This protocol is among the first to describe the use of an autoinjector apparatus in Drosophila tumor allotransplantation. A topic that has grown in popularity in recent years. The main advantage of this method is that it increases the efficiency of cancer research in Drosophila by improving the speed, consistency and yield of tumor allotransplantation.
Tumor allotransplantation in Drosophila is a delicate technique that can be difficult to master. Be patient and trust that practice makes perfect. After crossing the flies and incubating the eggs for six days at 18 degrees Celsius, transfer the vials containing the hatched larvae to an incubator set at 29 degrees Celsius.
Sort the anesthetized wild type or mutant adult flies based on their sexes. Secure a five centimeter long piece of fly tape to a microscope slide with the sticky side up. Immobilize the flies by adhering their wings to the tape with forceps.
Place the controller box and the autoinjector on opposite sides of the light microscope. Process one end of the glass capillary using a four-step micro pipette puller, heating it to a narrow closed end. Use forceps to clip the capillary at an approximately 60 degree angle to prepare a 3.5 inch glass capillary.
Lightly unscrew the cap of the injector. Hold down the empty button to advance the injector needle until 70 to 80%of it's total length is showing. Use a syringe to fill the glass capillary with mineral oil.
Select one of the larvae and transfer to a dissection plate filled with 100 microliters of Schneider's Medium to prepare for SG imaginal ring tumor dissection. Using one pair of forceps to hold the midsection of the larval body, pinch the larval head using another pair of forceps and apply a stretching force lengthwise. Locate and isolate the Y shaped SG of the larvae, then dissect the SG imaginal ring tumor by removing the adjacent tissue.
Fill the glass capillary with Schneider's Medium all the way to the top 0.5 centimeter segment by holding the fill button. Locate a primary tumor and press the fill button until the tumor is suctioned into the capillary. Ensure that the tumor sits at the tip of the capillary or several millimeters away from the tip of the capillary.
Locate an adult fly immobilized to the tape. Then use forceps to gently hold down the lower abdomen and pierce the lower lateral cuticle of the abdomen with the capillary. Press the empty button until the tumor enters the new host abdomen.
Using forceps, gently pinch the wings of the host up to remove it from the tape. Then place it into a new vial with fresh food. Screen for tumors that have grown in the host abdomens using a fluorescence microscope around 10 to 14 days post allograph and dissect the grown allografted tumor out of the host using two pairs of forceps.
Use one pair of forceps to hold down the abdomen and the other pair to incise open the abdominal cuticle, exposing the allografted tumor. Carefully isolate the tumor from the attached host tissues. Use sterile needles and dissect the tumors into smaller pieces that are appropriate for the capillary size.
Repeat this process for a new generation of adult hosts to complete the allotransplantation of the tumor. The process of tumor development was tracked by live imaging of a first generation SG imaginal ring tumor growing in an adult host abdomen on day 10, post-allotransplantation. A sixth generation SG imaginal ring tumor occupying a large portion of the host abdomen on day 10, post-allotransplantation is shown here.
Imaging at this stage may help reveal tumor growth patterns as well as its migration and invasion behaviors. Tumor transplantation is a great technique for longitudinal studies for tumor progression, evolution and a metastasis. Results of great importance, for studying tumor host interactions.
This proto has great potential in cancer drug screens using the drugs Temodal. Using this technique, researchers will be able to save significant time and effort in the investigation.
