Table 1.
Patient clinical and histopathological data.
Fig 1.
Expression of tumor markers in tissue samples from patients diagnosed with gastric adenocarcinoma. (A) Representative images of hematoxylin and eosin (H&E) staining and immunohistochemical staining (CEA, CK-7, E-cadherin, CA 19-9) in gastric adenocarcinoma and non-tumor tissues. Black arrows show signet-ring neoplastic cells, green arrows show tumor cell aggregates, and red arrows show endothelial cells. (B) Quantification of immunohistochemical analysis by the IHC Optical Density Score method in tumor tissue and non-tumor tissue samples. Data are shown as mean ± SD. Statistical significance is indicated by p-values (* p < 0.05). (C) Flow cytometric evaluation of tumor marker expression in cells isolated from gastric adenocarcinoma and non-tumor tissues.
Fig 2.
Infection of gastric cancer tumor cells by RV Wt 1-5.
Gastric tumor cells and explants were infected separately with RV Wt1-5 (MOI 0.8 or MOI 0.2) or mock-inoculated with PBS. (A) Representative images of HRP immunochemistry staining of rotavirus structural antigens and Alexa 488 fluorescent staining (green) at 12 h.p.i, MOI 0.8 after infection in tumor cells disaggregate, tumor explant and (B) non-neoplastic tissue explants. (C) Mean Fluorescence Intensity of the cell population infected with Wt1-5 (MOI 0.8) in comparison with the control cells at 12 h.p.i. (D) Quantification of RV antigens in isolated gastric adenocarcinoma cells from different tumors using flow cytometry at MOI of 0.2 (dark bar) and MOI of 0.8 (color bars) in comparison with non-tumor tissue control at 12 h.p.i. DT, diffuse-subtype gastric adenocarcinoma; IT, intestinal-subtype gastric adenocarcinoma. (E) Representative images of the immunochemistry assay for viral structural antigens from gastric tumor and non-neoplastic tissue explants inoculated with RV Wt1-5 at different times of infection (12, 24, 48, and 60 h.p.i). (F) HRP immunochemistry staining of RV structural antigens at 12, 24, 48, and 60 h.p.i was quantified according to the IHC optical density score as the number of cells positive for rotavirus structural antigens using HRP immunochemistry assay. (G) Evaluation of rotavirus antigens in RV-infected gastric cancer and non-neoplastic tissue at an MOI of 0.2 (black bar) or MOI of 0.8 (color bars) at 12 h.p.i by Capture ELISA. (H) As in G, except that cells positive for non-structural proteins NSP2, NSP3, NSP4, NSP5, NSP6, structural antigen VP7 (Ab159), and rotavirus structural antigens were recorded. (I) Western blot evaluation of the RV structural protein VP6 in gastric tumor cells previously infected with RV Wt1-5 (MOI 0.8) for 12 h.p.i. (J) Analysis by flow cytometry of infection by RV Wt 1-5 (MOI 0.2 or 0.8) of tumor cells expressing tumor markers CEA and cytokeratin 8/18 at 12 h.p.i. (K) Representative image of IHC evaluation of infection of NCI-N87 cell line. Tumor explants were inoculated with RV Wt1-5 (MOI 0.8) or with PBS, and at 12 h.p.i the supernatant was collected, centrifuged, and then used to inoculate the NCI-N87 cell line. The presence of rotavirus antigens was evaluated using hyperimmune sera against RV. (L) The quantification is shown in the bar chart. (IT2 intestinal-subtype GC, DT3 diffuse-subtype GC). Data from three independent experiments performed in duplicate are shown as mean percentages ± standard deviation (SD). Mann-Whitney U test with a value *p<0.05.
Fig 3.
Cell-surface protein expression in gastric adenocarcinoma and non-neoplastic adjacent tissues.
(A) Representative immunofluorescence staining of membrane proteins in gastric tumors and (B) adjacent non-neoplastic gastric tissues. Membrane protein expression was observed at a higher frequency and intensity in gastric tumors. Magnification: ×40. Scale bar: 50 μm. (C) Expression levels of proteins on the cellular membrane of gastric cancer cells and non-tumor cells were measured using flow cytometry. DT: diffuse-subtype gastric adenocarcinoma; IT: intestinal-subtype adenocarcinoma. (D) The median fluorescence intensity (MFI) was determined using flow cytometry to evaluate the expression of membrane proteins, as described in (C). Data from three independent experiments performed in duplicate are shown as mean percentages ± standard deviation (SD). Two-way ANOVA test with a value p<0.0001. (****p<0.0001, ***p<0.001, **p<0.01, *p<0.1).
Fig 4.
Binding of rotavirus particles to Hsp90, Hsp70, Hsp60, Hsp40, Hsc70, PDI, and integrin β3 and blocking of RV binding to the tumor cell membrane-enriched fractions surface by antibodies directed to these proteins.
A. Tumor cell membrane-enriched fractions were incubated with F(ab)2` fragments against synthetic peptides derived from Hsp90, Hsp70, Hsc70, Hsp60, Hsp40, or mAbs against β3 or PDI, followed by incubation with RV Wt1-5 (MOI 0.8). After incubation, were solubilized in RIPA buffer and captured in ELISA plates coated with guinea pig-generated capture antibodies against rotavirus structural proteins (TLPs). Goat polyclonal primary antibodies against Hsps, β3 integrin, and PDI were used for detection. The reaction was measured using an HRP-conjugated donkey anti-goat IgG secondary antibody and developed using an OPD substrate. Fractions that were unblocked with antibodies were used as binding controls. B. Inhibition of rotavirus infection by antibodies against Hsp90, Hsp70, Hsp60, Hsp40, Hsc70, β3 integrin, or PDI. NCI-N87 cells were separately incubated with the indicated dilutions of the F(ab)`2 fractions of antibodies against the different proteins for 1 h at 37°C. After incubation, cells were washed with RPMI and incubated with RV WT1-5 for 45 min at 4°C and then washed with medium and incubated in RPMI without FBS for 12 h at 37°C and 5% CO2. Rotavirus structural antigens were determined by immunocytochemistry. Cells without antibody pretreatment and incubated with the RV and cells without rotavirus and without antibody were used as controls. Data are presented as the percentage of infected cells. C. Representative images of the previous immunocytochemistry for RV Wt1-5 (Scale bar = 50 μm). Data of three independent experiments performed in duplicate are shown as mean percentages ± standard deviation (SD). The Mann-Whitney U-test was used, with a value of p<0.05.
Fig 5.
Genotoxic and apoptotic effects induced by Wt1-5 rotavirus infection.
Gastric tumors were infected with Wt1-5 (MOI 0.2 or 0.8) for 12 h.p.i. and non-infected tumor tissue and non-tumor tissue were used as controls. (A) Flow cytometry was used to evaluate the expression of PARP, Bcl-2, Cyt C, BAX, caspase 3, caspase 9, and BID. The percentages of cells showing the expression of apoptotic proteins and DNA damage are shown. Data are shown as mean ± SD of three independent experiments performed in duplicate. PARP expression was compared between RV-inoculated groups and their corresponding PBS-inoculated groups. The two-way ANOVA test was used, with a value of p****<0.0001 (B. C) Cells were stained with DAPI and analyzed by epifluorescence microscopy at the indicated times. Representative images from the cells analyzed in (A) are shown. The scale bar is 50 μm. (D) Quantitative analysis of images shown in (C) is expressed in terms of percentages of cells being positive for cell death proteins or Wt1-5 structural proteins. Cells positive for both viral proteins and cellular proteins are denoted as (+/+), while cells negative for both viral proteins and cellular proteins are denoted as (-/-). Data of three independent experiments performed in duplicate are shown as mean percentages ± standard deviation (SD). The Mann-Whitney U-test was used with a value of p*<0.05. (E) Western blot analysis of apoptosis-associated proteins pro-caspase-3 and pro-caspase-9 and cleaved caspase-9 and cleaved caspase-3 in rotavirus-infected gastric cancer tissues (12 h.p.i, MOI 0.8) was performed. Non-infected cells were used as a control. (F) The apoptosis and infection were evaluated using anti-Bcl-2, anti-BID, anti-PARP, and anti-RV staining in gastric cancer cells inoculated with Wt1-5 (MOI 0.2-12 h.p.i). Fluorescence was analyzed by flow cytometry. (G) Gastric tumors and adjacent non-cancerous tissues were infected with rotavirus (MOI 0.8) for 12, 24, or 60 h. Microphotographs of immunohistochemical staining of p53 protein with nuclear or predominantly nuclear staining (revealed with diaminobenzidine, seen as brown in the positive nuclei, with hematoxylin contrast giving a blue color to the negative nuclei) and (H) expression of Bcl-2 in the cytoplasm are shown. Original magnifications include x10, x20, x40 and x60, and black arrows show tumor cells. (I) The expression of p53 and Bcl-2 was quantified by the IHC Optical Density Score as the number of protein-positive cells using the HRP immunochemistry assay. Data are shown as mean ± SD of three independent experiments performed in duplicate. RV-inoculated groups were compared with the corresponding PBS-inoculated groups. The two-way ANOVA test was used, with a value of p****<0.0001. (J) Representative microphotographs of nuclear condensation and nuclear budding as the events inducing apoptosis-mediated cell death assessed at 48 h.p.i, MOI 0.8. Arrows are showing micronuclei, degenerated nuclei, and fragmented apoptotic nuclei in infected gastric cancer cells. (Scale bar = 50 μm).
Fig 6.
Gastric adenocarcinoma tissues were processed for histological hematoxylin and eosin (H&E) staining.
(A) Tumors from mock-treated tissue showed no signs of necrosis. (B and C) Tumors from Wt 1-5—treated (60 h.p.i MOI 0.8) had areas of necrosis of corresponding to layers in the stomach walls. (D) The blue arrows show necrosis of the smooth muscle, (E) tumor cell necrosis, and (F) signet ring cell necrosis.
Fig 7.
The immune microenvironment in cancer and adjacent non-cancerous tissues after infection with Wt 1-5 (24 or 48 h.p.i, MOI 0.8).
(A) Representative photographs of H&E staining of gastric tumors treated with PBS and inoculated with RV Wt1-5 (24 h.p.i) are shown. (B) Palatine tonsil tissue was used as a positive control for immunocytochemical analysis. (C) Immunohistochemical analysis of lymphocytic infiltration in the peritumoral area and non-tumor tissues treated with PBS (24 h). The black arrows show tumor cell aggregates; the red arrow shows signet ring cells, and the blue arrow shows a blood vessel. (D) Immunohistochemical analysis of lymphocytic infiltration in the peritumoral area and non-tumor tissues after Wt1-5 infection (24 h.p.i, MOI 0.8). (E) The numbers of stained immune cells in tumors and adjacent-noncancerous tissues were quantified according to IHC Optical Density Score as the number of protein-positive cells using the HRP immunochemistry assay. (F) Samples were analyzed by H&E staining and rotavirus structural proteins staining. In H&E-stained sections, infiltrating cells appear in nervous tissue. Immunohistochemical reveals a strong cytoplasmic signal in invasive cancer and immune cells, including those for muscle invasion, vascular penetration, and nerve invasion.