Reagents and mAbs
Anti-mouse CD73 mAb (clone TY/23) and Rat IgG2a isotype control (clone 2A3) were purchased from BioX-Cell (West Lebanon, NH, USA). Anti-mouse mAbs for flowcytometric analysis were used as described here. FITC-conjugated anti-CD4 (GK1.5), anti-CD8α (53 − 6.7), anti-CD11b (M1/70), and PE-conjugated anti-CD39 (Duha59), anti-IFN-γ (XMG1.2), APC-conjugated anti-CD73 (TY/11.8), anti-CD3 (17A2), and FITC-conjugated anti-CD45 (30-F11), and mouse recombinant Interleukin-2 (r-IL-2) were purchased from BioLegend (San Diego, CA USA). FcR blocking reagent was obtained from Miltenyi Biotec GmBH (Bergisch Gladbach, Germany). 7AAD (7-Aminoactinomycin D) and FVS-780 were purchased from Thermo-Fisher Scientific (Waltham, MA USA) and BD Biosciences (Franklin Lakes, NJ USA), respectively.
Cell culture and animal experiments
LuM-1, a highly metastatic sub-clone of murine colon cancer, colon26 (31) was kindly obtained from Dr. Oguri, Aichi Cancer Center, Japan., and maintained in DMEM supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin (Sigma, St. Louis, MO, USA). After achieving > 80% confluence, cells were removed by treatment with 0.25% (w/v) trypsin solution containing 0.04% (w/v) EDTA, and then used. The cultured cells were tested by the Mycoplasma Detection Kit (R&D Systems) in every 3 months and cells with passages 3 to 5 were used for experiments. Female Balb/c mice age 7–8 weeks were purchased from CLEA Japan (Fujinomiya, Japan) and housed in specific pathogen-free (SPF) conditions.
LuM-1 cells (1 × 106) were subcutaneously (sc) injected in the right flank of 8–9 weeks-old female Balb/c mice. When the primary tumors reached a volume of 100 to 150 mm3 at day12, the mice were divided into groups with each group containing 5 ~ 8 mice to enable the statistical evaluation. Local RT was delivered using MX-160 Labo (mediXtec), as described previously (32). In short, anesthetized mice were held in the decubitus position, and X-ray irradiation was delivered only to the subcutaneous tumor with the remainder of the body of the mouse including the lung covered with a 5 mm lead plate. We confirmed the effectiveness of shielding by this method. Mice received 3 fractions of 4 Gy every other day (days 12, 14, 16). For immunotherapy, mice received intraperitoneal (ip) injection of 200 µg anti-CD73 mAb or Rat IgG2a isotype control on days 12, 14, 16, 19, 22 and 25. All of the mice were sacrificed with cervical dislocation on day 28, and the weight of the sc tumor and number of macroscopic metastatic nodules in lung were evaluated. All the procedures were approved by Animal Care Committee of Jichi Medical University (No 17005-02) and performed according to the Japanese Guidelines for Animal Research.
Flow cytometry
LuM-1 cells were cultured at a density of 1 × 106 cells/10 cm dish and 10 Gy RT given with the MX-160 Labo and incubated for an additional 24 hours. The cells were harvested, incubated with 10 µl FcR blocking reagent for 10 min at 4℃ and incubated with PE-conjugated anti-CD39 (Duha59) and APC anti-CD73 mAb for 30 min at a final concentration of 2.5 µg/mL. After washing twice with staining buffer, the cells were incubated with 7-AAD for 15 min on ice and staining intensity analyzed in 7AAD (-) live cell population using FACS Calibur (BD Bioscience, NJ). For in vivo experiments, LuM-1 (1 × 106) cells were sc injected in Balb/c mice and treated with 2 fractions of 4 Gy RT as described above. Two days later, tumors were excised and digested using the Tumor Dissociation Kit, mouse (Miltenyi Biotec) with gentleMACs Dissociators (Miltenyi Biotec). After lysis of red blood cells (RBC) with RBC lysis buffer, cells were passed through a 40-∝m filter and single cell suspensions stained with APC conjugated anti-CD73 mAb and FITC conjugated anti-CD45 mAb, and the expression level of CD73 was analyzed in live tumor cell population defined in 7AAD(-) CD45(-) gated area.
T cells producing IFN-γ were identified by intracellular staining. Mice bearing sc LuM-1 tumors received 3 fractions of 4 Gy local RT and an intraperitoneal injection of 200 µg anti-CD73 mAb or Rat IgG2a isotype control every other day (days 12, 14, 16). The mice were sacrificed on day 18, and splenocytes (1 × 106) were cultured in RPMI-1640 + 10% FCS for 6 hours in the presence of 1 µl/mL brefeldin A for the last 2 hours. The cells were harvested, fixed, permeabilized using the fixation / permeabilization buffer (BD Bioscience) according to the manufacturer’s instructions and stained with PE-conjugated IFN-γ or isotype control and FITC-conjugated mAbs to CD4 or CD8 as well as FVS780 to exclude dead cells. The ratio of IFN-γ positive cells were calculated in CD4(+) or CD8(+) gated area using LSRFortessa (BD Bioscience).
Cytotoxicity
Splenocytes (5 × 106) from treated mice (as described above) were cultured with 1 × 106 irradiated (50 Gy) LuM-1 cells in 24-well tissue culture plates in 2 ml 10% FCS + PRMI-1640 medium supplemented with 20 ng/ml mouse rIL-2 for 12 days. Activated splenocytes were incubated with LuM-1 at an E/T ratio of 20:1 for 4 hours and all cells stained with FITC-conjugated Annexin-V, 7-AAD and anti-CD45-APC mAb. The ratio of 7-AAD positive dead cells was calculated in the tumor cell population defined in the FSC/SCC and CD45(-) gated areas.
Quantification of adenosine levels in tumor tissue
Quantitative analysis of adenosine, adenosine monophosphate (AMP) and inosine was performed using an LC-MS system consisting of Nexera X2, LCMS-8060 and a LC/MS/MS Method Package for Primary Metabolite Version 2 (Shimadzu Corp, Kyoto, Japan) as described previously (33). In brief, sc tumors irradiated as described above (4 Gyx2) were resected at 12, 24 and 48 hour after treatment and dissociated using a tumor dissociation kit (Miltenyi Biotec). Chromatographic separation was performed at 40 °C on a Discovery® HS F5-3 column, 150 × 2.1 mm, 3 µm, (Sigma-Aldrich) with a flow rate of 0.25 mL/min. A gradient elution of mobile phase A consisting of 0.1% of formic acid in water and mobile phase B consisting of 0.1% of formic acid in acetonitrile. The mobile phase B concentration was programmed as follows: 0% (0 min) – 0% (2.0 min) – 25% (5.0 min) – 35% (11 min) – 95% (15 min) – 95% (20 min) – 0% (20.1 min). Nitrogen gas was used as the nebulizer gas with drying gases at flow rates of 3.0 and 10 L/min, respectively. Dry air for the heating gas was at 10 L/min. Collision-induced dissociation (CID) was conducted by argon gas (purity, > 99.9995%). Interface, heat block, and desolvationline temperatures were set at 300, 400, and 250 °C, respectively. Multiple reaction monitoring (MRM) transitions for adenosine, AMP, and inosine were m/z 268.1 > 136.05, m/z 384.0 > 136.05 and m/z 269.1 > 137.05, respectively, in positive ion mode. MRM transition for 2-MES was m/z 194.0 > 80.15 in negative ion mode. The polarity switching time of the instrument was 5 ms (10 ms/cycle).
Immunohistochemistry of patient samples
Between 2008 and 2015, 64 patients with locally advanced RC received neoadjuvant chemoradiotherapy (CRT) in the Department of Surgery, Division of Gastroenterological General and Transplant Surgery, Jichi Medical University Hospital. Patients were treated with long-course RT (a dose of 50.4 Gy in 25 fractions) using 4-field box techniques. Some patients received concurrent chemotherapy with oral UFT or S1. Radical resections were performed at 8–10 weeks after the end of CRT. The excised tumors were immediately fixed in 10% buffered formalin, and consecutive formalin-fixed paraffin-embedded 4-µm sections prepared for immunohistochemical evaluation.
After treatment with xylene and ethanol and washing with phosphate-buffered saline (PBS), tumor specimens were subjected to heat-induced antigen retrieval in citrate buffer (Muto Pure Chemicals Co., Ltd, Tokyo, Japan) followed by endogenous peroxidase blocking by Peroxidase-Blocking solution (DAKO, Santa Clara, CA). The tissues were washed with PBS and incubated with 5% bovine serum albumin for 30 minutes to block nonspecific antibody binding. The slides were then incubated overnight at 4 °C with monoclonal antibodies against CD73 (D7F9A, Rabbit IgG, Cell Signaling Technology) at a dilution of 1:200 in humid chambers overnight at 4℃. After three 5-min washes with PBS, sections were incubated with anti-rabbit secondary antibody conjugated with peroxidase for 30 min at room temperature. After washing, the enzyme substrate 3,30-diaminobenzidine (Dako REAL EnVision Detection System, DAKO) was used for visualization and counterstained with Meyer’s hematoxylin.
Staining intensities in tumor cells or stroma were independently scored from 0 to 3 (Supplementary Fig.S1) by two different evaluators who were unaware of the clinical findings, and the cases were divided into high (score = 2 or > 2) and low (score < 2) expression groups by the mean score of the two evaluators. This study protocol was approved by the institutional IRB of Jichi Medical University (Rin A17-164) and conducted in accordance with the guiding principles of the Declaration of Helsinki. Written informed consent was obtained from all participants.
Statistical Analysis
Data are presented as the means ± SEM or median (min-max). Statistical differences were analyzed by student-t-tests, the Mann-Whitney test, or one-way ANOVA with post hoc test with Tukey’s or Dunnett’s procedure and p values less than 0.05 were considered significant. Recurrence-free survival rates were calculated using the Kaplan-Meier method and differences were evaluated using the log-rank test.