Asbestos accelerates disease onset in a genetic model of malignant pleural mesothelioma

Abstract

Hypothesis: Asbestos-driven inflammation contributes to malignant pleural mesothelioma beyond the acquisition of rate-limiting mutations. Methods: Genetically modified conditional allelic mice that were previously shown to develop mesothelioma in the absence of exposure to asbestos were induced with lentiviral vector expressing Cre recombinase with and without intrapleural injection of amosite asbestos and monitored until symptoms required euthanasia. Resulting tumours were examined histologically and by immunohistochemistry for expression of lineage markers and immune cell infiltration. Results: Injection of asbestos dramatically accelerated disease onset and end-stage tumour burden. Tumours developed in the presence of asbestos showed increased macrophage infiltration. Pharmacological suppression of macrophages in mice with established tumours failed to extend survival or to enhance response to chemotherapy. Conclusion: Asbestos-driven inflammation contributes to the severity of mesothelioma beyond the acquisition of rate-limiting mutations, however, targeted suppression of macrophages in established epithelioid mesothelioma showed no therapeutic benefit.

Keywords: GM mouse model; asbestos; cancer therapy; macrophages; mesothelioma.

FIGURE 1

Asbestos injection reduces survival of CNP mice (A) Schematic of the alleles and induction strategies employed in this study. Black arrows deonte LoxP sites. (B) Kaplan Meier plot of overall survival of lenti-Cre induced CNP mice with (N = 17) or without (N = 9) asbestos injection. *** denotes p < 0.001 (Mantel Cox logrank test). (C) Photographs showing representative examples anatomic distribution of visible MPM lesions (arrowheads) in mice from (B). Dashed lines circumscribe areas of lateral mesothelioma growth; arrowheads indicate individual mesothelioma tumours. Scale bars = 4 mm.

FIGURE 2

Histology of mesothelial lesions in CNP mice induced with lenti-Cre and/or asbestos (A) Representative images of indicated H&E stained tissues from mice induced with lenti-Cre (N = 9), Cre + asbestos (N = 17), both at end-stage, or asbestos without Cre-mediated allele inactivation, harvested 10 months after asbestos injection (N = 3). Arrows indicate neoplastic disease; arrowheads indicate reactive mesothelium. Scale bars = 100 μm. (B) H&E-stained examples of epithelioid and sarcomatoid histologies of tumours from CNP mice induced with lenti-Cre + asbestos. Scale bars = 100 μm.

FIGURE 3

Asbestos does not alter the tumour phenotype of induced CNP mice A) Representative IHC staining of tumours arising from diaphragm mesothelium in CNP mice induced by lenti-Cre, with and without asbestos. Scale bars = 100 μm.

FIGURE 4

Asbestos drives increased macrophage infiltration in CNP tumours (A) Representative IHC images show detection of tumour infiltration by major leukocyte cell types in CNP mice induced with lenti-Cre with and without asbestos. Scale bars = 100 μm. (B) Quantification of tumour-infiltrating leukocytes as per (A) in 6 mice from each cohort. **** denotes p < 0.0001 (T-test); ns = not significant.

FIGURE 5

Suppression of macrophages fails to impact survival of CNP mice (A) IHC validation of macrophage suppression in situ by AZD7507. Left panels show representative images of F4/80-stained diaphragm tumours from end-stage CNP mice induced with lenti-Cre + asbestos and treated with AZD7507 (right) or vehicle control (left). Scale bar = 100 μm. (B) Quantification of diaphragm tumour F4/80 staining in mice treated with AZD7507 (N = 5) or vehicle (N = 6). Mean ± SEM shown. (C) Normalised RNA-SEQ read counts for selected genes, grouped by function, from end-stage tumours of CNP mice treated with or without AZD7507 from day 60 post-induction (N = 3 per cohort). Individual values, Mean and SEM shown. Adjusted p values (T-test) were calculated in R: *** denotes p < 0.001; ** denotes p < 0.01; * denotes p < 0.05; ns = not significant. (D) Kaplan Meier plot of overall survival of mice treated with AZD7507 (N = 7) or vehicle control (N = 8) from 60 days post tumour initiation. Ns = Not significant (Mantel Cox logrank test). (E) Kaplan Meier plot of overall survival of mice treated with 2 rounds (brown arrows) of Cisplatin + Pemetrexed (Cis/Pem) with (N = 12) or without (N = 10) AZD7507. Ns = Not significant (Mantel Cox logrank test).