Description

• 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-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. LMP2A is a transmembrane protein that inhibits normal B-cell signal transduction by mimicking an activated B-cell receptor (BCR). The N-terminus domain of LMP2A is tyrosine phosphorylated and associates with Src family protein tyrosine kinases (PTKs) as well as spleen tyrosine kinase (Syk). PTKs and Syk are associated with BCR signal transduction.
• 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 and LMP2A CDS. Human HLA-A2.1 is highly expressed on the surface of B-HLA-A2.1/hLMP2 MC38. LMP2 is highly expressed on the surface of B-HLA-A2.1/hLMP2 MC38 cells.
• Tumorigenicity: Confirmed in B-HLA-A2.1 mice.
• Application: B-HLA-A2.1/hLMP2 MC38 tumor models can be used for preclinical evaluation of cancer vaccines.

Protein expression analysis

HLA-A2.1 and LMP2 expression analysis in B-HLA-A2.1/hLMP2 MC38 cells by flow cytometry and western blot, respectively. Single cell suspensions from wild-type MC38 and B-HLA-A2.1/hLMP2 MC38 #2-A05 cultures were stained with species-specific anti-human HLA-A2.1 antibody (Biolegend, 343306), anti-LMP2 (Santa Cruz, sc-101315). Both human HLA-A2.1 (A) and LMP2 (B) were detected on the surface of B-HLA-A2.1/hLMP2 MC38 cells but not wild-type MC38 cells.

Tumor growth curve & body weight changes

Subcutaneous tumor growth of B-HLA-A2.1/hLMP2 MC38 cells. B-HLA-A2.1/hLMP2 MC38 cells (1×10⁶) and wild-type MC38 cells (5×10⁵) were subcutaneously implanted into B-HLA-A2.1 mice (female, 7-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/hLMP2 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

Protein expression analysis of tumor tissue

HLA-A2.1 expression evaluated on B-HLA-A2.1/hLMP2 MC38 tumor cells by flow cytometry and western blot, respectively. B-HLA-A2.1/hLMP2 MC38 cells were subcutaneously transplanted into B-HLA-A2.1 mice (n=7). Upon conclusion of the experiment, tumor cells were harvested and assessed for human HLA-A2.1 (Biolegend, 343306) by flow cytometry. As shown, human HLA-A2.1 was highly expressed on the surface of tumor cells. Therefore, B-HLA-A2.1/hLMP2 MC38 cells can be used for in vivo efficacy studies evaluating novel LMP2 therapeutics.