Abstract B165: Investigating in vivo synergistic effect of checkpoint blockade and radiation therapy against chordomas in a humanized mouse model
Authors: Ishida Wataru , McCormick Kyle L. , Mahajan Aayushi , Feldstein Eric , Lim Michael , Bruce Jeffrey N. , Canoll Peter D. , Lo Sheng-fu L 
Published: February 2019

Abstract
Introduction: With the advent of immunotherapy (IT) against various cancers, its applications to other cancers have been extensively investigated. However, it has been a challenge to apply IT to chordomas, due to lack of clinically translatable in vivo models. Currently, there are no well-established murine chordoma cell lines that can be injected to syngeneic mice or no transgenic mouse models that develop chordomas spontaneously, which would allow us to study the interaction between murine chordomas and murine immune cells. Hence, we aimed to develop a humanized mouse model, where human immune cells are engrafted into immunodeficient mice, to study the interaction between human immune system and human chordomas. We also sought to utilize it to investigate synergistic effect between IT and radiation therapy (RT) against chordoma. Materials and Methods: Fifteen 10-12-week-old NSG mice, which lack mouse T-cells, B cells, and NK cells as well as functional mouse macrophages, were sublethally (1.5Gy) irradiated and then implanted with fetal thymic tissue and CD34+ stem cells that had been harvested from a fetus, whose HLA-types were partially-matched with those of the U-CH1 chordoma cell line. Reconstitution of immune cells in NSG mice was confirmed eight weeks post-transplantation, and then each animal (15 humanized NSG mice and 12 naïve NSG mice) was injected with U-CH1 cell suspension bilaterally and subcutaneously. Next, they were treated for 4 weeks as follows: A) control, isotype antibodies (Abs) injection (n=3), B) anti-human-PD-1 Abs (n=4, 10 mg/kg, 3 times/week for 4 weeks), C) RT + isotype Abs (n=3, unilaterally to the left-sided tumor, 8Gy x 4), D) anti-human-PD-1 Abs and RT (n=5), E) naïve NSG mice (n=6, without the engraftment of human immune cells) + isotype, and F) naïve NSG mice (n=6) + anti-human-PD-1 Abs. During and after the treatment, anti-tumor activities were monitored via tumor size, flow cytometry, qRT-PCR, and immunohistochemistry. Results: Eight weeks after stem cell engraftment, human peripheral blood mononuclear cells (PBMCs) of 43.8% among all PBMCs (human + mouse), human T-cells of 23.4% among human PBMCs, human CD8+ T-cells of 24.3% among human T-cells, and other lymphocytes such as B cells, macrophages, and NK cells were observed in peripheral blood of humanized mice via flow cytometry, which confirmed humanization. One week after the treatment, on the irradiated side, (D) demonstrated lowest tumor volume, highest number of human PBMCs, highest % of CD8+ human (cytotoxic) T-cells, highest % of CD45RO+CD4+ human (memory) T-cells, and lowest % of PD-1+CD8+ human (exhausted) T-cells in the tumors via flow cytometry, highest IFN-gamma in the tumors via qRT-PCR, and highest CD8+ human (cytotoxic) T-cells via immunohistochemistry, compared to the other five groups with statistical significance. Of note, on the nonirradiated side, a similar trend was observed with D) harboring the smallest tumor compared to the others (P=0.09), suggesting the abscopal effect. Finally, there were no statistically significant differences amongst (A) humanized NSG mice with isotype-control antibodies, (E) naïve NSG mice with isotype-control antibodies, and (F) naïve NSG mice with anti-PD-1 antibodies on either sides, indicating that HLA-partially-mismatched immune cells derived from the fetus donor were not able to eradicate U-CH1 chordoma cells. Conclusions: We demonstrated that this humanized mouse model could be a revolutionary platform to investigate IT against rare cancers such as chordomas, where murine equivalent cell lines are not available to date, which hinders us from utilizing syngeneic or transgenic mouse models to study IT. The direct synergistic effect between IT and RT against chordoma as well as the potential abscopal effect was observed, evidenced by lowest tumor volume and highest cytotoxic T-cells and memory T-cells.
Citation Format: Wataru Ishida, Kyle L. McCormick, Aayushi Mahajan, Eric Feldstein, Michael Lim, Jeffrey N. Bruce, Peter D. Canoll, Sheng-fu L Lo. Investigating in vivo synergistic effect of checkpoint blockade and radiation therapy against chordomas in a humanized mouse model [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B165.