Intra-adrenal murine TH-MYCN neuroblastoma tumors grow more aggressive and exhibit a distinct tumor microenvironment relative to their subcutaneous equivalents

Intra-adrenal murine TH-MYCN neuroblastoma tumors grow more aggressive and exhibit a distinct tumor microenvironment relative to their subcutaneous equivalents Michiel Kroesen 0 1 2 4 5 Ingrid C. Brok 0 1 2 4 5 Daphne Reijnen 0 1 2 4 5 Maaike A. van Hout-Kuijer 0 1 2 4 5 Ingrid S. Zeelenberg 0 1 2 4 5 Martijn H. Den Brok 0 1 2 4 5 Peter M. Hoogerbrugge 0 1 2 4 5 Gosse J. Adema 0 1 2 4 5 0 D. Reijnen Central Animal Laboratory, Radboud University Medical Center , Nijmegen , The Netherlands 1 M. Kroesen P. M. Hoogerbrugge Department of Pediatric Oncology, Radboud University Medical Centre , Nijmegen , The Netherlands 2 M. Kroesen I. C. Brok M. A. van Hout-Kuijer I. S. Zeelenberg M. H. Den Brok G. J. Adema ( 3 ) Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences/278 TIL, Radboud University Medical Center , Post Box 9101, 6500 HB Nijmegen , The Netherlands 4 P. M. Hoogerbrugge Princes Maxima Center for Pediatric Oncology , De Bilt , The Netherlands 5 Present Address: I. S. Zeelenberg Institute of Applied Sciences, HAN University of Applied Sciences , Nijmegen , The Netherlands In around half of the patients with neuroblastoma (NBL), the primary tumor is located in one of the adrenal glands. We have previously reported on a transplantable TH-MYCN model of subcutaneous (SC) growing NBL in C57Bl/6 mice for immunological studies. In this report, we describe an orthotopic TH-MYCN transplantable model where the tumor cells were injected intra-adrenally (IA) by microsurgery. Strikingly, 9464D cells grew out much faster in IA tumors compared to the subcutis. Tumors were infiltrated by equal numbers of lymphocytes and myeloid cells. Within the myeloid cell population, however, tumor-infiltrating macrophages were more abundant in IA tumors compared to SC tumors and expressed lower levels of MHC class II, indicative of a more immunosuppressive phenotype. Using 9464D cells stably expressing firefly luciferase, enhanced IA tumor growth could be confirmed using bioluminescence. Collectively, these data show that the orthotopic IA localization of TH-MYCN cells impacts the NBL tumor microenvironment, resulting in a more stringent NBL model to study novel immunotherapeutic approaches for NBL. Neuroblastoma; Orthotopic; Mouse model; Bioluminescence; Immunotherapy - Abbreviations AA Amino acids BD Becton Dickinson CMV Cytomegalovirus DC Dendritic cells DMEM Dulbeccos modified Eagle medium EDTA Ethylenediaminetetraacetic acid FCS Fetal calf serum GFP Green fluorescent protein G-MDSC Granulocytic myeloid-derived suppressor cells IA Intra-adrenal IVIS In vivo imaging system M-MDSC Monocytic myeloid-derived suppressor cells NBL Neuroblastoma NK cell Natural killer cell PBS Phophate buffered saline PD-L1 Programmed death ligand 1 Rae1 Retinoic acid early transcript 1 SC Subcutaneous SEM Standard error of the mean TIL Tumor infiltration leukocytes WT Wild-type Neuroblastoma (NBL) is an aggressive malignancy of childhood with a dismal prognosis in high-risk patients. NBL arises from any of the parasympathetic nervous tissues in the body. In around half of patients, the primary tumor is located in one of the adrenal glands [1]. In a recent phase III trial, immunotherapy was added to the current standard therapy regimen, which consists of chemotherapy, surgery, radiation therapy and autologous stem cell transplantation followed by retinoic acid treatment [2]. The addition of this immunotherapy regimen to the standard treatment resulted in an improved survival of these highrisk NBL patients. However, since around half of these patients eventually did show progressive disease, a clear need remains to improve (immuno)therapy regimens for the treatment of NBL. Most tumors are highly infiltrated by cells of the immune system exerting either pro- or anti-tumor effects, depending on cell type and activation status [3]. In established tumors, however, there is a state of chronic inflammation, paradoxically resulting in local immune suppression [4, 5]. Regulatory immune cell subsets are actively attracted to the tumor that interacts with each other and create an immunosuppressive microenvironment [6]. For example, M2 macrophages infiltrating tumors can actively suppress anti-tumor immune responses via the production of anti-inflammatory cytokines [7, 8]. In NBL tumors, the presence of tumor-associated macrophages was shown to be an independent prognostic factor associated with an adverse prognosis8. The immunosuppressive environment also contributes to progressive or recurrent disease during or after immunotherapy. Counteracting the tumorinduced immune suppression therefore is an important step in improving cancer immunotherapy [9]. To achieve this, there is a need for a more comprehensive understanding of the mechanisms involved in the process of tumor-induced immune suppression. To get a better understanding of the immunosuppressive environment of NBL tumors and to find novel ways to counteract the local immune suppression, relevant preclinical tumor models are needed [10]. Genomic amplification of the proto-oncogene MYCN is consistently associated with a poor prognosis and occurs in around 20 % of primary NBL tumors [11]. The THMYCN transgenic mouse model of NBL is driven by the over expression of MYCN in the developing sympathetic nervous system, eventually resulting in paraspinous NBL [12]. In our transplantable model, we inject the tumor cells into the adrenal gland, which is a different location than the tumors arising in the TH-MYCN transgenic, but more like in human adrenal NBL [13]. The spontaneous arising NBL tumors in the TH-MYCN model are very similar to high-risk human NBL with respect to tumor histology and genetics [14, 15]. Cell lines were derived from NBL tumors that arose in transgenic TH-MYCN mice. Transplantation of these cell lines in syngeneic mice also resulted in NBL tumors with similar histology and genetics compared to human NBL [16, 17]. We recently set up and described such a transplantable TH-MYCN model by transplanting the TH-MYCN-derived NBL cell line 9464D in C57Bl/6 mice [18]. In these studies, we found that the immunological properties of the transplantable TH-MYCN model were similar to those of high-risk human NBL, rendering this model a powerful tool in the preclinical development of novel immunotherapies for NBL [19]. In our previous studies, the TH-MYCN 9464D NBL cells were injected subcutaneously (SC), allowing for external measurement of tumor growth [18]. In this study, we established an orthotopic transplantable TH-MYCN model using 9464D NBL cells and a luciferase expressing 9464D variant. Interestingly, the orthotopic intra-adrenal (IA) NBL tumors grew out much faster compared to SC NBL tumors and were more heavily infiltrated by macrophages exhibiting an immunosuppressive M2 phenotype. We conclude that this orthotopic transplantable TH-MYCN model represents a highly relevant model to develop and understand the mechanisms o (...truncated)