• Origin: The MC38 cell line is derived from C57BL/6 murine colon adenocarcinoma cells. The cell line is a commonly used murine model for colorectal carcinoma.
• Background information: HLA-A belongs to the HLA class I heavy chain paralogues. This class I molecule is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). The heavy chain is anchored in the membrane. Class I molecules play a central role in the immune system by presenting peptides derived from the endoplasmic reticulum lumen so that they can be recognized by cytotoxic T cells. Glypican-3 (GPC3) is a cell-surface oncofetal protein, encoded by the GPC3 gene, that is highly overexpressed in hepatocellular carcinoma (HCC) but absent in healthy adult liver tissue. It functions as a diagnostic biomarker and a therapeutic target for immunotherapy, including antibodies and CAR-T therapies, due to its role in regulating Wnt, Hedgehog, and YAP pathways.
• Gene targeting strategy: The B2M gene (Exon2 to Exon3) of mouse were replaced by the sequence encompassing the human B2M CDS, HLA-A*0201 gene that included leader sequence, α1 and α2 domains ligated to a fragment of the murine H-2Db gene containing the α3, transmembrane and cytoplasmic domains in B-HLA-A2.1/hGPC3 MC38 cells. The human GPC3 gene was inserted to murine rosa26 site in B-HLA-A2.1/hGPC3 MC38 cells. Human HLA-A2.1 and GPC3 are highly expressed on the surface of B-HLA-A2.1/hGPC3 MC38.
• Application: B-HLA-A2.1/hGPC3 MC38 tumor models can be used for preclinical evaluation of cancer vaccines.

HLA-A2.1 and human GPC3 expression analysis in B-HLA-A2.1/hGPC3 MC38 by flow cytometry, respectively. Single cell suspensions from wild-type MC38 and B-HLA-A2.1/hGPC3 MC38 cultures were stained with species-specific anti-HLA-ABC antibody (Biolegend, 343306) and anti-human GPC3 antibody (in house). Human HLA-A2.1 was detected on the surface of B-HLA-A2.1/hGPC3 MC38 cells but not wild-type MC38 cells (A). Human GPC3 was highly expressed on the surface of the tumor cells (B). The 1-H05 clone of B-HLA-A2.1/hGPC3 MC38 cells was used for in vivo experiments.

Subcutaneous homograft tumor growth of B-HLA-A2.1/hGPC3 MC38 cells. B-HLA-A2.1/hGPC3 MC38 cells (1×10⁶) and wild-type MC38 cells (5×10⁵) were subcutaneously implanted into B-HLA-A2.1 mice (male, 7-week-old, n=6). Tumor volume and body weight were measured three times a week. (A) Average tumor volume ± SEM. (B)  Body weight (Mean± SEM). Volume was expressed in mm³ using the formula: V=0.5 × long diameter × short diameter². As shown in panel A, B-HLA-A2.1/hGPC3 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

Human HLA-A2.1 and human GPC3 expression evaluated in B-HLA-A2.1/hGPC3 MC38 tumor cells by flow cytometry, respectively. B-HLA-A2.1/hGPC3 MC38 cells were subcutaneously transplanted into B-HLA-A2.1 mice (male, 7-week-old, n=6), and on 20 days post inoculation, tumor cells were harvested and assessed for human HLA-A2.1 expression (Biolegend, 343306) and human GPC3 expression (in house) by flow cytometry, respectively. As shown, human HLA-A2.1 was highly expressed on the surface of tumor cells (A). Human GPC3 was highly expressed in the tumor cells (B). Therefore, B-HLA-A2.1/hGPC3 MC38 cells can be used for in vivo efficacy studies of novel GPC3 therapeutics.