Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors

This protocol outlines the generation of Human Immune System, or HIS mice for preclinical immuno-oncology studies. Mouse cancer models are limited in their diversity and translate poorly to the clinic. This technique serves as a preclinical model for immunotherapy studies by evaluating the treatment of human tumors in a mouse with a human immune system.

The humanized mouse model can be used to study multiple aspects of human immunology, including responses to many infections, or cancers of the human immune system. Acquiring a reliable and robust source of cord blood and maintaining the health of the immunodeficient mouse colony can be challenging. Expertise in flow cytometry and immunological analysis is essential.

Demonstrating the procedure will be Kristina Larsen, a research services professional from my laboratory. To begin, resuspend the CD34-positive cell pellet isolated from cord blood in 300 microliters of magnetic cell separator buffer per one times 10 to the eighth cells. Add 100 microliters of FCR blocking reagent per one times 10 to the eighth cells, followed by 100 microliters of CD34-positive magnetic beads per one times 10 to the eighth cells, and incubate at four degrees Celsius for 30 minutes.

Next, add five milliliters of magnetic cell separator buffer per one times 10 to the eighth cells, and spin at 360 g for 10 minutes at four degrees Celsius. Repeat the wash step and resuspend the pellet in 500 microliters per 100 million cells of magnetic cell separator buffer in a 15 milliliter conical tube labeled unfractionated. Label two more 15 milliliter tubes CD34-negative and CD34-positive Place the three tubes on a cooling rack.

Separate the cells using the two column positive selection program on an automatic magnetic cell separator in a biosafety cabinet. Next, for expanding and freezing CD34-positive human stem cells, prepare cord blood, or CB medium as described in the manuscript, and pass through a 0.22 micron filter. Resuspend the CD34-positive cells at 100, 000 per milliliter of CB medium, and incubate at 37 degrees Celsius.

On day three, add an equal volume of CB medium without cytokines to the flask. On day five, harvest the expanded CD34-positive cells. Pipette the cell suspension up and down, and collect in a 50 milliliter conical tube.

Add enough CB medium to cover the bottom of the flask. Using a cell scraper, scrape the entire bottom of the flask. Collect all the media into the same 50 milliliter tube, and centrifuge.

Resuspend the cells in two milliliters of CB medium, and centrifuge. Aspirate the medium down to the pellet, and resuspend the pellet in a freezing medium. Next, begin CD34-positive cell preparation three hours post-pup irradiation.

Warm 10 milliliters of CB media in a 50 milliliter tube. Retrieve one vial of in vitro expanded and frozen CD34-positive cells for every four to six pups to inject. Rapidly thaw at 50 to 55 degrees Celsius until just a small amount of ice is visible, and add the cells to the warmed CB medium.

Use one milliliter of medium to rinse each vial, and spin the cells at 360 g for 12 minutes at four degrees Celsius. Aspirate the medium carefully. Resuspend the pellet in two milliliters of CB media and count the cells.

Again, centrifuge the cells, aspirate the medium, and resuspend the pellet in 100 microliters of sterile PBS per n plus one pups to inject, resulting in 250, 000 to 450, 000 CD34-positive cells per mouse. For PBMC isolation from mouse blood, mix the blood heparin by gently pipetting up and down, and slowly overlay on top of 500 microliters of 1.077 gram per milliliter density gradient being careful not to disturb the interface. Centrifuge the tube at 1, 220 g for 20 minutes at room temperature with no breaks.

Remove as many cells as possible from the buffy coat with a 200 microliter pipette, and add to the new 1.5 milliliter tube containing 750 microliters of harvest medium. Centrifuge at 360 g for 11 minutes. Aspirate the medium to 50 microliters, and resuspend the pellet in 750 microliters of harvest medium.

Acquire the data for 100 microliters of each sample on a flow cytometer. Then import the fcs file to the flow data editing software, and apply a polygon gate to an FSC-A versus SSC-A plot. Change the axes to FSC-A versus FSC-H, and gate the cells on the linear diagonal, excluding the doublets that protrude from the line.

Select these cells and change the axes to hCD45 versus mCD45. Apply a polygon gate to the hCD45-positive population, and apply the name human. Apply a polygon gate to the mCD45-positive population, and apply the name mouse.

Create a counting statistic for human and mouse populations. Next, select the human population, and change the axes to CD19 versus CD3. Apply a polygon gate to the CD19-positive cells, and name them B cells.

Apply a polygon gate to the CD3-positive population, and name it T-cells. Then apply a polygon gate to the double negative population, and name it non-TB. Select the T-cell population and change the axes to CD4 versus CD3.

Apply a polygon gate to the CD4-positive population, and name it CD4-positive. Apply a polygon gate to the CD4-negative population, and name it CD8. Select the non-TB population, and change the axes to CD56 versus myeloid.

Apply a polygon gate to the total CD56-positive population, and name it NK cells. Then apply a polygon gate to the CD56-negative and myeloid-positive population, and name it myeloid. Create a table for the percentage and count statistics of all populations, and export it to a spreadsheet.

Calculate percent hCD45 chimerism using the indicated formula. After growing tumor cells in the mouse and administering drug treatments, harvest the tissues. Place the mouse on a foam dissection board with pins to hold them in place and the arms and legs extended at 45 degrees.

Make an incision up the middle of the torso starting near the pelvis and extending to the chin. Pull the skin to the edge, and hold it in place with pins. Extract the lymph nodes using fine forceps in the order axillary, cervical, mesenteric, inguinal, and hiatal.

Place the lymph nodes on one side of the frosted glass slide in a Petri dish in eight milliliters of harvest medium. Holding the slides at perpendicular angles with the frosted edges inward, gently press the tissues until the cellular contents are released. Rinse the slide several times by pulling them apart and together to release the maximal number of cells.

Collect the cells with a five inch glass pipette, and filter them through a nine inch cotton-plugged pipette into a labeled 15 milliliter conical tube. Next, extract the tumor from the open flank by holding the tumor with forceps while slowly snipping at the tumor margins with dissection scissors. Weigh the tumor and remove 1/4 of the tumor for RNA and immunohistochemistry processing.

Place the remaining 3/4 of the tumor into a six centimeter dish, and mince it into one millimeter pieces using a scalpel blade. Transfer the tumor pieces into a dissociation tube. Then rinse the dish with incomplete TIL medium, and add it to the tube.

Add collagenase preparation, and dissociate the tissue using mechanical dissociation at 37 degrees Celsius for 30 minutes to one hour. Pass the suspension over a 100 micron filter into a 50 milliliter conical tube and rinse the filter with 10 milliliters of TIL complete media. Centrifuge and resuspend the pellet in just enough harvest medium with DNase, so that the cells suspension can easily pass through a P1000 pipette tip and record the volume for downstream analysis.

In PDX CRC 307P, the combination treatment slowed the growth of the tumor, as determined by tumor growth volumes over time, tumor weights, and the specific growth rate. PDX CRC 307M tested in a different cohort of mice was less affected by the same combination treatment in HIS BRGS mice. Based on overall human hCD45-positive and human T-cell hCD3-positive chimerism in the blood before tumor implantation, both parameters increased in the peripheral immune system and the TIL in combination with treated CRC 307P-bearing mice, but not the CRC 307M model.

Investigation of the human T-cells revealed more activated T-cells in the CRC 307P tumors, but not the lymph from combination treated mice. Also, there were more effector memory CD8-positive T-cells and fewer TIM-3-positive T-cells in the combination treated CRC 307P tumors. In contrast, this difference was not noted in the CRC 307M model.

Further, no changes in the frequencies of cytotoxic T-cell populations were observed among the combination treated mice, although higher cytotoxic T-cells were observed in untreated. Combination treatment did not affect the frequencies of Tregs in either CRC 307P lymph organs, or tumors, although the CRC 307M data showed a trend of reduced Tregs. Immune related changes in tumor cells were elevated using flow cytometry.

Increased expression of MHC class I and class II was observed on the CRC 307P tumor cells excised from combination treated HIS BRGS mice. In the CRC 307M model, the same drug treatment induced HLA class II expression on the tumor cells. Similarly, the combination treatment resulted in increased PD-L1 expression on the EpCAM-positive CRC 307P tumor cells.

Finally, correlations of immune responses with tumor growth were investigated. Increased CD4-positive T-cells showed a significant correlation with smaller tumor growth, and more specifically the HLA-DR-positive activated T-cells in the combination treated HIS mice. Sterile technique while isolating the CD34-positive cells, as the mice are extremely immunodeficient.

The resulting chimerism is variable within and between cord blood donors, and T-cells have the largest variation. Unlimited immunotherapy combinations can be performed, as well as mechanistic and drug dosing kinetic studies, for example, deletion of CD8 T-cells to confirm their role. Importantly, drug related toxicities can also be studied.

It provided a more relevant and accessible preclinical model to test human immunotherapies for translation into the clinic. Several clinical trials are in progress now based on these data.