Establishment of peripheral blood mononuclear cell-derived humanized lung cancer mouse models for studying efficacy of PD-L1/PD-1 targeted immunotherapy

Abstract

Animal models used to evaluate efficacies of immune checkpoint inhibitors are insufficient or inaccurate. We thus examined two xenograft models used for this purpose, with the aim of optimizing them. One method involves the use of peripheral blood mononuclear cells and cell line-derived xenografts (PBMCs-CDX model). For this model, we implanted human lung cancer cells into NOD-scid-IL2Rg-/- (NSI) mice, followed by injection of human PBMCs. The second method involves the use of hematopoietic stem and progenitor cells and CDX (HSPCs-CDX model). For this model, we first reconstituted the human immune system by transferring human CD34+ hematopoietic stem and progenitor cells (HSPCs-derived humanized model) and then transplanted human lung cancer cells. We found that the PBMCs-CDX model was more accurate in evaluating PD-L1/PD-1 targeted immunotherapies. In addition, it took only four weeks with the PBMCs-CDX model for efficacy evaluation, compared to 10-14 weeks with the HSPCs-CDX model. We then further established PBMCs-derived patient-derived xenografts (PDX) models, including an auto-PBMCs-PDX model using cancer and T cells from the same tumor, and applied them to assess the antitumor efficacies of anti-PD-L1 antibodies. We demonstrated that this PBMCs-derived PDX model was an invaluable tool to study the efficacies of PD-L1/PD-1 targeted cancer immunotherapies. Overall, we found our PBMCs-derived models to be excellent preclinical models for studying immune checkpoint inhibitors.

Keywords: Non-small-cell-lung cancer; anti-PD-L1/PD-1 monoclonal antibody; humanized mouse model; immunotherapy; patient-derived-xenograft.

Figure 1.

Human T cells reconstitution in NSI mice. Representative FACS profiles of human T cell reconstitution in NSI mice transplanted with CD34+ HSPCs (A) and PBMCs (B) at indicated time points.

Figure 2.

Evaluating the antitumor effect of anti-PD-L1/PD-1 monoclonal antibodies in PBMCs-CDX mouse model. (A) Real-time PCR analyzes PD-L1 (top) and ACTIN (bottom) in indicated lung cancer cell lines. (B) Representative FACS profiles of PD-L1 expression in indicated cell lines. (C) Experimental design to evaluate the antitumor effects of anti-PD-L1/PD-1 monoclonal antibodies in PBMCs-CDX mouse model. (D) Tumor growth curve showing reduced tumor growth in H460-bearing PBMCS-CDX mice by treating with atezolizumab (blue), and pembrolizumab (green); n = 6. (E) Tumor weight showing reduced tumor growth in PBMCS-CDX mice by treating with atezolizumab, and pembrolizumab; n = 6. Summary of percentages of human T cells in tumor tissues (F) and peripheral blood (G) from H460-bearing PBMCs-CDX mice treated with atezolizumab, and pembrolizumab. (H) Tumor growth curve showing reduced tumor growth in A549-bearing PBMCS-CDX mice by treating with atezolizumab (blue) and pembrolizumab (green); n = 5. (I) Tumor weight showing reduced tumor growth in A549-bearing PBMCS-CDX mice by treating with atezolizumab and pembrolizumab; n = 5. Summary of percentages of human T cells in tumor tissues (J) and peripheral blood (K) from A549-bearing PBMCs-CDX mice treated with atezolizumab and pembrolizumab. Means ± SEM are shown in graphs. *p < 0.05, **p < 0.01, and ***p < 0.001; One-way ANOVA. Statistics on tumor growth curve (d, h), p-values are significance for measurements of tumor volume before sacrificing mice.

Figure 3.

Evaluating the antitumor effect of anti-PD-L1/PD-1 monoclonal antibodies in HSPCs-CDX mouse model. (A) Experimental design to evaluate the antitumor effects of anti-PD-L1/PD-1 monoclonal antibodies in HSPCs-CDX mouse model. (B) Summary of percentages of human T cells in peripheral blood (PB) of H460 tumor-bearing mice before treated with atezolizumab or pembrolizumab. (C) Tumor growth curve and tumor weight (D) showing no reduced tumor growth in H460-bearing HSPCs-CDX mice by treating with atezolizumab or pembrolizumab; n = 7. Summary of percentages of human T cells in tumor tissues (E) and peripheral blood (F) from H460-bearing HSPCs-CDX mice treated with atezolizumab or pembrolizumab. (G) Summary of percentages of human T cells in peripheral blood (PB) of A549 tumor-bearing mice before treated with atezolizumab or pembrolizumab. (H) Tumor growth curve and tumor weight (I) showing no reduced tumor growth in A549-bearing HSPCs-CDX mice by treating with atezolizumab or pembrolizumab; n = 5. Summary of percentages of human T cells in tumor tissues (J) and peripheral blood (k) from A549-bearing HSPCs-CDX mice treated with atezolizumab or pembrolizumab.

Figure 4.

Evaluating the antitumor effect of anti-PD-L1 monoclonal antibodies in PBMCs-PDX mouse model. (A) Immunochemical analyzes PD-L1 in LC2 and LC23 and matched PDX; Scale bar: 100 μm. Tumor growth curve showing reduced tumor growth in PBMCS-PDX2 (B) and PBMCS-PDX23 (C) mice by treating with atezolizumab (red), atezolizumab (N298A mutation, green), and MSB2311 (N298A mutation, blue). Summary of percentages of human T cells in tumor tissues (D) and peripheral blood (E) from PBMCS-PDX2 mice treated with atezolizumab, atezolizumab (N298A mutation), and MSB2311 (N298A mutation). Summary of percentages of human T cells in tumor tissues (F) and peripheral blood (G) from PBMCS-PDX23 mice treated with atezolizumab, atezolizumab (N298A mutation), and MSB2311 (N298A mutation). (H) Experimental design to evaluate the antitumor effects of anti-PD-L1 monoclonal antibody in auto-PBMCs-PDX50 mouse model. Tumor growth curve (I) and tumor weight (J) showing reduced tumor growth in auto-PBMCs-PDX50 mice by treating with atezolizumab; n = 6. Summary of percentages of human T cells in tumor tissues (K) and peripheral blood (L) of auto-PBMCs-PDX50 mice treated with atezolizumab. Means ± SEM are shown in graphs. *p < 0.05, **p < 0.01, and ***p < 0.001; One-way ANOVA (b, c), and unpaired two-tailed t-test (i, j). Statistics on tumor growth curve (b, c and i), p-values are significance for measurements of tumor volume before sacrificing mice.