The mouse Spn gene was replaced by the human SPN coding sequence in B-hSPN MC38 cells. Human SPN is highly expressed on the surface of B-hSPN MC38 cells.
Gene targeting strategy
Gene targeting strategy for B-hSPN MC38 cells. The exogenous promoter and human SPN coding sequence was inserted to replace part of murine exon 2. The insertion disrupts the endogenous murine Spn gene, resulting in a non-functional transcript.
Protein expression analysis
Species-specific SPN protein expression analysis in B-hSPN MC38 cells. Single cell suspensions from wild-type MC38 and B-hSPN MC38 cultures were stained with species-specific anti-SPN antibodies. Murine SPN protein was detected on the surface of MC38 cells, while human SPN protein was detected on the surface of B-hSPN MC38 cells. The 1-A05 clone of B-hSPN MC38 cells was used for in vivo experiments.
Tumor growth curve & Body weight changes
Subcutaneous homograft tumor growth of B-hSPN MC38 cells. B-hSPN MC38 cells (2×105) and wild-type MC38 cells (5×105) were subcutaneously implanted into C57BL/6 mice (female, 9-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-hSPN MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.
Cell Line Inoculation Precautions:
The cell inoculation amount is between 5E5-1E6.
Tumor growth curve of individual mice
B-hSPN MC38 tumor cell growth in individual mice. B-hSPN MC38 cells (2×105) and wild-type MC38 cells (5×105) were subcutaneously implanted into C57BL/6 mice (female, 9-week-old, n=6). As shown in panel, B-hSPN MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.
Tumor volume and weight measurements
Protein expression analysis post-inoculation
B-hSPN MC38 cells were subcutaneously transplanted into C57BL/6 mice (n=6), 42 days post inoculation, tumor cells were harvested and assessed for human SPN protein expression by flow cytometry. As shown, human SPN was highly expressed on the surface of tumor cells. Therefore, B-hSPN MC38 cells can be used for in vivo efficacy studies to test novel SPN therapeutics.