Cells and culture
All cell lines were purchased from The American Type Culture Collection (ATCC), Manassas, VA, USA. CT26, a murine colon cancer cell line, was cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Rockville, MD, USA) medium. The medium was supplemented with 10% FBS (Gibco), 100 μg/ml penicillin, and 100 μg/ml streptomycin. The cell lines SW620, HCT116, Lovo, HT29, CaCo-2, HCA-7 were cultured in RPMI-1640 (Gibco) medium with 10% FBS (Gibco), 100 μg/ml penicillin, and 100 μg/ml streptomycin.
This study was approved by the ethics the Committee of Shaanxi Provincial People’s Hospital, and informed consent was written and based on the ethical guidelines of the 1975 Declaration of Helsinki. Written informed consent was obtained from all patients. Furthermore, human subjects’ private rights were routinely respected. CRC specimens and matched adjacent tissues were used to construct a tissue microarray (Shanghai Biochip Co, Ltd. Shanghai, China). The tissue microarray was stained for HOXA7 (Abcam, ab211521), CD11b (Abcam, ab6640), CD8 (Abcam, ab4055) expression.
Paraffin-embedded sections were continuously sliced when cutting the tissue by microtome. Immunohistochemistry was carried out on 4-μm thick paraffin-embedded sections, and was performed according to standard procedures as outlined in our previous study . Briefly, after baking on a panel at 60 °C for an hour, the tissue sections were deparaffinized with xylene and rehydrated through gradient ethanol immersion. Endogenous peroxidase activity was quenched by 3% (vol/vol) hydrogen peroxide in methanol for 12 min, followed by three 3-min washes with phosphate-buffered saline (PBS). Then the slides were immersed in 0.01 mol/l citrate buffer solution (pH = 6.0) and placed in a microwave oven for 30 min. After washing in PBS, the sections were incubated in a moist chamber at 4 °C overnight with the primary antibody diluted in PBS containing 1% (wt/vol) bovine serum albumin. Negative controls were performed by replacing the primary antibody with preimmunized mouse serum. After three 5 min washes with PBS, the sections were treated with a peroxidase-conjugated second antibody (Santa Cruz) for 30 min at room temperature, followed by an additional three 5 min washes with PBS. The reaction product was visualized with diaminobenzidine for 2 min. Images were obtained under a light microscope (Olympus, Japan) equipped with a DP70 digital camera.
Analyses were performed by two independent observers who were blinded to the clinical outcome. The immunostaining intensity was scored on a scale of 0 to 3: 0 (negative), 1 (weak), 2 (medium), or 3 (strong). The percentage of positive cells was evaluated on a scale of 0 to 4: 0 (negative), 1 (1–25%), 2 (26–50%), 3 (51–75%), or 4 (76–100%). The final immuno-activity scores were calculated by multiplying the above two scores, resulting in an overall score of 0–12. Each case was ultimately considered “negative” if the final score ranges from 0 to 3 and “positive” if the final score ranges from 4 to 12.
In vivo metastatic model and bioluminescent imaging
Our implantation tests were under the approval of the ethics of the Committee of Shaanxi Provincial People’s Hospital. All the mice were male. All efforts were made to minimize the animals’ suffering during the experiments. BALB/C mice (5 weeks old) were housed under standard conditions and cared for according to the institutional guidelines for animal care. A metastatic colorectal cancer model in mice was established according to the existing protocol. Luciferase labeled mouse CRC cells (4.0 × 106) were injected into the cecal wall in mice under anesthesia (n = 10 for each group). Briefly, the caecum was gently exteriorized and was placed on a scalpel holder, flattened, and stabilized with forceps. This maneuver is crucial to prevent leakage of tumor cells into the caecal lumen or peritoneal cavity. A volume of 100 μl (4.0 × 106) cells was injected into the caecal wall. Then, the caecum was returned to the peritoneal cavity, peritoneum and skin were closed by running sutures and wound clips.
To investigate whether SB265610 can improve CRC responsiveness to anti-PD-L1 blocking in KRAS mutant CRC, SB265610 (2 mg/kg body weight, i.p. every day), or anti-PD-L1 antibody (200 μg, i.p., every three days), or Vehicle (i.p., every day) was injected. For combined treatment, mice were treated with SB265610 (2 mg/kg body weight, i.p. every day) and anti-PD-L1 antibody (200 μg, i.p., every three days).
Luciferase lentivirus was purchased from Shanghai Genechem Co., Ltd. Concentrated luciferase lentivirus was transfected into the CRC cells with a multiplicity of infection (MOI = 50) in the presence of polybrene (6 μg/ml). Seventy-two hours after infection, CRC cells were selected for 2 weeks using 2.5 μg/ml puromycin (OriGene).
The in vivo tumor formation and metastases were imaged by bioluminescence. d-luciferin (Xenogen, Hopkinton, MA) at 100 mg/kg was injected intraperitoneally into the mice. To anesthetize the mice, the mice were inhaled 2.5% isoflurane in the special volatilization chamber for 5–6 min. Then, bioluminescence was detected using an IVIS 100 Imaging System (Xenogen). After acquiring photographic images of each mouse, luminescent photos were captured using various (1–60 s) exposure times. The resulting grayscale photographic and pseudocolored luminescent images were automatically superimposed using the IVIS Living Image (Xenogen) software. This superimposition was performed to facilitate the matching of the observed luciferase signal with its location on the mouse. The survival of the mice was recorded daily.
At the 9 weeks, the mice were sacrificed by injecting excessive pentobarbital sodium for anesthesia (100 mg/kg, Merck, Germany), and the livers and lungs were collected and underwent histological examination.
Preparation of single cell suspensions
Mice were perfused with PBS and anesthetized, and tumors were dissected using a clean razor. Then, the tumor tissues were digested with DNase I (20 mg/ml, Sigma-Aldrich) and collagenase IV (1.5 mg/ml, Sigma-Aldrich) and placed on a thermomixer (Thermo Scientific), 37 °C, 40 rpm, for 1 h to digest the tissue adequately. At the end of 1 h, we filtered the dissociated cells through 70 μm pore filters rinsed with fresh media. Cell staining buffer was added to the separation tube to 15 ml, then centrifuged at 350g for 5 min, and the supernatant was discarded. The red cell lysis (Biolegend, #420301) was added to pelleted disintegrated tissue and incubated for 5 min to lysis the red blood cell. Cell staining buffer (10 ml) was added to the tube to stop lysis. The supernatant was then discarded after centrifugation at 350g for 5 min, and repeat this step again.
Flow cytometric analysis
Cells were incubated with anti-mouse CD16/CD32 purified antibody (#101302, clone 93, Biolegend) for 10 min to block nonspecific antibodies. Then, the cells were stained with fluorophore-conjugated antibodies. Matched isotype antibodies were used as control. All antibodies were purchased from Biolegend. Antibodies against CD45 (PE, #103105, 0.25 µg/106 cells), CD11b (FITC, #101205, 0.25 µg/106 cells), CD45 (PE/Cy7, #103113, 0.30 µg/106 cells), Ly-6G/Ly-6C (Gr-1) (PE, #108407, 0.25 µg/106 cells), CD3 (FITC, #100203, 1.0 µg/106 cells), CD8 (PE, #100707, 0.20 µg/106 cells) were used. Data were analyzed by Flowjo_V10 software (TreeStar, Ashland, OR).
Statistics were calculated with SPSS software (version 20.0). P values were statistically analyzed by the χ2 test for categorical variables and by Student’s test for quantitative data. The recurrence and survival data were analyzed by the Kaplan–Meier method. Cox proportional hazards model was used for univariate and multivariate analyses. Differences were considered statistically significant when P < 0.05.