PDXCF Core specific operations:
1.1 PDX development: project design and standard operational procedures
To generate PDX models, the PDXCF and Project Leader will meet to discuss the goals of the project and develop a plan of action. This discussion will identify 1) a defined source of tissue procurement; 2) a detailed time line for project execution; and 3) the number and specific type of PDXs desired to be developed. The PDXCF will assist in tissue procurement via submission of research projects to the WVU Tissue Bank, and prompt delivery of freshly procured tissue (2-12h) to PDXCF. Alternatively, if the specific type of cancer/tissue is not available via the WVU Tissue Bank, the Research Project PI will be advised to 1) submit a new IRB protocol designed specifically for procurement of the tissue of interest to the WVU IRB or 2) submit a protocol for tissue procurement via the Cooperative Human Tissue Network (CHTN). The CHTN is an NIH-supported resource, which aids NIH-funded investigators across the country and allows for fast, inexpensive tissue procurement. The PDXCF will assist PIs with submission of IRB or/and CHTN protocols to enable PDX production.
The tissue procured from either of the above-mentioned sources will come to PDXCF with 1) paraffin-embedded tissue from the same area as the fresh tissue used for grafting (to have a reference control for future PDX); 2) a deidentified pathology report (needed for molecular classification and history of treatments); and 3) if more tissue is available, it will be cryopreserved to allow for future re-implantation in case of animal death or infections. The tissue will be immediately grafted into two NSG mice. Each fragment of 2x2x2mm or tumor mesh of 100 microliters with or without matrigel will be surgically transplanted or injected depending on the specific protocol design (which is different for each type of cancer) into NSG mice. Following wound closure and post-operative care, animals bearing patient’s tumors will be housed in the PDXCF animal holding room in microisolators and inspected weekly for tumor growth either via palpation (if subcutaneous or mammary gland housed) or via ultrasound imaging using the Vevo2000 imaging system (AMIF support). Once detected, tumors will be closely followed until they reach 1 cm in diameter or the animal is found in distress with signs of pain, limited mobility or other clinical symptoms as evaluated by a clinical veterinarian. The tumors will be collected, cut into fragments and stored in liquid nitrogen for further experiments planned by the PI. These tumors are designated as PDX p0 (patient-derived xenograft passage zero since they have yet to be passaged but demonstrated successful grafting). Two to three fragments of PDX p0 will be immediately transplanted in naïve NSG mice in accordance with the developed and tested transplantation technique specific for each type of tumor. If the graft successfully grows, the resultant tumors will be collected, fragmented and delivered to the PI as both fresh and cryopreserved samples, and some PDX sample will be stored in the WVUCI Biospecimen Processing Core Facility for banking and further distribution to other PIs.
The time line for project execution needs to be discussed with PDXCF in advance since PDX development takes a significant amount of time (up to 1 year). The arrangements for tissue procurement should be placed up to one year before the actual experimental procedures using these PDXs are planned so that PDXCF will be confident in delivering the requested PDXs to the PIs. Not every procured tissue will generate PDX. Thus, more tissue and NSG mice will be required to develop a number of PDXs for PIs. The success rates depend upon many parameters and are highly dependent on the type of cancer, quality of tissue, and site of transplantation. The PDXCF has significant experience with establishment and growth of brain, head and neck, and breast PDXs. The number of tissue samples to be procured and NSG mice injected will be determined based on the specific project needs and tumor type.
1.2 PDX propagation
PDXs established at WVU have already been made available to investigators. For example, Dr. Pugacheva’s laboratory has a panel of breast cancer PDXs that are currently used by multiple investigators. Alternatively, the PDXCF offers services to propagate/grow PDXs established elsewhere. In this case, the PDXCF will assist with the search, negotiation and development of MTA agreements to get the established PDXs released for research purposes to the WVU PIs. Both types of sample procurement (internal and external sources) are available for a fee-for-service fixed price, which ranges from $0-$500, while costs to procure tissues externally from NIH-subsidized sources are approximately $100. The PDX-bearing animals either will be transferred to a PI’s animal protocol for further studies or will be analyzed/treated/processed by PDXCF staff (for a fee) if the Research PI has no experience working with animals. The propagated PDXs or resultant product will be delivered to the PI. The PDXs will be cryopreserved and submitted to the Biospecimen core for further storage and archiving for potential future use.