An improved cell line-derived xenograft humanized mouse model for evaluation of PD-1/PD-L1 blocker BMS202-induced immune responses in colorectal cancer

Abstract

The establishment of an in vivo mouse model mimicking human tumor-immune environments provides a promising platform for immunotherapy assessment, drug discovery and clinical decision guidance. To this end, we construct humanized NCG mice by transplanting human hCD34 ⁺ hematopoietic progenitors into non-obese diabetic (NOD) Cg- Prkdc ^scidIL2rg ^tm1Wjl /Sz (null; NCG) mice and monitoring the development of human hematopoietic and immune systems (Hu-NCG). The cell line-derived xenograft (CDX) Hu-NCG mouse models are set up to assess the outcome of immunotherapy mediated by the small molecule BMS202. As a PD-1/PD-L1 blocker, BMS202 shows satisfactory antitumour efficacy in the HCT116 and SW480 xenograft Hu-NCG mouse models. Mechanistically, BMS202 exerts antitumour efficacy by improving the tumor microenvironment and enhancing the infiltration of hCD8 ⁺ T cells and the release of hIFNγ in tumor tissue. Thus, tumor-bearing Hu-NCG mice are a suitable and important in vivo model for preclinical study, particularly in cancer immunotherapy.

Keywords: BMS202; PD-1/PD-L1 blocker; cancer immunotherapy; cell-derived xenograft; humanized mouse model.

Figure 1

Illustration of establishment of the Hu-CDX mouse model (A) Graphical abstract of the establishment of the humanized mouse model. Fresh hCD34 + stem cells were injected into irradiated mice via the tail vein. (B) Flow cytometric analysis of mouse immune cells in the peripheral blood after irradiation. (C) Comparison of the levels of mCD117 + cells in NCG mice with or without irradiation.

Figure 2

Antitumour activity of BMS202 in Hu-CDX cells and a human colorectal cancer model (A) Survival curves and (B) body weight changes of NCG mice and NCG mice transplanted with CD34 + HSCs. (C,D) Twelve weeks after hCD34 + stem cell implantation, mice were injected subcutaneously with HCT116 cells to develop the CDX model. Two weeks later, the Hu-CDX model was treated with BMS202 at 60 mg/kg twice a day for 21 days. Antitumour activity of BMS202 against HCT116 tumors in Hu-NCG mice in vivo. (E) Changes in tumor volume during treatment. (F) and (G) Photographs and weights of the excised tumors. (H) TGI of the treatment group. Data are presented as the mean±SD. **P<0.01, n=5.

Figure 3

Monitoring the progress of the humanized mouse model by detecting human immune cells in mouse organs (A) The gating strategy of flow cytometry to detect differentiated human immune cells in mouse organs. Human CD45 + lymphocytes were gated out from all cells, and human T, B, and NK cells were gated out from the human CD45 + lymphocyte subpopulation. Analysis of humanization progress by human immune cells in the peripheral blood at (B) 4 weeks, (C) 8 weeks, and (D) 12 weeks after CD34 + stem cell implantation. Approximately 100 μL of peripheral blood was collected for analysis. At the same time, immune analyses were performed in the peripheral blood, spleen, and bone marrow. Three independent experiments were conducted, and the results were similar. Data are presented as the mean±SD. **P<0.01, *** P<0.001, n=3.

Figure 4

The antitumour immune response of BMS202 in humanized NCG mice bearing colorectal cancer (A) The gating strategy of flow cytometry for detecting human immune cells in the mouse spleen. Mouse spleens were processed into single cells and analyzed by flow cytometry, as described in the Materials and Methods. The percentages of (B) hCD4 +/hCD8 + T cells, (D) hCD4 +CD25 +Foxp3 + T cells, and (F) hCD8 +IFN-γ + T cells are shown. (C,E,G) Statistical analyses of these cell subsets. * P<0.05; ** P<0.01; *** P<0.001.

Figure 5

Comparison of the human immune microenvironment in BMS202 treated Hu-NCG mice and untreated Hu-NCG mice (A) Representative immunohistochemistry images of Ki67 + tumor cells in tumor tissues. (B) Quantitative analysis of Ki67 + tumor cells in both groups. The tumor cells expressing Ki-67 were counted with Image-Pro-Plus. Ki-67 positive (%)=(cell expressing Ki-67/total cells)×100%. Immunofluorescence images for (C) CD8 + T cells and (E) IFN-γ on CDX (HCT116) tissue developed in humanized mice. Quantitative analysis of human CD8 + T cells and IFN-γ in (D) and (F). Quantitative analysis of CD8 + T and IFN-γ + using Aperio ImageScope software. (G) The infiltration of CD69 + CD 8 T cells in tumor tissues was identified using an anti-CD69 antibody (brown staining). (H) The statistical and quantitative analysis of (G). (I) The infiltration of TIM-3 + CD 8 T cells in tumor tissues was identified using an anti-TIM-3 antibody (brown staining). (J) The statistical and quantitative analysis of (I). The relative number of cells was calculated with Image-Pro-Plus. (K) H&E staining of tumor tissue sections. Scale bar =50 μM. * P<0.05; ** P<0.01; *** P<0.001.