Schuelke et al. Journal for ImmunoTherapy of Cancer (2019) 7:188 https://doi.org/10.1186/s40425-019-0673-2 RESEARCHARTICLE Open Access Diverse immunotherapies can effectively treat syngeneic brainstem tumors in the absence of overt toxicity Matthew R. Schuelke1,2, Phonphimon Wongthida3, Jill Thompson3, Timothy Kottke3, Christopher B. Driscoll4, Amanda L. Huff4, Kevin G. Shim1,2, Matt Coffey5, Jose Pulido6, Laura Evgin3 and Richard G. Vile1,3,7* Abstract Background: Immunotherapy has shown remarkable clinical promise in the treatment of various types of cancers. However, clinical benefits derive from a highly inflammatory mechanism of action. This presents unique challenges for use in pediatric brainstem tumors including diffuse intrinsic pontine glioma (DIPG), since treatment-related inflammation could cause catastrophic toxicity. Therefore, the goal of this study was to investigate whether inflammatory, immune- based therapies are likely to be too dangerous to pursue for the treatment of pediatric brainstem tumors. Methods: To complement previous immunotherapy studies using patient-derived xenografts in immunodeficient mice, we developed fully immunocompetent models of immunotherapy using transplantable, syngeneic tumors. These four models – HSVtk/GCV suicide gene immunotherapy, oncolytic viroimmunotherapy, adoptive T cell transfer, and CAR T cell therapy – have been optimized to treat tumors outside of the CNS and induce a broad spectrum of inflammatory profiles, maximizing the chances of observing brainstem toxicity. Results: All four models achieved anti-tumor efficacy in the absence of toxicity, with the exception of recombinant vaccinia virus expressing GMCSF, which demonstrated inflammatory toxicity. Histology, imaging, and flow cytometry confirmed the presence of brainstem inflammation in all models. Where used, the addition of immune checkpoint blockade did not introduce toxicity. Conclusions: It remains imperative to regard the brainstem with caution for immunotherapeutic intervention. Nonetheless, we show that further careful development of immunotherapies for pediatric brainstem tumors is warranted to harness the potential potency of anti-tumor immune responses, despite their possible toxicity within this anatomically sensitive location. Keywords: Immunotherapy, Brainstem, Toxicity, DIPG Background unfortunately, decades of clinical research have not in- Brain tumors are the leading cause of pediatric cancer creased median overall survival beyond 9–11 months [2]. death [1]. Ten to 15 % of these tumors occur in the Cancer immunotherapy is among the most promising brainstem, most of which are a uniformly fatal disease areas of biomedical research, with recent FDA approval classified as an H3K27M-mutant diffuse midline glioma, of the first chimeric antigen receptor (CAR) T cell [4] or historically, diffuse intrinsic pontine glioma (DIPG) and oncolytic virus [5] therapies. In addition to strong [2, 3]. Radiation extends survival by several months and anti-tumor activity, these treatments have the potential dexamethasone is used for symptomatic control, but to provide long-term cancer immunosurveillance through the generation of immunologic memory [6, 7]. Furthermore, while traditional chemotherapy and radi- * Correspondence: [email protected] ation have long-term, developmental effects on pediatric 1Department of Immunology, Mayo Clinic, Rochester, MN 55905, USA 3Department of Molecular Medicine, Mayo Clinic, Rochester, MN 55905, USA patients [8], clinical studies in adults suggest that im- Full list of author information is available at the end of the article munotherapies may have favorable long-term safety © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Schuelke et al. Journal for ImmunoTherapy of Cancer (2019) 7:188 Page 2 of 13 profiles [9, 10], though ongoing studies are validating pcDNA3.1OVA and maintained in 10% DMEM with 5 mg/ this in children. mL G418 selection media. The B16-EGFRvIII cell line was However, brainstem gliomas like DIPG provide unique generated by retroviral transduction with pBABE PURO challenges for immunotherapy. Physiologically, the brain- encoding the murine EGFRvIII modified by the deletion of stem controls vital functions [11], requiring that therapies 500 aa from the intracellular domain of the protein using a avoid damage to healthy tissue. Infectious or autoimmune construct given as a kind gift from Dr. Luis Sanchez-Perez inflammation of the brainstem carries high morbidity and and Dr. John Sampson (Duke University, Durham, NC) mortality [12], raising concerns for cancer immunother- [18]. A clonally derived cell line was subsequently main- apies in this location. Additionally, DIPGs contain few tained in 1.25 μg/mL of puromycin. adaptive immune cells, suggesting a lack of functional Cell lines were authenticated by morphology, growth immunosurveillance and a need for induction of de novo characteristics, PCR for melanoma specific gene expres- immune responses [13, 14]. sion (gp100, TYRP-1 and TYRP-2) and biologic behavior, Thesedataraisethequestionofwhetherpotentiallycura- tested mycoplasma-free, and frozen. EGFRvIII-positive tive immunotherapy for brainstem tumors may generate cells were verified by flow cytometry staining with an anti- unacceptable toxicity. Preclinically, cancer vaccines [15] EGFRvIII primary antibody (L8A4). Cells were cultured and CAR T cells [16] have been studied in DIPG xenograft less than 3 months after thawing. Cells were tested for models, establishing evidence of anti-tumor efficacy. How- mycoplasma using the MycoAlert Mycoplasma Detection ever, while these human xenograft models allow for proof- Kit (Lonza Rockland, Inc. ME, USA). of-concept efficacy, exploration of immune-mediated tox- For recombinant vaccinia virus (VV) production, CV-1 icity and associated inflammation is best recapitulated in cells were infected by vaccinia virus (WR strain) and trans- an immunocompetent animal model. To this end, we used fected with the pSC65 plasmid transfer vector (a generous previously-validated syngeneic gliomas and melanomas to gift from Dr. Bernard Moss, NIAID) containing murine establish brainstem tumor models to assess a diverse range granulocyte monocyte colony stimulating factor (GM-CSF) of immunotherapies. Our goal was not to compare relative cDNA [19]. Recombinant viruses were isolated and then efficacies of each therapy, but rather to ascertain the poten- bulked up in Hela cells (ATCC, Manassass, VA), followed tial for immune-mediated toxicity in the brainstem. From by sucrose cushion purification. Purified virus was titered these studies, we demonstrate that although the possibility on Hela cells and stored at -80C. Clinical-grade reovirus of therapy-related inflammatory toxicity exists, a diverse was acquired from Oncolytics Biotech (Calgary, Canada). range of immunotherapies can extend survival of mice GL261 cells were infected with reovirus or VV- bearing syngeneic brainstem tumors without generating GMCSF at an MOI of 10 followed by exposure to Cell overt neurologic toxicity. These results suggest greater con- Titer Blue (Promega) for survival assessment or harvest- sideration of clinical immunotherapy trials for pediatric ing for replication assessment using a plaque assay on brainstem tumors. Hela cells (vaccinia) or L929 cells (reovirus). All vesicular stomatitis viruses (VSV) were generated Materials and methods as previously described [20]. Briefly, VSV (Indiana sero- Cell lines and viruses type) expressing human gp100 or chicken ovalbumin GL261 and GL261-QUAD cells were obtained from Dr. was generated by cloning the respective antigen into the Aaron Johnson (Mayo Clinic). GL261-QUAD cells were pVSV-XN2 plasmid by inserting between the VSV G and originally created by John Ohlfest, et al [17], and stably L proteins. VSV was titered by plaque assay on BHK express the model tumor antigens chicken OVA257–264, cells and stored at -80C. chicken OVA323–339,humangp10025–33,andthemouseal- loantigen I-Ea – .BothGL261tumorlinesweregrownin 52 68 Mice DMEM (HyClone, Logan, UT, USA) + 10% FBS (Life Tech- Female C57BL/6 mice at 6–8 weeks of age (Jackson nologies, Carlsbad, CA). B16.F1 parental murine melanoma Labs) were used for all in vivo experiments. OT-I [21] cells were obtained from the ATCC (Manassas, VA). B16tk and pmel [22] mice were bred at the Mayo Clinic, and cells were derived from a B16.F1 clone stably infected with harvested between 8 and 14 weeks of age for adoptive a lentivirus expressing the Herpes Simplex Virus thymidine transfer experiments. kinase (HSV-1 TK) gene. Following stable selection in 1.25 μg/mL puromycin, these cells were shown to be sensi- tive to Ganciclovir (APP Pharmaceuticals, Barceloneta, PR) Transgenic T cell preparation at 5 μg/ml. B16tk cells were grown in DMEM (HyClone, Pmel or OT-I T cells were harvested from transgenic Logan, UT, USA) + 10% FBS (Life Technologies, Carlsbad, pmel or OT-I mouse spleens, respectively, and under- CA) + 1.25 μg/mL puromycin (Sigma,