전임상 환자 유래 종양 소설 안티 - 암 치료의 조사를위한 이종 이식 모델의 개발 및 유지 보수

The overall goal of this subcutaneous Patient Derived Tumor Xenograft procedure is to the study the efficacy of novel therapies predictive biomarker discovery and drug resistant pathways in tumors. This method can help answer key questions in the oncology field about whether a particular drug exhibits activity against a specific tumor type. The main advantage of this technique is that Patient Derived Tumor Xenographs maintain the molecular, genetic and histologist heterogeneity typical of the tumors of origin, making the model more clinically relevant.

Demonstrating the procedure will be Stacey Bagby, a professional research assistant and certified veterinary technician from our laboratory. After collecting one to two milliliters of blood in a blood cell separation tube containing sodium citrate, centrifuge the samples and transfer the peripheral blood mononuclear cell layer into a 1.5 milliliter micro-centrifuge tube. Fill the tube to the top with sterile PBS.

Then, spin down the cells again. Followed by a quick wash with one milliliter of PBS, being careful not to disturb the PMBC pellet. Then, use a pipette to transfer the plasma from the blood cell collection tube to a 1.5 milliliter sterile cryogenic vial, and store the plasma and the PBMC pellet at negative 80 degrees Celsius.

Transfer the human tumor tissue to the animal facility in a sterile cup containing complete medium and place the cup in a laminar flow hood. As soon as possible, after harvesting, transfer the tissue into a sterile plastic cutting dish in a small amount of the retrieval medium. Then, using autoclave sterilized small scissors and forceps, cut the tissue into approximately three by three by three milliliter pieces and place 10 to 12 pieces into an autoclave sterilized 1.5 milliliter micro-centrifuge tube containing 300 microliters of gelatinous protein mixture solution on ice for the implantation.

For flash frozen storage, transfer the appropriate number of tumor pieces into a new 1.5 milliliter micro-centrifuge tube and place the vial in a liquid nitrogen doer. Then, place in a minus 80 degrees Celsius freezer. For formal infixing and paraffin embedding, place three small tumor pieces into a 10 milliliter 10%formalin cup for 24 hours.

After the tissues are processed into paraffin embedded blocks, transfer any remaining tissue pieces into a cryogenic tube. Then, add one milliliter of complete medium supplemented with 10%DMSO to the tissue and store the tubes on ice until they can be transferred into an isopropyl alcohol freezing container. And put that container in a minus 80 degrees Celsius freezer.

To inject the tumor tissue, load the pieces of tumor stored in the gelatinous protein mixture solution into autoclaved 12-gauge trocars, taking care that each tumor piece is completely inserted into the trocar. Next, confirm a lack of response to toe pinch in a six to eight week old athymic, nude mouse and transfer the mouse onto a clean field in a sterile laminar flow hood. Deliver the F0 tumor subcutaneously to the dorsal neck region and to each side of the mouse's flank, followed by the subcutaneous administration of analgesia distal to one of the flank sites of tumor injection.

Then, place the mouse back in its cage with monitoring until it is fully recovered. Monitor the growth and health of the xenograph-implanted mice at least once per week, recording the tumor sizes, tumor generation, date of tumor injection, and health of the mice in an appropriate tracking system. When a tumor reaches approximately 1, 500 to 2, 000 cubic millimeters in size, use autoclaved scissors and forceps to excise the tumor and passage the F1 tumor into the next generation of five animals, as just demonstrated, taking care to collect flash frozen formalin and viable tumor samples as demonstrated for each tumor.

When the remaining mice from the F0 tumor group exhibit tumors approximately 1, 500 to 2, 000 cubic millimeters in size, collect these tumors as just demonstrated for collection. When the stage F15 tumor generation is reached, use the earliest generation of viable tumor pieces available in liquid nitrogen storage for the next series of tumor injections. When the desired tumor is very large, 1, 500 to 2, 000 cubic millimeters, follow the procedures above to collect the tumor to expand it into the desired amount of mice.

Dose the mice with the drug and weigh and measure them twice per week. Randomize the mice into treatment groups with 10 tumors per group, and a group tumor volume average of 100 to 300 cubic millimeters within a few standard deviations of each other. Then, sort the mice into the appropriate groups for treatment.

Dual color fluorescence in situ hybridization for the human and mouse caught one DNA demonstrates that the human stromal cells in the F0 tumor are replaced by the mouse stromal cells in the F1 tumor. Whereas, all of the human-derived hepatocyte growth factor in the F0 generation is replaced with mouse hepatocyte growth factor in F1 generation, the vascular endothelial growth factor ligand consists of both human and mouse protein in the F1 generation. The treatment of different generations of the same tumor does not change the treatment response.

With the resistance or sensitivity of the tumor appearing to be tumor strain rather than tumor generation specific. Further, the frequency of mutations in these commonly mutated genes is very similar between the cancer genome atlas and the colorectal patient-derived tumor xenograph bank. Suggesting that the common mutations observed in the colorectal patient population are well represented in the colorectal patient-derived xenotransplant model.

Once mastered, this technique can be completed in one to two hours, if performed properly. While attempting this procedure, it is important to have a cohesive clinical team for identifying and obtaining consent from the cancer patients and for the removal and grossing of the tumor tissue. Following this procedure, other methods, like the investigation of anti-cancer compounds as single or combination agents, can be performed to answer additional questions about the viability of these strategies for patients with a particular tumor type.

After its development, this technique paved the way for researchers in the field of oncology to explore novel cancer therapeutics, tumor heterogeneity, treatment resistant mechanisms, cancer stem cell biology, and tumor stromal interactions. After watching this video, you should have a good understanding on how to develop and maintain a patient derived tumor xenograph model for evaluating novel cancer therapeutics.