Surgical explants.

Human gastric specimens were obtained from six patients with primary gastric adenocarcinomas who underwent radical gastrectomy at the Hospital Universitario de la Samaritana (Bogotá, Colombia) between 2016 and 2019. All subjects in the study were Colombians with primary gastric cancer, preoperatively diagnosed by endoscopy, who did not receive chemotherapy before surgery and showed no signs of distant metastases. Gastric cancer tissues and corresponding non-tumor normal tissues (<5 cm) were collected. Each biopsy sample was divided into two sections: one was submitted for routine histological diagnosis, and the remaining section was used to evaluate infection with RV Wt1-5. Pathological evaluation was performed on the resected specimens after they were fixed in 10% neutral buffered formalin and sectioned into 4-mm-thick segments. The evaluation determined various characteristics of the tumor, including its diameter, location, differential degree, depth of invasion, and lymphovascular invasion. This study was approved by the Ethics Committee of Hospital Universitario de la Samaritana (approval no.2; 18 February 2016), and all experiments were conducted in compliance with the Declaration of Helsinki and guidelines for ethical principles for medical research involving human subjects. The ethics committee waived the need for consent from the participants.

Tissue preparation and cell viability.

Human gastric cancer specimens obtained from radical gastrectomy were immediately placed in transport media RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 50 IU/mL penicillin, and 50 μg/mL streptomycin after resection. The surgical specimens were washed twice with sterile phosphate-buffered saline (PBS) with pH 7.4. Tumor tissue was transferred to a 100 mm-Petri dish containing 20 mL of antibiotic-containing medium under aseptic conditions in a laminar flow hood, using sterile forceps. The tissue was then cut into ∼2 mm³ tissue explants using scissors and a number 22 scalpel blade, and any necrotic tissue was removed. Samples were enzymatically disaggregated after incubation with trypsin (5 mg/mL; Invitrogen, Madison, WI, USA) for 10 min at 37°C in a humidified atmosphere containing 5% CO₂. The resulting tissue slices were further processed by washing them twice with RPMI 1640 without FBS. The tissue was then divided into portions of ∼200 mg each and placed in 50-ml centrifugation tubes (Biologix Group Ltd.). To obtain single-cell suspensions from the tumors, mechanical disaggregation was carried out using the PRO 200 Micro Homogenizer (ProScientific Inc.). The homogenizer was set at 1,000 rpm for 10 min, with RPMI without serum used as the medium. The tumor fragments and fluid underwent filtration through a Corning^® 100 μm Cell Strainer (Corning, NY, USA). The resulting single-cell suspension was then centrifuged at 700 x g for 7 min, and an aliquot was taken from each tube to determine the number of viable nucleated cells using a cell counter (Countess™ II Automated Cell Counter). For this, 10 μL of the sample was mixed with 10 μL of trypan blue (Sigma Chemical Co., St Louis, MO, USA). The cell suspension was then maintained in RPMI medium without FBS and penicillin-streptomycin.

Infection of disaggregated tumor cells.

The tumor cell-adapted rotavirus Wt1-5 was obtained as previously described [15]. The Multiplicity of infection (MOI) was determined by analyzing the focus-forming units (FFU) through a serial dilution assay of the stock in NCI-N87 cells (ATCC^® CRL-5822™). The Infection was confirmed by immunocytochemistry using hyperimmune rabbit serum against RV structural proteins, and positive cells (an average between 100 and 150) were counted under an inverted microscope using a 40x objective. Finally, a viral titer of 1.2 x 10⁶ FFU/mL was determined [29].

To examine whether gastric tumor cells are susceptible to infection by RV Wt1-5, approximately 5 X 10⁴ cells were obtained from disaggregated tumors or non-tumor tissues and washed twice with RPMI 1640 medium. The cells were then inoculated with either PBS or RV Wt1-5 (MOI 0.8) activated with trypsin (10 μg/mL) (Sigma-Aldrich) in RPMI medium without FBS. The cells were incubated for 12 h at 37°C with 5% CO₂. After incubation, the cells were fixed with 4% (vol./vol.) paraformaldehyde in PBS for 30 min at room temperature. The cells were washed twice with PBS, resuspended in PBS with 0.02% (weight/vol) sodium azide, and stored at 4°C until further assessment of tumor cell susceptibility to infection using various assays, including immunocytochemistry, epifluorescence, capture ELISA, flow cytometry and Western blot.

To determine whether viral particles with infectious capacity are generated when infecting tumor cells isolated from patients, 5x10⁴ cells/well were seeded and inoculated with RV Wt1-5 (MOI 0.8) for 1 h at 37°C with 5% CO₂. The cells were washed twice with RPMI 1640 medium to remove the unbound viruses. After adding RPMI without FBS, the cells and supernatant were separately collected at 12 h.p.i. Three freeze-thaw cycles were performed, with cells being centrifuged at 700 x g during each cycle, and trypsin (10 μg/mL) was added to the supernatant for 30 min. The resulting supernatant was then inoculated into the NCI-N87 gastric cancer cell line (ATCC^® CRL-5822™) and incubated at 37°C with 5% CO₂. After 12 h.p.i, cells were fixed with 4% paraformaldehyde (PFA) and analyzed by immunocytochemistry using rabbit hyperimmune serum to detect the presence of rotavirus antigens. The assay was performed three times in duplicate, with uninfected cells serving as the control.

Ex vivo infection of gastric adenocarcinoma.

Previously reported protocols [30, 31] were used with slight modifications to examine the effects of oncolytic virus in gastric cancer explants. Briefly, human gastric adenocarcinoma samples and non-tumor gastric tissue collected by radical gastrectomy were immediately transported for processing to the Virus Molecular Biology Laboratory of Universidad Nacional de Colombia. The tissues were stored in two sterile 150 mL DURAN^® borosilicate glass flasks (DWK Life Sciences Tennessee, USA) with culture medium (RPMI 1640 with 10% FBS, 50 IU/mL penicillin, and 50 μg. /mL streptomycin). Under sterile conditions in the laboratory, tumor tissue was first placed in a 100 mm-Petri dish containing supplemented medium (RPMI 1640 with 10% FBS, 50 IU/ml penicillin, and 50 μg/ml streptomycin) using sterile forceps. Necrotic tissue was removed with sterile scissors and a 22-number scalpel blade, and then the tissue explants were cut into ∼ 2 mm³ thick sections (∼0.1g). Each explant was transferred to a new 100 mm-Petri dish with 20 mL of medium to prevent tissue desiccation. Using sterile forceps, each explant was distributed to a different well of a Corning™ 12-well flat-bottom cell culture plate containing 3 mL of RMPI 1640 medium and penicillin-streptomycin without FBS. The selected wells were inoculated with either PBS or rotavirus Wt1-5 (MOI 0.2 or 0.8) diluted in 25 μL of medium, directly onto each explant. The explants were then incubated at 37°C with 5% CO₂ for 1 h. The unattached virus was removed by washing three times with RPMI 1640 medium. Tissue sections were harvested at 0, 12, 24, 48, and 60 h.p.i and then fixed for 1 h at room temperature with fresh 10% neutral-buffered formalin with sodium acetate or 4% (v/v) paraformaldehyde at RT, or frozen at -80°C, depending on the assay to be carried out later. The volume ratio between the sample to be fixed and fixative solution was 1:20. After fixation, three washes of at least 5 min each with PBS were performed. Cell viability was assessed at each time point, as previously described.

Antibodies and reagents.

Primary rabbit polyclonal antibodies against rotavirus structural proteins (SPs) or non-structural proteins (NSP2-NSP6) were produced in our animal facilities. Monoclonal mouse anti-human BCL2 (catalog # IS614), mouse anti-human cytokeratin 7 (catalog # M7018), mouse anti-human carcinoembryonic antigen (catalog # GA622) mouse anti-human E-cadherin (catalog # GA059), mouse anti-human CA 19-9 (catalog # M3517), mouse anti-human CD4 (cat # M7310), mouse anti-human mutant and wild-type p53 (catalog # IS616), and polyclonal rabbit anti-human CD3 (catalog # IS503) antibodies were obtained from Dako (Agilent Technologies, Inc. Santa Clara, CA, USA). Polyclonal goat anti-human antibodies against Hsp90α/β (sc-1055), Hsp70 (sc-1060), Hsp60 (sc-1052), Hsp40 (sc-1801), Hsc70 (sc-1059), integrin β3 (sc-6626), PDI (sc-17222), and monoclonal mouse anti-human against cleaved PARP-1 (sc-56196), caspase-3 (sc-65496), caspase-9 (sc-56077), Bax (sc-20067), cytochrome c (sc-13561), Hsp90 (sc-13119), Hsp70 (sc-32239), Hsc70 (sc-7298), Hsp60 (sc-59567), Hsp40 (sc-7298), PDI (sc-376369), ERp-57 (sc-166680), integrin β3 (sc-46655), integrin β2 (sc-8420), integrin β1 (sc-374429), and monoclonal rabbit anti-human Bcl-2 (SC-783) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Monoclonal mouse antibodies against cytokeratin 8/18 (MA5-43995) were obtained from Thermo Fisher Scientific (Waltham, MA). As secondary antibodies, the following were obtained from Santa Cruz, CA, USA: mouse anti-goat IgG-PE (sc-3752), donkey anti-rabbit IgG PerCP-Cy5.5 (sc-45106), rabbit anti-goat IgG-PE (sc-3755), rabbit anti-mouse IgG-PE (sc-358926), bovine anti-goat IgG-FITC (sc-2348), mouse anti-rabbit IgG-488 (sc-516248), mouse anti-rabbit IgG-FITC (sc-2359), and donkey anti-goat or anti-rabbit antibodies conjugated with FITC (sc-362255 and sc-362261, respectively), or HRP (sc-2020 and sc-2313, respectively). 4,’6-diamidino-2-phenylindole (DAPI) was purchased from Invitrogen (Carlsbad, CA, USA).

Immunohistochemistry.

After fixation, the tissue samples were dehydrated using an ethanol/xylene series and embedded in fresh paraffin wax at a temperature of 55–60°C. Histopathological and immunohistochemical characterization of both the tumor and non-tumor tissues were then performed for each block. The formalin-fixed paraffin-embedded (FFPE) specimen blocks were sliced into 5 μm-thick sections and subjected to immunohistochemical analyses. For the immunohistochemical staining system, a fully automatic staining device, the Ventana BenchMark XT from Ventana Medical Systems Inc. Tucson, USA (Roche Diagnostics Division) was used. The device uses a biotin-free, HRP multimer-based hydrogen peroxide substrate and 3, 3’-diaminobenzidine tetrahydrochloride (DAB) chromogen (ultraView ^TM Universal DAB Detection Kit, Catalog number 760-500, Ventana Medical Systems, Tucson, USA). Primary antibodies against CEA (1:100 dilution), Ck7 (1:100 dilution), E-cadherin (1:100 dilution), CA 19-9 (1:100 dilution), Bcl-2 (1:100 dilution), mutant and wild-type p53 (1:100 dilution), CD3 (1:100 dilution) or CD4 (1:100 dilution), and hyperimmune rabbit serum against rotavirus structural proteins (1:1000 v/v) were used. The slides were counterstained with hematoxylin and eosin. In addition to internal positive controls, positive control slides were included in each experiment. Antibody specificity was determined using a matched IgG isotype antibody as a negative control. The slides were examined using a Bx51 TF light microscope (Olympus Corporation) at magnification of 2x, 10x, 20x, 40x, or 60x, and image acquisition was performed using CellSens software (Olympus Corporation). Immunohistochemical reactivity was blindly interpreted by two independent investigators. For scoring assessments, cells were counted in five separate intratumoral regions under 40x magnification, and 10 representative photographs of each coverslip were taken. The percentage of antigen-positive cells was recorded using ImageJ2 2.3.0/1.53f. The area of staining was evaluated as follows: semiquantitative scoring - score 0 if < 5% of cells are positive, score 1 if 5-24% are positive, score 2 if 25-49% are positive, score 3 if 50-75%, are positive, and score 4 if > 75% are positive. Semi-quantitative scoring was also performed using IHC Profiler, with high positive, positive, low positive, and negative categories. Quantitative scoring was performed using the IHC optical density score, which ranges from 1 to 4 [32].

ELISA.

Infection of gastric tumor cells or non-tumor cells with RV Wt1-5 (MOI 0.2 or MOI 0.8, 12 h.p.i) was assessed using the double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). For this, hyperimmune sera recognizing rotavirus structural proteins (dilution 1:2000) or non-structural proteins (NSP2-NSP6) (dilution 1:2000), or monoclonal antibody 159 (nMAb159, dilution 1:2000) recognizing rotavirus structural protein VP7 [33] were used. The plates were then incubated with HRP-conjugated goat anti-rabbit IgG antibodies (0.13 μg/ml in PBS containing 1% BSA; SC-2301 Santa Cruz Biotechnology Inc.). The reaction was developed using o-phenylenediamine dihydrochloride substrate (Thermo ScientificTM Pierce^®, Wilmington, DE, USA) in 50 mM citrate buffer (pH 5.0) containing 0.05% H₂O₂. The reaction was stopped by adding 2N H₂SO₄ and incubating for 10 min at room temperature. The plates were read using an Ultramark™ microplate reader (model 8422; Bio-Rad, Hercules, Cal, USA). The net optical density (OD) value at 490 nm was calculated by subtracting the negative control value, and the values ​​were expressed as ΔOD. To account for background signals, lysates of cells that were not infected (negative control for rotavirus detection) and incubated with all antibodies in the absence of lysates were used. Absorbance values were obtained from three independent experiments, each performed in duplicate.

Fluorescent IHC staining of frozen tissue sections.

The procedure involved the following steps: (1) cryopreservation of fresh gastric tumor and non-tumor tissues, infected or not infected with RV Wt1-5 (MOI 0.8, 12 h.p.i). (2) Embedding and sectioning of the tissues in the Embedding Compound (OCT) and freezing at -20°C, followed by cutting tissue sections of 5 – 6 μm using a cryostat. (3) Thawing the samples and mounting them on loaded histological slides to improve tissue adhesion. (4) Drying the slides for 30 min at 37°C, followed by detection of rotavirus antigens using hyperimmune rabbit serum against rotavirus structural proteins (1:2000 vol/vol) diluted in PBS and 1% BSA for 1 h at 37°C, and then washing twice with PBS. FITC-conjugated anti-rabbit IgG secondary antibody (0.88 μg/mL Santa Cruz, CA, USA, SC 362261) diluted in 1% BSA was then incubated with 0.15 μg/mL 4’,6-diamidino-2 -phenylindole (DAPI) for 30 min at room temperature. Cells incubated in PBS-BSA without antibodies and isotype controls were used as controls. For evaluation, six representative photographs of each slide were taken using a Nikon C1 4-channel inverted confocal laser scanning microscope using the EZ-C1 software (see Gold, 3.90.).

Western blot.

Gastric tumor cells were seeded at a density of 1 × 10⁵ cells/dish and inoculated with PBS or RV WT1-5 (MOI 0.8) for 12 h at 37° C. Twenty-five μg of total proteins from cell lysates were extracted using an SDS-loading buffer, separated by SDS-polyacrylamide gel electrophoresis (PAGE), and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, CA, USA) using a semi-dry transfer apparatus (Bio-Rad). After blocking with 5% skimmed milk at room temperature for 1h, the membranes were incubated with primary antibodies against specific proteins (VP7 for infection and caspases 3 and 9 for apoptosis; 0,2 μl/mL) for 1 h at 37°C and then incubated with HRP-conjugated secondary antibodies (0.13 μL/ml, Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 1 h at 37°C. The membrane was washed three times, and peroxidase activity was developed using luminol reagent (Santa Cruz Biotechnology, CA, USA) according to the manufacturer’s instructions.

Flow cytometry analysis (FACS) and epifluorescence.

The tumor cells were subjected to flow cytometry analysis to determine the expression of various proteins using, including cell surface membrane proteins such as CEA, CK-7, CK 8/18, CA 19-9, Hsp90, Hsp70, Hsp60, Hsp40, Hsc70, β1, β2, and β3 integrin; PDI and ERp57, as well as intracellular expression of rotavirus antigens and apoptotic proteins such as PARP, cytochrome C, BAX, BID, Bcl-2, caspase 3, and 9. The cells were washed twice with 50 mL of sterile PBS and centrifuged at 800 x g for 5 min at 4°C using a Beckman Coulter J2-MC High-Speed Centrifuge. The cells were fixed in methanol at -20°C and treated with NH₄Cl (50 mM). The cells were then resuspended in PBS containing the primary antibody (0.2 μg/ml) in 0.1% saponin and incubated at 37°C in the dark for 1 h. After washing twice with sterile PBS, the cells were incubated with the secondary antibody (0.88 μg/mL) for 1 h at room temperature in the dark. Cells were resuspended in 300 μL of PBS for flow cytometry analysis. Uninfected cells and cells incubated without antibodies or with secondary antibodies were used as controls. The analysis was performed using a FACScanto II^TM flow cytometer (Becton-Dickinson, Sydney, Australia), and data were analyzed using FACSDiva^TM software (version 8.0, Becton-Dickinson, Franklin Lakes, NJ, USA).

For epifluorescence analysis of protein expression, the same protocol was used, except that the cells were fixed on coverslips with 4% PFA. To evaluate the images, six representative photographs were taken of each group using a 40x objective on a Nikon C1 inverted confocal laser scanning microscope. The NIS-Elements Advanced Research Program from Nikon Corp. was used to obtain the images, and the analysis was performed using ImageJ2 Version 2.3.0/1.53f. For nuclear counterstaining, 0.1 μg/mL of 4’,6-diamidino-2-phenylindole (DAPI) was added for 30 min in a humid chamber at 37°C, protected from light.

Isolation of cell membrane‐enriched fractions.

Cell membrane-enriched fractions were isolated according to a previously described method (Lin et al., 1987) [34]. Briefly, tumor cells isolated from patients with gastric subtype-intestinal adenocarcinoma (Ti2) (1 × 10⁸) were washed with PBS and treated with a hypotonic solution (5 mM HEPES, pH 7.4-, and 50-mM sucrose) for 5 min. The cells were then lysed using a glass homogenizer (DWK Life Sciences Tenbroeck, 40 ml, Fisher Scientific ^®, USA) on ice. Cell membrane aggregates were sedimented by adding CaCl₂ (final concentration 10 mM) and centrifuging the mixture at 5,000 x g for 15 min at 4°C. The supernatant was further centrifuged at 20,000 x g for 30 min. The supernatant was discarded, and the pellet was resuspended in sterile PBS and stored at 4°C for up to 24 h until use.

Binding assay.

The binding of rotavirus particles to tumor cells was characterized by treating tumor cell membrane-enriched fractions with goat anti-human polyclonal antibodies (0.2 μg/mL Santa Cruz, CA, USA) against Hsp90, Hsp70, Hsp60, Hsp40, Hsc70, integrin β3, and PDI. Tumor cell membrane-enriched fractions were incubated with RV Wt1-5 (MOI 0.8) overnight at 4°C, followed by solubilization with RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0,5% DOC, and 0.1% SDS) and 1mM protease inhibitor phenylmethanesulfonylfluoride (PMSF) for 30 min at 37°C. RIPA buffer-solubilized cell membrane-enriched fractions were added to guinea pig hyperimmune serum-coated ELISA plates as capture antibodies (1:1000) to rotavirus structural proteins in duplicate and incubated overnight at 4°C. After three washes with washing buffer (PBS with 0.05% Tween 20 [PBS-T]), non-specific binding sites were blocked by incubation with 5% skimmed milk in PBS-T for 1 h at 37°C. Next, goat primary antibodies (0.2 μg/mL Santa Cruz, CA, USA) against Hsp90 (SC-1055), Hsp70 (SC-1060), Hsp60 (SC-1052), Hsp40 (SC-1801), Hsc70 (SC-1059), integrin β3 (SC-6626), or PDI (SC-17222) were added in PBS containing 1% BSA and incubated for 1 h at 37°C. After three washes with PBS-T, secondary HRP-conjugated donkey anti-goat IgG antibodies (0.133 μg/mL, SC-2020) were added and incubated for 1 h at 37°C. After washing three times with PBS-T, the reaction was developed using o-phenylenediamine (OPD) and H₂O₂, and then stopped by adding 2 N H₂SO₄. The plates were read using an Ultramark™ Microplate Reader (model 8422, Bio-Rad). The net value of optical density (OD) at 490 nm was obtained after subtraction of the negative control value, and values were expressed as ΔOD. The assays were performed three times in duplicate.

Antibody blocking assay.

The tumor cell membrane-enriched fractions were incubated separately for 1 h at 37°C with rabbit F(ab’)2 fragments against Hsp90, Hsp70, Hsp60, or Hsp40 (0,04 mg/ml), or with antibodies against Hsc70 (SC-1059), integrin β3 (SC-6626), or PDI (SC-17222) (4 μg/ml). The F(ab’)2 fragments were prepared using a solid-phase method as previously described [35]. The peptide sequences used to induce the antibodies were as follows:

Hsp90 (620-RDNSTMGYMAAKKHLEINPDHS-641)

Hsp70 (705-QIQQYMKIISSFKNKEDQYDHLD-727)

Hsp70 (646-NSFTLKLEDTENWLYEDGDQPKQ-668)

Hsp70 (741-AMEWMNNKLNLQNKQSLTMDP-761)

Hsp60 (393-RLAKLSDGVAVLKVGGTSDVEVN-415)

Hsp40 (251-GSDVIYPARISLREALCGCTVNV-273).

Excess F(ab’)2 fragments and antibodies were removed by diluting the fractions with 1 mL of ice-cold PBS and centrifuging at 650 x g for 5 min. The supernatant was then removed, and the precipitate was incubated with RV Wt1-5 (MOI 0.8), as described above. Tumor cell membrane-enriched fractions without F(ab’)2 fragment pre-treatment and incubated with RV Wt1-5, as well as tumor cell membrane-enriched fractions inoculated with ovalbumin instead of the virus, were used as controls.

In vitro RV Wt 1-5 infectivity and antibody blocking assay.

The role of cell surface proteins PDI, integrin β3, and Hsps in rotavirus infection was tested by treating NCI-N87 cells with antibodies against these cellular proteins. Cells (1×10⁵ cells/100 μl of RPMI without FBS) were separately incubated with different dilutions (0.025, 0.0125, and 0.0031 μg/mL) of F(ab’)2 fragments against Hsp90, Hsp70, Hsp60 or Hsp40, or antibodies to Hsc70 (SC-1059), integrin β3 (SC-6626), or PDI (SC-17222) in RPMI 1640 containing 1% BSA, for 1 h at 37°C. The cells were then washed twice with RPMI 1640 to remove unbound antibodies, incubated on ice for 5 min, and immediately inoculated with 100 μL of ice-cold RPMI 1640 containing RV Wt1-5 at MOI 0.8. The inoculated cells were then incubated for 45 min at 4°C, washed with RPMI 1640 to remove the unbound virus, and incubated in RPMI without FBS for 12 h at 37°C in a 5% CO2 environment. After incubation, the cells were fixed with 4% PFH for 30 min at room temperature, washed twice with PBS, and processed for an immunochemistry assay to determine the percentage of infected cells, as indicated above. Cells without antibody pretreatment and incubated with rotavirus, as well as cells inoculated with ovalbumin instead of the virus, were used as controls.

Statistical analysis.

Statistical analysis was performed for multiple comparisons using Mann-Whitney U, Kruskal-Wallis, and two-way analysis of variance (ANOVA) with Dunnett´s multiple comparison tests. The data were presented as mean ± standard deviation (SD) from three biological replicates, and p-values less than 0.05 or 0.0001 were considered statistically significant. GraphPad Prism V.8 (GraphPad Software, La Jolla, California, USA) and Stata 12 software (StataCorp, Texas, USA) were used to calculus statistical values. Significance was indicated with an asterisk.