Mesothelioma-Associated Fibroblasts Modulate the Response of Mesothelioma Patient-Derived Organoids to Chemotherapy via Interleukin-6

Abstract

Malignant pleural mesothelioma (MPM) remains an incurable disease. This is partly due to the lack of experimental models that fully recapitulate the complexity and heterogeneity of MPM, a major challenge for therapeutic management of the disease. In addition, the contribution of the MPM microenvironment is relevant for the adaptive response to therapy. We established mesothelioma patient-derived organoid (mPDO) cultures from MPM pleural effusions and tested their response to pemetrexed and cisplatin. We aimed to evaluate the contribution of mesothelioma-associated fibroblasts (MAFs) to the response to pemetrexed and cisplatin (P+C). Organoid cultures were obtained from eight MPM patients using specific growth media and conditions to expand pleural effusion-derived cells. Flow cytometry was used to verify the similarity of the organoid cultures to the original samples. MAFs were isolated and co-cultured with mPDOs, and the addition of MAFs reduced the sensitivity of mPDOs to P+C. Organoid formation and expression of cancer stem cell markers such as ABCG2, NANOG, and CD44 were altered by conditioned media from treated MAFs. We identified IL-6 as the major contributor to the attenuated response to chemotherapy. IL-6 secretion by MAFs is correlated with increased resistance of mPDOs to pemetrexed and cisplatin.

Keywords: IL-6; PDO; chemoresistance; cisplatin; cocultures; mesothelioma patient-derived organoids; mesothelioma-associated fibroblasts; pemetrexed.

Figure 1

Validation of mesothelioma patient-derived organoid (mPDO) cultures. (A) Representative micrographs of passage two organoids formed from pleural effusions of malignant pleural mesothelioma patients, as described in Section 4. Size bar: 200 μm. Magnification of the circled PDO is shown to the right of each panel. (B) Flow cytometry analysis of cells expressing mesothelin (MSLN), calretinin (CALB2), keratin 5/6 (KRT5/6), and podoplanin (PDPN) at passage 0 (immediately after Dispase II treatment, p0) and after three passages (p3). (C) Scatter plot of p0 vs. p3. Percentage of marker expressing cells from mPDOs in (B) at passage 3 (y-axis) and those at passage 0 (x-axis) were reported in the graph (triplicate experiments). Linear correlation analysis results reported in the figure. Statistics: * p < 0.05; note that differences between p0 and p3 expression of markers were not significant except where indicated.

Figure 2

A large proportion of mPDOs were resistant to cisplatin + pemetrexed (C+P) treatment. Representative graphs of eight mPDO cultures treated with cisplatin (2 ugr/mL) (C) or cisplatin + pemetrexed (213 ng/mL) (C+P) for 96 h (with drug washout after 24 h). The effect of the drug was assessed by an empirically defined response score (RS) according to the formula: number of organoids formed × mean maximum diameter at day 0/number of organoids × mean maximum diameter at day 4. An RS ≤ 1 indicates resistance to treatment. Mean + SE of triplicate experiments.

Figure 3

Co-culturing of mPDOs with matched MAFs increased resistance to C+P treatment. (A) Representative micrographs of MAFs isolated and propagated as described in the methods. Size bar: 50 μm. (B) Mesothelioma-associated fibroblasts (MAFs) did not express mesothelial markers. MAFs were analysed for the expression of mesothelial markers and known cancer-associated fibroblast markers by qRT-PCR. The mean of triplicate experiments is expressed as fold over control (FOC). (C) Briefly, passage 3 mPDO#1-2-5-6 showing decreased sensitivity to C+P treatment were treated with C+P for 96 h as single cultures or as co-cultures with MAFs as indicated in the methods (1:1 ratio). RS score is reported. Mean + SE of quadruplicate experiments. Statistics: * p < 0.05; ** p< 0.01.

Figure 4

The conditioned medium (CM) of MAFs altered the organoid-forming ability and the size of the formed mPDOs. (A,B) Organoid-forming ability was recorded after culturing the recipient mPDOs with different ratios of organoid growth medium (OGM) to MAF-conditioned medium (MAF-CM) (medium obtained 24 h after C+P washout). (B) Upper panel. A representative image of mPDO#1 treated at passage 3 with either unconditioned OGM medium (n-CM) or MAF-CM. Size bar: 200 μm. Bottom panel: graph showing the effect of MAF-CM on the mean maximum diameter of mPDO#1 at each passage (0–4). Note that passage 0 refers to 5 days after seeding. (C) MAF-CM increased the expression of stem cell markers. A representative heat map of the expression levels of the indicated mRNAs, assessed by qRT-PCR, at the indicated time after the addition of MAF-CM. Values are expressed as fold over ctrl (n-CM treated mPDO#1). The mean of two independent experiments is reported. Statistics: * p < 0.05; ** p < 0.01.

Figure 5

IL-6 was increased in the medium of mPDO + MAF co-cultures after C+P challenge and mediated increased OFA. (A) A Luminex-based assay for eight cytokines was used to detect inflammatory chemokines in the conditioned media of mPDO+MAF co-cultures. The histogram shows the average levels of the indicated cytokines from two independent experiments. (B) IL-6 is mainly secreted by MAFs after C+P. mPDOs, MAFs, and co-cultured mPDO+MAFs were assayed for IL-6 secretion 24 h after the C+P challenge by indirect ELISA. Results are expressed as fold-over controls, where controls are ctrl (vehicle)-treated cells. (C) Increased IL-6 may mediate the resistance of mPDOs to C+P. Y axis: OFA score (number of organoids formed/1000 live cells) was calculated in mPDO cultures challenged with ctrl (MAF-CM, not treated with C+P) or with MAF-CM (treated with C+P), in the presence of anti-IL-6 neutralising antibody (IL-6-IgG) or a control antibody (ctrl-IgG). Histograms represent the mean + SE of three independent experiments. Statistics: ** p < 0.01; ns = not significant.