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Crosstalk between the mesothelium and lymphomatous cells: insight into the mechanisms involved in the progression of body cavity lymphomas

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 epithelial–mesenchymal 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 α-smooth muscle actin (α-SMA), a mesenchymal protein. A progressive thickening of serosal membranes was observed in vivo, accompanied by loss of cytokeratin staining and appearance of α-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 activity on PEL cells was retained after transdifferentiation, and was impaired by interferon-α2b 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.


Antibodies, flow cytometry and IFA
To detect pan cytokeratin (pCK) and α-smooth muscle actin (α-SMA), human mesothelial cells (HMC) were fixed, rendered permeable and incubated with a mouse anti-pCK monoclonal antibody (mAb) mixture (mouse ascites fluid, Sigma-Aldrich) and a rabbit polyclonal antibody to α-SMA (Abcam, Cambridge, UK), and then with an Alexa 594-conjugated goat anti-mouse immunoglobulin G (IgG; heavy and light chains [H+L]) and an Alexa 488-conjugated chicken antirabbit IgG (H+L) (Molecular Probes, Eugene, OR, USA). HMC were also stained with a goat polyclonal Ab to E-cadherin (R&D Systems), and then with an Alexa 594-conjugated rabbit antigoat IgG (H+L) (Molecular Probes). To evaluate the expression of mesothelin and fibroblast surface protein 1 (Fsp-1), a mouse mAb to mesothelin or to Fsp-1 (GeneTex, San Antonio, TX, USA) was used as primary Ab, and an Alexa 488-conjugated goat anti-mouse IgG (H+L) or IgM (Molecular Probes), respectively, was used as secondary Ab. HMC staining was also performed using a phycoerythrin (PE)-conjugated mouse mAb anti-CD14 (AbD Serotec, Oxford, UK), a PEconjugated mouse mAb to CD54/ICAM-1 (eBioscience, San Diego, CA, USA), and a PEconjugated rat mAb to CD44 (BioLegend, San Diego, CA, USA). All antibodies were used diluted according to the manufacturers' instructions in PBS-3% BSA. Isotype-matched irrelevant mAbs or secondary mAbs were always used in parallel as negative controls. Analyses were done using a LSR-II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) equipped with a 488-, 405-, 640-, and 561-nm laser and an EPICS XL flow cytometer (Coulter Electronics, Hialeah, FL, USA).
To assess the expression of pCK and α-SMA by IFA, HMC were seeded on coverslips previously coated with poly-L-lysine and cocultured with PEL cells or treated with TGF-β1/IL-1β or exposed to the combined condition. Cells were fixed, rendered permeable and incubated with primary antibodies diluted in PBS-2% FCS for 40 minutes at 37°C, washed three times with PBS, and incubated with appropriate secondary antibodies, followed by 4',6-diamino-2-phenylindole (DAPI, Sigma-Aldrich) counterstaining. Stained cells were visualized using a fluorescence microscope (Vico, Nikon, Melville, NY, USA) with a 20x/0.50 NA objective and images were acquired as stated above.

Apoptosis analysis and proliferation assay
Spontaneous and induced apoptosis was evaluated at different time points in CRO-AP/3, BCBL-1 and CRO-AP/2 cells suspended at 0.5-1 x 10 5 /mL and cocultured with or without HMC, or HMC undergoing EMT (EMT-HMC). EMT-HMC were obtained by TGF-β1/IL-1β treatment for 96 hours. Apoptosis was induced in PEL cell lines by serum deprivation (complete medium with 5-10% FCS). To assess the proapoptotic activity of IFN-α 2 b in coculture, HMC or EMT-HMC were treated with 6000 international units (IU)/mL recombinant IFN-α 2 b (IntronA, Schering Plough, Kenilworth, NJ, USA) for 6 hours, then washed and cocultured with CRO-AP/3 cells for 24 hours.
CRO-AP/3 cells were sensitized to TRAIL-mediated apoptosis by treatment with 10 µg/mL azidothymidine (AZT, Sigma-Aldrich, Munich, Germany) for 48 hours before coculture. To mimic in vivo conditions, IFN-α 2 b was added at the beginning of the coculture and apoptosis was measured after 24 hours. Apoptosis was analyzed by flow cytometry after staining with annexin V (Annexin-V-FLUOS staining kit, Roche Diagnostic, Indianapolis, IN, USA). To block TRAILinduced apoptosis, cocultures were performed by adding an anti-TRAIL mAb or an isotype control (5 µg/mL) [clone RIK-2, low-endotoxin azide-free (LEAF)-purified anti-human TRAIL, or LEAFpurified isotype control; BioLegend, San Diego, CA, USA] to IFN-α 2 b-treated HMC or EMT-HMC before the coculture. The proliferative capability of PEL cell lines was analyzed by 5-bromo-2'deoxyuridine (BrdU) incorporation measured at different time points using an Europium-labeled anti-BrdU antibody (Delfia cell proliferation assay, PerkinElmer, Cambridge, UK) by time-resolved fluorescence using a VICTOR™ X4 Multilabel Plate Reader (PerkinElmer) at 615 nm. Proliferation was measured in PEL-derived cell lines (2-4 x 10 4 /mL) plated in empty wells and on HMC or EMT-HMC seeded in 96-well flat-bottomed plates (ViewPlate, PerkinElmer). Proliferation was also measured in HMC and EMT-HMC alone. Each culture condition was set up in triplicate or quadruplicate and incubated with BrdU at each time point. To analyze the cytostatic activity of IFNα 2 b, experiments were set up as described before, and IFN-α 2 b (6000 IU/mL) was added to the coculture. Results were reported as the mean of Europium counts ± SEM of three independent experiments.

Supplemental Table S1
Table S1: Designation and sequence of primers used in qualitative and quantitative RT-PCR.

Primer
Sequence (   HMC acquired a myofibroblastic phenotype, with elongated shape and multi-layered growth after coculture with CRO-AP/2 and BCBL-1 cells. (b) Relative expression 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 was measured in HMC cocultured with CRO-AP/2 and BCBL-1 cells, suggesting that all PEL cell lines may induce EMT in cocultured HMC. Measurement of TGF-β1 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 independent measurements set up in duplicate. PEL cells express and release high levels of TGF-β1, suggesting that EMT in HMC may be induced by this factor.