Basic Information
Description
The mouse Pdl1 gene was replaced by human PD-L1 coding sequence in B-hPD-L1/hHER2 MC38 cells. Human PD-L1 is highly expressed on the surface of B-hPD-L1/hHER2 MC38 cells.
The mouse Erbb2 gene was replaced by human ERBB2 coding sequence in B-hPD-L1/hHER2 MC38 cells. Human ERBB2 is highly expressed on the surface of B-hPD-L1/hHER2 MC38 cells.
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Gene targeting strategy
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Gene targeting strategy for B-hPD-L1/hHER2 MC38 cells. The exogenous promoter and human PD-L1 coding sequence was inserted to replace part of murine exon 3. The insertion disrupts the endogenous murine Pdl1 gene, resulting in a non-functional transcript.
The exogenous CAG promoter and human ERBB2 coding sequence was inserted to replace part of murine exon 2 and all of exons 3-7. The insertion disrupts the endogenous murine Erbb2 gene, resulting in a non-functional transcript.
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Protein expression analysis
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PD-L1 and HER2 protein expression analysis in B-hPD-L1/hHER2 MC38 cells. Single cell suspensions from wild-type MC38 and B-hPD-L1/hHER2 MC38 cultures were stained with species-specific anti-PD-L1 and anti-HER2 antibodies. Human PD-L1 and HER2 were detected on the surface of B-hPD-L1/hHER2 MC38 cells but not wild-type MC38 cells. The 3-B08 clone of B-hPD-L1/hHER2 MC38 cells was used for in vivo tumor growth assays.
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Tumor growth curve in wild-type mice
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Subcutaneous homograft tumor growth of B-hPD-L1/hHER2 MC38 cells. B-hPD-L1/hHER2 MC38 cells (5×105) and wild-type MC38 cells (5×105) were subcutaneously implanted into C57BL/6 mice (female, 9-week-old, n=6). Tumor volume and body weight were measured twice a week. (A) Average tumor volume ± SEM. (B) Body weight (Mean ± SEM). Volume was expressed in mm3 using the formula: V=0.5 X long diameter X short diameter2. As shown in panel A, B-hPD-L1/hHER2 MC38 cells were able to form tumors in vivo, which can be used for efficacy studies.
Tumor volume and weight measurements
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Protein expression analysis post-inoculation
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B-hPD-L1/hHER2 MC38 cells were subcutaneously transplanted into C57BL/6 mice (n=6), 39 days post inoculation, tumor cells were harvested and assessed for human PD-L1 and HER2 protein expression by flow cytometry. As shown, human PD-L1 and HER2 were highly expressed on the surface of tumor cells. Therefore, B-hPD-L1/hHER2 MC38 cells can be used for in vivo efficacy studies to test novel PD-L1 and HER2 therapeutics.
