Mechanism Of CD155 Molecule Transfer From Tumor Cells To Host Cells And Mediating Tumor Immune Escape

Background:Since 2013,tumor immunotherapy has gained significant attention,particularly after being named the most important breakthrough of the year by the journal Science.This type of therapy employs checkpoint inhibitors and adoptive cell therapy to regulate the immune system and identify and kill tumor cells.Some of the defining features of tumor immunotherapy include the selective targeting of the tumor microenvironment,the normalization of tumor immunity,and the remodeling of immune responses in the tumor microenvironment.While immune checkpoint inhibitors,such as anti-CTLA-4 and PD-1,have shown promise in the treatment of various tumors by alleviating immunosuppression or promoting the activation of immune responses,the complexity of the process of tumor occurrence and development,along with our incomplete understanding of its mechanism,has limited the efficacy of tumor immunotherapy in many tumor types.Single immune checkpoint inhibitors often lead to new immune tolerance,resulting in tumor non-response or recurrence.However,double blocking of PD-1/CTLA-4co-inhibitory receptors on T cells has demonstrated good therapeutic efficacy in patients with melanoma.In their long-term interaction with the immune system,tumor cells can use various mechanisms to resist the killing of immune cells,including up-regulation of various immune checkpoint molecules.Aside from the classic PD-L1/PD-1 pathway,many other inhibitory molecules collectively regulate the immune status and intensity of the tumor microenvironment.Combining the suppression of multiple immune regulatory sites has been shown to enhance the therapeutic effect of tumors in many studies.CD155（NECL-5/PVR/TAGE4）,a member of the Nectins/Nectin like molecule family,plays a crucial role in cell adhesion,motility,proliferation,and survival.While poorly expressed in most adult organs,it is abnormally highly expressed in tumor cells of many cancer types and has been linked to a poorer prognosis in tumor patients.CD155 can interact with cell surface receptors,including co-inhibitory receptors TIGIT and CD96,as well as co-stimulatory receptor CD226.The balance between these three receptors regulates the antitumor immune response.Like PD-L1,CD155 is low expressed in antigen-presenting cells and can be up-regulated by inflammatory factors such as LPS,IL-6,and IFN-γ,and highly expressed in tumor cells.Ligands TIGIT and PD-1 corresponding to CD155 and PD-L1,respectively,are up-regulated on tumor-infiltrating immune cells,exerting an immunosuppressive function as immune checkpoint molecules.Antibodies that block PD-1 or its ligand PD-L1 have been approved for the treatment of various solid cancers such as non-small cell lung cancer,hepatocellular carcinoma,Hodgkin’s lymphoma,and other hematologic malignancies.However,CD155 has not been studied adequately,and further investigation and exploration of CD155 are necessary.Objective:The purpose of this study was to perform a preliminary exploration of the function of the CD155 gene by utilizing CRISPR/Cas9 genome editing technology to construct CD155 knockout B16F10 cell line and MC38 cell line.The study aimed to investigate the impact of CD155 molecules on the growth of multiple tumor cells in vitro and to uncover the effect of CD155 molecules on the immune cell status in the tumor microenvironment in both tumor cells and host cells.By studying the CD155immune checkpoint molecule and mechanisms related to immune escape,this research provides a theoretical basis for the development of targeted immunothe-rapies.Methods:We employed CRISPR/Cas9 genome editing technology to generate CD155knockdown B16F10 mouse melanoma and MC38 mouse colon cancer cell lines.The sorted B16F10-CD155KO and MC38-CD155KO cells were amplified in vitro to obtain high-purity cell populations.We co-cultured equal numbers of wild-type and CD155KO tumor cells in vitro and analyzed the impact of CD155 on the growth of multiple tumor cells by flow cytometry.In vivo experiments were conducted by subcutaneously injecting wild-type and CD155KO B16F10 cells into WT and CD155^-/-mice,respectively.After tumor removal and digestion,the percentage and expression levels of DNAM-1 and CD96molecules on tumor-infiltrating NK,CD4⁺T and CD8⁺T cells were assessed by flow cytometry.We also examined the immune cell status in the tumor microenvironment by analyzing the host myeloid cells and infiltrating NK,CD4⁺T,and CD8⁺T cells using flow cytometry.These findings contribute to understanding the role of CD155in immune checkpoint mechanisms and the potential development of targeted immunotherapies.Results:The CD155 gene was successfully knocked out using CRISPR/Cas9,and B16F10-CD155KO and MC38-CD155KO cell lines with high purity were established through flow detection.Co-culture of equal numbers of CD155-positive and CD155-negative cells revealed no significant growth advantage for either type of cell.Subcutaneous tumors were established in WT mice using either B16F10-control or B16F10-CD155KO cells,and after 15 days,flow cytometry analysis showed that CD155 deletion in tumor cells increased the proportion of DNAM-1-positive NK cells and CD96-positive NK,CD4⁺T and CD8⁺T cells in the tumor microenviro-nment.Additionally,the expression level of CD96 on CD8⁺T cells in the tumor microenvironment was upregulated following CD155 deletion.Flow cytometry analysis of tumor-infiltrating host myeloid cells and NK,CD4⁺T,and CD8⁺T cells in the B16F10 tumor microenvironment revealed that CD155molecules could be transferred from CD155-positive tumor cells to CD11b⁺myeloid cells in CD155^-/-mice.Furthermore,CD155 deletion in both tumor cells and host cells increased the expression level of DNAM-1 on NK cells,CD4⁺T cells,and CD8⁺T cells in the tumor microenvironment.Conclusion:1.In vitro experiments revealed that CD155 does not significantly impact the growth of tumor cells.2.The absence of CD155 in tumor cells was found to disrupt the balance between relevant receptors within the tumor microenvironment.3.Deletion of CD155 in both tumor cells and host cells was observed to synergistically upregulate DNAM-1 expression levels on immune cells within the tumor microenvironment.