Objective To investigate the influence of bacterial outer membrane vesicles (OMVs) tumor vaccine on tumor cell proliferation and CD8⁺ T cell infiltration of mouse with pancreatic cancer.

Methods The experimental study was conducted. The ovalbumin (OVA) lentivirus vector plasmid pLV‑EF1a‑hluc‑P2A‑mNeongreen‑CMV‑OVA‑3Xflag‑P2A‑puro was used to construct the mouse pancreatic cancer Pan02‑OVA cells. The ClyA‑Catchers‑OMVs (CC‑OMVs) originated from Escherichia coli and labeled antigenic peptide SpyTag‑OVA were used to construct the OMVs tumor vaccine. Mouse CD8⁺ T cells were stimulated by OMVs tumor vaccine, and the effects of OMVs tumor vaccine on inhibiting pancreatic cancer cells proliferation and stimulating CD8⁺ T cell infiltration were analy-zed by in vitro cell killing assay, including the OMVs tumor vaccine stimulated T cell group and the control T cell group, subcutaneous pancreatic cancer model, including the OMVs tumor vaccine group and the control group, and immunohistochemical staining. Observation indicators: (1) identification of mouse pancreatic cancer Pan02‑OVA cells; (2) morphological observation of CC-OMVs; (3) inhibi-tion of mouse pancreatic cancer Pan02‑OVA cells by OMVs tumor vaccine specific T cells; (4) inhibi-tion of mouse pancreatic cancer by OMVs tumor vaccine; (5) CD8⁺ T cell infiltration in pancreatic cancer tissue of mouse stimulated by OMVs tumor vaccine. Measurement data with normal distribu-tion were represented as Mean±SD, and comparison between groups was analyzed using the t test. Count data were described as absolute numbers or percentages.

Results (1) Identification of mouse pancreatic cancer Pan02‑OVA cells. Results of laser scanning confocal microscopy showed that the mNeongreen fluorescence was expressed in Pan02‑OVA cells infected with the OVA lentivirus vector plasmid of pLV‑EF1a‑hluc‑P2A‑mNeongreen‑CMV-OVA‑3Xflag-P2A-puro. Results of Flow cytometry showed that using the mouse pancreatic cancer Pan02 cells as references, the protein expression rate of Flag on the Pan02‑OVA cells was 90.7%. (2) Morphological observation of CC‑OMVs. Results of transmission electron microscopy analysis showed that the CC‑OMVs were in spherical shape, with a diameter <50 nm. (3) Inhibition of mouse pancreatic cancer Pan02‑OVA cells by OMVs tumor vaccine specific T cells. Results of cell proliferation toxicity test showed that the absorbance at 450 nm of mouse pancreatic cancer Pan02‑OVA cells was 0.41±0.12 and 1.05±0.15 in the OMVs tumor vaccine‑stimulated T cell group and the control T cell group, respectively, showing a significant difference between the two groups (t=9.54, P<0.05). (4) Inhibition of mouse pancreatic cancer by OMVs tumor vaccine. The weight of subcutaneous tumor tissue in the back of mouse was (81±10)g and (153±17)g in the OMVs tumor vaccine group and the control group, respectively, showing a significant difference between the two groups (t=8.26, P<0.05). (5) CD8⁺ T cell infiltration in pancreatic cancer tissue of mouse stimulated by OMVs tumor vaccine. Results of immuno-histochemical staining showed that the numbers of CD8⁺ T cells staining in the mouse back subcu-taneous tumor tissues was 28.7±3.5 and 9.3±1.5 in the OMVs tumor vaccine group and the control group, respectively, showing a significant difference between the two groups (t=8.74, P<0.05).

Conclusion Bacterial OMVs tumor vaccine can inhibit proliferation of pancreatic cancer cells and increase the numbers of CD8⁺ T cells infiltrated in pancreatic cancer tissue of mouse.