Description
The mouse B7h3 gene was replaced by human B7-H3 coding sequence in B-hB7-H3 MC38 cells. Human B7-H3 is highly expressed on the surface of B-hB7-H3 MC38 cells.
Targeting strategy
Gene targeting strategy for B-hB7-H3 MC38 cells. The exogenous promoter and human B7-H3 coding sequence was inserted to replace part of murine exon 3 and all of exon 4. The insertion disrupts the endogenous murine B7h3 gene, resulting in a non-functional transcript.
Protein expression analysis
B7-H3 expression analysis in B-hB7-H3 MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hB7-H3 MC38 cultures were stained with species-specific anti-B7-H3 antibody. Human B7-H3 was detected on the surface of B-hB7-H3 MC38 cells but not wild-type MC38 cells. The 2-F02 clone of B-hB7-H3 MC38 cells was used for in vivo experiments.
Protein expression analysis of tumor cells
B-hB7-H3 MC38 cells were subcutaneously transplanted into C57BL/6 mice (n=5). At the end of the experiment, tumor cells were harvested and assessed for human B7-H3 expression by flow cytometry. As shown, human B7-H3 was highly expressed on the surface of tumor cells. Therefore, B-hB7-H3 MC38 cells can be used for in vivo efficacy studies of novel B7-H3 therapeutics.
FMO Control 1: MC38 tumor cells only stained anti-mB7-H3 antibody;
FMO Control 2: B-hB7-H3 MC38 tumor cells only stained anti-mB7-H3 antibody.
Tumor growth curve & Body weight changes
Subcutaneous homograft tumor growth of B-hB7-H3 MC38 cells. B-hB7-H3 MC38 cells (1x106) and wild-type MC38 cells (5x105) were subcutaneously implanted into C57BL/6 mice (female, 8-week-old, n=5). 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-hB7-H3 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.
Recommendations for inoculation in other mouse strains:
Cell inoculation amount is recommended to be tried between 1x106-1x107;
Inoculated cells can be tried to be suspended with DMEM stock solution.
Subcutaneous homograft tumor growth of B-hB7-H3 MC38 cells. B-hB7-H3 MC38 cells and wild-type MC38 cells were subcutaneously implanted into B-h4-1BB mice (female, 8-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 X long diameter X short diameter2. As shown in panel A, B-hB7-H3 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.
