The impact of Charcot-Leyden Crystal protein on mesothelioma chemotherapy: targeting eosinophils for enhanced chemosensitivity

Abstract

Background: In mesothelioma (MPM), clinical evidence indicates that the absolute eosinophil count negatively correlates with overall survival and response to standard chemotherapy. Since eosinophils poorly infiltrate MPM tumours, we hypothesised that endocrine rather than paracrine pathways mediate the therapeutic response. We thus studied the effect of eosinophil-associated factors on response to chemotherapy in mesothelioma.

Methods: The culture supernatant conditioned by primary human eosinophils was added to mesothelioma cells in presence of the standard chemotherapeutic regimen. The effectiveness of an anti-eosinophil treatment was evaluated in a preclinical model of C57BL/6 mice transplanted with mesothelioma tumour cells.

Findings: Supernatant of eosinophils differentiated from EOL1 cells or directly isolated from peripheral blood inhibited apoptosis induced by cisplatin and pemetrexed in 2D cultures and in spheroids. Transcriptomic analysis indicated that the anti-apoptotic effect mediated by eosinophils involved molecular interactions with the Charcot-Leyden Crystal protein or Galectin-10 (CLC-P/Gal10). The functional relevance of CLC-P/Gal10 was demonstrated by antibody-mediated depletion. Recombinant human CLC-P/Gal10 mimicked the anti-apoptotic activity of eosinophil-derived supernatants. In the mouse model, eosinophilia did not significantly affect tumour growth but altered the response to chemotherapy. Finally, pretreatment of eosinophilia with the anti-Siglec-F antibody before chemotherapy restored the effectiveness of the treatment.

Interpretation: This study provides a mechanistic rationale to clinical evidence correlating the poor outcome of patients with mesothelioma and with eosinophil-derived CLC-P/Gal10, opening new prospects for intervention in this fatal solid tumour.

Funding: Belgian Foundation against Cancer, Fonds National de la Recherche Scientifique (FNRS), Télévie, Foundation Léon Fredericq, ULiège.

Keywords: Charcot-leyden crystal protein; Chemoresistance; Eosinophils; Mesothelioma.

Fig. 1

Eosinophils differentiated from EOL1 cells inhibit the response to cisplatin and pemetrexed. (a) Valproate-differentiated EOL1 (Dif-EOL1) supernatant (SN) was added to M14K mesothelioma cells at 25% v/v for 48 h. M14K were then treated with 10 μM cisplatin and 10 μM pemetrexed (C + P) for 48 h. After fluorescent labelling of IL-5Rα and CCR3, EOL1 progenitors and Dif-EOL1 were analysed by flow cytometry. The relative Median Fluorescence Intensity (rMFI) corresponds to the ratio of fluorescence intensities associated with IL-5Rα (b) and CCR3 (c) with control isotypes. Bars represent mean ± standard deviation (SD) from 6 independent experiments. (d) Dif-EOL1 were labelled for CCR3 and actin, stained with DAPI and analysed by confocal microscopy (magnification 40×). (e) Flow cytometry was used to discriminate EOL1 progenitors from Dif-EOL1 based on size (forward scatter; FSC) and granulometry (side scatter; SSC). (f) Eosinophil peroxidase activity in EOL1 and Dif-EOL1 cells (stimulated with mock or IL-5; 100 ng/mL). Absorbance of the chromogenic substrate (OPD) was measured at 492 nm with a spectrophotometer. (g) CD63-positive cells (number/mm²) were recorded by time-lapse microscopy (Incucyte imaging S3 Live-Cell system equipped with a 20X objective) in Dif-EOL1 cultures in presence or not of IL-5 (100 ng/mL). (h) Percentages of apoptotic M14K cells (2D) were determined by flow cytometry after Annexin V/propidium iodide (PI) staining. Early (Annexin V⁺ PI⁻) and late (Annexin V⁺ PI⁺) apoptotic cells were counted for each condition. (i) After ethanol permeabilization and PI staining, the cell cycle profiles (2D) were analysed by flow cytometry. The percentages of cells with fragmented genomic DNA (i.e., Sub-G1) were evaluated. (j) M14K spheroids were generated by the liquid overlay method for 72 h in presence or absence of Dif-EOL1 supernatant. After treatment with C + P for 48 h, the proportion of M14K cells in Sub-G1 was determined by flow cytometry after spheroid dissociation, cell permeabilization and PI staining. (k) After transfer of the spheroids into an adherent 24-well plate, cell migration was monitored with an Olympus CKX41 microscope. (l) The surface occupied by the cell culture in mm² was measured after 24 h. Data are expressed as means ± SD, each point representing an independent test. Normality was checked by Shapiro–Wilk and equality of the variances was checked by Brown–Forsythe. Variance of the means was compared by t-test (panels b and c) or by one-way ANOVA followed by Tukey's multiple comparison test (panels f, h, i, j and l).

Fig. 2

Conditioned media of differentiated EOL1 cultures induce transcriptomic changes in M14K cells. (a) Unsupervised heatmap of the 25 most significant up-regulated (red) and down-regulated (blue) genes in the transcriptome of M14K cells. Experimental data with the control (Mock), Dif-EOL1 supernatant (SN) and/or cisplatin + pemetrexed (C + P) were deduced from 3 independent replicates. (b) Volcano plot of differentially expressed genes (DEGs) in conditions C + P and SN Dif-EOL1 vs. C + P. Genes with |Log₂(FC)| > 1 and -Log₁₀pvalue >1.3 (p-adj. threshold: 0.05) are marked in red. (c) Venn diagram of significant DEGs in the different conditions (Mock, C + P, Dif-EOL1 SN, C + P and Dif-EOL1). The numbers of genes impacted by SN Dif-EOL1 are in bold. (d) Representative chord diagram of the most significant pathways affected in Gene Ontology Molecular Functions (GO:MF) in conditions C + P and SN Dif-EOL1 vs. C + P. Pathways (right side) are linked to the genes (left side) according to their Log₂(FC). The names of the pathways are provided below the diagram. (e) Representative chord diagram of the most significant pathways associated with CLC-P/Gal10 in GO:MF.

Fig. 3

CLC-P/Gal10 inhibits MPM cell response to chemotherapy. (a) Incucyte time-lapse imaging of M14K cells (black) and CFSE-stained Dif-EOL1 cells (green). The white arrow shows a CFSE-labelled granule interacting with M14K cells. (b) Confocal microscopy of primary human eosinophils (Eos) co-cultured with CFSE-labelled M14K cells. After 24 h, cells were fixed, permeabilized, stained with DAPI and labelled with an anti-CD63 APC conjugate (in red). Images were acquired using a Zeiss LSM 880 AiryScan Elyra confocal microscope equipped with a x63–1.4 oil immersion objective. White arrows indicate eosinophils. (c) Confocal analysis of Dif-EOL1 cells labelled with DAPI (blue), with an anti-CLC-P/Gal10 antibody and with an AlexaFluor488 conjugate (green). Images were acquired using a Zeiss 880 or 980 Airyscan Elyra confocal microscope equipped with a x63–1.4 oil immersion objective. (d) Representative histogram plot of CLC-P/Gal10 expression acquired by flow cytometry. Dif-EOL1 cells were labelled as described in panel c. (e) Apoptosis of M14K cells in presence of SN Dif-EOL1, SN Dif-EOL1 depleted in CLC-P/Gal10 and/or C + P. CLC-P/Gal10 was depleted from the supernatant by antibody-mediated depletion. Then M14K cells were cultured for 48 h in presence of SN Dif-EOL1 depleted in CLC-P/Gal10. After treatment with C + P for 48 h, cells were labelled with Annexin V/PI and analysed by flow cytometry. (f) Recombinant human CLC-P/Gal10 (0.1, 0.5, 1 and 5 μg/mL) was added in M14K culture medium for 48 h before treatment with C + P. Apoptosis of M14K cells was determined by flow cytometry after Annexin V/PI labelling. (g) Representative images of senescent M14K cells in presence of conditioned medium of differentiated EOL1 (SN Dif-EOL1). M14K cells were cultured for 48 h in presence of mock, SN Dif-EOL1 (25% v/v) or recombinant human CLC-P/GAL10 (1 μg/mL). M14K cells were then treated with 10 μM cisplatin and 10 μM pemetrexed for an additional 2 days. Senescence-associated β-galactosidase (SA-β-gal) activity at pH 6.0 was visualized with an Olympus CKX41 inverted microscope equipped with a 20X objective. (h) The percentages of SA-$\mathrm{\beta }$-gal positive cells were counted in ten different fields. Data are expressed as median ±95% CI, each dot representing an independent test. Normality was checked by Shapiro–Wilk and equality of the variances was verified by Brown–Forsythe and Welch. Variance of the means was compared by one-way ANOVA followed by either Tukey's (panels e and f) or Dunnett's T3 (panel h) multiple comparison test.

Fig. 4

CLC-P/Gal10 expressed by primary eosinophils impairs the cytotoxic activity of cisplatin and pemetrexed. (a) Schematic representation of the experimental protocol for the isolation of primary human eosinophils. Primary eosinophils were purified from the polymorphonuclear cell (granulocytes)-rich fraction of peripheral blood by Ficoll gradient centrifugation and positively selected by magnetic-activated cell sorting using an anti-CCR3 antibody. The culture supernatant of CCR3-positive eosinophils (SN Eos) was collected after 24 h and added at a ratio of 25% (v/v) to M14K cells for 48 h. Then, M14K cells were treated with C + P for 48 h and analysed by flow cytometry after Annexin V/PI labelling. (b) Purified CCR3⁺ eosinophils were fixed, permeabilized and labelled with DAPI (blue), CLC-P/Gal10 (green) and MBP (red). Images were acquired using a Zeiss 980 Airyscan Elyra confocal microscope equipped with a x63–1.4 oil immersion objective. (c) Representative histogram plot of CLC-P/Gal10 expression acquired by flow cytometry. Primary eosinophils were labelled with an anti-CLC-P/Gal10 antibody and an AlexaFluor488 conjugate. (d) Apoptosis of M14K cells in presence of SN Eos and/or C + P. M14K cells were cultured with SN Eos for 48 h. After treatment with C + P for 48 h, cells were labelled with Annexin V/PI and analysed by flow cytometry. (e) Same as in panel d except that CLC-P/Gal10 protein was depleted from SN Eos by using an anti-Gal10 antibody. (f) Representative images of senescent M14K cells in presence of SN Eos. M14K cells were cultured for 48 h in presence of SN Eos treatment with 10 μM cisplatin and 10 μM pemetrexed for an additional 2 days. Senescence-associated $\mathrm{\beta }$-galactosidase (SA-β-gal) activity at pH 6.0 was visualized with an Olympus CKX41 inverted microscope equipped with a 20X objective. (g) The percentages of SA-β-gal positive cells were counted in ten different fields. Data are expressed as median ±95% CI, each dot representing an independent test. Normality was checked by Shapiro–Wilk and equality of the variances were determined by Brown–Forsythe and Welch. Variance of the means was compared by one-way ANOVA followed by Tukey's (panel e) or Dunnett's T3 (Panels d and g) multiple comparison test. (h) Graphical summary of conclusions drawn from cell culture experiments.

Fig. 5

CLC-P/Gal10 is present in the tumour microenvironment and correlates with poor survival. (a) Histochemical analyses of fixed tumour biopsies from MPM patients with low and high absolute eosinophil counts (AEC). Samples were fluorescently labelled for CCR3 (APC in red) and CLC-P/Gal10 (in green) and DAPI (in blue). Images were acquired using a Zeiss 880 Airyscan Elyra confocal microscope equipped with a x63–1.4 oil immersion objective. Scale bars are 10 μm. (b) Representative image of an eosinophil with a characteristic bilobed nucleus (blue) expressing CLC-P/Gal10 (green) analysed with Imaris. (c) CLC-P/Gal10 externalized by a degranulating eosinophil (white arrow). (d) M14K cells were incubated either with culture medium (mock), 25% (v/v) SN Eos or recombinant h-CLC-P/Gal10 for 48 h. Cells were fixed, labelled for CLC-P/Gal10 (green) and stained with DAPI (blue) and CellMask (red). Images were acquired using a Zeiss 980 Airyscan Elyra confocal microscope equipped with a x63–1.4 oil immersion objective. Representative images and 3D representations of CLC-P/Gal10 interaction with MPM cells were computed with Imaris. (e) ELISA titration of CLC-P/Gal10 (in ng/mL) in pleural effusions (n = 79) and sera (n = 37) from MPM patients. (f) Overall survival analysis of patients displaying high (>123.8 ng) and low (<123.8 ng) CLC-P/Gal10 levels in MPM pleural fluids.

Fig. 6

Peripheral blood eosinophilia inhibits chemotherapy in mice while an anti-eosinophilic treatment restores effectiveness. (a) Experimental design. C57BL/6 mice were implanted subcutaneously with epithelioid AK7 mesothelioma cells (1.5 × 10⁶ cells/flank). When the tumour reached ∼150 mm³, eosinophilia was increased with IP injections of IL-5 and/or IL-33 as indicated. When the tumour reached ∼500 mm³, mice were given C + P chemotherapy and tumour growth was assessed until tumour reached 1000 mm³. (b) Absolute counts (number of cells/μL of blood collected from the tail vein) at Day 10 of the different leukocyte populations measured with an hematocytometer just before C + P treatment. (c) The tumour volume (mm³) was regularly determined by using the hemi-ellipsoid formula (L x H x W x π/6), where L=length, W=width, H=height. (C+P, C+P + IL-5, C+P + IL-5 + IL-33 n = 5). (d) Eosinophilic mice were injected with anti-Siglec F antibody 2 days prior to chemotherapy administration. Absolute leukocyte counts were measured before (grey) and concomitantly (green) with chemotherapy administration, and at the end of the experiment (yellow). (e) Tumour growth was determined as in panel c (C + P + IL-5/IL-33 + anti-Siglec F n = 4). Growth curves were constructed based on median and range. Normality of the populations and homogeneity of variances were checked by Shapiro–Wilk and by Levene's test, respectively. Growth curves were compared by using the “growth index” method followed by two-way ANOVA. (f) Perspectives for an improved mesothelioma therapy. Based on the mouse model, the reduction of eosinophils is predicted to ameliorate the survival in human mesothelioma.