Establishment of Humanized Mouse Models to Study Drug Function

The testing of new therapies against cancer traditionally uses a combination of cell culture and mouse models. While many aspects of drug anti-cancer activity and function can be studied using these models they lack the ability to model the effects of a drug on human tissues in vivo. Humanized mouse models have been developed to overcome this limitation. Humanized mouse models are broadly defined as a mouse model which expresses human genes or harbors human tissues. Recently, the use of mice with a humanized immune system have been used more commonly to model the effects of antibody based immune therapies (checkpoint blockade), or the effects of small molecules on the human immune system. Mice with a humanized immune system can be combined with human patient derived xenograft (PDX) tumor tissues to study how therapies can modulate the human immune response to human cancer.

With the support of the OVPRI, Massey Comprehensive Cancer Center, VCU SOM and the VIMM the Landry Lab has developed methods and protocols to create mice with a humanized immune system. Several mouse backgrounds are available including NSG, IL-15 NSG. SGM3 NSG, FLT3 NSG to create mice with enrichments of select innate immune cells including both natural killer cells and differentiated and active cells of the myeloid lineage. The purpose of these studies is to provide more advanced humanized models to the VCU research community for use in characterizing the effects of anti-cancer therapies.

Figure 1. Scheme for Constructing Humanized Mice.

(A) CD34+ cells were isolated from hUCB using a Ficoll-Paque gradient followed by an Ultrapure Human CD34 MicroBead Kit from Miltenyi Biotech. Busulfan was used to condition the mice 48 and 24 hrs. before transplanting purified CD34+ cells via IV injection. Mice were housed in an ultra-barrier facility until experimental endpoint was achieved. The humanized blood, bone marrow, and spleen were removed and analyzed using a flow cytometer at set time points. (B) Analysis of representative human CD34+ cell purification from hUCB using anti-human CD34+ and 7AAD by flow cytometry. (C) Effectiveness of busulfan treatment for the engraftment of human CD34+ cells and expansion of hCD45+ cells from in the blood, bone marrow and spleen were determined in NSG mice. N=2 per group.

Highlights of the study:

• This study establishes the ability to create mice with a humanized immune system on either an NSG, IL15 NSG or SGM3 NSG background.

• Mice from the IL15 NSG background can generate mice with differentiated and active natural killer cells.

• Mice from the SGM3 NSG background can generate differentiated and active cells of the myeloid lineage, but at low abundance.

Humanized mouse models have improved biomedical research by providing a tractable system with which to perform in vivo experiments on human tissues. Use of irradiators is the standard method for establishing high levels of stem cell engraftment, however not all institutes have access to this instrumentation in the animal facility. The use of busulfan has been successfully used to precondition for stem cell engraftment on a limited number of mouse backgrounds. In this report we further test the utility of busulfan to successfully engraft hIL15-Tg-NSG and SGM3-NSG mouse stains which are capable of establishing the innate NK cell and myeloid immune compartments. Results from our studies show that busulfan can successfully precondition hIL15-Tg-NSG mice but not SGM3-NSG mice for high levels of human immune cell engraftment. SGM3-NSG mice preconditioned with busulfan exhibited only 10-20% human CD45 cells in the bone marrow or spleen, where as NSG and hIL15-Tg-NSG mice routinely achieved ∼80%. Busulfan preconditioned SGM3-NSG mice showed elevated levels of granulocytic MDSC, and cDC1 and cDC2 myeloid populations. This is in contrast with hIL15-TG-NSG which showed robust reconstitution of mature CD16 expressing NK cells. We conclude from our studies that busulfan is an effective means to precondition mice for CD34+ stem cell engraftment, but it may have limitations when used to precondition the SGM3-NSG model.

This work was supported by seed money from VCU OVPRI, Massey Cancer Center and the VCU SOM (JWL).

Publication:

Alhawiti O, Hu B, Koblinski J, Guo C, Landry J.W. (2022) Limitations of Busulfan to Create Humanize Mice with an Innate Immune System. BioRxIV, doi: https://doi.org/10.1101/2022.07.15.500220

https://www.biorxiv.org/content/10.1101/2022.07.15.500220v1

Funding:

The technology of humanized mice at VCU was recently used to generate several preliminary data sets which were used in several grant applications to the DoD, and NIH. As a result of these efforts several grants were recently scored in the fundable range and are awaiting funding from the NIH.

Title: Development of Mouse and Humanized Models to Study Sex Disparities in NSCLC Tumor Progression and Treatment

Sponsor: NIH/NCI – R21

Title: Enhancing Tumor Cell Immunogenicity using Improved Molecules Targeting Chromatin Remodeling

Sponsor: NIH/NCI – R21

Title: Modulating growth, progression and metastasis in breast cancer by inhibiting MDA-9.

Sponsor: NIH/NCI – R01

About the Investigators: Joseph W. Landry is an associate professor in the Department of Human and Molecular Genetics, a full member of the Massey Comprehensive Cancer Center and the VIMM. Chunqing Guo is an assistant professor in the Department of Human and Molecular Genetics, a member of the Massey Cancer Center and the VIMM. Bin Hu is core manager of the Cancer Mouse Models Shared Resource of the Massey Comprehensive Cancer Center. Jennifer Koblinski is an associate professor in the Department of Pathology, Full Member of the Massey Cancer Center, Director Cancer Mouse Models Core, and Co-Director Tissue and Data Acquisition and Analysis Core.