Study On Anti-tumor Effect And Immunity Mechanism Induced By NDV HN Gene Recombinant Lactobacillus On Colon Cancer-bearing BALB/c Mice

Colorectal cancer (CRC) is a common digestive tract malignancy with higherincidence. Colorectal cancer self is likely to induce immune suppression, in addition,factors of patient’s age and nutritional status are also linked with immune suppression.Colon cancer potential immunogenicity and host immune respect are connected withhost survival. Therefore, enhancing tumour immunogenicity, stimulating or rebuildingimmunity function, any of which could restrain immune suppression, all contribute torepel tumor cell. This is very important to supplement for cancer immunologicaltherapy.Newcastle disease virus surface protein-HN is the important instrument of virusinfection to host. Interestingly, it is also the functional protein of being anti-tumoreffect. With the development of prokaryotic expression system of Lactobacillus, itpresent highlights benefit on mediating intestinal local immunity and systemimmunity as functional protein gene delivering vector. Additionally, Lactobacilluspossesses immuno-stimulatory and adhension activity to intestinal mucosa, which willbe act as representative strain among diverse strains of probiotics.Therefore, NDV HN gene was cloned onto Lactobacillus shuttle vector, whichcontained anchoring sequence to express the foreign protein onto cell surface, toconstruct a recombinant named HN-pSIP409. Then HN-pSIP409plasmid waselec-transformed into Lactobacillus plantarum NC8competence. SDS-PAGE andwestern blotting were used to detect the expression of HN protein. In order to observeif the expression of foreign gene from Lactobacillus anchored cell surface, flowcytometry was used to detect the expression of HN protein on Lactobacillus strain cellsurface. To facilitate the stability determination for recombinant Lactobacillus, weinsert an additional green fluorescent protein gene (GFP) in the downstream of HNprotein.The result displayed that recombinant NDV HN gene Lactobacillus wasconstructed. Result of SDS-PAGE and western blotting showed a nearly66KDa band,which has the ability to react with HN protein antibody. Importantly, the HN proterincould express on the surface of Lactobacillus strain. The result of fluorescence effectshowed GFP could be expressed on the cell surface, which also could be testified thefluorescence effect after30passages. Epidemiological studies have reported that consumption of fermented dairyproducts may elicit anti-tumor effects and are associated with a lower incidence ofCRC. Although the mechanism of Lactobacillus on preventing CRC is unclear, manystudies demonstrated Lactobacillus can induce tumor cell apoptosis, preventcancerogen mutation and stimulate antitumor immunity. So Lactobacillus possesspotential candidate for preventing and retaining tumor growth. While NDV can attachonto the surface of tumor cell and hydrolyze receptor by HN protein molecule,importantly, it possesses strong immunostimulatory properties to enhance innateimmunity to repel tumor.In order to ascertain antitumor function of NDV HN gene Lactobacillus, coloncancer tumor-bearing BALB/c mice was pre-inoculating mice with HN generecombinant Lactobacillus（namely HN/Lab）or Lactobacillus plantanrum (namelyL.p) for2weeks by gavage as109CFU. Then we constructed colon primarysubcutaneous implantation tumor model, orthotopic implantation tumor model andextra-intestinal metastasis model by CT26.WT induction, respectively. To strengthenthe immunity, lactobacilli was continued inoculating for another3weeks, as one timeper week, after tumor induction. The result demonstrated as follows:(1) HN/Lab recombinant lactobacilli (CT26+HN/Lab group) significantlyinhibited CT26growth at25d and30d post-inoculation (p<0.05) compared withPBS (CT26alone group). However, L. plantarum (CT26+L.p group) did notsignificantly restrain CT26growth after25d compared with the PBS (CT26alonegroup). The subcutaneous tumor-bearing mice from the CT26+HN/Lab groupsurvived much longer than mice from other groups (p<0.05). The tumor volume ofsubcutaneous tumor form CT26+HN/Lab were significant decreased compared withCT26group (p<0.05). Histopathology showed obvious evidence of mitosis and cellpolymorphisms in all groups, prominent lymphocytic infiltration was observed only inthe CT26+HN/Lab group.(2) For orthotopic CRC xenografts. After7d of post-implantation, a significantdecrease in tumor volume was observed in the CT26+HN/Lab group compared to theCT26alone group (p<0.05). The tumor size in the CT26+L. plantarum group wasreduced without significance. The tumor weight in the CT26+HN/Lab group at21dpost-implantation was reduced by43.13%compared to the CT26alone group, whichin CT26+L. plantarum was down-regulated by23.03%. Histolopathology results showed obvious necrosis and lymphocyte infiltration in the CT26+HN/Lab group.However, necrosis was also observed in CT26groups with less lymphocyteinfiltration. In addition, the intestinal score from CT26+HN/Lab group mice waslower than that from CT26(3) For extra-intestinal metastasis tumor-bearing mice, we observed the lesstumour node in group of CT26+HN/Lab than that in CT26, and the number of tumournode in CT26+L.p was also lower than CT26. This result demonstrated HN/Lab couldbetter restrain the metastasis of colon cancer cell than L.p.During studying the anti-tumor effect induced by lactobacilli, we observed theinhibitory effect on colon cancer cell growth for subcutaneous, orthotopic ormetastasis tumor model after inoculation with lactobacilli (HN/Lab). Maybelactobacilli displayed the effect via simulating system immunity. Study has testifiedHN protein can induce strong I type interferon (including interferon-,-β or TNF-)production to activate NK cell, which did not depend the replication of virus.Meanwhile, oral administration with lactobacilli could induce dendritic cell (DCs)maturation, and the latter further activate NK cell, regulate differentiation of Th cell.For NK activation, NK could produce interferon-γ to kill tumor cell and also activatedDCs further to form a tight cross talk. The differentiation of Th cell was likely linkedwith strain-dependant. So we presumed that lactobacilli can induce anti-tumorimmunity.To further ascertain this hypothesis，we determined the number and activation oftumor infiltration lymphocyte, that of NK cell or DCs or CD4+T or CD8+T insplenocyte, polarization of CD4+T cell through flow cytometry, ELISA andimmunohistochemistry. The results were demonstrated as follows:(1) HN/Lab recombinant lactobacilli could promote I type cytokine, such asIFN-γ and TNF-through ELISAtest. And it can promote the NK cell proliferation invivo and also increase the cytotoxicity to kill target cell in vitro. Importantly, wedetermined a significant increase of IFN-γ and granzyme secretion by FCM (p<0.05).(2) We determined that HN/Lab recombinant lactobacilli could significantlyincrease the ratio of CD4+T cell (p<0.05) and elevate the ratio of CD8+T cell withoutsignificance. It diaplayed that HN/Lab recombinant lactobacilli mainly stimulate Thcell respect. For find out the orientation of Th cell differentiation, we detected thatHN/Lab induced the DCs activation by elevating the expression of CD80and CD86 firstly, then we detected the matured DCs could induce CD4+Th1differentiation byup-regulating of IFN-γ (but not IL-4or IL-17) production. Interestingly, HN/Labrecombinant lactobacilli elevated the ratio of CD8+T cell secreting IFN-γ and Grb+.In summary, our results suggest that HN/Lab recombinant lactobacilli can enhance theanti-tumor immune response to delay tumor formation.(3) Detection of flow cytometry and immunohistochemistry showed that,HN/Lab could enhance CD8+T cell or NK cell infiltrating into tumor, especiallypromote CD8+T cell or NK cell to produce granzyme B. It demonstrated that HN/Labcan influence the tumor micro-environment to enhance the cytotoxicity aganist tumor.In concluntion, we constructed the NDV HN gene recombinant Lactobacillus.It can delay tumor formation for subcutaneous or orthotopic or extra-intestinalmetastasis tumor-bearing mice. It mainly elevated the NK cell, CD4+Th1and CD8+Tcell proliferation and cytotoxity to display the anti-tumor effect. It also promoted NKand CD8+T cell inflatrating into tumor tissue to change the tumor micro-environmentto restrain tumor growth.