High-Throughput Dissociation and Orthotopic Implantation of Breast Cancer Patient-Derived Xenografts

Research in my laboratory is focused on understanding the underlying mechanisms that promote muscle fatigue in patients with breast cancer. Specifically, we want to understand the communication between a tumor localized in the breast tissue with peripheral skeletal muscle, and then the molecular responses within that muscle that lead to fatigue. The development of 3D cell culture models is enabling high-throughput studies where functional responses such as muscle fatigue can be studied under controlled conditions.

Functional cell culture experiments can be conducted in parallel and require less time than equivalent animal models, facilitating rapid discovery and innovation. Assessing relevant functional phenotypes using traditional patient derived xenograft models is both time consuming and resource intensive. This protocol facilitates simultaneous injection of PDXs into mice, allowing researchers to study systemic responses to human tumors using well-powered animal experiments.

This protocol allows the orthotopic injection of patient-derived breast tumor cells at an improved scale over existing methods facilitating rapid injection of up to several dozen mice per day by a single researcher. Additionally, dissociation of the tumor generates a homogenous cell suspension that evenly distributes cell types between animals. Data from my laboratory has identified similar muscle molecular responses to breast cancer in the PDX mouse and human cancer patients.

Therefore, we want to utilize this preclinical mouse model to screen candidate drugs and identify therapies with the potential to mitigate fatigue in patients with breast cancer. To begin, thaw the enzymes H, R and A from the human tumor dissociation kit. Add 200 microliters of enzyme H, 100 microliters of enzyme R and 25 microliters of enzyme A in a sterile tube C.Add 4.7 milliliters of sterile RPMI 1640 medium to the C tube to adjust the final volume to approximately five milliliters.

Then transfer the thawed tumor fragments and the accompanying freezing medium onto one half of a sterile 100-millimeter culture dish. Using sterile forceps, transfer the tumor fragments to a sterile five-milliliter micro tube and wash them with one to three milliliters of sterile PBS. Using sterile forceps, transfer the disinfected fragments to a new five-milliliter micro tube.

After a second wash with sterile PBS, transfer the fragments to the dry half of the 100-millimeter culture dish. Using two sterile number 10 scalpel blades, thoroughly mince the tumor tissue. Transfer the minced tumor fragments onto one blade and deposit them into the C tube containing the prepared enzyme solution.

Invert the C tube several times to ensure an even distribution of tumor fragments within the enzyme solution. Now place the C tube in the mechanical dissociator and invert the tube to ensure that all contents are submerged in the medium. Select the 37CHTDK3 program from the touchscreen interface.

Upon completion of the dissociation program, inspect the tube contents. Once adequate dissociation is achieved, centrifuge the C tube at 200G for five to eight minutes at four degrees Celsius. Using vacuum aspiration or a pipette, carefully remove the supernatant and re-suspend the cell pellet in RPMI 1640 to a volume equivalent to half the desired final injection volume.

In a two-milliliter round-bottom micro centrifuge tube, combine the desired amount of diluted cell suspension in RPMI with an equal volume of Matrigel. Gently mix the suspension by repeated pipetting to ensure homogeneity. To begin, dissociate human breast tumors into a single-cell suspension.

Then, using a one-milliliter syringe without an attached needle, gently aspirate the cell suspension up and down to ensure homogeneity. Attach a 26-gauge needle to the syringe and withdraw the desired injection volume. Then gently tap or flick the syringe to eliminate any air bubbles.

Apply a thin layer of depilatory cream to the target injection site on the mouse. After cleaning the injection area, grasp the dorsal skin firmly at the nape and back to securely restrain the mouse and place the mouse in a supine position. Insert the needle at a 45-degree angle pointing toward the hip approximately 0.5 to one centimeter caudal to the fat pad, adjusting for needle length.

Advance the needle into the fat pad and, in a controlled manner, inject the desired volume of cell suspension. Post-injection, rotate the needle 180 degrees and maintain the position briefly before slowly withdrawing it to minimize cell loss from the injection site.