Abstract P6-14-02: An anti-PD1 antibody-based therapy results in dramatic reduction of TNBC PDX tumors in humanized mice models
Authors: Rosato RR , Davila-Gonzalez D , Choi DS , Dave B , Chang JC 
Published: March 2017

Abstract
Recently, the field of cancer immunotherapy has seen a rapid growth based on a better understanding of the complex interplay between the tumor and the immune system. Although for long time breast cancer has been considered non-immunogenic and patients have seen limited options to immunotherapies, new strategies have changed this paradigm. In the present study, we aimed to test the in vivo activity of a human anti-PD1 antibody against the TNBC tumor line MC1. One of the main limitations of performing laboratory-based in vivo studies resides in the availability of the appropriate animal models. To circumvent these obstacles, we used patient-derived breast cancer tumor lines xenografts (PDX) from our existing collection previously established in immuno-compromised SCID/beige mice. Low-passage fresh xenograft tumor fragments of the TNBC tumor lines MC1 and HM#2147 were transplanted into the cleared fat pad of recipient non-humanized (non-hNSG) and humanized NSG (hNSG) mice. Humanized mice were obtained by i.v. injecting 3-4 weeks old NSG mice with CD34+ hematopoietic stem cells (HSC) following whole body radiation. Flow cytometry and immuno-histochemistry analyses of hNSG blood, spleen and bone marrow showed the presence of human CD45+ (15.1% ± 10.3; 61.5% ± 19.1; 71.9% ± 17.9; respectively), CD20+, CD3+, CD8+, CD68+, and CD33+ cells. BC tumor engraftment was then evaluated by comparing the growth of the MC1 tumor line in non- and hNSG mice, showing a slower growth in the corresponding humanized mice. Importantly, the presence of hCD45+ cells was readily detectable in all the hNSG-derived tumors, localizing both toward the periphery of the tumors and inside them. Analysis of hCD45+ subpopulation cells showed also the tumor presence of hCD20+ cells (B cells), hCD8+ T-cells and CD68+ (macrophages) cells. To determine whether BC PDX may have conserved the capability to metastasize to the lung, hNSG mice were engrafted with the tumor line HM#2147. Once the primary tumor reached the maximum volume allowed by humane standards, mice humanization levels, tumor engraftment and lung metastasis were evaluated. Humanized engrafted mice showed same levels of human cells and primary tumor engraftment as those harboring MC1 PDXs. Macroscopically, lungs displayed clear evidence of metastases. IHC assays using Ki67 and CK19 identified the microscopic region corresponding to its localization. Importantly, as described in the primary breast tumor, the presence of hCD45+ was also observed infiltrating the lung metastatic tumor. The efficacy of an anti-PD1 therapy was then evaluated. Levels of tumor PD-L1 were determined by western blot showing high levels of expression. Animals were weekly i.p.-administered either the human anti-PD1 antibody or vehicle. Evaluation of tumor volumes showed a significant reduction in anti-PD1- vs. vehicle-treated animals at day 18 of treatment (i.e. 457.8 mm3 vs. 1074.24 mm3, respectively; P= 0.001). The present study show encouraging results associated with anti-PD1 immunotherapy to treat TNBC tumors. In addition, our results provide evidence supporting the use of humanized mice as key animal model that may allow to overcome some of the technical difficulties associated with the investigation of immune-based therapies.
Citation Format: Rosato RR, Davila-Gonzalez D, Choi DS, Dave B, Chang JC. An anti-PD1 antibody-based therapy results in dramatic reduction of TNBC PDX tumors in humanized mice models [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-14-02.