Spatial Landscape of Malignant Pleural and Peritoneal Mesothelioma Tumor Immune Microenvironments

Abstract

Immunotherapies have demonstrated limited clinical efficacy in malignant mesothelioma treatment. We conducted multiplex immunofluorescence analyses on tissue microarrays (n = 3) from patients with malignant pleural mesothelioma (MPM, n = 88) and malignant peritoneal mesothelioma (MPeM, n = 25). Our study aimed to elucidate spatial distributions of key immune cell populations and their association with lymphocyte activation gene 3 (LAG3), BRCA1-associated protein 1 (BAP1), neurofibromatosis type 2 (NF2), and methylthioadenosine phosphorylase (MTAP), with MTAP serving as a cyclin-dependent kinase inhibitor 2A/2B (CDKN2A/B) surrogate marker. Additionally, we examined the relationship between the spatial distribution of major immune cell types and prognosis and clinical characteristics of patients with malignant mesothelioma. We observed a higher degree of interaction between immune cells and tumor cells in MPM compared with MPeM. Notably, within MPM tumors, we detected a significantly increased interaction between tumor cells and CD8+ T cells in tumors with low BAP1 expression compared with those with high BAP1 expression. To support the broader research community, we have developed The Human Spatial Atlas of Malignant Mesothelioma, containing hematoxylin and eosin and multiplex immunofluorescence images with corresponding metadata.

Significance: Considering the limited therapeutic options available to patients with malignant mesothelioma, there is substantial translational potential in understanding the correlation between the spatial architecture of the malignant mesothelioma tumor immune microenvironment and tumor biology. Our investigation reveals critical cell-cell interactions that may influence the immune response against malignant mesothelioma tumors, potentially contributing to the differential behaviors observed in MPM and MPeM. These findings represent a valuable resource for the malignant mesothelioma cancer research community.

Figure 1

Profiling the spatial landscape of malignant mesothelioma using mIF. A, Schematic diagram outlining the acquisition of multiplexed immunofluorescence images from patients with malignant mesothelioma generated using BioRender.com. B, Waterfall plot depicting the distribution of cell populations as a percentage of all cells in the TIME, categorized by CD8⁺ T cells and sorted across histologic subgroups within MPM and MPeM. Vertical bars represent cell percentages, with distinct colors corresponding to various cell lineages. C, Pie charts depicting the mean cellular composition in samples with MPM and MPeM, including relative frequencies of B cells, CD4⁺ T cells, CD8⁺ T cells, Tregs, macrophages, DCs, NK cells, pan-CK⁺ tumor cells, and unidentified cells. D, Assessment of the prevalence of CD8⁺ T cells and pan-CK⁺ tumor cells in MPM and MPeM as a fraction of the total cell population. P values were calculated using the one-sided Mann–Whitney U test. RPCI, Roswell Park Cancer Institute; UPENN, University of Pennsylvania; UPITT, University of Pittsburgh.

Figure 2

Interactions between immune cells and tumor cells in malignant mesothelioma. A, Heatmap depicting cell–cell contacts in patients with MPM or MPeM. Columns represent normalized pairwise cell–cell contact scores, and rows represent samples. B, Comparison of contact scores between tumor cells and the overall immune cell population and contact scores specifically involving tumor cells and CD4⁺ T cells, CD8⁺ T cells, macrophages, and Tregs between cohorts of patients with MPM and MPeM. Statistical analysis was performed using FDR-adjusted one-sided multiple Mann–Whitney U tests. Each dot represents the cell contact score of an individual core. C, Representative cases from two samples with MPM and two patients with MPeM demonstrating mIF, H&E staining, and cell-type analyses. Images were captured at 10× magnification. Scale bar, 100 μm. D, Heatmap depicting types of CNs (rows) and relative enrichment above or below the mean across neighborhoods for B cells (CD20⁺), CD8⁺ T cells, CD4⁺ T cells, Tregs (FOXP3⁺), tumor cells (Pan-CK⁺), and macrophages (CD68⁺). Likelihood of enrichment was calculated as log odds ratio values normalized between 5 and −5. E, Pie charts representing median relative contributions of CNs in MPM (top) and MPeM (bottom) biologically independent samples.

Figure 3

Immune cell phenotypes associated with clinical features. A, Prevalence of all cell types in MPM epithelioid (n = 45), biphasic (n = 16), and sarcomatoid (n = 7) subtypes (left) and MPeM epithelioid (n = 12) and biphasic (n = 3) subtypes (right) presented as the proportion of total cells. Statistical analysis was performed using one-sided Multiple Mann–Whitney nonparametric U tests. *, P < 0.05. B, Bubble plot illustrating significance levels, in which circle size corresponds to the level of significance, and circle color indicates which of the two comparisons on the y-axis shows higher levels of the cell type plotted on the x-axis. C, Box plots displaying cell-type proportion across different clinical subgroups in patients with MPM and MPeM. Statistical analysis was conducted using one-sided multiple Mann–Whitney U tests. P < 0.05 was considered significant. The center of the box plot represents the median, with the box boundaries indicating the 25th and 75th percentiles. Whiskers extend to the minimum and maximum values in the dataset.

Figure 4

Association of BAP1, NF2, MTAP, and LAG3 intensities with immune cell–type intensity in MPM or MPeM. A, Heatmaps showing correlations between the mean intensity of immune cell markers and expression levels of BAP1, NF2, MTAP, and LAG3 across biologically independent tumors in MPM and MPeM. B, Correlation between mean CD56 (NK cell) intensity and mean BAP1, NF2, MTAP, and LAG3 expression. Dots are color-coded by histologic subtypes.

Figure 5

Analysis of cell types in MPM BAP1-high and BAP1-low tumor groups. A, Correlation between mean CD8⁺ (cytotoxic T-cell) and CD11c⁺ (DC) intensities and mean BAP1 expression levels. Dots are color-coded by histologic subtypes. B, Ridge density plot illustrating tumor and CD8⁺ T cell contact scores among BAP1-high and BAP1-low pan-CK–enriched cores (cores with CK percent>20%). Density values >0.6 quantile and <0.4 quantile were used as thresholds to categorize cores as BAP1-high and BAP1-low, respectively. C and D, Segmented images show increased interaction between cancer cells and CD8⁺ T cells in BAP1-low MPM (D) compared with BAP1-high MPM (C). Top, TMA core sections stained with H&E, in which BAP1 is represented in Opal 690 (red); NF2 in Opal 780 (orange); MTAP in Opal 620 (green); and LAG3 in Opal 570 (magenta). Scale bars, 100 μm, 10× original magnification. Middle, Zoomed-in area from the boxed segment in the top, stained with DAPI for DNA (cyan); Opal 480 for CD8⁺ T cells (yellow); and Opal 780 for pan-CK cells (blue). Scale bars, 10 μm, 100× original magnification. Bottom, Composite images. Scale bars, 30 μm, 30× original magnification.