FIGURE 6 from Low Exposures to Amphibole or Serpentine Asbestos in Germline <i>Bap1</i>-mutant Mice Induce Mesothelioma Characterized by an Immunosuppressive Tumor Microenvironment

Expression of IL6 and IL10 in ascites and Bap1 and p16Ink4a in MM tumors from chrysotile-exposed Bap1+/− mice. A, Concentration of IL6 in ascites of Bap1+/− mice compared with that from Bap1+/+ mice with MMs induced by chrysotile. Ascitic fluids were obtained from 5 Bap+/+ mice and 4 Bap1+/− mice with chrysotile-induced MMs. B, Expression of IL10 in ascites of 4 Bap1+/− mice versus that in ascites of 5 Bap1+/+ mice with MMs induced by chrysotile. C, Immunoblot analysis of tumor suppressors Bap1, Nf2, and p16Ink4a in MM tumors from 1 chrysotile-exposed Bap1+/+ mouse and 6 chrysotile-exposed Bap1+/− mice. NMC, normal (mouse) mesothelial cells; Vinculin, loading control.</p

FIGURE 5 from Low Exposures to Amphibole or Serpentine Asbestos in Germline <i>Bap1</i>-mutant Mice Induce Mesothelioma Characterized by an Immunosuppressive Tumor Microenvironment

IHC staining for CD39 (top; diffuse brown stain) and Ccr2 (bottom; punctate stain) in crocidolite-induced and chrysotile-induced MMs from Bap1+/− mice. Arrows indicate examples of cells positively stained for Ccr2.</p

FIGURE 1 from Low Exposures to Amphibole or Serpentine Asbestos in Germline <i>Bap1</i>-mutant Mice Induce Mesothelioma Characterized by an Immunosuppressive Tumor Microenvironment

MM formation induced by crocidolite or chrysotile asbestos in mice with germline Bap1 heterozygous mutation. MM formation induced by minimal exposure to crocidolite (A and B). A,Bap1+/− and Bap1+/+ (WT) littermates injected with a total of 0.8 mg of crocidolite fibers. Left, Kaplan–Meier curves depicting survivals in Bap1+/− and Bap1+/+ groups. Right, Summary of MM incidence and median survival times. B, Results for Bap1+/− and Bap1+/+ mice injected with a total of 0.1 mg of crocidolite. Left, Kaplan–Meier curves. Right, Summary of MM incidence and median survival times. MM formation induced by high (C) and minimal (D) exposure to chrysotile. C,Bap1+/− and Bap1+/+ mice were injected with a total of 3.2 mg of chrysotile. Left, Kaplan–Meier survival curves. Right, MM incidence and median survival times. D,Bap1+/− and Bap1+/+ mice injected with a total of 0.4 mg/mouse of crocidolite. Left, Kaplan–Meier curves. Right, MM incidence and median survival times. For all Kaplan–Meier survival curves, deaths due to all causes are included. Some mice (especially WT animals) succumbed because of adhesion-related intestinal obstructions and/or liver fibrosis.</p

Supplementary Figure S6 from Low Exposures to Amphibole or Serpentine Asbestos in Germline <i>Bap1</i>-mutant Mice Induce Mesothelioma Characterized by an Immunosuppressive Tumor Microenvironment

IHC staining for WT1 (purple) and SMA (brown in top panels; teal in bottom panels) in representative crocidolite-induced and chrysotile-induced MMs from Bap1+/- mice. WT1 stains MM cells, whereas SMA stains CAFs, the latter typically more abundant in chrysotile-induced than crocidolite-induced MMs, as in the examples shown here.</p

FIGURE 2 from Low Exposures to Amphibole or Serpentine Asbestos in Germline <i>Bap1</i>-mutant Mice Induce Mesothelioma Characterized by an Immunosuppressive Tumor Microenvironment

TIME of crocidolite-induced MMs from Bap1+/− mice. A, Serial sections of a crocidolite-induced MM from Bap1-mutant mouse stained with several different immune cell markers: CD3 (T cells; arrows); CD45R (B cells); F4/80 (macrophages; arrows); and NK1.1 (natural killer cells), clockwise starting from top left. B, Serial sections showing distribution of macrophages in granuloma and TIME of MM from Bap1+/− mouse exposed to crocidolite. Left, Proinflammatory CD11c-positive M1 macrophages. Right, Protumorigenic CD206-positive M2 macrophages. Blue circles, granulomatous lesions; red squares, MM tumor areas. Note numerous crocidolite fibers (yellow block arrows) in granulomatous lesions.</p

FIGURE 4 from Low Exposures to Amphibole or Serpentine Asbestos in Germline <i>Bap1</i>-mutant Mice Induce Mesothelioma Characterized by an Immunosuppressive Tumor Microenvironment

Triple indirect IF of crocidolite- and chrysotile-induced MMs from Bap1+/− mice. IF staining showing tumor cells as well as M1 and M2 macrophages in crocidolite- and chrysotile-induced MMs from 2 Bap1+/− mice. Histologic sections were subjected to triple indirect sequential IF using three different antibodies: WT1 to detect MM tumor cells (red circles; Red 555 fluorescence); CD11c to stain M1 macrophages (green circles; green 488 fluorescence); and CD163 marker to stain M2 macrophages (yellow circles; Cy5 yellow fluorescence).</p

Supplementary Figure S4 from Low Exposures to Amphibole or Serpentine Asbestos in Germline <i>Bap1</i>-mutant Mice Induce Mesothelioma Characterized by an Immunosuppressive Tumor Microenvironment

Tumor immune microenvironment of chrysotile-induced MM from a Bap1+/- mouse. Serial sections of MM stained with several different immune cell markers: CD3 (T cells), CD45R (B cells), F4/80 (macrophages), and NK1.1 (natural killer cells), clockwise starting from upper left. T, B, and NK cells are depleted in tumor, whereas numerous cells in and around the tumor are stained with the macrophage marker F4/80.</p

Supplementary Table S1 from Low Exposures to Amphibole or Serpentine Asbestos in Germline <i>Bap1</i>-mutant Mice Induce Mesothelioma Characterized by an Immunosuppressive Tumor Microenvironment

Primary antibodies used for IHC, IF, and immunoblot analyses</p

FIGURE 3 from Low Exposures to Amphibole or Serpentine Asbestos in Germline <i>Bap1</i>-mutant Mice Induce Mesothelioma Characterized by an Immunosuppressive Tumor Microenvironment

Quantification of M2 macrophages in the TIME of asbestos-induced MMs from Bap1+/− mice using an Aperio scanner, and IHC depicting arginase and iNOS staining of MM from asbestos-treated Bap1+/− mice. A, Dot plot showing percentages of CD206-stained (left) and CD163-stained (center) cells in representative histologic images from crocidolite-induced and chrysotile-induced MMs from Bap1+/− mice. Right, CD163 IHC staining of representative MMs from crocidolite-induced and chrysotile-induced MMs. Arrows indicate examples of cells positively stained for CD163. B, Serial sections of a crocidolite-induced and chrysotile-induced MMs from Bap1+/− mice stained for arginase (Arg-1) and iNOS. Arrows indicate Arg-1-stained M2 macrophages.</p

Supplementary Figure S2 from Low Exposures to Amphibole or Serpentine Asbestos in Germline <i>Bap1</i>-mutant Mice Induce Mesothelioma Characterized by an Immunosuppressive Tumor Microenvironment

H&E images of liver fibrosis in Bap1+/- mice chronically injected i.p. with asbestos. A) Interlobular liver fibrosis in a chrysotile-exposed mouse. B) Liver fibrosis in between liver and spleen in chrysotile-injected mouse. C) Fibrosis on surface of liver in a crocidolite-exposed mouse. D) Fibrosis between liver and pancreas of a crocidolite-exposed mouse. All original images are at 200X magnification. Abbreviations: F, fibrosis; L, liver; M, mesothelioma; S, spleen; P, pancreas.</p