Llc1 cells

B16F10 mouse melanoma cells (ATCC CRL-6475) and LLC1 cells (ATCC CRL-1642) were cultured in DMEM supplemented with glucose, L-glutamine, sodium pyruvate, 10% FBS, penicillin, and streptomycin. Cells were split once they reached 70% confluency and were not used for mouse challenge past a fifth passage. T cells were cultured in RPMI 1640 supplemented with 10% FBS, 1 mM sodium pyruvate, 50 μM β-ME, penicillin, streptomycin, 2 mM l-glutamine, 100 mM nonessential amino acids, 5 mM HEPES, free acid and β-mercaptoethanol. For tumor inoculations, 5 × 10⁴ B16F10 cells or 5 × 10⁵ LLC cells were injected subcutaneously on the right flank of each mouse on day 0. Tumor volume was estimated using the formula (L × W²) ÷ 2. Survival endpoint was reached once the tumors measured 500 mm³, around day 15. Mice were euthanized by isoflurane inhalation and subsequent cervical dislocation, and tumors were harvested for further prospecting.

KPC-1 and KPC-2 cell lines^{70 (link)} were obtained from pancreas tissue from p48^creTrp53^LSL-R172HKras^LSL-G12D mice^{71 (link)}. Pan02^{72 (link),73 (link)} (DCTD Tumour Repository) cells were provided by D. Lyden. The Colon adenocarcinoma cell line MC38^{73 (link),74 (link)} and melanoma cell line B16F10^{75 (link)} (ATCC, CRL-6475 was a gift from J. D. Wolchok). Lewis lung carcinoma line LLC1 cells^{76 (link)} (ATCC, CRL-1642) were purchased from ATCC. KPC-1, KPC-2 and PANC-1 cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin (Invitrogen). Panc02 cells, LLC1 cells, MC38 cells and B16F10 cells were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin (Invitrogen) and incubated at 37 °C in 5% CO₂.

RAW264.7 cells and LLC1 cells were purchased from ATCC (Rockville, MD, USA) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 50 mg/mL penicillin/streptomycin and 2 mM glutamine, respectively. RAW264.7 cells at ~90% confluence received treatment in serum-free medium. The thioglycollate-elicited peritoneal macrophages were collected from WT mice as described 49 (link). All cells were incubated in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C.
DC 2.4 cells (a murine dendritic cell line) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and cultured in DMEM medium supplemented with 10% FBS, 50 mg/mL penicillin/streptomycin and 2 mM glutamine, respectively.
Bone marrow cells (BMCs) were isolated by flushing from mouse femur with pre-cooled PBS, and cultured in RPMI 1640 medium supplemented with 10% FBS, 50 mg/mL penicillin/streptomycin and 2 mM glutamine, respectively. To induce differentiation of BMCs into DCs, after BMCs attached dishes well, half of the medium was replaced with fresh complete RPMI 1640 medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF, 20 ng/mL) and interleukin 4 (IL-4, 10 ng/mL). After 6 days of culture, the immature DCs were collected, counted and re-distributed equally in 6-well culture plates for the subsequent experiments.

Madin-Darby canine kidney cells (MDCK) (ATCC CCL-34), human lung adenocarcinoma epithelial cells (A549) (ATCC CCL-185), human embryonic kidney HEK293T cells (293T) (ATCC CRL-3216), and Lewis lung carcinoma LL/2 (LLC1) cells (ATCC CRL-1642) of C57BL were purchased. The 293T-IAV-Luc cell line is constructed by our previous study [70 (link)]. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Lonsera, 711-001S, www.lonsera.cn), 100 U/mL penicillin, and 100 ug/mL streptomycin at 37°C in an atmosphere containing 5% CO₂. The influenza A/WSN/1933 (WSN), influenza A/California/2009/04 (CA04), and influenza A/Puerto Rico/8/1934 (PR8) strains were propagated in MDCK cells. A/Darwin/6/2021/H3N2, A/chicken/Hebei/LC/2008(H9N2) (HB08) strain and Sendai virus (SeV) was propagated in 9-day-old SPF chicken embryonated eggs purchased from Beijing Vital River Animal Technology Co., Ltd. (licensed from Charles River) at 37°C for 72 h.

C57Bl/6NHsd female mice immunized with DNP-BSA and CFA/IFA adjuvants received a single subcutaneous (SQ) injection of 10⁶ murine Lewis lung carcinoma (LLC1) cells (ATCC CRL-1642) in the right flank on day 1. On day 3, mice were randomized into groups and agents including DNP-pHLIP, DNP-Peg4-pHLIP or DNP-Peg12-pHLIP (50 µM 250 µl per injection) were administered every second day by IP injections. Control mice (untreated group) did not receive any injections. All mice in the control and treated groups were euthanized at day 14, tumors were collected and weighed.

Lewis lung carcinoma (LLC1) cells were obtained from the American Type Culture Collection (ATCC, Baltimore, U.S.A.). Cultures were maintained in RPMI-1640 supplemented with 10% FCS. All experiments were carried out within 6 months of resuscitation. LLC1 cells were incubated with etoposide (400 μM), curcumin (160 μM), tamoxifen (40 μM), staurosporine (1 μM), cisplatin (400 μM) or 5-fluorouracil (160 μM) (all from Enzo Life Sciences) for 24 hrs to induce apoptosis. Apoptotic bodies were purified as described by Fransen et al., 2009 (28 (link)); briefly, cells were centrifuged at 1500 g for 10 mins at 20°C. The supernatant was centrifuged at 15700 g for 50 mins at 20°C. Apoptotic bodies were resuspended in PBS.
4 x 10⁶ LLC1 cells were incubated with 100 µl RIPA buffer containing a protease inhibitor cocktail (20 µl/ml; Calbiochem), phosphatase inhibitor (Thermo Scientific) and 1% Triton X-100 for 20 mins at 4°C. Supernatants, collected after centrifugation at 18000 g for 30 mins at 4°C, were employed as cell lysate.