Cell lines and drug treatment. Human CRC cell lines (HT29, HCT116, HCT15, DLD1, LOVO) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All the above cell lines were authenticated by Biowing Biotech (Shanghai, China). HT29, HCT116, and LOVO were cultured in DMEM (Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS, VACCA, Shanghai, China) and 100 µg/mL penicillin/streptomycin (Invitrogen). The rest of the cell lines were grown in RPMI-1640 medium (Gibco) with the same supplements. All cells were cultured in a humidified incubator with 5% of carbon dioxide (CO2) and 95% air at 37 °C and were routinely checked for mycoplasma by PCR. Palbociclib and erlotinib were purchased from MedChemExpress Chemicals. Drugs were dissolved in DMSO at 200 µM and 10 mM stock solution concentration and stored in aliquots at − 80 °C.
Animals. Mice were housed and handled according to institutional guidelines complying with local legislation. All experiments with animals were approved by the animal experiment committee of the Nanjing Medical University. NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt (NCG, female, 3–4 weeks, 18–20 g) mice were purchased from Jiangsu GemPharmtech co., ltd (Nanjing, China) and were adapted to the environment for a week before the experiment.
Anti-proliferation and clonogenic assays. The cells were seeded in 96-well plates at a density of 2000 cells per well one night before 72 h drug treatment. Proliferation rates of CRC cells were determined using an Alamar Blue assay (Yeasen, Shanghai, China). Drug interaction analysis was performed using the Chou-Talalay method . The combination index (CI) was calculated by the CompuSyn program (ComboSyn Inc). For the clonogenic assay, cells were seeded in 12-well plates at a density of 400–1000 cells per well. Drug containing medium was refreshed every 3–4 days. Cells were fixed after 10–14 days of treatment and stained with a Giemsa staining solution (KeyGEN Biotech, Nanjing, China). The number and density of colonies were quantified using Image J.
Flow Cytometry Analysis of Cell Cycle, Apoptosis, and ROS level of CRC cells. Cells were seeded at 1 × 105 cells per well in 6-well plates and were incubated overnight. For cell cycle analysis, the cells were synchronized by starving in serum-free DMEM for 24 h before treatment. Then the cells were treated with palbociclib, erlotinib, or their combination for 72 h and were collected by trypsinization into ice-cold PBS followed with brief centrifugation. For cell cycle analysis, the above-collected cell pellets were fixed in 75% ethanol for 2 h and resuspended in 1% (w/v) bovine serum albumin in PBS. Next, the cells were stained with propidium iodide (PI) at a final concentration of 0.1 mg/ml together with 0.1 mg/mL RNaseA (20 g/mL) at 37 °C for 15 min in the dark. For apoptosis analysis, 1 × 105 drug-treated cells were stained with Annexin V-APC and propidium iodide (PI) using an Apoptosis Detection Kit (Yeasen Biotech, Shanghai) at room temperature for 15 min. For ROS measurement, the cells were incubated with 10 µM DCFH-DA (Solarbio Life Science, Beijing, China) at 37 °C for 30 min in the dark and then washed with PBS. The stained cells from the above treatments were subjected to flow cytometric analysis using a FACS Calibur (BD Biosciences) flow cytometer, and data were analyzed by FlowJo 7.6.
Western blotting. Cells were lysed in RIPA buffer supplemented with protease inhibitors (50 mmol/L Tris-HCl, pH 8.0, 150 mmol/L sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS). The equal amount of proteins (15–20 µg) were loaded on SDS-PAGE gels and separated by electrophoresis. After transferring the protein to a PVDF membrane (Merck Millipore, MA, USA), it was blocked with 3% BSA and incubated with appropriate primary and secondary antibodies. The following primary antibodies were used in the study: pEGFR (Tyr1068) (#3777T), pAKT (Ser473) (#4060), AKT (#9272), pERK1/2 (Thr202/204) (#4370), pMEK1/2 (#2338), pRB (Ser807/811) (#8516), RB (#9309), GSK3β (#12456), pGSK3β (Ser9) (#9323) and FoxM1 (#5436) (Cell Signaling Technology); p4EBP1 (Ser65) (#SC293124, Santa Cruz Biotechnology, CA, USA), GAPDH (#AC002) (ABclonal, Hubei, China). At last, the blots were detected after exposure to the enhanced chemiluminescence kit using a ChemiDoc MP imaging system.
Patient-derived CRC organoids and xenografts. CRC specimens were acquired from patients in routine operation after obtaining fully informed consent according to Jiangsu Cancer Hospital. The CRC organoids were established according to the protocol developed by Hans Clevers lab . In brief, fresh surgically resected CRC tissues were minced into 1 mm3 fragment and digested with 1 mg/mL collagenase A (Sigma, #C0130, USA) at 37 °C for 30 min. After brief centrifugation, the pellet was resuspended in PBS and was mechanically dissociated by repetitive pipetting. The isolated cells/fragments were passed through a 70 µm cell strainer, centrifuged, and resuspended in matrigel (Corning, #354230, USA) at 1 × 106 cells per mL. The mixture was dispensed as 25 µl per drop and seeded into each well in 24-well plates and was solidified in a 37 °C incubator for 10 min. 500 µl of culture medium composed of DMEM/F-12 (Hyclone) supplemented with 1 × penicillin/streptomycin, 10 mM HEPES (Invitrogen), 2 mM GlutaMAX (Invitrogen), 1 × B27 (Invitrogen), 1 × N2 (Gibco), 1 mM N-Acetylcysteine (Sigma) together with niche factors: 50 ng/mL for EGF (Thermo Fisher Scientific/Gibco #PHG0313), 500 nM for TGF-β receptor type I inhibitor A83-01 (MCE, #HY-10432) and 10 µM for p38 MAP kinase inhibitor SB202190 (MCE, # HY-10295) was added into each well. 10 mM Y-27632 dihydrochloride kinase inhibitor (MCE, # HY-10071) was also added for the first 2 days. After the successful formation of CRC organoids, palbociclib, erlotinib, or their combination was added into the culture medium to evaluate their effects.
For the PDX model, 4–5 weeks female immunodeficient NCG mice  were used as the recipient of patient-derived CRC tissue. Fresh surgically resected CRC tissues from two CRC patients (P328 and P44) were minced into approximately 3 mm3 fragments and subcutaneously engrafted into the right region of each mouse. When tumors reached a volume of 80–120 mm3, the mice were separated randomly into 4 groups. The P328 PDX cohort including 20 tumor-bearing mice (five in each group) received erlotinib (resuspended in 6% Captisol; 50 mg/kg), palbociclib [resuspended in sodium(S)-lactate buffer (50 mmol/L, pH 4.0); 25 mg/kg], or their combination and the P44 PDX cohort including 37 tumor-bearing mice (8–10 in each group) received erlotinib (25 mg/kg), palbociclib (25 mg/kg), or their combination, respectively, by oral gavage 5 days a week for more than 4 weeks. The mice were weighed every 2 days and monitored for tumor volume (Length × Width2 × 0.5) until the tumor reached the maximum authorized ethical volume of 2500 mm3 or if their health severely deteriorated (censored event).
β-Gal staining. The intracellular β-galactosidase level was determined using a Cell Senescence Testing Kit (GenMed Scientifics Inc. MN, USA). The staining procedure was followed by the manufacture’s instructions. Briefly, CRC cells pre-seeded in 12-well plates were treated with palbociclib, erlotinib, or their combination for 72 h and incubated with the Fixative Solution, washed, and incubated with Staining Solution for 16 h at 37 °C. Then the cells were washed with PBS three times and observed with a Zeiss microscope. β-galactosidase positive cells were counted and the numbers from eight representative fields of three independent experiments were calculated.
Statistical analysis. The in vitro data in figures are represented as the mean ± SD and the data of PDX tumor growth are presented as mean ± SEM (standard error of the mean). Statistical significance was calculated by Student's t-test or ANOVA using GraphPad Prism. In vitro experiments were performed in triplicate and replicated in more than two independent experiments. The statistical significance of differences is indicated in figures by asterisks as follows: *, p < 0.05; **, p < 0.01; and ***, p < 0.001.