P-276 Robust and cost effective in-vivo CAM models for drug combination study in colorectal cancer during COVID-19 pandemic

COVID-19 pandemic has affected research activities, as a direct consequence of lockdown measures and the closure of most university research facilities. We have utilized CAM (Chick Chorio-Allantoic Membrane) assay over mouse models for our drug combination study. CAM assay is minimally invasive to the chicken embryo and hence is a refinement model for animal research. CAM is formed by the fusion of the mesodermal layers of two developmental structures: the Allantois and the Chorion of the chick embryo. The embryo of the chicken develops over 21 days and the CAM forms on day 3 to day 4 and serve as a respiratory organ with a rapidly developing vascular system. The CAM assay was performed using four cell lines DLD1 (KRAS/PIK3CA mutated) CACO2 (KRAS/PIK3CA wild-type), LS1034 (KRAS mutated) and SNUC4 (PIK3CA mutated) to demonstrate the anti-tumour effects of Ribociclib (R), Alpelisib (A), the combination of Ribociclib and Alpelisib (R+A) or DMSO as matched vehicle control (VC). On D(day)1 cleaned eggs put in incubator then on D3 egg windows were opened and inspected for vascularization. After five days of incubation, the windows were reopened and silicon rings were gently placed on the CAM. 2 x 106 cells were then mixed 1:1 with Matrigel and slowly dropped within the silicon ring. On D10, Treated with either R, A, the combination of R+ or VC. Tumor extraction for histology examination was done on D14 and immunohistochemically stained for Ki67 and cytokeratin. In the CACO2 model, none of the CAMs treated with the combination of R and A had visible tumours (VT) by the study endpoint. In the DLD1 group 2/6 (33%) of CAMs treated with the combination of R and A had VT, while 3/6 (50%) of CAMs in the LS1034 group and 5/9 (56%). CAMs in the SNUC4 group had VT following treatment with the combination. At least 80% of DMSO treated CAMs in all groups, other than the DLD1 group 3/6 (50%), had VT 7/8 (80%) in the CACO2 group, 5/5 (100%) in the LS1034 group and 9/11 (82%) in the SNUC4 group. In the CACO2-CAM, 5/8 (62%) of tumours in the R and A combination treated group were negative for cytokeratin and demonstrated Ki67 staining less than 50%. In the DLD1-CAM, 3/6 (50%) of tumours in the R and A combination treated group did not have any expression of cytokeratin and demonstrated Ki67 staining less than 50%. In the LS1034-CAM, 4/5 (75%) of tumours in the R and A combination treated group. In the SNUC4-CAM, 3/9 (34%) of tumours in the R and A combination treated group were negative for cytokeratin expression and demonstrated Ki67 staining less than 50%. The CAM assay is a robust and cost effective in-vivo model. We noted significant both macroscopic and microscopic evidence of best response with the R and A combination as compared to those treated with single agent drugs or DMSO vehicle control was in the CAM xenografts. These findings provide novel insight into a possible therapeutic strategy for patients with relapsed refractory CRC.