The patient-derived xenograft (PDX) is a powerful model of human cancer designed with the focus on personalized medicine. It has the potential to become part of a cancer patient?s standard care (cancer avatars) and a superior platform for drug discovery, mechanistic studies on cancer drug resistance, and screening of the potential biomarkers of cancer progression/metastasis and drug response. In combination with current work on developing humanized mice, it will be able to explain interactions within the patient?s immune system as well. However, the methodology and tools for genetic manipulations in PDX are currently unavailable, representing a significant barrier for usage of PDX models in the research community. Our objective is to validate developed in our laboratory PD-VivoS toolkit and methodology to enable genetic manipulation of PDXs. This methodology will allow for self-inactivating lentivirus-based delivery of inducible shRNAs/miRNA, or cDNA and luciferase directly into the PDX growing in a mouse along with the ability to select for the infected tumor cells via diphtheria toxin administration. Such a system will allow for the robust tumor cell genomic manipulation in vivo, enabling the inclusion of PDX models as a standard cancer research platform.
The specific aims of the current application are: 1) to validate the system developed in our laboratory for the ability to successfully infect, select and express the desired cDNA/or shRNA in multiple different cancer PDXs available via our PDX core facility; 2) to systematically examine the PDX biology (growth, pathology, genomic/transcriptome integrity) upon introduction of the empty cassette/s and selection with diphtheria toxin for any potential changes it might induce. This analysis will allow us to account for any non-specific effects this methodology might possess and define its limitations. To achieve these goals, two PD-VivoS vectors (to express cDNA or sh/miRNA) were generated for production of self-inactivating lentivirus, and coding for firefly luciferase fused with shRNA against DPH2 and multiple cloning site for cDNA or sh/miRNA insertion under doxycycline inducible promoter. PDXs, grown in NSG mice, will be infected through direct intratumoral injections of concentrated viral stock and selected via intraperitoneal injections of diphtheria toxin. The luciferase signal will be used to control for successful infection and to follow up the growth/selection of infected tumor cells. The resultant PD-VivoS modified PDX tumor will be collected, frozen for longer term storage or could be directly re-transplanted into nave mice to induce cDNA/shRNA expression with doxycycline food/water. The PD-VivoS-PDX tumor will be used for genomic and RNA sequencing to compare to parental PDX and define the effects of transgene transduction. The rational for this work is that there is no system currently available for in vivo genetic manipulation and selection, and that such a system will significantly increase the range of application for PDX.
The proposed project seeks to develop novel methodology to allow for genetic manipulations to be conducted in patient-derived xenograft (PDX) models, which is currently the most clinically relevant model of cancer. The successful completion of this project will allow researchers to use PDX models for robust genetic manipulation and detection of patient-derived cancer cells in mice. Further, this will facilitate the discovery and validation of cancer biomarkers, novel targeted therapy and increase the knowledge gained from studying cancer biology in the relevant context.
