Description

• Origin: The CT26.WT cell line is derived from BALB/c murine colon adenocarcinoma cells. The cell line is a commonly used murine model for colorectal carcinoma.
• Background Information: Glypican-3 (GPC3) is a core protein of 580 amino acids that has two heparan sulfate (HS) side chains located near its C-terminal. GPC3 can complex with growth factors like Wnt, fibroblast growth factor (FGF), and hepatocyte growth factor (HGF) through its HS side chains. GPC3 (Glypican-3) is highly overexpressed in various malignant tumors (such as hepatocellular carcinoma, malignant melanoma, ovarian cancer, etc.), while it is barely expressed in normal adult tissues (except in a few tissues like the placenta). This tumor-specific expression profile makes GPC3 an ideal therapeutic target.
• Gene targeting strategy: The exogenous promoter, human GPC3 coding sequence and puromycin resistance gene were inserted to replace part of murine Gpc3 exon 1. The insertion disrupts the endogenous murine Gpc3 gene, resulting in a non-functional transcript.
• Tumorigenicity: Confirmed in BALB/c mice.
• Application: B-hGPC3 CT26.WT plus tumor models can be used for preclinical evaluation of monoclonal antibodies, bispecific antibodies, antibody-drug conjugates (ADCs)—that targeting human GPC3.
• Notes:

        Inoculated cell lines can be suspended with DMEM stock solution.

        Before implementing the project, it is recommended to perform tumor growth experiments. The recommended cell inoculation amount is between 1E5.

        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.

Protein expression analysis

GPC3 expression analysis in B-hGPC3 CT26.WT plus cells by flow cytometry. Single cell suspensions from wild-type CT26.WT and B-hGPC3 CT26.WT plus #1-G01 cultures were stained with GC33-hIgG1 analog (in-house). Human GPC3 was detected on the surface of B-hGPC3 CT26.WT plus cells but not wild-type CT26.WT cells.

Tumor growth curve & body weight changes

Subcutaneous tumor growth of B-hGPC3 CT26.WT plus. B-hGPC3 CT26.WT plus (1×10⁵) and wild-type CT26.WT cells (1×10⁵) were subcutaneously implanted into BALB/c mice (female, 7-week-old, n=6). Tumor volume and body weight were measured three times 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-hGPC3 CT26.WT plus 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

GPC3 expression evaluated on B-hGPC3 CT26.WT plus tumor cells by flow cytometry. B-hGPC3 CT26.WT plus cells were subcutaneously transplanted into BALB/c mice (n=6). Upon conclusion of the experiment, tumor cells were harvested and assessed for human GPC3 expression by flow cytometry (GC33-hIgG1 analog in-house). As shown, human GPC3 was highly expressed on the surface of tumor cells. Therefore, B-hGPC3 CT26.WT plus cells can be used for in vivo efficacy studies evaluating novel GPC3 therapeutics.