitial cells) decreased while K-Ras protein expression levels gradually increased, which indicated the same trend with Western and qPCR.5visual fields (up, middle, down, left and right) were selected for statistical positive rate analysis of cells under the microscope. The results showed that PTEN-positive rate decreased significantly in the second group (NE+CI), while the positive rate of K-Ras in the second group (NE+CI) increased significantlyl, the results were statistically significant (P<0.05).2.6Cell cycle detection result showed that, for epithelial cells co-cultured with cancer interstitial cells, their G0/G1phase proportions dropped while S and G2/M phase cells increased, indicating that cells in this group divided more exuberant with accelerated proliferation (P<0.05).2.7With real-time quantitative PCR (RT-PCR), the downstream gene CyclinDl was detected. The result showed that epithelial cells co-cultured with adenocarcinoma interstitial cells had higher expression level of Cyclin D1, suggesting accelerated cell proliferation (P<0.05).2.8The expression levels of p21and p27genes were observed with real-time quantitative PCR method. The results showed that there were no significant difference of p21and p27gene expression levels between the two co-cultured groups (P>0.05), indicating that the changes in cell cycle of epithelial cells co-cultured with adenocarcinoma interstitial cells were not significantly associated with the roles of p21and p27.2.9The result of nude mouse transplantation tumor experiment showed that the proliferation rate of epithelial cells co-cultured with adenocarcinoma interstitial cells was faster than those co-cultured with normal endometrial interstitial cells, and the former had large tumors. During the observation period, the tumor volume and weight of epithelial cellsco-cultured with adenocarcinoma interstitial cells were significantly greater than those co-cultured with normal endometrial interstitial cells.3. Paracrine role of endometrial adenocarcinomainterstitial cells3.1Enzyme-linked immunosorbent assay (ELISA) experiments detected the level of HGF and PGF2a with cell culture medium which had been co-cultured for72hr. The results showed that the HGF concentration in the co-culture medium of the second group (NE+CI) was significantly higher than the first group (NE+NI)(P<0.001); in addition, PGF2a concentration was also significantly higher thanthe second group (P <0.001).3.2Real-time quantitative PCR and Western blotting were used to detect the gene expression levels of HGF in two kinds of interstitial cells. The results showed that HGF gene and protein expression levels of adenocarcinoma interstitial cells were significantly higher than in normal endometrial interstitial cells, suggesting that more HGF can then be synthesized and secreted to extra cellular due to the upregulation of HGF within adenocarcinoma interstitial cells (P<0.01). Since PGF2a was an unsaturated fatty acid, its gene expression cannot be detected.3.3HGF expression within the endometrial adenocarcinoma xenografts was detected using real-time quantitative PCR and Western blotting. The result indicated that the expression levels of HGF gene and protein within the endometrial adenocarcinoma xenografts formed by epithelial cells co-cultured with adenocarcinoma interstitial cells were significantly higher than those with normal endometrial interstitial cells(P <0.05). The results further displayed that expression of HGF significantly increased during the co-culture process between cancer interstitial cells and epithelial cells, which would be the possible cause for accelerated epithelial cell proliferation.3.4Immunohistochemical detection method was used to detect the HGF expression levels of normal and endometrial adenocarcinoma. The results showed that HGF expression level of endometrial adenocarcinoma was significantly higher than normal endometrium.3.5To observe the effects of HGF on normal endometrial epithelial cells, cell proliferation curves were used.The results showed that the addition of cytokine HGF into the culture medium can promote the proliferation of normal endometrial epithelial cells which were cultured alone. This showed similar trends with co-culture situation, indicating cytokine HGF can stimulate epithelial cell proliferation.4. HGF mechanism in epithelial cell proliferation4.1Western blot (WB) method was used to screen the signaling pathway nodes. We detected the activation level of several common signaling nodes (β-catenin, Akt, ERK, NF-kB) which were related to proliferation. After screening, we found that Akt phosphorylation levels were increased during the process of co-culture with cancer mesenchyme, while other signaling pathways did not change significantly, indicating HGF paracrine secretionmay play a role through the activation of Akt signaling pathway.4.2Western blot (WB) was used to observe the phosphorylation of the signaling pathway molecule-Akt. The result showed that HGF stimulation can cause activation of Akt signaling pathways, while addition of Met inhibitor can reduce the level of Akt phosphorylation. However, the overall phosphorylation level of signaling pathway molecule Akt of adenocarcinoma co-culture group was higher than the group co-cultured with normal interstitial cells. And after adding Met inhibitor, Akt phosphorylation level was reduced, indicating that the proliferation promotion effect of adenocarcinoma interstitial cells was implemented through paracrine factor HGF and HGF-Akt signaling pathway. Meanwhile, as detected by cell growth curve which can show cell proliferation ability, the results observed that inhibition of HGF signaling pathway can reverse the proliferation promotion effect of adenocarcinoma interstitial cells, which can further confirm that the proliferation promotion effect of adenocarcinoma interstitial cells was realized through paracrine factor HGF signaling pathway.4.3Western blot (WB) and PCR experiment were used to extract thetotal proteins from tumor tissues to detect changes in Akt phosphorylationlevels. The results showed that Akt phosphorylation level of the tumor co-cultured with adenocarcinoma interstitial cells was significantly higher than those epithelial cells co-cultured with normal endometrial interstitial cells, indicating a higher intratumoral activation level of Akt signaling pathway (P<0.01).4.4The real-time quantitative PCR method was used to further test in vivo Cyclin D1gene expression levels in mouse transplanted tumors. The results showed that, in the interstitial cells co-cultured with adenocarcinoma xenografts, Cyclin D1expression levels were higher, suggesting accelerated cell proliferation (P<0.05).[Conclusion]1. In vivo experimental studies demonstrated that co-culturing endometrial adenocarcinoma interstitial cells with normal endometrial epithelial cells can promote the proliferation of normal epithelial cells.2. The down regulation of PTEN and increased expression of K-Ras in normal epithelial cells were observed when co-culture with stromal cells from endometrial cancer.3. Endometrial adenocarcinoma interstitial cells can play a role on the proliferation process of normal endometrial epithelial cells by gene upregulation, synthesis and secretion of cell growth factor HGF.4. The overexpression of HGF and c-Met are closely related to the carcinogenesis and development of endometrial adenocarcinoma.5. HGF and c-Met can promote tumor cell proliferation. 6. Endometrial adenocarcinoma interstitial cells can regulate proliferation of normal epithelial cells through HGF/AKT/CyclinDl signaling pathways.7. Blocking of HGF/c-Met expression or signal transduction system can be used as a new therapeutic target of endometrial adenocarcinoma. |
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