Crizotinib-induced immunogenic cell death in non-small cell lung cancer

Immunogenic cell death (ICD) converts dying cancer cells into a therapeutic vaccine and stimulates antitumor immune responses. Here we unravel the results of an unbiased screen identifying high-dose (10 µM) crizotinib as an ICD-inducing tyrosine kinase inhibitor that has exceptional antineoplastic activity when combined with non-ICD inducing chemotherapeutics like cisplatin. The combination of cisplatin and high-dose crizotinib induces ICD in non-small cell lung carcinoma (NSCLC) cells and effectively controls the growth of distinct (transplantable, carcinogen- or oncogene induced) orthotopic NSCLC models. These anticancer effects are linked to increased T lymphocyte infiltration and are abolished by T cell depletion or interferon-γ neutralization. Crizotinib plus cisplatin leads to an increase in the expression of PD-1 and PD-L1 in tumors, coupled to a strong sensitization of NSCLC to immunotherapy with PD-1 antibodies. Hence, a sequential combination treatment consisting in conventional chemotherapy together with crizotinib, followed by immune checkpoint blockade may be active against NSCLC.

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Policy information about availability of computer code Data collection Fluorescent images were collected by using a robot-assisted Molecular Devices IXM XL BioImager (Molecular Devices) equipped with a Sola light source (Lumencor), adequate excitation and emission filters (Semrock) a 16-bit monochrome sCMOS PCO.edge 5.5 camera (PCO) and a 20 X PlanAPO objective (Nikon); Flow cytometry data was obtained on a LSRFortessa (BD Biosciences) or on a CyAn ADP cytofluorometer (Beckman Coulter); Luminescence and absorbance were measured using a SpectraMax I3 multi-mode microplate reader (Molecular Devices); qPCR data was obtained with a StepOnePlus Real-Time PCR System (Applied Biosystems); Western blotting signal was obtained on a ImageQuant LAS 4000 software-assisted imager (GE Healthcare); Extracellular acidification rate was analyzed by the Wave Desktop software assisted Seahorse XFe96 Analyzer (Agilent Technologies); Mass spectrometry data was obtained with a RRLC 1260 system (Agilent Technologies) coupled to a 6500+ QTRAP MS (Sciex) equipped with an electrospray ion source; In vivo luciferase photons were acquired on a Xenogen IVIS 50 bioluminiscence in vivo imaging system (Caliper Life Sciences Inc.,); Confocol fluorescent images were acquired on a HR-SP8 Confocal Microscope (Leica); Lung lobes of urethane induced model were imaged with a stereo microscope (Motic SMZ168TP) equipped with a high-resolution camera (Sony HD 1/1.8 inch color CCD); Histological images were obtained with the NanoZoomer 2.0-RS slide scanner system (Hamamatsu). Quantification of clonogenicity was obtained with Image J (https://imagej.nih.gov/ij/) Data analysis Fluorescent images were processed and segmented with the MetaXpress software (Molecular Devices); FSC files were processed with the FlowJo software (Tree Star); Integration and quantification of the mass spectrometry analytes were performed using the MultiQuant quantitative software (Version 3.0.3); In vivo luciferase photons were quantified with the living Image® software V3.2; stereo microscope images were quantified with the supporting software EZ-NET™; Aperio ImageScope Viewer (Leica Biosystems) was used for histological image quantification; GraphPad Prism 7 (https://www.graphpad.com/), Microsoft Excel 2010, and the freely available software R

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