Basic Information
Description
The mouse Pdl1 gene and Ror1 gene were replaced by human PD-L1 and ROR1 coding sequence in B-hPD-L1 plus/hROR1 MC38 cells. Human PD-L1 and ROR1 are highly expressed on the surface of B-hPD-L1 plus/hROR1 MC38 cells.
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Targeting strategy
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Gene targeting strategy for B-hPD-L1 plus/hROR1 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 promoter and human ROR1 coding sequence was inserted to replace part of murine exon 2 and all of exon 3. The insertion disrupts the endogenous murine Ror1 gene, resulting in a non-functional transcript.
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Protein expression analysis
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PD-L1 and ROR1 expression analysis in B-hPD-L1 plus/hROR1 MC38 cells by flow cytometry. Single cell suspensions from B-hPD-L1 plus/hROR1 MC38 cultures were stained with species-specific anti-PD-L1 and anti-ROR1 antibodies. Human PD-L1 and ROR1 were detected on the surface of B-hPD-L1 plus/hROR1 MC38 cells. The 1-D08 clone of B-hPD-L1 plus/hROR1 MC38 cells was used for in vivo experiments.
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Tumor growth curve & Body weight changes
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Subcutaneous homograft tumor growth of B-hPD-L1 plus/hROR1 MC38 cells. B-hPD-L1 plus/hROR1 MC38 cells (5×105) and wild-type MC38 cells (5×105) were subcutaneously implanted into C57BL/6N mice (female, 6-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 plus/hROR1 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.
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Tumor growth curve of individual mice
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B-hPD-L1 plus/hROR1 MC38 tumor cells growth of individual mice. B-hPD-L1 plus/hROR1 MC38 cells (5×105) and wild-type MC38 cells (5×105) were subcutaneously implanted into C57BL/6Nmice (female, 6-week-old, n=6). As shown in panel, B-hPD-L1 plus/hROR1 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.
Tumor volume and weight measurements
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Protein expression analysis of tumor cells
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B-hPD-L1 plus/hROR1 MC38 cells were subcutaneously transplanted into C57BL/6N mice (n=6), and on 35 days post inoculation, tumor cells were harvested and assessed for human PD-L1 and ROR1 expression by flow cytometry. As shown, human PD-L1 and ROR1 were highly expressed on the surface of tumor cells. Therefore, B-hPD-L1 plus/hROR1 MC38 cells can be used for in vivo efficacy studies of novel PD-L1 and ROR1 therapeutics.
