Recently, the chicken ChorioAllantoic Membrane (CAM) assay has been used as an in vivo model for cancer research, In fact, the CAM assay is a highly vascularized extraembryonic membrane surrounding the embryo, which is a good soil for tumors to grow and provides the condition for studying the tumor metastatic invasion in different organs and tissues. Besides, at the Embryonic Development Day (EDD) 9 when tumor cells are xenografted, the embryo is an “adult” embryo and has all organs formed and functional, including an active immune system. Therefore, this model is suited for the development of different types of anti-cancer therapies. Here we used the CAM assay to investigate the therapeutic activity of different RNA-based approaches.

On EDD 9, a small window is made in the eggshell and allows easy access to the CAM. Cancer cell lines are then grafted onto the CAM and allowed to form tumors. From EED10 to EDD18 (end of the experiment) tumors can be treated with RNA-based therapeutics. Different therapeutics approaches have been used in ovo: (1) either through cell modification in vitro followed by graft in ovo or (2) by treating tumors with RNA directly in ovo. The effect of RNA mutation (1) or RNA therapeutic’s treatment (2) is evaluated through different parameter: the tumor weight, the histopathological analyses of tumors (classical staining or immunohistochemistry), the immune cell infiltration (by RT-qPCR), the detection of metastases in specific target tissues, the analysis of the angiogenic network surrounding the tumor and the analysis of the toxicity (mortality rate and/or abnormal development of the embryo).

In the first approach, the efficacy of siRNA, miRNA, RNAi and anti-sens RNA in various cancer types was studied: - On PC9 non-small cell lung cancer model, the expression of a siEx8 reversed the resistance of tumors to gefitinib by blocking a specific variant of the autophagic gene ATG16L1b. - On LNCaP prostate cancer model, the miR-135a inhibited ROCK1 expression and led to a significant regression in tumor cell invasion ability. In contrast, the inhibition of mTOR by a combination of 2 RNAi induced an increase in metastatic invasion. - On A375 melanoma cancer model, the suppressive effect of the anti-sens RNA (LADON) in the TGFβ pathway inhibited the cancer cell invasion. In the second approach, the efficacy of miRNAs was evaluated: - On SK-OV-3 ovarian cancer model, the combination of one miRNA with doxorubicin showed an additive effect of both compounds. - On BT-474 breast cancer model, the anti-miRNA loaded in lipid nanoparticle shows no effect on tumor, while reduced by 50% the metastatic invasion in the lower CAM.

The CAM model is a relevant alternative in vivo model that can be used for evaluating the efficacy of RNA-based therapies, in just a few days, with various interpretations on tumor growth, metastasis and as well as deeper analyses, like transcriptomics or proteomics.