Differential Susceptibility of Human Pleural and Peritoneal Mesothelial Cells to Asbestos Exposure

Abstract

Malignant mesothelioma (MM) is an aggressive cancer of mesothelial cells of pleural and peritoneal cavities. In 85% of cases both pleural and peritoneal MM is caused by asbestos exposure. Although both are asbestos-induced cancers, the incidence of pleural MM is significantly higher (85%) than peritoneal MM (15%). It has been proposed that carcinogenesis is a result of asbestos-induced inflammation but it is not clear what contributes to the differences observed between incidences of these two cancers. We hypothesize that the observed differences in incidences of pleural and peritoneal MM are the result of differences in the direct response of these cell types to asbestos rather than to differences mediated by the in vivo microenvironment. To test this hypothesis we characterized cellular responses to asbestos in a controlled environment. We found significantly greater changes in genome-wide expression in response to asbestos exposure in pleural mesothelial cells as compared to peritoneal mesothelial cells. In particular, a greater response in many common genes (IL-8, ATF3, CXCL2, CXCL3, IL-6, GOS2) was seen in pleural mesothelial cells as compared to peritoneal mesothelial cells. Unique genes expressed in pleural mesothelial cells were mainly pro-inflammatory (G-CSF, IL-1β, IL-1α, GREM1) and have previously been shown to be involved in development of MM. Our results are consistent with the hypothesis that differences in incidences of pleural and peritoneal MM upon exposure to asbestos are the result of differences in mesothelial cell physiology that lead to differences in the inflammatory response, which leads to cancer.

Keywords: ASBESTOS; INFLAMMATION; MASSIVELY PARALLEL SEQUENCING; MESOTHELIAL CELLS; MESOTHELIOMA; RNA-SEQ.

Figure 1

Asbestos is more cytotoxic to human peritoneal mesothelial cells compared to human pleural mesothelial cells. Two peritoneal (LP9 and HM3) and 2 pleural (HPM3 and HPM4) mesothelial cells were exposed to asbestos (5 µg/cm² or 75) for 24 h and cells were counted using hemocytometer. (n=3) *p≤0.05 as compared to untreated control.

Figure 2

Principal component analysis indicates common response to asbestos exposure. PCA plot reflects Treatment (triangles), Cell lines (colors), and Cell source (ellipses; red and purple = pleural; green and blue = peritoneal) in three components representing 83% of the overall variation. The x-axis differentiates the Cell source (purple vs. teal ellipses), and possibly immortalization based on the distance between the LP9 cell line as compared to the other three lines. The y-axis captures the response to Treatment, and is the same direction for all four cell lines, but to a greater extent in the pleural cell lines, as indicated by the distance between control (squares) and treated (triangle) samples (scalar data not shown).

Figure 3

Control-centered principal component analysis. Control-centered principal component analysis also indicates a common response to asbestos exposure but to a greater extent in the pleural cell lines. PCA plot centered on the Control samples for each cell line. Variation from the Control proceeds almost linearly from LP9 -> HM3 -> HPM4 -> HPM3, or peritoneum to pleural cavity.

Figure 4

Transcripts commonly or uniquely differentially expressed in primary cell lines in response to asbestos exposure. A Venn diagram illustrating the number of transcripts commonly or uniquely differentially expressed based on the binary filter for the three primary cell lines.

Figure 5

Patterns of differential expression between pleural and peritoneal cell lines in response to asbestos exposure. Scatter plots of the log-fold changes for differentially expressed transcripts when A) pleural cell line HMP4 is compared to peritoneal cell line HM3, linear regression indicates a correlation coefficient of r = 0.70, and B) pleural cell line HPM3 is compared to peritoneal cell line HM3 (linear regression indicates a correlation coefficient of r = 0.72).

Figure 6

Transcripts from the IL-17, IL-6, IL-10 signaling pathways commonly differentially expressed in response to asbestos. Both were identified by pathway analysis as the most enriched in all cell lines.

Figure 7

Twenty-nine transcripts uniquely differentially expressed between pleural and peritoneal mesothelial cells. These transcripts represent the interaction between Cell Source and Treatment. Samples and transcripts were clustered based on Euclidean distance.

Figure 8

RNA-Seq expression data was validated at the protein level. (A) HPM3 cells were exposed to asbestos (1(15 × 10⁶) or 5 (75 × 10⁶) µg/cm²) for 8 h. Medium was collected and concentrated as described in the method section. Concentrated medium was analyzed for IL-1β (ELISA assay), IL-6 or IL-8 (Western blot analysis). N=3 samples/group. *p≤0.05 as compared to untreated control. (B) Validation of some highly expressing genes by qRT-PCR.