All experimental procedures were approved by Institutional Animal Care and Use Committees. Six-week old female nude mice (NU/J) were purchased from Jackson Laboratories. The animals were subjected to a 12 h light cycle, relative humidity of 55%, and temperature of 21 ± 2 °C. After one week of adaptation, animals were inoculated with HTB-43 tumor cells (2 × 106 cells in 100ul volume of PBS) in tongue. HTB-43 xenograft model induces aggressive HNSCC tumors [35]. Tumor volume was measured continuously from day 4 post implant to following the development of tumors. Ten days post implantation of tumor cells, 24 mice were randomized to three group of control (PBS injection), LNA- miR-34a-5p or LNA-miR-34a-5p (n = 8 per group; n = 24 total). Mice were dosed with LNA- control mimic or LNA-miR-34-mimic (20 mg/kg/ intraperitoneal (i.p)) every week for two weeks at day 10 post tumor injection. Tumor volume measurements were quantified by digital calipers and calculated using the formula (π)/6 × (large diameter) × (small diameter)2. The mice were anesthetized by CO2 chamber (70% CO2/30% O2) and cervical dislocation. No adverse events and change in the mice well-being were observed in any of the treatment groups. Tumors were retrieved from animals and went through tumor dissociation protocol for flow cytometry analysis or single cell isolation using a gentleMACS™ Tissue dissociator (Miltenyi) and tumor dissociation kit (Miltenyi) as recommended by the manufacturer. Anti-EpCAM magnetic beads (Miltenyi) were used for isolation of squamous epithelial tumor cells. CD11b magnetic beads (Miltenyi) were used for isolation of TAMs after tumor dissociation as recommended by the manufacturer. Sections of tumors were kept in RNAlater® for RNA isolation or stored at − 80 °C for protein analysis.

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