Calu 1

The immortalized human ovarian surface epithelial cells T29 and their transformed counterparts T29Kt1 (with the introduction of KRAS^V12 into the immortalized cells) were reported previously28 (link) and were cultured in medium consisting of 1:1 MCDB105 medium and M199 medium (Sigma-Aldrich Co.) with 10% fetal bovine serum (HyClone) in the presence of 1% penicillin and streptomycin (HyClone). HCT-116 (p53^+/+) and HCT-116 (p53^−/−) were kindly provided by Dr Chuangui Wang (East China Normal University, Shanghai, China) and were maintained in DMEM with the same supplements. The culture medium of other cells used is listed in Supplementary Table 2. A549, H441, H358, H1299, H1975, PC9, H661, H522, HCT-15, LoVo, SW480, SW620, DLD-1, HT29, CaCo2, HCT-8, CCD-18Co, CCD841CoN, Panc-1, CFPAC-1 and BxPC-3 were obtained from the American Type Culture Collection (Manassas, VA, USA); Calu-1, Calu-3, WI-38, MRC-5, T84, LS174T, SW1116 and AsPC-1 were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). KRAS status identification by sequencing and limited genotyping in cancer cells was shown in Supplementary Table 2. Most of these cells were grown at 37 °C under a humidified 95:5 (%; v/v) mixture of air and CO₂. SW480 and SW620 cells were grown without CO₂. All of the cell lines were authenticated by short tandem repeat analysis.

Calu‐6, SW1573, SW480, T84, SK‐LU‐1, DLD‐1, HCT8, NCI‐H2122, NCI‐H1299, and NCI‐H460 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). LoVo, NCI‐H358, LS174T, and Calu‐1 cells were purchased from Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Science (Shanghai, China). A549, HCC2998, and NCI‐H23 were kindly provided by National Institute of Biological Sciences (Beijing, China). Calu‐6, LS174T, LoVo, SW1573, T84, and SK‐LU‐1 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Gaithersburg, MD, USA). A549, DLD‐1, HCT8, SW480, HCC2998, NCI‐H2122, NCI‐H23, NCI‐H1299, NCI‐H460, and NCI‐H358 cells were maintained in RPMI‐1640 (Gibco). Calu‐1 cells were maintained in McCoy’s 5A medium (Gibco). All growth media were supplemented with 10% FBS (Thermo Scientific, Waltham, MA, USA), 100 units·mL⁻¹ penicillin (Gibco), and 0.1 mg·mL⁻¹ streptomycin (Gibco). Cell lines were reinstated from frozen stocks and passaged no more than thirty times.

Lung cancer cell lines (H838, A549, HCC 827, Calu-1, H322, H1975, H2347, H1299, SK-mes-1, and LLC) and BEAS-2B (human normal lung epithelial cells) were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences. The cells were grown at 37 °C in a humidified atmosphere with 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 μg/ml streptomycin.

The HBE cell line was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The A549, H292, H1299, Calu-1, SK-MES-1 and SPC-A-1 (SPC) cell lines were obtained from the Shanghai Cell Bank (Shanghai, China). The LK2 cell line was a gift from Dr. Hiroshi Kijima (Department of Pathology and Bioscience, Hirosaki University Graduate School of Medicine, Japan). All cells were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) supplemented with10% fetal bovine serum (Invitrogen), 100 IU/ml penicillin (Sigma), and 100 μg/ml streptomycin (Sigma), and passaged every other day using 0.25% trypsin (Invitrogen).

Human NSCLC cell lines (A549 and H460) were obtained from the Cell Resource Center, Peking Union Medical College (Beijing, China), H1299 was purchased from the ATCC, and other human NSCLC cell lines (H292, H2228, HCC827, H1703, and Calu-1) were purchased from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). The murine Lewis lung carcinoma (LLC) cell line was obtained from the ATCC. The LLC-OVA cell line expressing a single polypeptide encoding H-2Kbb2-M, and the OVA SIINFEKL peptide was kindly gifted by Amer A. Beg (Moffitt Cancer Center, Tampa, FL). H460, H1299, H292, H2228, HCC827 and H1703 cell lines were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Calu-1 cells were cultivated in McCoy’s 5A medium supplemented with 10% FBS and 1% penicillin-streptomycin. A549 and LLC-OVA cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin. All the cell lines were cultured in a 37 °C humidified incubator with 5% CO₂.
Anlotinib was kindly provided by Chia Tai Tianqing Pharmaceutical Group Co., Ltd (Nanjing, Jiangsu, China) and was prepared as a 10 mmol/L stock solution in dimethyl sulfoxide (DMSO) for in vitro experiments or in normal saline for in vivo studies.