Extracavitary PEL onset during the course of intracavitary effusion likely represents the outcome of this process 35C37. of pan cytokeratin (pCK) and Snail1 transcripts in HMC after coculture with PEL cell lines. Quantitative RT-PCR analyses were performed on RNA extracted from HMC cultured for 6 days in standard conditions, and from HMC cocultured for 6 days with BCBL-1 or CRO-AP/2 cells. Human PBGD was used as housekeeping gene. Expression levels of pCK and Snail1 transcripts are reported relative to expression levels measured in HMC. Data are reported as mean SD. Downregulation of pCK and upregulation of Snail1 were measured in HMC cocultured with CRO-AP/2 and BCBL-1 cells, suggesting that all PEL cell lines may induce EMT in cocultured HMC. Physique S3. Analysis of expression and release of TGF-1 in PEL-derived cell lines. (A) Relative expression of TGF1 was analyzed by quantitative RT-PCR in RNA extracted from CRO-AP/3, CRO-AP/2, and BCBL-1 cell lines. PBGD was used as housekeeping gene and expression levels are Methylnaltrexone Bromide reported relative to levels measured in 239 H cells. Data are expressed as mean SD. (B) Measurement of TGF-1 levels in culture supernatants of PEL cell lines. Data are expressed as picograms per milliliter, and each histogram represents the mean SEM of data obtained in two impartial measurements set up in duplicate. PEL cells express and release high levels of TGF-1, suggesting that EMT in HMC may be induced by this factor. Methylnaltrexone Bromide Table S1. Designation and sequence of primers used in qualitative and quantitative RT-PCR. cam40003-0001-sd1.pdf (210K) GUID:?D8B0F987-9896-4CEB-A83F-B0F9A7F14C88 Abstract The peculiar localization of body cavity lymphomas implies a specific contribution of the intracavitary microenvironment to the pathogenesis of these tumors. In this study, primary effusion lymphoma (PEL) was used as a model of body cavity lymphoma to investigate the role of mesothelial cells, which line the serous cavities, in lymphoma progression. The crosstalk between mesothelial and lymphomatous cells was studied in cocultures of primary human mesothelial cells (HMC) with PEL cells and a xenograft mouse model of peritoneal PEL. PEL cells were found to induce type 2 epithelialCmesenchymal transition (EMT) in HMC, which converted into a myofibroblastic phenotype characterized by loss of epithelial markers (pan cytokeratin and E-cadherin), expression of EMT-associated transcriptional repressors (Snail1, Slug, Zeb1, Sip1), and acquisition of -easy Ki67 antibody muscle actin (-SMA), a mesenchymal protein. A progressive thickening of serosal membranes was observed in vivo, accompanied by loss of cytokeratin staining and appearance of -SMA-expressing cells, confirming that fibrosis occurred during intracavitary PEL development. On the other hand, HMC were found to modulate PEL cell turnover in vitro, increasing their resistance to apoptosis and proliferation. This supportive Methylnaltrexone Bromide activity on PEL cells was retained after transdifferentiation, and was impaired by interferon-2b treatment. On the whole, our results indicate that PEL cells induce type 2 EMT in HMC, which support PEL cell growth and survival, providing a milieu favorable to lymphoma progression. Our findings provide new clues into the mechanisms involved in lymphoma progression and may indicate new targets for effective treatment of malignant effusions growing in body cavities. test was used to estimate statistical significance of differences between two groups. One-way analysis of variance (ANOVA) was applied to assess the statistical significance of differences between groups. values <0.05 were considered significant. Details about other techniques and reagents are reported in Data S1. Results Coculture with PEL cells induces a myofibroblastic morphology in mesothelial cells As mesothelium may go through different architectural alterations in response to numerous stimuli, we first assessed whether coculture with the CRO-AP/3 PEL-derived cell line could affect HMC morphology. To this end, subconfluent primary cultures of HMC were cocultured with CRO-AP/3 cells for up to 12?days. HMC were found to spindle and pile up, reaching a pattern of multilayered crisscross growth (Fig.?1A). The transition to a myofibroblastic morphology occurred in 5C6?days. Parallel control cultures of HMC, plated at the same concentration and maintained in complete medium, reached a typical flat, cobblestone morphology, without signs of three-dimensional outgrowth. As a positive control, HMC were treated with TGF-1 and IL-1, two potent inducers of EMT in HMC 13. Although TGF-1 alone Methylnaltrexone Bromide induces EMT, IL-1 was shown to potentiate EMT by stimulation.
- For three of the four experiments included in Fig
- Comparative gene expression levels were analyzed using the two 2?Cq technique (27)
- Finally, MSCs in ePL or FBS, following 28 days of chondrogenic media exposure, showed increased proteoglycans simply by Alcian Blue staining weighed against the undifferentiated group (Fig
- Phenotypic identity of MSC was verified using flow cytometry for the current presence of CD44, CD117 and CD90, as described by all of us previously
- Data Availability StatementThe datasets used and/or analyzed during the current study are available from your corresponding author on reasonable request