The Role Of Tumor Microenvironment In The Prognosis And Chemoresistance Of Cancer Patients

Background and aimEsophageal cancer is one of the most common malignant solid tumors and was ranked the sixth leading cause of cancer-related mortality. The 5-year survival rate of esophageal cancer was only approximately 20% despite of the advancement in multi-disciplinary treatment including surgery, radiation therapy and chemo therapy. There were two main histological subtypes of esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC). China was one of the high risk areas of esophageal cancer with ESCC accounts for 90% of all cases. Therefore, the study of prognostic factors of ESCC is very important.Solid tumors were composed with tumor part and stroma part. Tumor stroma was also named tumor microenvironment (TME) and was composed with elements including cancer associated fibroblasts (CAFs), extracellular matrix (ECM), and tumor associated macropahges and so on. The interaction between stromal cells, such as CAFs, and tumor cells plays important roles in promoting tumor growth, invasion and metastasis and was gaining more and more attention. Caveolin-1 (Cav-1) was proved by multiple researches to be essential in tumor-stromal interactons.Cav-1 was an integral transmembrane protein that was essential for Caveolae. It could bind with signaling molecules such as tyrosine kinase receptor and G-protein coupled receptor and regulates signal transduction. Multiple studies had confirmed Cav-1 as an important regulator of tumor growth, invasion, metastasis and drug resistance. Recent research has further revealed the role of Cav-1 in regulating the interaction between tumor cells and stromal cells. In addition to that, the down regulation of Cav-1 in many human cancer types was found to be related with poor prognosis. However, the study on expression of Cav-1 in ESCC was rare. To our best knowledge, there was no study about the correlation between Cav-1 expression and clinicopathological parameters in ESCC.The aim of our current study, therefore, is to examine the Cav-1 expression in both tumor and stromal part of ESCC by immunohistochemical staining. Moreover, we would evaluate its association with clinicopathological factors and prognosis in ESCC patients.Methods1. Patients and follow-up We chose the patients who went to the Department of thoracic surgery in Qilu hospital of Shandong University for curative surgical resection between December 2006 to December 2007 as the subjects of the current study. Those who had undergone radio/chemo therapy before surgery or died within 30 days after surgery were excluded.110 were finally included in our study, with 89 males and 21 females. Clinical data of all patients were obtained from the digital medical history system of our hospital and retrospectively analyzed. TNM classification were performed according to American Joint Committee on Cancer Staging Manual (7th ed,2010) for all patients.Follow-up started since the day after surgery and lasted until January 2013. The median follow-up period was 49.2 months (range,3.5-72.9 months). Regular physical examination and history review were performed every 3 months within the first 2 years after surgery. After 2 years, the follow-up schedule was carried out every 6 months. Cancer related serum markers were checked every 1-3 months. Medical imaging exam were performed when necessary.All patients had signed formal consent and all the procedures in our study were admitted by the ethical committee of Qilu Hospital of Shandong University.2. Patient sample preparation and immunohistochemical stainingTumor samples of all patients were obtained from the Pathology Department of our hospital. All tissue samples were fixed, imbedded and cut into 5μm slides. Immunohistochemical staining was performed on these slides with anti-Cav-1 antibody. Positive and negative controls were set during the staining.3. Evaluation of the staining10 hot spots were identified under low magnification (10*10). Cav-1 positive tumor and stromal cells were counted under high magnification (10*40), respectively. The slides were then graded based on Cav-1 expression level. The results of the staining were analyzed by two pathologists independently.ResultsCav-1 expression was found in the cytoplasm of both tumor and stromal cells. Tumor Cav-1 overexpression was observed in 37.3% tumors, which correlated to deeper tumor invasion (p=0.038). Downregulation of stromal Cav-1 expression was observed in 40.9% tumors. The stromal Cav-1 downregulation group had more lymph node metastases and more locoregional recurrence than those with higher expression (p=0.020 and p=0.002, respectively). In addition, downregulation of stromal Cav-1 expression was associated with shorter disease free survival (DFS, p<0.001) and overall survival (OS, p<0.001). Multivariate analysis showed that downregulation of stromal Cav-1 expression was an independent prognostic factor for both DFS (p =0.028) and OS (p=0.007).ConclusionsDownregulation of stromal Cav-1 expression in ESCC had high malignant potential. It predicts high risk of lymph node metastases, locoregional recurrence, and could be a powerful prognostic marker for patients with ESCC.Background and aimThe ratio of M2-like macrophages versus Ml-like macrophages has been identified as a novel prognostic parameter in various human cancers recently. This study was aimed to evaluate the clinicopathological significance of M2/M1 ratio and examine its prognostic value in operable ESCC patients after long-term follow-up.Methods110 ESCC surgical resection specimens were immunostained with CD40 (for Ml macrophages) or CD 163 (for M2 macrophages). The number of Ml and M2 macrophages was counted under microscope and the M2/M1 ratio was evaluated. Correlation of M2/M1 ratio with clinicopathologic characteristics and prognosis in ESCC patients was analyzed.ResultsNeither M1 nor M2 count correlated with prognosis in ESCCs. High M2/M1 ratio was significantly associated with lymph node metastasis and locoregional recurrence. The median survival time (MST) of high M2/M1 ratio group and low M2/M1 ratio group was respectively 25.4 months (range 3.5-102.3 months) and 62.8 months (range 7.5-103.9 months). Kaplan-Meier analysis showed that high M2/M1 ratio correlated with poor 5-year OS (p< 0.001) and poor 5-year DFS (p< 0.001). Multivariate analysis indicated that M2/M1 ratio was an independent prognostic factor for both 5-year OS (p= 0.016) and 5-year DFS (p= 0.010).ConclusionsHigh M2/M1 ratio in ESCCs is a predictor of high malignant potential. High M2/M1 ratio is significantly correlated with lymph node metastasis and locoregional recurrence, and could be a reliable predictor of poor prognosis for patients with ESCC.Background and aimDespite the advancement in treatment strategies including surgery, radiation therapy, chemo therapy, hormone therapy and targeted therapy, the mortality of breast cancer patients were unacceptably high, especially for those patients with recurrence. Therefore, breast cancer is a big threat for female health. For breast cancer patients, the major obstacle in improving their outcome was the drug resistance after repeated therapy.Many previous studies about the drug resistance of breast cancer had been focusing on the gene mutations of cancer cells during treatment, and tried to explain the underlying mechanism by such studies. Their findings led to the discovery of new breast cancer related genes and the development of novel target therapy. However, no cure for the drug-resistant breast cancer had been found so far. It seems that the mechanism of drug resistance in breast cancer is more complicated than we thought. Focusing on the cancer cells themselves could not solve the problem.Tumor microenvironment (TME) was the main component of solid tumors. TME significantly affects the biological behavior of cancer cells by cell-cell interaction and signaling molecules secretion. Multiple studies had demonstrated that TME could promote the survival of cancer cells after chemo therapy. Mikala Egeblad et al. had provided visual evidence of such notion by the in vivo imaging of breast cancer right after chemo therapy.It had been proved by many researchers that drug-resistant cancer cells are often low differentiated. Our previous study in Egeblad lab showed that the recurrent breast cancer cells in CCR2 knock-out mice are more differentiated than that in the wild-type mice. It indicated that CCR2 in the TME of breast cancer could affect the differentiation of cancer cells as well as the drug resistance. Our further study showed that among multiple genes that were regulated by CCR2, Multidrug and toxin extrusion protein 1(MATE1) and GATA binding protein-3 (GATA3) were the most prominent candidate genes.MATE1 could transport the cationic drugs such as Doxorubicin and Cisplatin out of cells, while GATA3 were essential in the differentiation, prognosis and metastasis in breast cancer. Both of them were worthy to be studied since they were regulated by CCR2 in TME of breast cancer and were likely to be related with drug resistance.The aim of our study is to investigate the role of MATE1 and GATA3 in the drug resistance in breast cancer and explore the underlying mechanism, so that novel targets could be found to improve the treatment outcome for drug-resistant breast cancers.Methods1. Evaluate the effect of MATE1 inhibitor on Doxorubicin resistance in breast cancerHuman breast cancer cell lines Cal-51 and MDA-MB-231 were used for the study. Four groups were set for each cell line:the control group, the MATE1 inhibitor (PYR) group, the Doxorubicin group and the Doxorubicin plus MATE1 inhibitor group. Perform MTS assay on each group of cells after 48 hours of drug treatment. Evaluate the effect of MATE1 inhibitor on Doxorubicin resistance by calculating the survival rates of each group of cells.2. Study of the MATE1 expression in recurrent breast cancer cells in MMTV-PyMT mice108-weeks old female MMTV-PyMT mice were randomly assigned to two groups, the control group and the study group,5 mice for each group. Inject Doxorubicin into the mice of the study group, once a week for three weeks. Use same amount of PBS instead of Doxorubicin in the control group. Follow up for another three weeks without any treatment after the Doxorubicin injection. Then sacrifice the mice and take out the recurrent tumors. Make slides and perform immunohisochemical staining with MATE1 antibody, so that the MATE1 expression could be evaluated.3. Construction of GATA3 over-expression breast cancer cellsExtract pBabePuro-GATA3 plasmid and perform retroviral transfection on Cal51 and Py2T cell lines. Puromycin were used for selection of transfected cells. Use pBabePuro vector as control. After selection, perform Western Blot on GATA3 protein to make sure that the transfection was successful.4. Study of morphological changes and growth rates in GATA3 over-expression breast cancer cellsObserve the morphological changes in Cal51 and Py2T GATA3 over-expression cells compared to the control group cells. Then count and calculate the growth rates of GATA3 over-expression cells and the control group cells.5. Test the response of GAT A3 over-expression eells to Doxorubicin and Cisplat inThree groups were set for both GATA3 over-expression cells and the control group cells, including the control group, the Cisplatin group and the Doxorubicin group. After 48 hours treatment with drugs, perform MTS assay on each group of cells. Then calculate the survival rates of each group and evaluate the effect of GATA3 expression on drug resistance of breast cancer.6. Evaluate GAT A3 expression in the recurrent breast cancer in MMTV-PyMT mice15 female MMTV-PyMT were randomly assigned to three groups, including the control group, the Doxorubicin group and the Cisplatin group,5 mice for each group. Inject drugs once a week for three weeks, then follow up for three weeks without any treatment. Sacrifice the mice and take the tumors out for RNA in-situ hybridization, so that GATA3 expression could be evaluated in each group of breast cancer.Results1. The effect of MATE1 inhibitor on Doxorubicin resistance in breast cancerThe growth rates of Cal51 and MDA-MB-231 cells were significantly decreased after 48 hours treatment of MATE1 inhibitor. The effect of MATE1 inhibitor increases with higher concentrations. However, the effect of combing MATE1 inhibitor and Doxorubicin was not stronger than Doxorubicin alone.2. MATE1 expression in recurrent breast cancer cells in MMTV-PyMT miceMATE1 was mainly expressed on cell membrane. H score of the expression of MATE1 protein was not significantly different between the recurrent breast cancer cells and the control group.3. Morphological changes and growth rates in GAT A3 over-expression breast cancer cellsCal51 GATA3 and Py2T GATA3 cells were more basal-like, while the control group cells were more luminal. In addition to that, the growth rate of GATA3 over-expression cells was obviously lower than that of the control group cells.4. The response of GATA3 over-expression cells to Doxorubicin and Cisplat inThe response of Cal51 GATA3 to Doxorubicin was not significantly different with the control group cells. However, Cal51 GATA3 were obviously less sensitive than the control group cells, which indicate that GATA3 could affect the response of breast cancer cells.5. GATA3 expression in the recurrent breast cancer in MMTV-PyMT miceGATA3 was significantly upregulated in recurrent breast cancer cells after Doxorubicin treatment (P=0.034). No obvious changes in the expression of GATA3 in the breast cancer cells after Cisplatin treatment.ConclusionsMATE1 was not significantly correlated with the drug resistance in breast cancer, which was different from what we had expected. The up regulation of GATA3 could promote the resistance of breast cancer cells to cationic drugs. In addition to that, the GATA3 expression was up regulated in the recurrent breast cancer cells. The underlying mechanism, however, remains unclear. Therefore, further study on the downstream signals of GATA3 should be performed.