B-HLA-A2.1/hAFP MC38

Basic Information

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. This gene encodes alpha-fetoprotein, a major plasma protein produced by the yolk sac and the liver during fetal life. Alpha-fetoprotein expression in adults is often associated with hepatocarcinoma and with teratoma, and has prognostic value for managing advanced gastric cancer. However, hereditary persistance of alpha-fetoprotein may also be found in individuals with no obvious pathology. The protein is thought to be the fetal counterpart of serum albumin, and the alpha-fetoprotein and albumin genes are present in tandem in the same transcriptional orientation on chromosome 4. Alpha-fetoprotein is found in monomeric as well as dimeric and trimeric forms, and binds copper, nickel, fatty acids and bilirubin. The level of alpha-fetoprotein in amniotic fluid is used to measure renal loss of protein to screen for spina bifida and anencephaly.
• 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 AFP CDS. Human HLA-A2.1 is highly expressed on the surface of B-HLA-A2.1/hAFP MC38. Human AFP is highly expressed on the surface of B-HLA-A2.1/hAFP MC38 cells.
• Tumorigenicity: Confirmed in B-HLA-A2.1 mice.
• Application: B-HLA-A2.1/hAFP MC38 tumor models can be used for preclinical evaluation of cancer vaccines.

Protein expression analysis

HLA-A2.1 and AFP expression analysis in B-HLA-A2.1/hAFP MC38 cells by flow cytometry and western blot, respectively. Single cell suspensions from wild-type MC38 and B-HLA-A2.1/hAFP MC38 #1-C11 cultures were stained with species-specific anti-human HLA-A2.1 antibody (Biolegend, 343306), anti-human AFP (abcam, ab169552), and anti-mouse AFP (abcam, ab213328). Both human HLA-A2.1 (A) and human AFP (B) were detected on the surface of B-HLA-A2.1/hAFP MC38 cells but not wild-type MC38 cells. In addition, both wild-type MC38 and B-HLA-A2.1/hAFP MC38 cells didn’t express the mouse AFP (C).

Tumor growth curve & Body weight changes

Subcutaneous tumor growth of B-HLA-A2.1/hAFP MC38 cells. B-HLA-A2.1/hAFP 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=7). 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/hAFP 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 and AFP expression evaluated on B-HLA-A2.1/hAFP MC38 tumor cells by flow cytometry and western blot, respectively. B-HLA-A2.1/hAFP 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) and human AFP (abcam, 169552) expression by flow cytometry and western blot, respectively. As shown, both human HLA-A2.1 (A) and human AFP (B) were highly expressed on the surface of tumor cells. Therefore, B-HLA-A2.1/hAFP MC38 cells can be used for in vivo efficacy studies evaluating novel AFP therapeutics.