Establishment of an in vivo small animal model of human tumor and human immune system interaction would enable preclinical investigations into the mechanisms underlying cancer immunotherapy. To this end, nonobese diabetic (NOD).Cg-PrkdcscidIL2rgtm1Wjl/Sz (null; NSG) mice were transplanted with human (h)CD34+ hematopoietic progenitor and stem cells, which leads to the development of human hematopoietic and immune systems [humanized NSG (HuNSG)]. HuNSG mice received human leukocyte antigen partially matched tumor implants from patient-derived xenografts [PDX; non–small cell lung cancer (NSCLC), sarcoma, bladder cancer, and triple-negative breast cancer (TNBC)] or from a TNBC cell line-derived xenograft (CDX). Tumor growth curves were similar in HuNSG compared with nonhuman immune-engrafted NSG mice. Treatment with pembrolizumab, which targets programmed cell death protein 1, produced significant growth inhibition in both CDX and PDX tumors in HuNSG but not in NSG mice. Finally, inhibition of tumor growth was dependent on hCD8+ T cells, as demonstrated by antibody-mediated depletion. Thus, tumor-bearing HuNSG mice may represent an important, new model for preclinical immunotherapy research.—Wang, M., Yao, L.-C., Cheng, M., Cai, D., Martinek, J., Pan, C.-X., Shi, W., Ma, A.-H., De Vere White, R. W., Airhart, S., Liu, E. T., Banchereau, J., Brehm, M. A., Greiner, D. L., Shultz, L. D., Palucka, K., Keck, J. G. Humanized mice in studying efficacy and mechanisms of PD-1-targeted cancer immunotherapy.

No abstract

Mouse models are frequently used to test the therapeutic efficacy of anti-cancer drugs. However, the translation of murine experimental data to treatments for patients with cancer often fails due to significant differences between the species, including the differences in the immune system. Our goal is to bridge this gap and to establish an in vivo preclinical model of human tumor immunotherapy by engrafting immunodeficient mice expressing a partial human immune system with human tumor implants. Humanized NOD-scid IL2Rγ (null) (hu-NSG) mice were initially generated by transplanting NSG mice with human CD34+ hematopoietic stem and progenitor cells (HSPCs) which support human hematopoietic and immune system development. Hu-NSG mice develop functional human T cells and B cells with high levels of TCR excision circles, complex TCR repertoire diversity and antigen-specific T cell proliferative responses. Several types of patient-derived tumors (non small cell lung cancer, sarcoma, triple negative breast cancer and invasive bladder cancer) were successfully implanted into HLA mismatched hu-NSG mice. Tumor growth curves show a delay in tumor growth in hu-NSG compared to non-humanized NSG mice. In a colon cancer xenograft model, treatment with chemotherapy agent (5-FU) or with a therapeutic antibody directed against VEGF (Avastin) resulted in decreased tumor growth. In addition to PDX tumors we have also tested human cancer cell lines. Tumor growth was observed in all hu-NSG mice implanted with human ovarian tumor cell line SKOV3-Luc-D3 cells at different time points post HSPC engraftment, showing no evidence of tumor rejection. Thus, our model of humanized mice bearing tumor-derived xenografts provides opportunities to study both the safety and efficacy of current cancer therapies. Citation Format: Minan Wang, James G. Keck, Mingshan Cheng, Danying Cai, Leonard Shultz, Karolina Palucka, Jacques Banchereau, Carol Bult, Rick Huntress. Patient-derived tumor xenografts in humanized NSG mice: a model to study immune responses in cancer therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-050. doi:10.1158/1538-7445.AM2015-LB-050

Background: PDX have become critical elements of preclinical drug development as they better reflect the heterogeneity, molecular and histopathologic signatures of the original tumor than cell lines or genetically engineered mouse models, and their drug response profiles correlate with clinical response. While PDX models have become a powerful tool in drug discovery and development, limitations include low throughput for broad drug screening, lack of dose-response curves, high cost and progressive loss of human-derived stromal elements over serial passages, restricting utility for certain therapeutic classes. A potential mechanism to overcome the low throughput and high cost of PDX models is the incorporation of ex vivo 3D (EV3D) DRP on cells isolated from early passage PDX models. Thus, we correlated DRP results using PDX with genetic mutations and drug response of PDX tested in vivo. Materials & Methods: Cells were isolated from low-passage triple negative breast, invasive bladder, and non-small cell lung PDX tumors propagated in NSG mice and cultured as 3D spheroids. 3D spheroid cultures were exposed to 15 clinically-relevant chemotherapy and targeted agents and assayed for cell viability over a range of concentrations. Non-linear regression curves were generated and relative IC50s estimated. In vivo response with limited numbers of agents at clinically relevant concentrations (3 including controls) was assessed. Results: 3D cultures and testing were successfully established across all PDX and IC50s were successfully generated in 98% of drugs tested. EV3D DRP of PDX tumors differentiated activity of cytotoxic and targeted agents across tumors of similar histologic site of origin. Gemcitabine (IC50 = .007 versus 27 uM) and docetaxel (0.2 versus 40uM) activity was highly correlated with in vivo response in bladder and breast cancers, respectively, whereas cisplatin was equally active across all tumor types (IC50 = 3-8uM). hENT1 mRNA expression was not predictive of gemcitabine activity. EV3D DRP data correlated with PDX and clinical outcome. It identified Erlotinib as being relatively inactive (3 uM) against lung cancer PDX with an EGFR e19del, T790M mutation which correlated with the outcome seen in the PDX mouse and the clinical patient outcome in which the patient became nonresponsive to erlotinib. Trametinib was highly active against lung cancer PDX with a KRAS G12C mutation (IC50 6.7 × 10-6 versus 1.1 × 10-3) and will be used to perform efficacy studies in the KRAS mutant lung PDX model Conclusions: EV3D DRP predicts in vivo response and correlates with pathway activating mutations. EV3D DRP using PDX may represent a novel high throughput and predictive drug response platform that enables compound ranking for preclinical and clinical applications. Citation Format: Tessa M. DesRochers, Christina Mattingly, Stephen Shuford, Matthew Gevaert, David Orr, Carol Bult, Susie Airhart, Mingshan Cheng, Minan Wang, James Keck, Howland Crosswell. Enhancing drug discovery and development throughput without sacrificing predictivity: ex vivo 3D drug response profiling (DRP) using patient-derived xenografts (PDX). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 318. doi:10.1158/1538-7445.AM2015-318

Humanized mice engrafted with tumors enable in vivo investigation of the interactions between the human immune system and human cancer. We have recently found that humanized NOD-scid IL2Rγnull (NSG) mice bearing patient-derived xenografts (PDX) allow efficacy studies of check-point inhibitors. Next generation NSG strains include triple transgenic NSG mice expressing human cytokines KITLG, CSF2, and IL-3 (NSG-SGM3). Here we provide a direct comparison of check-point inhibitors evaluation in NSG and NSG-SGM3 mice engrafted with CD34+ human hematopoietic progenitor cells (HPCs) from the same donor and implanted with PDX tumors. Corroborating earlier studies, reconstitution of human immune system in the blood was faster and more robust in NSG-SGM3 compared to NSG recipients throughout the course of the study (18 weeks). Human CD45+ cells reached 25% of total blood cells at week 4 in hu-NSG-SGM3 mice and at week 9 in hu-NSG mice. A majority of blood hCD45+ cells in hu-NSG-SGM3 at week 4 were CD33+ myeloid cells. Circulating hCD3+ T cells reached 10% at week 9 and included regulatory T cells (Tregs), consistent with earlier studies. Hu-NSG mice displayed comparable hCD3+ T cells in the blood only at 12-15 weeks and did not contain Tregs. PDX tumors were then engrafted into partially HLA-matched hu-NSG-SGM3 mice at 9 weeks post engraftment. Two PDX models previously shown to respond to anti-PD1 therapy in hu-NSG mice, BR1126 and LG1306, were used. Treatment with the anti-PD-1 receptor antibody pembrolizumab (Keytruda) significantly reduced tumor growth in both models. Thus, PDX-bearing hu-NSG-SGM3 mice might serve as a new and improved platform for preclinical immuno-oncology efficacy studies. Citation Format: Li-Chin Yao, Mingshan Cheng, Minan Wang, Jacques Banchereau, Leonard Shultz, Karolina Palucka, James G. Keck. Patient-derived tumor xenografts in humanized NSG-SGM3 mice: A new immuno-oncology platform. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C01.