Journal: eBioMedicine

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

doi: 10.1016/j.ebiom.2024.105418

Figure Lengend Snippet: 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- 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 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.

Article Snippet: 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.

Techniques: Imaging, Staining, Confocal Microscopy, Cell Culture, Microscopy, Expressing, Flow Cytometry, Recombinant, Activity Assay, Inverted Microscopy, Comparison

Journal: eBioMedicine

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

doi: 10.1016/j.ebiom.2024.105418

Figure Lengend Snippet: 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 β -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.

Article Snippet: 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.

Techniques: Activity Assay, Isolation, Purification, Gradient Centrifugation, FACS, Flow Cytometry, Microscopy, Expressing, Cell Culture, Inverted Microscopy, Comparison

Journal: eBioMedicine

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

doi: 10.1016/j.ebiom.2024.105418

Figure Lengend Snippet: 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.

Article Snippet: 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.

Techniques: Microscopy, Expressing, Incubation, Recombinant, Staining, Enzyme-linked Immunosorbent Assay, Titration