Mast cells (MCs) have been identified in various tumors; however the

Mast cells (MCs) have been identified in various tumors; however the role of these cells in tumorigenesis remains controversial. revealed that MCs are elevated in MPEs compared with benign effusions. Moreover MC abundance correlated with MPE formation in a human malignancy cell-induced effusion model. Treatment of mice with MGCD-265 the c-KIT inhibitor imatinib mesylate limited effusion precipitation by mouse and human adenocarcinoma cells. Together the results of this study indicate that MCs are required for MPE formation and suggest that MC-dependent effusion formation is usually therapeutically addressable. = 3). In addition MC abundance was correlated with the volume of experimental effusions (Physique 1B). MPE MCs displayed common morphology and TB/c-KIT staining but they were easily overlooked when MGG Wright or other conventional staining was employed (Physique 1 C and D and Physique 2A). MPE MCs were identified as CD45+c-KIT+Sca1+Lin- by flow cytometry (27-29) were reduced in c-KIT-defective mice (30) and were completely absent from MC-eradicated mice (15) – a mouse IL1B model of more complete and selective MC deficiency as compared with mice – that were challenged with pleural adenocarcinoma cells MGCD-265 (Physique 2B). In mice with MPEs MCs were preferentially located in parietal and mediastinal but not visceral pleural tumors; most commonly resided in viable but not necrotic tumor tissue; and aggregated near or at the tumor front forming chains or clusters (Physique 3). Hence pleural MC accumulation is usually associated with MPE development in humans and mice. Moreover MPE MCs appear to stream into the malignancy-affected pleural space via the parietal and mediastinal pleural surfaces. Physique 2 Characterization of MCs from mouse MPEs. Physique 3 MC topology in experimental MPEs. Physique 1 MCs in human and murine MPEs. Dynamic MC accumulation in the pleural space. To test MC kinetics during MPE development we cultured murine BM-derived MCs (BMMCs) using c-KIT ligand (KITL) and interleukin-3 (IL-3) according to previously published protocols (31). BMMCs of C57BL/6 mice stained TB+ (>90%) CD45+c-KIT+Sca1+Lin- (>80%) and CD25+ (>50%) – and BMMCs of red-fluorescent mice (32) – formed pseudopodia and moved confirming the nature of these cells (Physique 4 A-C and Supplemental Videos 1 and 2; supplemental material available online with this article; doi:10.1172/JCI79840DS1). BMMCs of luminescent CAG-luc-EGFP mice (33) emitted light proportional to cell number and BMMCs of green fluorescent CAG-EGFP mice (34) were green fluorescent (Physique 4 D and E). When pulsed i.v. into irradiated C57BL/6 recipients adoptively reconstituted with BM (35) these tracer BMMCs distributed diffusely. However when chimeras were challenged exclusively with pleural adenocarcinoma cells BMMCs accumulated in the thorax concomitant with MPEs (Physique 5 A and B). Comparable results were obtained with nonirradiated mice pulsed s.c. with tracer BMMCs (Physique 5C). Hence pleural adenocarcinomas MGCD-265 remotely mobilize/recruit MCs via circulating messengers. Physique 4 Isolation and characterization of BMMCs. Physique 5 Dynamic MC trafficking to the pleural space. MGCD-265 CCL2 as an adenocarcinoma-derived mastokine. To identify these messengers effusion-competent and effusion-incompetent tumor cells were transcriptionally profiled on 2 different occasions (biological = 2) by microarray analysis. Although 39 genes were overrepresented in MPE-competent adenocarcinoma cells on both occasions only 2 RNAs possessed cytokine/chemokine activity required for systemic MC recruitment and were selected for further study: and (encoding osteopontin or secreted phosphoprotein 1 [SPP1] and CCL2 respectively; Physique 6A and Supplemental Tables 1 and 2). ELISA of tumor cell-conditioned media (CM) validated the microarray and serum ELISA of pleural tumor-bearing C57BL/6 mice identified a significant difference in serum CCL2 but not SPP1 between adenocarcinoma- and melanoma-bearing mice (Physique 6 B and C). In altered mastotaxis assays (36) tracer BMMCs migrated toward LLC cells MGCD-265 expressing random and anti-shRNA (sh) but not toward B16F10 cells or LLC cells.