Chronic exposure to intraperitoneal asbestos triggered a marked response in the mesothelium well before tumor development. Macrophages, mesothelial precursor cells, cytokines, and growth factors accumulated in the peritoneal lavage. Transcriptome profiling revealed YAP/TAZ activation in inflamed mesothelium with further activation in tumors, paralleled by increased levels of cells with nuclear YAP/TAZ. Arg1 was one of the highest upregulated genes in inflamed tissue and tumor. Inflamed tissue showed increased levels of single-nucleotide variations, with an RNA-editing signature, which were even higher in the tumor samples. Subcutaneous injection of asbestos-treated, but tumor-free mice with syngeneic mesothelioma tumor cells resulted in a significantly higher incidence of tumor growth when compared to naïve mice supporting the role of the environment in tumor progression.

Defining the ontogeny of tumor-associated macrophages (TAM) is important to develop therapeutic targets for mesothelioma. We identified two distinct macrophage populations in mouse peritoneal and pleural cavities, the monocyte-derived, small peritoneal/pleural macrophages (SPM), and the tissue-resident large peritoneal/pleural macrophages (LPM). SPM rapidly increased in tumor microenvironment after tumor challenge and contributed to the vast majority of M2-like TAM. The selective depletion of M2-like TAM by conditional deletion of the Dicer1 gene in myeloid cells (D ^−/− ) promoted tumor rejection. Sorted SPM M2-like TAM initiated tumorigenesis in vivo and in vitro, confirming their capacity to support tumor development. The transcriptomic and single-cell RNA sequencing analysis demonstrated that both SPM and LPM contributed to the tumor microenvironment by promoting the IL-2-STAT5 signaling pathway, inflammation, and epithelial–mesenchymal transition. However, while SPM preferentially activated the KRAS and TNF-α/NFkB signaling pathways, LPM activated the IFN-γ response. The importance of LPM in the immune response was confirmed by depleting LPM with intrapleural clodronate liposomes, which abrogated the antitumoral memory immunity. SPM gene signature could be identified in pleural effusion and tumor from patients with untreated mesothelioma. Five genes, TREM2, STAB1, LAIR1, GPNMB , and MARCO , could potentially be specific therapeutic targets. Accordingly, Trem2 gene deletion led to reduced SPM M2-like TAM with compensatory increase in LPM and slower tumor growth. Overall, these experiments demonstrate that SPM M2-like TAM play a key role in mesothelioma development, while LPM more specifically contribute to the immune response. Therefore, selective targeting of monocyte-derived TAM may enhance antitumor immunity through compensatory expansion of tissue-resident TAM.

Increasing evidence indicates that local hypofractionated radiotherapy (LRT) can elicit both immunogenic and immunosuppressive local and systemic immune responses. We thus hypothesized that blockade of LRT-induced immunosuppressive responses could augment the antitumor effects and induce an abscopal response. In this study, we found that the upregulation of Foxp3⁺ regulatory T cells (Tregs) in the mesothelioma tumor microenvironment after nonablative oligofractionated irradiation significantly limited the success of irradiation. Using DEREG mice, which allow conditional and efficient depletion of Foxp3⁺ Tregs by diphtheria toxin injection, we observed that transient Foxp3⁺ Treg depletion immediately after nonablative oligofractionated irradiation provided synergistic local control and biased the T cell repertoire toward central and effector memory T cells, resulting in long-term cure. Furthermore, this combination therapy showed significant abscopal effect on the nonirradiated tumors in a concomitant model of mesothelioma through systemic activation of cytotoxic T cells and enhanced production of IFN-γ and granzyme B. Although local control was preserved with one fraction of nonablative irradiation, three fractions were required to generate the abscopal effect. PD-1 and CTLA-4 were upregulated on tumor-infiltrating CD4⁺ and CD8⁺ T cells in irradiated and nonirradiated tumors, suggesting that immune checkpoint inhibitors could be beneficial after LRT and Foxp3⁺ Treg depletion. Our findings are applicable to the strategy of immuno-radiotherapy for generating optimal antitumor immune responses in the clinical setting. Targeting Tregs immediately after a short course of irradiation could have a major impact on the local response to irradiation and its abscopal effect. Key Points Tregs rapidly rise in the tumor microenvironment after nonablative irradiation. Transient Treg depletion increases the efficiency of nonablative irradiation. Treg depletion after three fractions of radiation induces systemic antitumor immunity.

Malignant pleural mesothelioma (MPM) is an intractable disease with an extremely poor prognosis. Our clinical protocol for MPM of subablative radiotherapy (RT) followed by radical surgery achieved better survival compared to other multimodal treatments, but local relapse and metastasis remain a problem. This subablative RT elicits an antitumoral immune response that is limited by the immunosuppressive microenvironment generated by regulatory T (T_reg) cells. The antitumor effect of immunotherapy to simultaneously modulate the immune activation and the immune suppression after subablative RT has not been investigated in MPM. Herein, we demonstrated a rationale to combine interleukin-15 (IL-15) superagonist (IL-15SA) and glucocorticoid-induced tumor necrosis factor receptor–related protein (GITR) agonist (DTA-1) with subablative RT in mesothelioma. IL-15SA boosted the systemic expansion of specific antitumoral memory CD8⁺ T cells that were induced by RT in mice. Their effect, however, was limited by the up-regulation and activation of T_reg cells in the radiated tumor microenvironment. Hence, selective depletion of intratumoral T_reg cells through DTA-1 enhanced the benefit of subablative RT in combination with IL-15SA. The addition of surgical resection of the radiated tumor in combination with IL-15SA and DTA-1 maximized the benefit of RT and was accompanied by a reproducible abscopal response in a concomitant tumor model. These data support the development of clinical trials in MPM to test such treatment options for patients with locally advanced or metastatic tumors.

Cancer cell repopulation during chemotherapy or radiotherapy is a major factor limiting the efficacy of treatment. Cancer stem cells (CSC) may play critical roles during this process. We aim to demonstrate the role of mesothelioma stem cells (MSC) in treatment failure and eventually to design specific target therapies against MSC to improve the efficacy of treatment in malignant mesothelioma. Murine mesothelioma AB12 and RN5 cells were used to compare tumorigenicity in mice. The expression of CSC-associated genes was evaluated by quantitative real-time PCR in both cell lines treated with chemo-radiation. Stemness properties of MSC-enriched RN5-EOS-Puro2 cells were characterized with flow cytometry and immunostaining. A MSC-specific gene profile was screened by microarray assay and confirmed thereafter. Gene Ontology analysis of the selected genes was performed by GOMiner. Tumor growth delay of murine mesothelioma AB12 cells was achieved after each cycle of cisplatin treatment, however, tumors grew back rapidly due to cancer cell repopulation between courses of chemotherapy. Strikingly, a 10-times lower number of irradiated cells in both cell lines led to a similar tumor incidence and growth rate as with untreated cells. The expression of CSC-associated genes such as CD24, CD133, CD90 and uPAR was dramatically up-regulated, while others did not change significantly after chemoradiation. Highly enriched MSC after selection with puromycin displayed an increasing GFP-positive population and showed typical properties of stemness. Comparatively, the proportion of MSC significantly increased after RN5-EOS parental cells were treated with either chemotherapy, γ-ray radiation, or a combination of the two, while MSC showed more resistance to the above treatments. A group of identified genes are most likely MSC-specific, and major pathways related to regulation of cell growth or apoptosis are involved. Upregulation of the gene transcripts Tnfsf18, Serpinb9b, Ly6a, and Nppb were confirmed. Putative MSC possess the property of stemness showing more resistance to chemoradiation, suggesting that MSC may play critical roles in cancer cell repopulation. Further identification of selected genes may be used to design novel target therapies against MSC, so as to eliminate cancer cell repopulation in mesothelioma.