Monday, October 2, 2023

A novel retinoic acid receptor-γ agonist antagonizes immune checkpoint resistance in lung cancers by altering the tumor immune microenvironment – Scientific Reports

Cell culture and chemicals

Human lung cancer cell lines (A549, HOP62, and H1299) were purchased and authenticated by American Type Culture Collection (ATCC). Murine lung adenocarcinoma cell lines (344SQ and 393P) were obtained from Dr. Jonathan Kurie at MD Anderson Cancer Center (Houston, TX) and were originally derived from K-rasLA1/+p53R172HΔg/+ engineered mice24. The ED1SQ4 murine lung cancer cells were derived in our laboratory from ED1 cells25. Murine ED1 lung cancer cells were isolated from cyclin E transgenic mice in our laboratory, as described25,26,27,28. Cells were cultured in RPMI1640 media (with L-glutamine) supplemented with 10% fetal bovine serum (FBS) in a humidified incubator maintained at 37 °C and with 5% CO2. IRX4647 and IRX6696 compounds were obtained from Io Therapeutics, Inc.

Proliferation assays

Logarithmically growing cancer cells were seeded at optimized densities (8 × 102 cells/well) for each examined cell line onto individual wells of 96-well polystyrene tissue culture-treated microplates (3598, Corning, Corning, NY). Cells were treated with IRX4647 or vehicle (dimethyl sulfoxide, DMSO) at desired concentrations 24 h later. The WST-1 cell proliferation assay (Takara Bio USA, Inc., San Jose, CA) measured cell growth over 5 days using a FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany). These assays were independently repeated at least three times with each biological replicate performed at least in triplicate. Data were normalized to baseline (0 h; no treatment) cell growth.

Lung cancer murine syngeneic models and drug treatments

All methods are performed and reported in accordance with Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines ( Animal experiments were conducted following study protocols: #100161 was approved by the National Cancer Institute (NCI) Animal Care and Use Committee (ACUC), and #06-14-04731 was approved by MD Anderson Cancer Center’s Institutional Animal Care and Use Committee (IACUC). Animal euthanasia was with carbon dioxide (CO2) inhalation and used the NIH Guideline for Euthanasia of Rodents ( Each mouse was observed for lack of respiration and faded eye color, which was followed by continued CO2 flow for a minimum of 1 min after respiration ceased. Murine adenocarcinoma lung cancer 344SQ cells (0.5 × 105 cells) were injected subcutaneously into male 129/Sv mice (Charles River Laboratories, Wilmington, MA). Murine colon adenocarcinoma MC38/gp100 cells (5 × 105) or melanoma BP cells (5 × 105) were injected into C57BL/6 mice (Charles River Laboratories, Wilmington, MA) subcutaneously. ED1SQ4 cells (0.5 × 105 cells) were injected into FVB/N or athymic mice (Charles River Laboratories, Wilmington, MA). The inbred mice were at least 8 weeks old and had at least a one-week acclimation period before experiments began.

Mice with palpable tumors following independent 344SQ or ED1SQ4 lung cancer cell subcutaneous injections were randomized based on similar basal tumor sizes to receive oral doses of vehicle, ATRA, IRX6696 or IRX4647 (1.5 mg/kg body weight; 5 days per week) with or without intraperitoneal injections of PD-L1 antibody (1 mg/ml; twice per week; BP0101; BioXCell, Lebanon, NH). Compound IRX4647 was formulated in 2.5% DMSO/30% PEG400/67.5% Phosal MCT53 solution and treatment results were compared to that of vehicle (2.5% DMSO/30% PEG400/67.5% Phosal MCT53). Body weights and tumor volumes were measured three times weekly. Tumor volumes were calculated as V = (length × width2)/2. Tumor, blood, and tissues were harvested at baseline (n = 10; no treatment), after 2 weeks (n = 40), 3 weeks (n = 40), 5 weeks (n = 41), and 8 weeks (n = 48) of treatments.

Pharmacokinetic studies

Inbred 8–12 weeks old female C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) were housed in ventilated cages on a regular 12 h light/dark cycle with ad libitum access to food and water. Animals acclimated for at least one week before a single dose administration of the studied retinoid via oral gavage. Compound IRX4647 was formulated in 2.5% DMSO/30% PEG400/67.5% Phosal MCT53 solution. Plasma and liver from compound-treated mice (n = 5 per time point) were individually harvested at 0.5, 1, 3, 6, 12, 24, and 48 h post-drug administration. Samples were flash frozen and stored at − 80 °C until analysis. Plasma was isolated as were liver homogenates and samples were extracted with acetonitrile and 0.1% formic acid for 30 min at room temperature. Extracts were separated by centrifugation and used for high-performance liquid chromatography (HPLC) analysis. HPLC analyses were performed with a Shimadzu 20AC-XR system. Separation was at room temperature with a Cortecs C18 column (Waters Corp., Milford, MA).

T cell depletion

Murine lung adenocarcinoma ED1SQ4 (0.5 × 105 cells) or colon adenocarcinoma MC38/gp100 cells (5 × 105 cells) were injected into FVB/N or C57BL/6 mice (Charles River Laboratories, Wilmington, MA) subcutaneously on day 0, respectively. Mice with palpable tumors were randomized based on similar basal tumor sizes to independently receive CD8+ T cell depletion antibody (clone HB129/116-13.1, Bioxcell), CD4+ T cell depletion antibody (clone GK1.5, Bioxcell, Lebanon, NH), or rat IgG 2b isotype control antibody (Bioxcell) independently on days 5, 7, 13 and 19. Mice were treated with vehicle or ATRA via oral gavage (1.5 mg/kg body weight) on days 7, 8, 9, 12, 13, 14, 15, 16, 19, 20, 21 and 22 before harvesting tumors on day 23. Tumor size was calculated as V = (length × width2)/2.

Flow cytometry

Blood, tissues, and tumors were each freshly harvested and stained with antibodies following the manufacturer’s protocols. Antibodies used were: CD3-PE-Cy7 (560591, BD Biosciences, San Jose, CA), CD4-BV786 (563727, BD Biosciences, San Jose, CA), CD8a-BB515 (564422, BD Biosciences, San Jose, CA), CD19-BB515 (564509, BD Biosciences, San Jose, CA), CD25-BV421 (564370, BD Biosciences, San Jose, CA), CD45-PerCP-Cy5.5 (550994, BD Biosciences, San Jose, CA), CD45RA-PE (553380, BD Biosciences, San Jose, CA), CD49b-BV421 (563063, BD Biosciences, San Jose, CA), CD197-BV650 (564356, BD Biosciences, San Jose, CA), F4/80-BV421 (565411, BD Biosciences, San Jose, CA), CD11b-BV650 (563402, BD Biosciences, San Jose, CA), CD11c-PE (565592, BD Biosciences, San Jose, CA), Ly-6C-BV785 (128041, BioLegend, San Jose, CA), Ly-6G-PE-Cy7 (560601, BD Biosciences, San Jose, CA), RORγt-BV650 (564722, BD Biosciences, San Jose, CA), Foxp3-PE (560408, BD Biosciences, San Jose, CA), or viability stain-510 (564406, BD Biosciences, San Jose, CA). For intracellular staining, cells were fixed and permeabilized with Foxp3/Transcription factor staining buffer set (00-5523-00, ThermoFisher Scientific, Waltham, MA). Data were collected by using a CytoFLEX S flow cytometer (Beckman Coulter Life Sciences, Indianapolis, IN) and analyzed with the CytExpert software (version 2.3; Beckman Coulter Life Sciences, Indianapolis, IN).

Cytokine analysis

Custom-designed U-Plex multiplex assay kits (Meso Scale Diagnostics, Rockville, MD) independently detected IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-10, IL-13, IL-17A, IL-21, and TNF-α cytokines in plasma and tumors following the manufacturer’s methods. Data were collected using a MESO QuickPlex SQ 120 instrument (Meso Scale Diagnostics, Rockville, MD) and analyzed with the Discovery Workbench software (version 4.0; Meso Scale Diagnostics, Rockville, MD). Standard curves for each cytokine were generated for calculations of concentrations.

qRT-PCR assays

Total RNA was isolated using RNeasy Mini Kit (QIAGEN, Hilden, Germany) from cultured cancer cells. The desired cDNA was synthesized from RNA using TaqMan™ reverse transcription reagents (N8080234, ThermoFisher Scientific, Waltham, MA) using the manufacturer’s methods. Real-time qPCR assays were with the TaqMan™ fast advanced master mix (4444963, ThermoFisher Scientific, Waltham, MA) on a QuantStudio™ real-time PCR assay system (ThermoFisher Scientific, Waltham, MA). The mRNA quantification was normalized to β-actin expression. Primers were purchased (ThermoFisher Scientific) for human RARα (Hs00940446_m1), murine RARα (Mm01296312_m1), human RARβ (Hs00233407_m1), murine RARβ (Mm01319677_m1), human RARγ (Hs01559234_m1), murine RARγ (Mm00441091_m1), human β-actin (Hs99999903_m1), and murine β-actin (Mm00607939_s1).


Harvested tumors were formalin-fixed and paraffin-embedded. Automated immunohistochemical staining was performed with the Leica Biosystems’ BondRX using Epitope Retrieval 2 (EDTA) for CD38 (ab216343, Abcam, Boston, MA, 1:1000) and CD4 (13-9766, eBioscience, San Diego, CA, 1:250), Epitope Retrieval 1 (Citrate) for CD8a (14-0195-82, eBioscience, San Diego, CA, 1:50) and PD-L1 (AF1019, R&D Systems, Minneapolis, MN, 1:300). The Bond Polymer Refine Detection Kit (DS9800, Leica Biosystems, Wetzlar, Germany) with omission of the PostPrimary Reagent was used. Anti-rat secondary antibody was used for CD4 and CD8; anti-goat secondary antibody was used for PD-L1. Matched isotype antibodies served as negative controls.

Multiplex fluorescent staining was with Leica Biosystems’ BondRX autostainer using the Bond Polymer Refine Kit (DS9800, Leica Biosystems, Wetzlar, Germany), with omission of the PostPrimary reagent, DAB, and Hematoxylin. Antigen retrieval was with Epitope Retrieval 2 (EDTA). Incubations were for 30 min with CD38 (ab216343, Abcam, Boston, MA, 1:1000) followed by the Polymer reagent and OPAL Fluorophore 690 (AKOYA Biosciences, Marlborough, MA). CD38 antibody complex was stripped by heating with Bond Epitope Retrieval 2. Sections were incubated for 30 min with panCytokeratin AE1/AE3 biotin conjugate (NBP2-33200B, NOVUS, Centennial, CO, 1:50), followed by Streptavidin HRP (434323, ThermoFisher Scientific, Waltham, MA) and OPAL Fluorophore 570. Sections were stained with DAPI and coverslipped with Prolong Gold AntiFade Reagent (ThermoFisher Scientific, Waltham, MA). Images were captured using the Aperio Scanscope FL (Leica Biosystems, Buffalo Grove, IL) whole slide scanner.

Image analysis was with Halo imaging analysis software (v3.4.2986.235; Indica Labs, Corrales, NM). Image interpretation was performed by a single reference pathologist. Folds, tears and necrotic regions were excluded from analysis using Densenet AI V2 (Plugin). CD4+ T cells, CD8+ T cells, and PDL-1 were each quantified using cytonuclear algorithm, v2.0.12. CD38 profiles were quantified using Area quantification FL v2.1.9 in HALO to score percent positive regions of interest.

Statistical analysis

Data were group means ± standard error of the mean (SEM). Data were analyzed by unpaired Student’s t-test and one-way analysis of variance (ANOVA) using GraphPad Prism (version 9.2.0). A P value of 0.05 was deemed as statistically-significant.

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