Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors

Abstract

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine cancer models are extremely limited in their diversity and suffer from poor translation to the clinic. To serve as a more physiological preclinical model for immunotherapy studies, this protocol has been developed to evaluate the treatment of human tumors in a mouse reconstituted with a human immune system. This unique protocol demonstrates the development of human immune system (HIS, "humanized") mice, followed by implantation of a human tumor, either a cell-line derived xenograft (CDX) or a patient derived xenograft (PDX). HIS mice are generated by injecting CD34+ human hematopoietic stem cells isolated from umbilical cord blood into neonatal BRGS (BALB/c Rag2^-/- IL2RγC^-/- NOD^SIRPα) highly immunodeficient mice that are also capable of accepting a xenogeneic tumor. The importance of the kinetics and characteristics of the human immune system development and tumor implantation is emphasized. Finally, an in-depth evaluation of the tumor microenvironment using flow cytometry is described. In numerous studies using this protocol, it was found that the tumor microenvironment of individual tumors is recapitulated in HIS-PDX mice; "hot" tumors exhibit large immune infiltration while "cold" tumors do not. This model serves as a testing ground for combination immunotherapies for a wide range of human tumors and represents an important tool in the quest for personalized medicine.

Figure 1:. Schematic illustrating the generation of HIS-BRGS mice and implantation of human CDX or PDX tumors for cancer immunotherapy studies.

The timeline is important to ensure the presence of T cells that take months to engraft in the CB-derived HIS mice. Created with Biorender.com.

Figure 2:. Analysis of tumor growth.

Tumor growth measurements for CRC307P PDX in control (black) or combination treated (red) HIS-BRGS mice quantified by (A) volume over time, (B) weights at the end of the study, or (C) specific growth rate (SGR). (D) SGRs for CRC307M PDX. Data include tumors implanted into both flanks of six vehicle HIS-BRGS mice, seven combination-treated BRGS mice for CRC307P, and only two vehicle and combination-treated mice for CRC307M due to high exclusion rates. Statistical analyses between two independent groups were performed using unpaired, parametric two-group Welch’s t-test. **p < 0.01, ****p < 0.0001.

Figure 3:. Human immune and T cell chimerism in lymph organs and tumors of CRC PDX tumor-bearing HIS-BRGS mice.

(A) Representative flow cytometry analysis of human (hCD45) and mouse (mCD45) hematopoietic and human T (CD3) cells in peripheral blood (PBMC) prior to tumor implantation, and in LNs and tumors (TIL) at the end of the study. (B,C) Equivalent human and T cell chimerism in blood at 14 weeks in HIS-BRGS mice subsequently injected with (B) CRC307P or (C) CRC307M PDXs and untreated or treated with combination immunotherapy (Tx). (D-F) Increased human T cells in lymph organs and tumors (TIL) of combination-treated HIS-BRGS-CRC307P mice but not HIS-BRGS-CRC307M mice. Data show human and T cell frequencies on the Y-axis as a percentage of the parent population in (D) lymph nodes (LNs), (E) spleens (SP), and (F) tumors of individual mice at the end of the study. Statistical analyses between two independent groups were performed using unpaired, parametric two-group Welch’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4:. Analysis of T cell activation by flow cytometry in peripheral lymph organs and tumors of CRC PDX bearing HIS-BRGS mice.

(A) Representative flow cytometry analysis of T cells in LNs, spleens (SPs), and tumors (TILs) excised from HIS-BRGS mice measuring the following populations: activated T cells (HLA-DR+), naïve T cells (CD45RA+ CCR7+), Tem (CD45RA-, CCR7-), Tregs (CD25+, FoxP3+), and cytotoxic T cells (Granzyme B+, TNFα+, and/or IFNγ+). (B-D) Frequencies of indicated T cell populations in lymph organs or TILs of HIS-CRC307P-BRGS and HIS-CRC307M-BRGS mice: (B) HLA-DR+ activated T-cells, (C) CCR7-CD45RA-Tem CD8+ T cells, (D) inhibitory receptor TIM-3 CD8+ T cells, (E) Granzyme B CD8+ T cells, and (F) CD25+FoxP3+ CD4+ Tregs. For the HIS-CRC307M-BRGS mice in (B), the first data set shows all mice analyzed at harvest, whereas the second data set includes only those with sufficient LN and splenic T cell chimerism. In all graphs, the filled symbols for 307M represent mice with sufficient chimerism, whereas open symbols are excluded HIS mice. Statistical analyses between two independent groups were performed using unpaired, parametric two-group Welch’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5:. Flow cytometry analysis of MHC class I, II, and PD-L1 on human tumors in HIS-BRGS mice.

(A) Representative flow cytometry dot plots, illustrating gating strategy for measuring expression levels of MHC Class I (HLA-ABC), Class II (HLA-DR), and PD-L1 on epithelial (EpCAM+) human tumors in HIS-BRGS mice. (B-D) Mean fluorescence intensity (MFI) of HLA-ABC (B), HLA-DR (B,C), and PD-L1 (D) on human (hCD45+) and tumor (EpCAM+) cells from dissociated CRC307P (B,D) or CRC307M (C) PDXs excised from HIS-BRGS mice. Statistical analyses between two independent groups were performed using unpaired, parametric two-group Welch’s t-test. * p <0.05. Abbreviation: Tx = Treatment

Figure 6:. Analysis of immunocorrelates of response.

The SGRs of CRC307P tumor growth in the flanks of untreated (Veh) or combination immunotherapy-treated (Tx) HIS-BRGS mice were plotted versus the frequency of (A) CD4+ or (B) HLA-DR+ T cells as an indicator of immune parameters that correlate with reduced tumor growth (i.e., smaller SGR). A simple linear regression analysis was performed to indicate significant correlations. R² values indicate degree of correlation. *p < 0.05, **p < 0.01, ***p < 0.001.