•  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. CEACAM5 encodes a cell surface glycoprotein that represents the founding member of the carcinoembryonic antigen (CEA) family of proteins. The encoded protein is used as a clinical biomarker for gastrointestinal cancers and may promote tumor development through its role as a cell adhesion molecule. Additionally, the encoded protein may regulate differentiation, apoptosis, and cell polarity. This gene is present in a CEA family gene cluster on chromosome 19. Alternative splicing results in multiple transcript variants.
• Gene targeting strategy: The B2m gene (Exon 2 to Exon 3) 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 and human CEACAM5 CDS. Human HLA-A2.1 is highly expressed on the surface of B-HLA-A2.1/hCEACAM5 MC38. Human CEACAM5 is detectable by flow cytometry.
• Tumorigenicity: Confirmed in B-HLA-A2.1 mice.
• Application: The B-HLA-A2.1/hCEACAM5 MC38 tumor model can be used for preclinical evaluation of cancer vaccines and TCR mimics.
•  Notes:

➢ Inoculated cell lines can be suspended with RPMI-1640 stock solution.

➢ Before implementing the project, it is recommended to perform tumor growth experiments. The recommended cell inoculation amount is between 5×105 - 1×106.

➢ 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-A2.1 and CEACAM5 expression analysis in B-HLA-A2.1/hCEACAM5 MC38 by flow cytometry, respectively. Single cell suspensions from wild-type MC38 and B-HLA-A2.1/hCEACAM5 MC38 #1-F03 cultures were stained with anti-human HLA-A2 antibody (BioLegend, 343306) and anti-human CEACAM5 (BioLegend, 342308), respectively. Human HLA-A2 and CEACAM5 were detected on the surface of B-HLA-A2.1/hCEACAM5 MC38 cells but not wild-type MC38 cells.

Subcutaneous tumor growth of B-HLA-A2.1/hCEACAM5 MC38 cells. B-HLA-A2.1/hCEACAM5 MC38 cells (1×10⁶) and wild-type MC38 cells (2×10⁵) were subcutaneously implanted into B-HLA-A2.1 mice (male, 8-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-HLA-A2.1/hCEACAM5 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

HLA-A2.1 and CEACAM5 expression were evaluated on B-HLA-A2.1/hCEACAM5 MC38 by flow cytometry, respectively. These cells were subcutaneously transplanted into B-HLA-A2.1 mice (n = 6). At the end of the experiment, tumor cells were harvested and analyzed with species-specific anti-HLA-ABC antibody (BioLegend, 311406) (A) and anti-human CEACAM5 (BioLegend, 342308) (B) expression by flow cytometry, respectively.