The CDS of human C5 was inserted to replace part of murine exon 2 in B-hC5 MC38 cells. Human C5 is highly expressed on the surface of B-hC5 MC38 cells.
B-hC5 MC38 cells have the capability to establish tumors in vivo, which can be used for efficacy studies.
The CDS of human C5 was inserted to replace part of murine exon 2. The insertion disrupts the endogenous murine C5 gene, resulting in a non-functional transcript.
Protein Expression Analysis
C5 expression analysis in B-hC5 MC38 cells by ELISA. Supernatant from wild-type MC38 and B-hC5 MC38 cultures were stained with an anti-human C5 antibody. Human C5 was detectable in the supernatant of B-hC5 MC38 cells but not in that of wild-type MC38 cells. The 1-G04 clone of B-hC5 MC38 cells was used for in vivo experiments.
Species-specific C5 and C5a expression analysis in serum and tumor tissues by ELISA. Wild-type MC38 cells and B-hC5 MC38 cells were subcutaneously inoculated into C57BL/6 mice. Serum and tumors were collected at two different timepoints. The first timepoint marks the tumor volume reaching 300-500 mm3 while the second timepoint marks the tumor volume reaching 3000 mm3. Mouse and human C5 and C5a expression was analyzed using a species-specific ELISA kit. Murine C5 and C5a protein was detectable in serum and tumor tissues collected from mice inoculated with wild-type MC38 cells and B-hC5 MC38 cells. Human C5 and C5a expression was detectable in tumor tissues collected when tumor volume reached 300-500 mm3 from mice inoculated with B-hC5 MC38 cells. Human C5 and C5a was not detectable in tumor tissues when tumor volume reached approximately 3000 mm3.
Species-specific CD88 (C5AR1) expression analysis in MC38 cells and tumor tissues by flow cytometry. Wild-type MC38 cells and B-hC5 MC38 cells were subcutaneously inoculated into C57BL/6 mice. Wild-type MC38 cells and tumor tissues were cultured after the tumor volume reached 3000 mm3, and analyzed by flow cytometry using an anti-mouse CD88 antibody. Mouse CD88 was not detectable in cultured MC38 cells, however tumor tissues expressed mCD88 in CD45+ cells and, to a lesser extent, CD45− cells.
Tumor Growth Curve
Tumor Growth Curve & Body Weight Changes in C57BL/6N Mice
Subcutaneous homograft tumor growth of B-hC5 MC38 cells. B-hC5 MC38 cells (1×106) and wild-type MC38 cells (1×106) were subcutaneously implanted into C57BL/6N mice (female, 7-week-old, n=5). (A) Average tumor volume ± SEM and (B) body weight (Mean± SEM) were measured twice a week. Three mice from each group were sacrificed on day 14 and the remaining mice were sacrificed at the endpoint. Volume was expressed in mm3 using the formula: V=0.5 X long diameter X short diameter2. As shown in panel A, B-hC5 MC38 cells were able to establish tumors in vivo, which can be used for efficacy studies.