Gentlemacs instrument

SG biopsy tissue (average of five minor SG/subject) was minced, digested with 750 U/mL Collagenase I (Sigma, St. Louis, MO, USA), 500 U/mL Hyaluronidase IV (Sigma), and 0.1 mg/mL DNAse I (Roche, Basel, Switzerland) in RPMI/10 mM Hepes/5% fetal calf serum (FCS, Gold Coast, Australia) for 1 h at 37 °C, then dissociated with program “B” on a gentleMACS instrument (Miltenyi Biotec, Bergisch Gladbach, Germany) or processed as previously described [28 (link)]. Cells were stained with monoclonal antibodies (CD3-PE, CD4-PECy5, CD8-Alexa 488, and CD45RA-V450, Becton Dickinson, Franklin Lakes, NJ, USA) as described [3 (link)]. CD3⁺CD4⁺CD45RA⁻ cells excluding propidium iodide were first bulk sorted at high purity using doublet discrimination on a FACSAria (Becton Dickinson). Then 200 cells in ~1 µL were sorted into 6.7 µL SuperAMP buffer (Miltenyi Biotec, Auburn, CA, USA) using a MoFlo-XDP (Beckman-Coulter, Indianapolis, IN, USA) and stored at −80 °C. Samples were shipped on dry ice to Miltenyi Biotec where mRNA isolated with paramagnetic oligo (dT) microbeads underwent proprietary bead-bound cDNA synthesis, 3′-end tailing and labeling, followed by single-primer amplification of cDNA and cDNA purification. Samples showing products of 200–1000 bp were hybridized to Agilent Whole Genome 8 × 60 K Oligo Microarrays, using the SuperAMP service.

Animals were housed in the University of South Florida comparative medicine facility at the Morsani College of Medicine and all protocols were reviewed and approved by the USF Institutional Animal Care and Use Committee (IACUC). All experiments were performed in accordance with IACUC approved guidelines and regulations. C57BL/6 mice were purchased from Envigo and NSG immunocompromised mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) were purchased from Jackson Laboratory. Subcutaneous tumors were grown in the flanks of mice by injecting one million LLC1 monolayer cells, 100,000 LLC1 tumoroids, or 5 million A549 cells. Tumors were allowed to grow and treatment was started when they became palpable (2–3 mm diameter by caliper). Drugs were injected intratumorally once every 3 days at the indicated concentrations. Drug solutions were made in PBS with 1% DMSO and this solution was used as vehicle control. Tumors were collected when controls reached 10 mm in diameter. Tumor volume was estimated by the formula V = ((W² * L)/2). To obtain single cell suspensions of tumors the tissue was digested using a mouse tumor digestion kit and Gentle MACS instrument (Miltenyi Biotech) according to manufacturer instructions.

Whole lungs were removed at 24 hours post-infection and dissociated into a single cell suspension per manufacturer’s protocol (Miltenyi 130-095-927). Briefly, separated lung lobes were placed in a C tube (Miltenyi 130-096-334) containing 2.4 ml 1X Buffer S, 100 µl enzyme D, and 15 µl enzyme A. Samples were mechanically disrupted using a GentleMACS instrument (Miltenyi), filtered, and red blood cells were lysed by incubation in Ammonium-Chloride-Potassium (ACK) buffer (Gibco A10492-01) (16 (link)). 10⁶ cells per sample were stained in PBS (Gibco) + 1% BSA (Quality Biological Inc K719500ML) with the following antibody cocktail: CD45-FITC (clone 30-F11, Biolegend 103108), CD11b-BV650 (clone M1/70, Biolegend 101259), CD11c-BV605 (clone N418, Biolegend 117334), F4/80-PerCP-Cy5.5 (clone BM8, Biolegend 123128), SiglecF-BV421 (clone E50-2440, BD Horizon 562681), and Ly6G-APC-Cy7 (clone 1A8, Biolegend 127624). Samples were resuspended in stabilizing fixative (BD 338036), run on an LSRII flow cytometer (BD Biosciences), and data were analyzed using FlowJo software v10.7.1 (BD Life Sciences).