D Luo1, S Y Hu2, G X Liu1. 1. Department of Respiratory Medicine, the First Hospital Affiliated to AMU, Chongqing 400038, China. 2. the First Surgical Department, Qingmuguan Central Hospital, Chongqing 400038, China.
Abstract
Objective: To observe the growth and metastasis of lung cancer promoted by bone marrow derived mesenchymal stem cells (BMSCs) in tumor microenvironment and investigate the underlined mechanisms. Methods: Specific chemotaxis of BMSCs towards lung cancer was observed, and the tumor growth and metastasis were assessed in vivo. Furthermore, CD34 expression determined by immunohistochemistry was used to assess the microvessel density (MVD), and the expressions of GFP and α-SMA determined by immunofluorescence were used to detect the BMSCs derived mesenchymal cells. We investigated the effect of BMSCs on migration, invasion of lung cancer cells including A549 and H446 cells by using scratch assays and Transwell Assay in vitro. We also explored the effect of BMSCs on epithelial mesenchymal transition of A549 and H446 cells by observing the phenotype transition and E-Cadherin protein expression detected by Western blot. At last, we screened the potentially key soluble factors by enzyme linked immunosorbent assay (ELISA). Results: In NOD mice, labeled BMSCs injected via tail vein were special chemotaxis to tumor cells, and promoted the tumor growth [the time of tumor formation in A549+ BMSCs and A549 alone was (5.0±1.5) days and (10.0±3.6) days, respectively, P<0.05; the time of tumor formation in H446+ BMSCs and H446 alone was (5.2±1.5) days and (12.0±2.0) days, respectively, P<0.05]. The tumor incidence of A549+ BMSCs was 100%, significantly higher than 66.7% of A549 alone (P<0.05), while the tumor incidence of H446+ BMSCs was 83.0%, significantly higher than 50.0% of H446 alone (P<0.05). The BMSCs also increased the tumor volume [the tumor volume of A549+ BMSCs and A549 alone was (193.0±42.3) mm(3) and (97.8±42.9) mm(3,) respectively, P<0.05; the tumor volume of H446+ BMSCs and H446 alone was (78.6±34.8) mm(3) and (25.3±12.7) mm(3,) respectively, P<0.05] and facilitated the tumor metastasis (the tumor metastatic incidence of A549+ BMSCs and A549 alone was 100.0% and 16.7%, respectively, P<0.05; the tumor metastatic incidence of H446+ BMSCs and H446 alone was 100.0% and 0.0%, respectively, P<0.05). Furthermore, BMSCs increased tumor vessel formation (the MVD of A549+ BMSCs and A549 alone was 53.2±11.4 and 25.3±11.5, respectively, P<0.05; the MVD of H446+ BMSCs and H446 alone was 56.8±12.5 and 24.8±10.0, respectively, P<0.05). BMSCs were able to differentiate to fibroblasts in the lung squamous cell carcinoma and promoted the migration and invasion of lung cancer cells (the A of cells in the migrated lower chambers of A549+ BMSCs and A549 alone was 1.9±0.2 and 1.1±0.1, respectively, P<0.05; the A of cells in the migrated lower chambers of H446+ BMSCs and H446 alone was 1.9±0.3 and 0.9±0.2, respectively, P<0.05). The cell shape was longer and sharper, the intercellular junctions were reduced and the relative expression level of E-Cadherin protein in A549 co-cultured with BMDCs was 0.36, significantly down-regulated when compared to 0.55 of A549 alone (P<0.05), and the relative expression level of E-Cadherin protein in H446 co-cultured with BMDCs was 0.28, significantly down-regulated when compared to 0.46 of H446 cells alone (P<0.05). The concentration of IL-6 in the conditional medium of BMSCs, A549 co-cultured with BMSCs and H446 co-cultured with BMSCs was 910.5, 957.2, and 963.8, respectively, significantly up-regulated when compared to 18.8 of control group (P<0.05). The expression level of PGE2 in A549 co-cultured with BMSCs and H446 co-cultured with BMSCs was 130.5 and 87.2, significantly up-regulated when compared to 13.8 of control group and 23.8 of BMSCs group (P<0.05). Conclusions: Our results suggest that BMSCs contribute to the tumor growth through facilitating angiogenesis, and promote tumor metastasis through paracrine manner and down-regulation of E-Cadherin protein. IL-6 and PGE2 produced by BMDCs might be the potentially important cytokines.
Objective: To observe the growth and metastasis of lung cancer promoted by bone marrow derived mesenchymal stem cells (BMSCs) in tumor microenvironment and investigate the underlined mechanisms. Methods: Specific chemotaxis of BMSCs towards lung cancer was observed, and the tumor growth and metastasis were assessed in vivo. Furthermore, CD34 expression determined by immunohistochemistry was used to assess the microvessel density (MVD), and the expressions of GFP and α-SMA determined by immunofluorescence were used to detect the BMSCs derived mesenchymal cells. We investigated the effect of BMSCs on migration, invasion of lung cancer cells including A549 and H446 cells by using scratch assays and Transwell Assay in vitro. We also explored the effect of BMSCs on epithelial mesenchymal transition of A549 and H446 cells by observing the phenotype transition and E-Cadherin protein expression detected by Western blot. At last, we screened the potentially key soluble factors by enzyme linked immunosorbent assay (ELISA). Results: In NOD mice, labeled BMSCs injected via tail vein were special chemotaxis to tumor cells, and promoted the tumor growth [the time of tumor formation in A549+ BMSCs and A549 alone was (5.0±1.5) days and (10.0±3.6) days, respectively, P<0.05; the time of tumor formation in H446+ BMSCs and H446 alone was (5.2±1.5) days and (12.0±2.0) days, respectively, P<0.05]. The tumor incidence of A549+ BMSCs was 100%, significantly higher than 66.7% of A549 alone (P<0.05), while the tumor incidence of H446+ BMSCs was 83.0%, significantly higher than 50.0% of H446 alone (P<0.05). The BMSCs also increased the tumor volume [the tumor volume of A549+ BMSCs and A549 alone was (193.0±42.3) mm(3) and (97.8±42.9) mm(3,) respectively, P<0.05; the tumor volume of H446+ BMSCs and H446 alone was (78.6±34.8) mm(3) and (25.3±12.7) mm(3,) respectively, P<0.05] and facilitated the tumor metastasis (the tumor metastatic incidence of A549+ BMSCs and A549 alone was 100.0% and 16.7%, respectively, P<0.05; the tumor metastatic incidence of H446+ BMSCs and H446 alone was 100.0% and 0.0%, respectively, P<0.05). Furthermore, BMSCs increased tumor vessel formation (the MVD of A549+ BMSCs and A549 alone was 53.2±11.4 and 25.3±11.5, respectively, P<0.05; the MVD of H446+ BMSCs and H446 alone was 56.8±12.5 and 24.8±10.0, respectively, P<0.05). BMSCs were able to differentiate to fibroblasts in the lung squamous cell carcinoma and promoted the migration and invasion of lung cancer cells (the A of cells in the migrated lower chambers of A549+ BMSCs and A549 alone was 1.9±0.2 and 1.1±0.1, respectively, P<0.05; the A of cells in the migrated lower chambers of H446+ BMSCs and H446 alone was 1.9±0.3 and 0.9±0.2, respectively, P<0.05). The cell shape was longer and sharper, the intercellular junctions were reduced and the relative expression level of E-Cadherin protein in A549 co-cultured with BMDCs was 0.36, significantly down-regulated when compared to 0.55 of A549 alone (P<0.05), and the relative expression level of E-Cadherin protein in H446 co-cultured with BMDCs was 0.28, significantly down-regulated when compared to 0.46 of H446 cells alone (P<0.05). The concentration of IL-6 in the conditional medium of BMSCs, A549 co-cultured with BMSCs and H446 co-cultured with BMSCs was 910.5, 957.2, and 963.8, respectively, significantly up-regulated when compared to 18.8 of control group (P<0.05). The expression level of PGE2 in A549 co-cultured with BMSCs and H446 co-cultured with BMSCs was 130.5 and 87.2, significantly up-regulated when compared to 13.8 of control group and 23.8 of BMSCs group (P<0.05). Conclusions: Our results suggest that BMSCs contribute to the tumor growth through facilitating angiogenesis, and promote tumor metastasis through paracrine manner and down-regulation of E-Cadherin protein. IL-6 and PGE2 produced by BMDCs might be the potentially important cytokines.