Epitope prediction and molecular docking
The OCT4 protein sequence was obtained from the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov). Using the epitope prediction program IEDB (http://www.iedb.org/), the OCT4 sequence was analyzed and peptides with high affinity for MHC class I was identified. The peptide sequence was: EAAGSPFSGGPVSFPLAPGPHFGTPGYGSPHF. The efficacy of this peptide fragment at suppressing mouse teratocarcinoma cell growth has been reported by our groups recently (Cancer Biol Med 2020. doi: 10.20892/j.issn.2095-3941). Discovery studio software (55) was used to design the optimal combination of Globo H-OCT4-T7 vaccines by combining energy and scoring functions.
Experimental animals, cell lines and human colorectal cancer specimens
Female BALB/c mice (4–6 weeks old) and female BALB/c nude mice (4–6 weeks old) were purchased from the Medical Laboratory Animal Center (Guangzhou, Guangdong Province, China). All animal treatments and experimental protocols for this study were performed with the approval of the Laboratory Animal Welfare and Ethics Committee, School of Medicine, Shenzhen University (Permit No. AEWC-20190003). All mice were housed under a 12 h light/dark cycle, at 23 ± 1°C, with 39%–43% relative humidity. Water and food were provided ad libitum. CT26 cells (mouse colorectal cancer) and F9 (mouse teratocarcinoma cells) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 100 U/mL penicillin-streptomycin (all from Hyclone Laboratories, Inc., South Logan, UT, USA). Colorectal carcinoma samples (n=16) were obtained from patients at Shenzhen People's Hospital in China. Data collected for each patient included their age, sex and tumor samples (Tables 1). All clinicopathological information was obtained by reviewing pathology reports. This study and the data obtained from clinical samples were approved by the Ethics Committee of Shenzhen People's Hospital.
Vaccine preparation and administration
The OCT4 peptides were synthetized at ChinaPeptides Co., Ltd. (Shanghai, China). The Globo H in the form of lyophilized powder was bought from Oligotech (Crolles, French). EDC (#22980, Thermo Fisher Scientific) and NHS (#24500, Thermo Fisher Scientific) were used to activate carboxylic acid as an active ester. The molar ratio of Globo H, OCT4 peptides, TLR7 agonist, EDC and NHS was 1:1:2.5:5:5 and this mixture was incubated at 4°C overnight. The final conjugate Globo H-OCT4-T7 was stable for at least 6 months at -20℃ at concentration of 1.5 µM.
Generation and stimulation of bone marrow derived dendritic cells (BMDCs) in vitro
BMDCs were isolated from hind limb bones of mice as previously described (56, 57). Briefly, both distal bone ends were excised and the marrow cells were flushed using RPMI 1640 (Gibco, Grand Island, NY). The red blood cells were lysed and the remaining cells were centrifuged at 450 x g for 10 min. Then, 2´106/mL cells were aliquoted in RPMI 1640 supplemented with 10% FBS, 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, 100 U/mL penicillin, 100 mg/mL streptomycin, 20 ng/mL GM-CSF (PeproTech) and 10 ng/mL IL-4 (PeproTech) and incubated at 37°C. The cells were cultured in six well plates in complete medium with cytokines at 4´106 cells/2 mL/well. On the fifth day of culturing, OCT4-T7, Globo H-T7 or Globo H-OCT4-T7 were added to a final concentration of 10 mg/mL, and the cells were cultured for two more days. On day 7, most of the non-adherent cells had acquired typical dendritic morphology, and these cells were used as the source of DCs in subsequent experiments. For flow cytometry, the cells were cultured for 7 days and then rinsed three times with PBS and briefly trypsinized to form a single cell suspension. The cells were stained for 30 min on ice with the following fluorescence-labeled antibodies: Anti-mouse MHCII APC (eBioscience: 17-5321-82), Anti-mouse CD86 PE (clone: GL1), Anti-mouse CD80 FITC (clone: 16-10A1). Finally, the cells were washed twice with cold PBS and analyzed on flow cytometry (BD FACSAria II cell sorter, BD Biosciences).
Tumor protection in BALB/c mice
Female BALB/c mice (6–10 mice per group) were vaccinated with untreated group (PBS), T7 (0.012 μmol), OCT4-T7 (0.012 μmol), Globo H-T7 (0.012 μmol) and Globo H-OCT4-T7 (0.012 μmol) via intraperitoneal (i.p.) injection on days 0, 14 and 21. To assess the memory response, a booster dose was given on day 28. Five days after the final vaccination, the mice were challenged with 1´ 105 CT26 cells (100 μl of cell mixture in 50% Matrigel; #354234, Corning) that were injected subcutaneously (s.c.) into the lower flank of the right back. Then, the tumor size was measured periodically with calipers. Starting from day 0 after cell inoculation, the tumor volume was measured every 2 days. The tumor size was calculated using the formula: 0.5 × length × width2 (cm3) (58). Mice were euthanized when the tumor size was > 1.5 cm3. Mouse blood samples were collected from retro-orbital tissues.
Enzyme-linked immunosorbent assay (ELISA)
Sera samples were collected at 3–5 days after four doses of the vaccine had been administered. An ELISA for multiple cytokines in peripheral blood was performed using a Ready-SET-GO! ELISA kit (eBioscience, Thermo Fisher Scientific), according to the manufacturer’s instructions.
Cytotoxicity assay
Splenocytes and lymph nodes (LN) of vaccinated mice were harvested for evaluating the cellular immune responses 4–5 days after the final vaccination. The cytotoxicity activity of the vaccinated mice was analyzed using a CytoTox96 Non-Radioactive Cytotoxicity Assay Kit (G1780, Promega, Madison, WI, USA). Briefly, splenocytes from the vaccinated mice were mixed at an effector: target (E: T) ratio of 12.5:1, 25:1, 50:1 with 5 ´ 103 CT26 cells. Cytotoxic T lymphocyte (CTL) activity was evaluated by analyzing the released LDH. The cytolysis rate (%) was calculated based on the equation: cytotoxicity (%) = (Experimental release ˗ spontaneous release)/ (maximum release ˗ spontaneous release) ´ 100.
Humoral immune response
In order to assess humoral responses to anti-OCT4 IgG and anti-Globo H IgG levels, ELISA was performed to measure antigen-specific total IgG, according to previously described (59). Briefly, each 96-well ELISA plate (Costar, Corning, New York, USA) was coated with 2 μg synthetic OCT4 peptide and 2 μg Globo H and incubated at 4°C overnight. The plates were then washed with PBST (0.01 M PBS containing 0.05% Tween 20, pH 7.4) three times. Serum samples were detected at a different dilution for 2 h. Alkaline phosphate-conjugated detection antibody for total IgG (Sigma-Aldrich) was added and incubated for 1 h at room temperature. Then, p-NPP substrate (Millipore) and stop solution (50 μL of 3 M NaOH) were added to each well, and the optical density (OD, 405 nm) was measured using a spectrophotometer (BioTek, Winooski, VT, USA).
Tumor-reactive IgG assay
To detect tumor-reactive serum IgG, CT26 and F9 cells were cultured with serum harvested from BALB/c mice immunized with different vaccines for 4 h, washed thoroughly with PBS, and stained with an GFP-labeled anti-mouse IgG antibody, before analysis by flow cytometry using a FACS Calibur flow cytometer (BD FACSAria II cell sorter, BD Biosciences).
Splenocyte and lymph node cell proliferation assay
One week after the last immunization, spleen and lymph node cells were isolated from BALB/c mice and plated into 96-well plates. The lymphocytes were then co-cultured with different vaccines. After 3 days, a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was performed to test the proliferation of the lymphocytes, as previously described (60).
Analysis of tumor infiltrating immune cells
CT26-bearing mice were sacrificed on day 28. Tumors were dissociated using a mouse tumor dissociation kit according to the manufacturer’s protocol (Miltenyi Biotec). The cells were stained for PE F4/80 (macrophage marker), APC CD86 (M1 marker), PE/Cy7 CD206 (M2 marker). Post-staining, the cells were washed and quantified by flow cytometry. The ratio of M1/M2 macrophages was calculated based on the % M1 positive cells to % M2 positive cells. Tregs were identified as CD4+ CD25+ Foxp3+.
RNA sequencing
Gene expression analysis of subcutaneous tumor samples from mice exposed to different vaccine immunizations was evaluated at Novo Gene Corporation (Beijing, China). The up-regulated or down-regulated genes were identified by filtering the RNA-seq data with the following cutoff values: 2-fold change in expression level and a false discovery rate (FDR) analog with a P value of < 0.05.
H&E and immunofluorescence staining
Tumor tissues were harvested and fixed in 10% buffered formalin, dehydrated, cleared and embedded in paraffin according to standard procedures (61). The tissues were then sectioned at 3 μm, mounted onto polylysine-coated slides and then the sections were stained with hematoxylin and eosin (H&E). Histopathological morphology was checked under a light microscope (Olympus BX51). For immunofluorescence assays, the sections were deparaffinized in xylene and rehydrated in graded ethanol. Antigen retrieval was performed in citrate buffer (pH 6.0) by boiling for 30 min in a microwave and cooling down to room temperature. Then, the sections were blocked with 3% bovine serum albumin in PBS for 1 h at room temperature. Following this, the sections were stained with antibodies against mouse CD8 (14-0081-82, Invitrogen), OCT4 (701756, Invitrogen) and SSEA3 (Globo H homologs) (1:200, 14-8833-80, eBioscience) at 4°C overnight. After washing, the sections were incubated with a secondary AlexaFluor 488 goat anti-rabbit antibody (1:200, ab16669, Abcam) for 1 h, and were then washed and mounted in Fluoroshield with DAPI (F6057, Sigma-Aldrich). Images were captured under a Nikon Eclipse E400 microscope using an Olympus DP73 camera and analyzed with cellSence Entry software.
The transfer experiment
The primary tumor was resected from a patient diagnosed with CSC. For the patient-derived xenograft (PDX) model, the fresh patient tumor specimen was cut into small pieces (less than 2-3 mm) and initially implanted subcutaneously in nude BALB/c mice. For the transfer experiment, PBMCs harvested from healthy people’s peripheral blood were stimulated with or without Globo H-OCT4-T7 vaccines and injected (i.p.) into the PDX once a week for 3 weeks. The tumor volumes were measured every one week.
TUNEL staining
Tissue sections were dewaxed with xylene and then hydrated with a gradient of ethanol using the TUNEL FITC Apoptosis detection kit (yazyme, A111). After washing three times with PBS, it was permeabilized with proteinase K. The TUNEL reaction mixture was prepared, and after completion of the reaction, the film was sealed with a DAPI-containing sealing liquid, and then observed under a fluorescence microscope.
Statistical analysis
We used Graphpad Prism 8.0 to conduct statistical analyses. For lifespan, Kaplan-Meier survival was utilized and P values were calculated using the log-rank test. To compare two groups, Student’s t-test was performed. One-way analysis of variance (ANOVA) with Duncan’s test was conducted for comparing multiple groups. P < 0.05 was considered statistically significant.