Our protocol can serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for immuno-oncology research. These procedures provide a cost-effective and highly reproducible approach for partially reconstituting human immunity in human tumor-bearing mice through subcutaneous and mixing of human PBMC with cancer xenografts. Demonstrating the procedures with me will be Xinxin Cui, Yanjuan Zhang, Huichen Bai and Xiaolong Yang, sent us from the Pharmacology Department.
For myeloid cell depletion intra peritoneally deliver 100mg per kilogram of cyclophosphamide and orally deliver 125mg per kilogram of disulfiram to six to eight week old NOD/SCID female mice once a day for two days. 24 hours after the second dose of cyclophosphamide and disulfiram, resuspend five times 10 of the six freshly isolated human PBMC and 2.5 times 10 of the six human tumor cell line cells and 200 microliters of PBS plus 50%metrogel per animal and inject the entire volume subcutaneously into the right flank of each experimental animal. Measure and record the primary tumor volume two times a week for four to six weeks.
When the tumors reach an average volume of 200 to 500 millimeters cubed, use optomic scissors to harvest whole tumor from each animal. PBMC donors tumor cell lines and patient derived xenografts that result in a moderate tumor growth with relatively high PD-1, PD-L1 and CD8 expression should be used for subsequent analysis. For immunohistochemical analysis of the harvested tumor tissues, fix the samples and formalin 24 to 72 hours before dehydration and embedding of the tissues in paraffin.
Obtain three micrometer sections of the embedded tissues on polylysine coated slides and deparaffinize the samples with three seven minute xylene immersions. After the third treatment, hydrate the sections with three minute graded alcohol immersions. Then rinse the slide in deionized water three times and remove any excess liquid from the slides.
To perform antigen retrieval, place the slides in a container and cover the slides with 10 millimolar sodiocitrate buffer. Heat the slides, container, in a microwave for three minutes before placing the slides in a 95 degrees Celsius water bath for 30 minutes. After cooling to room temperature, rinse the slides three times in deionized water and aspirate any excess liquid from the slides.
Incubate with 0.3%hydrogen peroxide for 10 minutes to block endogenous peroxidase activity followed by blocking with 3%BSA and PBS for one hour. At the end of the hydrogen peroxide incubation, label the slides with the appropriate primary antibodies at four degrees Celsius overnight. The next morning, treat the slides with horseradish peroxidase conjugated secondary antibody for one hour at room temperature before applying the substrate dropwise onto the slides until an appropriate level of brown staining is observed by light microscopy.
Next, stain the samples with hematoxylin. After one minute, stop the reaction with distilled water followed by a five second submersion in 0.5%hydrochloric acid alcohol and five seconds in 0.5%ammonia water. Then dehydrate the samples with three ascending three minute ethanol and three, five minute xylene immersion.
To assess the antitumor activity of a candidate drug of interest, initiate the treatment on the day of the cell inoculation according to the planned experimental protocol and monitor the primary tumor volume twice a week for four to six weeks. For PharmaCODE dynamic analysis of the tumor infiltrated immune cells, mince the tumor tissues into small pieces and digest the fragments with collagenase type one and DNase I and RPMI-1640 medium plus 5%FBS for 30 minutes at 37 degrees Celsius. At the end of the digestion, filter the digested tissue through a 40 micrometer cell strainer to obtain a single cell suspension and collect the cells by centrifugation.
Resuspend the pellet at a one times 10 of the seventh cells per milliliter of ice cold fax buffer concentration and transfer an equal volume of cells to the appropriate number of wells in a 96-well round bottom plate. Collect the cells by centrifugation and resuspend the pellets in 20 micrograms per milliliter of human IGG for 30 minutes to block any nonspecific binding. Then stain the cells with the appropriate primary antibodies for 30 minutes at four degrees Celsius before their analysis by flow cytometry according to standard protocols.
As demonstrated, 100 milligrams per kilogram cyclophosphamide and 125 milligrams per kilogram disulfiram myeloablation results in a significant depletion of neutrophils and monocytes four days post-treatment. After human PBMC and tumor transplantation, the presence of human immune cell infiltrates can be verified within the tumor microenvironment by immunohistochemistry. An in vivo screening of a panel of PBMC donors can be performed to confirm the presence of a relatively high immune cell infiltration into the tumor microenvironment in an acceptable tumor growth rate.
Human cancer cell lines in patient derived xenografts of different cancer types can also be evaluated to assess the tumor growth rate and immune cell infiltration. In this representative experiment, treatment of the PBMC and grafted humanized mice with humanized anti-PD1 antibody resulted in significant antitumor activities. Disulfiram decreases urotoxicity of cyclophosphamide, the combination may exert longer lasting neutropenia effects in mice.
The exact dose regimen of cyclophosphamide and disulfiram may need to be predetermined. Newer strains of more immunodeficient mice for easier human immunity reconstitution and decreased xenogenic GvHD effect have been established and are pending further investigations with our documented protocol. Our model is useful for evaluating T-cell engaging cancer immune therapies, particularly when working on short timelines or for selecting agents before moving to a more complex multilineage immunity model.
