Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line

The overall goal of this procedure is to produce xenograft mice with circulating human cytokines. This method can help answer key questions in the cancer and hematopoiesis fields such as, What is the role of a specific cytokine in normal and malignant lymphocyte development? The main advantage of this technique is that investigators can ingest the circulating concentrations of exogenous cytokine in their xenograft animals according to the needs of their experiment.

This eliminates the need to produce new and expensive transgenic mouse lines. This technique will be useful for researching new cancer therapies, because this preclinical model can be used to test anticancer drugs, similar to what is used in the clinic. Though this method can provide insight into normal hematopoiesis, it can also be used to study specific diseases such as leukemia.

I first developed this idea when we needed an in vivo model to evaluate the role of the TSLP cytokine in normal and malignant human B-cell production. Begin by plating freshly thawed control empty vector-transduced stroma in cytokine-producing cells in five milliliters of R-10 medium in individual T25 flasks. For their culture, at 37 degrees Celsius in five percent CO2 for 24 to 48 hours.

When the stroma are confluent, detach the cells with Trypsan in EDTA for replating in T150 culture flasks for three to four days. When the stroma achieve confluence in the T150 flasks, collect the supernatants and freeze them at 80 degrees Celsius for later evaluation of their cytokine production by ELISA. Then, passage each T150 flask culture into three new T150 flasks for their culture until confluency.

To store the cells after the appropriate number of passages, transfer the harvested cell suspensions to a conical tube for centrifugation and resuspend the pellets in a freezing medium. Then transfer 1.5E6 cell aliquots into long-term storage tubes for their storage in liquid nitrogen. To validate the supernatant produced from engineered stroma, thaw the supernatant samples harvested from the control in cytokine-expressing stroma, and plate three wells of leukemia cell lines per condition in Au-20 medium at a 2E5 cells per well concentration in a 96-well tissue culture plate.

Incubate the cells for two hours at 37 degrees Celsius to allow for the phosphorylation from their prior culture to be lost. Then transfer the cells from each well into individual four milliliter tubes and collect the cells by centrifugation. Resuspend the pellets in 600 microliters per experimental condition.

Plate 200 microliters of cells per well for each condition in triplicate. Incubate the cells for the appropriate period according to the specific commercial phosphoassay. At the end of the incubation, pellet the cells by centrifugation.

Label the cells via phospho-flow cytometry staining according to the manufacturer's protocol. Then analyze the cells by flow cytometry using standard flow cytometric analysis protocols to verify that cytokine in the stroma supernatant phosphorylates the leukemia cells. After a minimum of three post-thaw passages, transfer the harvested stromal cell suspensions into individual conical tubes for counting.

Pellet the cells by centrifugation and gently resuspend them in the appropriate volumes of sterile PBS for injection. Hold the cells at four degrees Celsius until immediately before the injection at which point the suspension should be warmed to at least room temperature. When the cells are ready, gently mix the first sample by inversion and draw 200 microliters of the cells into a tuberculin syringe.

Then restrain the first animal and inject the cells into the peritoneal cavity using standard intraperitoneal injection techniques. To collect a peripheral blood sample, first gently position the mouse in a disinfected small animal restrainer and secure the tube to minimize the animal's movements. Next, scrub the tail with fresh isopropyl alcohol followed by the application of topical anesthetic cream.

Using very sharp surgical scissors, snip about 5 to one millimeter from the tip of the tail and collect approximately 80 microliters of peripheral blood into heparinized capillary tubes. Use a bulb syringe to transfer the blood samples into labeled potassium EDTA microtainer tubes and invert the tube 20 times to prevent coagulation. After centrifuging blood collection tubes per manufacturer's instructions, carefully collect the plasma layer and store the aliquots at 20 degrees Celsius and aliquot the plasma for 20 degree Celsius storage.

On the day of the ELISA, thaw the frozen mouse plasma samples and serially dilute the ELISA standard into 120-microliter volumes. Now plate 40 microliters of each standard in triplicate into the appropriate wells of the ELISA plate followed by the addition of 40 microliters of each mouse plasma sample to the appropriate wells. Then complete the ELISA analysis according to the manufacturer's instructions.

24 hours after irradiating the mice, prepare the human hematopoietic cells for xenograft transplantation. When you resuspend the hematopoietic cells in sterile PBS, it is a good idea to prepare about 15 to 20 percent extra, in case you have a spill during the transplantation. Keep the cells at four degrees Celsius until transplant and transfer the first recipient animal into a warming cage for at least five minutes to facilitate tail-vein dilation.

Now warm the cells to room temperature. Gently mix the cell suspension and draw 200 microliters into a tuberculin syringe. Place the mouse in the restrainer and disinfect the tail with isopropyl alcohol.

Inject the cells into a lateral tail vein. Then monitor the mouse for at least five minutes to confirm a lack of adverse effects related to the transplant. Patient-derived xenografted, or PDX animals, injected with control stroma consistently demonstrate levels of human thymic stromal lymphopoietin, or TSLP, in mouse plasma that are below the threshold for ELISA detection.

The plasma levels of human TSLP in PDX mice injected with TSLP-transduced stroma is proportional to the amount of TSLP stromal cells injected during the previous one to two weeks. Despite being performed in simplicate, this weekly assessment of plasma TSLP levels using the modified ELISA gives results that are, in general, consistent over time. TSLP has been shown to increase the production of normal human B-cell precursors.

Indeed, B-lineage cells are significantly increased in TSLP-plus mice compared to the control PDX animals as early as the B-cell precursor stage, suggesting that human TSLP produced in TSLP-plus PDX animals exerts the expected in vivo functional effects on target cell populations. Once optimized, these procedures can generate xenograft mice with detectable levels of human cytokines in three to four weeks. It's important to monitor the cytokine concentration in the stroma cell supernatant over time.

This ensures that the cytokine production is sustained in the cytokine-producing stroma and absent in the control stroma. PDX mice can be used for preclinical studies to answer additional questions, such as, How is the efficacy of a specific drug of interest altered by varying the physiological levels of an experimental cytokine of interest in vivo? This innovative model provides a new way for researchers in the fields of hematology and oncology to study the role of TSLP in the development and progression of leukemia, as well as its role in normal hematopoiesis.

Provided that you have access to cytokine-transduced cell lines, you can use the techniques demonstrated in this video to supply any exogenous cytokine to patient-derived xenograft disease models using a wide range of immune-deficient mouse strains. Don't forget that working with human hematopoietic cells can be hazardous. You should always use precautions to avoid the transmission of bloodborne pathogens while working with these fluids and cells.