• Origin: The MC38 cell line is derived from C57BL6 murine colon adenocarcinoma cells. The cell line is a commonly used murine model for colorectal carcinoma.
• Background Information: HLA-G, or Human Leukocyte Antigen G, is a non-classical major histocompatibility complex (MHC) class I molecule that plays a critical role in immune tolerance and modulation. Its primary function involves suppressing immune responses to prevent rejection, particularly in contexts such as pregnancy and transplantation. HLA-G achieves this by interacting with inhibitory receptors on immune cells, including natural killer (NK) cells, T cells, and antigen-presenting cells, thereby inhibiting their cytotoxic activity and cytokine production. HLA-G is involved in tumor immune evasion, as many cancers upregulate its expression to avoid detection and destruction by the immune system.
• Gene targeting strategy: The mouse B2m gene was replaced by human B2M and HLA-G coding sequence in B-hB2M/HLA-G MC38 plus. Human HLA-G is highly expressed on the surface of B-hB2M/HLA-G MC38 plus cells.
• Tumorigenicity: Confirmed in B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4),Pirb KO mice.
• Application: B-hB2M/HLA-G MC38 plus cells have the capability to establish tumors in vivo and can be used for efficacy studies.
• Notes:

1. Inoculated cell lines can be suspended with DMEM stock solution.
2. Before implementing the project, it is recommended to perform tumor growth experiments. The recommended cell inoculation amount is between 2E5-5E5.
3. In the experiment, it is necessary to ensure that the number of animals inoculated subcutaneously is at least 1.6 times the actual grouping number.

HLA-G and human β2M expression analysis in B-hB2M/HLA-G MC38 plus cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hB2M/HLA-G MC38 plus cells cultures were stained with species-specific anti-HLA-G or anti-β2m antibody. Human HLA-G and β2m was detected on the surface of B-hB2M/HLA-G MC38 plus cells, but not on the surface of wild-type MC38 cells. The 1-D06 clone of B-hB2M/HLA-G MC38 plus cells was used for in vivo experiments.

HLA-G and human β2M expression evaluated on B-hB2M/HLA-G MC38 plus tumor cells by flow cytometry-hB2M/HLA-G MC38 plus (5×10⁵) were subcutaneously implanted into B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4),Pirb KO mice (female, 12-week-old, n=6). Upon conclusion of the experiment, tumor cells were harvested and assessed for human HLA-G (antibody in house) and β2M (Biolegend, 395712) expression by flow cytometry. As shown, human HLA-G and β2M were highly expressed on the surface of tumor cells. Therefore,  B-hB2M/HLA-G MC38 plus can be used for in vivo efficacy studies evaluating novel HLA-G therapeutics.

Subcutaneous tumor growth of  B-hB2M/HLA-G MC38 plus.  B-hB2M/HLA-G MC38 plus (5×10⁵) were subcutaneously implanted into B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4),Pirb KO mice (female, 12-week-old, n=6). Tumor volume and body weight were measured twice a week. (A) Average tumor volume. (B) Body weight. Volume was expressed in mm³ using the formula: V=0.5 × long diameter × short diameter². Results indicate that  B-hB2M/HLA-G MC38 plus were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

Subcutaneous tumor growth of  B-hB2M/HLA-G MC38 plus. B-hB2M/HLA-G MC38 plus (5×10⁵, 2×10⁵) were subcutaneously implanted into B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4), Pirb KO mice (female, 18-week-old, n=8). Tumor volume and body weight were measured twice a week. (A) Average tumor volume. (B) Body weight. Volume was expressed in mm³ using the formula: V=0.5 × long diameter × short diameter². Results indicate that  B-hB2M/HLA-G MC38 plus were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.