Multipotent cancer stem cells derived from human malignant peritoneal mesothelioma promote tumorigenesis

Abstract

During the progression of malignant peritoneal mesothelioma (MPeM), tumor nodules propagate diffusely within the abdomen and tumors are characterized by distinct phenotypic sub-types. Recent studies in solid organ cancers have shown that cancer stem cells (CSCs) play a pivotal role in the initiation and progression of tumors. However, it is not known whether tumorigenic stem cells exist and whether they promote tumor growth in MPeM. In this study, we developed and characterized a CSC model for MPeM using stably expandable tumorigenic stem cells derived from patient tumors. We found morphologically distinct populations of CSCs that divide asymmetrically or symmetrically in MPeM in vitro cell culture. The MPeM stem cells (MPeMSCs) express stem cell markers c-MYC, NES and VEGFR2 and in the presence of matrix components cells form colony spheres. MPeMSCs are multipotent, differentiate into neuronal, vascular and adipose progeny upon defined induction and the differentiating cells express lineage-specific markers such as TUBB3, an early neuronal marker; vWF, VEGFA, VEGFC and IL-8, endothelial markers; and PPARγ and FABP4, adipose markers. Xenotransplantation experiments using MPeMSCs demonstrated early tumor growth compared with parental cells. Limiting dilution experiments using MPeMSCs and endothelial lineage-induced cells derived from a single MPeMSC resulted in early tumor growth in the latter group indicating that endothelial differentiation of MPeMSCs is important for MPeM tumor initiation. Our observation that the MPeM tumors contain stem cells with tumorigenic potential has important implications for understanding the cells of origin and tumor progression in MPeM and hence targeting CSCs may be a useful strategy to inhibit malignant progression.

Figure 1. Live and immunofluorescence of malignant peritoneal mesothelioma stem cell (MPeMSC) division.

(A) Time-lapse images of asymmetric and symmetric cell division in MPeMSCs. Asymmetric cell division (arrows) generates two unequal cells (upper panel); symmetric cell division (arrows) generates two identical cells (lower panel). Cell size shown in µM. Time (hour : minute) noted on the lower left corner. Scale bars: 20 µM. (B) Fluorescent images of asymmetric (upper panel) and symmetric (lower panel) cell division in MPeMSCs. Hoechst nuclear staining shows asymmetric and symmetric division of cells. (C) DNA is segregated asymmetrically during cell division (upper panel). Cells are grown in BrdU for several passages (the pulse period) and after a long pulse BrdU is removed from the cell culture (the chase); BrdU retention in the dividing cells was studied using anti-BrdU antibody. In an asymmetric cell division, the template DNA labeled with BrdU segregated within the new daughter stem cell whereas the newly synthesized DNA without BrdU label condensed within the differentiating cell. During symmetric cell division BrdU labeled DNA segregated randomly within the daughter cells (lower panel).

Figure 2. Malignant peritoneal mesothelioma stem cells (MPeMSCs) with distinct morphology.

(A) Formation of MPeMSCs with capsular covering and possible asymmetric and symmetric stem cell division. a, b, CSCs originate from the parental cells as dark bubble-like protrusions (arrows). Cells detach and float in the culture and MPeMSCs ‘hatch-out’ of the capsule (arrows). c, Asymmetric cell division in capsular stem cells. Asymmetrically dividing cells originate from the capsule (upper panel, arrows) or cells divide unequally along with the capsule generating two non-identical cells (lower panel). d, Symmetric cell division of a capsular stem cell. Scale bars: 100 px. e, f, Fluorescent images of asymmetric (e) and symmetric (f) cell division of encapsulated cells. Arrows indicate the outer capsular covering of the cell (B) a, MPeMSCs originate from the nucleus of a multinucleated cell; the newly formed cell moves through the cytoplasm and is released from the parental cell. b, Origin of multiple CSCs from a multinucleated cell. c, Fluorescent images of a multinucleated cell.

Figure 3. Expression of a mesothelioma marker gene and stem cell markers in malignant peritoneal mesothelioma stem cells (MPeMSCs).

(A) Relative mRNA expression of mesothelin (MSLN) and (B) the stem cell marker genes in parental MPeM cells and in MPeMSCs. c-MYC expression was significantly higher (*p<0.05) in MPeMSCs whereas other stem cell markers showed comparatively similar pattern of expression in both groups. (C) Flow cytometry evaluation of human CD133, CD31 and CD144 demonstrated that the proteins are not expressed in MPeMSCs. Bar diagrams are expressed as mean ±SEM.

Figure 4. Multilineage differentiation of malignant peritoneal mesothelioma stem cells (MPeMSCs).

(A) Differentiation of MPeMSCs into neuron-like cells in the neural differentiation medium (arrow). Immunofluorescence and relative mRNA expression studies shows that differentiated neuronal cells express the neural stem cell marker nestin and an early neuronal marker βIII-tubulin (*p<0.01). (B) Endothelial differentiation of MPeMSCs on a Matrigel surface form tubular networks (arrows). Fluorescent imaging shows that cells express the endothelial stem cell marker, VEGFR2, and uptake of Dil-AcLDL by differentiated endothelial cells. Relative mRNA expression studies demonstrates that during endothelial differentiation VEGFR2 and VEGFR3 expression decreased significantly (*p<0.05) whereas the expressions of endothelial cell markers, vWF, VEGFA, VEGFC and IL-8 significantly increased (*p<0.001). (C) A single MPeMSC form a colony sphere on a Matrigel surface followed by extensive proliferation of MPeMSCs. Scale bars 100 px. (D) MPeMSCs differentiated into adipocytes (Oil Red) and (F) express adipocyte markers PPARγ and FABP4 (*p<0.01) in adipose differentiated cells compared to controls. Bar diagrams are expressed as mean ±SEM; ND, not detectable.

Figure 5. Malignant peritoneal mesothelioma stem cells (MPeMSCs) and endothelial lineage-induced cells promote early tumor growth.

(A) Parental cells from which MPeMSCs derived were injected subcutaneously (n = 8) into the right flank of athymic nude mice (nu/nu). First palpable tumor appeared on day 30 after tumor cell injection. Similar injections of MPeMSCs (n = 8) generated palpable tumors on day 15 (end-stage tumors are shown). In limiting dilution experiments, cells derived from a single MPeMSC were divided and grown either in a Matrigel plate to induce endothelial differentiation or in a regular cell culture plate with LIF containing medium. Five hundred cells dissociated from the first group were resuspended in Matrigel and injected subcutaneously (n = 8). Tumors were first found by palpation on day 59 (mice with 60 days of tumor growth after tumor palpation are shown) whereas similar injections of MPeMSCs (n = 10) resuspended in PBS did not generate tumors. (B) Hematoxylin and eosin (H&E) staining and immunofluorescence of tumor tissues. Early tumors generated from endothelial lineage-induced cells express neuronal markers; NES and TUBB3.