Abstract NG11: Autologous humanized PDX modeling for immuno-oncology recapitulates the human tumor microenvironment
Authors: Chiorazzi Michael , Martinek Jan , Krasnick Bradley , Zheng Yunjiang , Robbins Keenan , Qu Rihao , Kaufmann Gabriel , Skidmore Zachary , Henze Laura , Brösecke Frederic , Adonyi Adam , Zhao Jun , Shan Liang , Sefik Esen , Mudd Jacqueline , Bi Ye , Goedegebuure S Peter , Griffith Malachi , Griffith Obi , Oyedeji Abimbola , Fertuzinhos Sofia , Garcia-Milian Roland , Boffa Daniel , Detterbeck Frank , Dhanasopon Andrew , Blasberg Justin , Judson Benjamin , Gettinger Scott , Politi Katerina , Kluger Yuval , Palucka A Karolina , Fields Ryan , Flavell Richard A. 
Published: April 2023

Abstract
The immune milieu within tumors, consisting of diverse cell types including adaptive immune cells as well as macrophages, dendritic cells, natural killer and other innate immune cells, is critical to determining cancer outcome. However, the immune tumor microenvironment (TME) has been challenging to model, owing to inherent inter-species differences. While humanized mice can support human immune cells, the hematopoietic stem and progenitor cells (HSPCs) used for transplantation have been largely limited to fetal or neonatal stem cell sources, necessitating allogeneic experiments with limited applicability. We sought to develop a method to pre-clinically model an individual adult cancer patient, capturing the unique features of an individual such as germline genetic determinants of immune function and somatic tumor heterogeneity, and creating an autologous system.
MISTRG6 may be engrafted with low numbers of HSPCs. When engrafted with equivalent numbers of CD34+ cells from human fetal liver (FL), neonatal cord blood (CB), adult mobilized peripheral blood (MPB), or adult bone marrow (BM), MISTRG6 mice harbored greatly increased human hematopoietic cells as a proportion of total hematopoietic cells in peripheral blood compared with NOD-scid-gamma (NSG) and MISTRG mice (p&lt;0.0001). We found that MISTRG6 mice could be engrafted with as few as 1,000 human HSPCs, arguably 100x more efficient than other models, and achieve robust hematopoietic transplantation after 10-12 weeks, indicating the efficiency of this strain in supporting the growth of hematopoietic cells. To better elucidate the mechanism responsible for this enhanced human engraftment, we enumerated human and mouse hematopoietic progenitors in BM of NSG, MISTRG, and MISTRG6 mice. Human progenitors, including CD34+ and CD34+CD38+ cells, were significantly increased in both frequency and absolute numbers in MISTRG and MISTRG6 mice compared with NSG mice (p&lt;0.001), and mouse hematopoietic lin(-)cKit+ (LK) and lin(-)Sca1+cKit+ (LSK) progenitor populations were significantly diminished (p&lt;0.0001), suggesting that the enhanced hematopoietic engraftment observed in MISTRG6 is, in part, a consequence of increased human progenitor frequency and reduced mouse competition.
MISTRG6 allows efficient engraftment of patient derived HSPCs. We sought to apply this improved engraftment prospectively to model individual patients’ TME through collection of BM-derived CD34+ cells from patients under active treatment along with tumor tissue from the same patient. At two cancer centers, we enrolled patients with melanoma, NSCLC, PDAC, and HNSCC to provide BM aspirate, peripheral blood, and tumor tissue. CD34+ cells were isolated from BM aspirates and tumor tissue was utilized to generate PDXs. Overall, 71 patients were enrolled, 46 melanoma, 19 NSCLC, 4 PDAC, 2 HNSCC, ages 22-85, 39% females. These yielded autologous, immune-reconstituted MISTRG6 hosts from 14 melanoma, 5 NSCLC, 2 PDAC, and 1 HNSCC patients. Autologously engrafted MISTRG6 mice displayed the gamut of human immune cells of adaptive and innate types in PB at 7 weeks of age. Notably, this included CD33+ myeloid cells such as CD14+CD16− classical, CD14+CD16+ intermediate, and CD14−CD16+ non-classical monocytes. Moreover, human dendritic cells (DCs), key innate immune cells for initiation of anti-tumor responses were readily detected by flow cytometry in spleens of autologously-engrafted mice, including cDC1, cDC2, and pDC cells.
MISTRG6 mice bearing a patient’s hematopoietic cells support autologous PDX growth. Having achieved successful engraftment of patient hematopoietic systems in MISTRG6 hosts, we next subcutaneously introduced the patient’s matched PDX tumor tissue to generate autologously engrafted PDX mice. For most patients, tumors grown in autologous HSPC-engrafted hosts were significantly larger than in non-engrafted hosts. Multicolor immunofluorescence staining of PDX tumors demonstrated that human immune cells, including CD3+ T cells, CD14+ and HLA-DR+ myeloid cells, penetrated deeply into the tumor and co-localized with tumor cells as well as with other engrafted immune cells. Indeed, HLA-DR+CD14+macrophages and HLA-DR+CD14(-) dendritic cells were present, and direct physical interaction between T cells and macrophages was evident. Using whole-exome sequencing, we found that 225 somatic changes were shared between patient Mel738’s surgical resection sample, two PDX tumors from non-engrafted mice lacking human immune cells, and two PDX tumors from mice with autologous engraftment. 5 additional changes were shared among the tumor samples and absent from the cell line, with 36 additional mutations being specific to the cell line. These data underscore the capacity of the autologous PDX method to recapitulate the somatic heterogeneity that the patient tumor possesses.
Autologous MISTRG6 mice display diverse human immune cell populations and recapitulate an immunosuppressive TME. To fully characterize the autologous MISTRG6 model and investigate mechanisms by which autologous human immune cells enhance tumor growth, we performed single cell transcriptomics on hCD45+-enriched cells from blood and tumor isolated from autologous mice. This revealed 16 distinct cell subtypes, including 3 myeloid, 2 NK cell, 2 CD8 T cell, 3 CD4 T cell, 2 cycling lymphocyte, 1 B cell, and 3 melanoma cell clusters. Subclustering of myeloid cells revealed 9 distinct clusters including 4 monocyte, 4 macrophage, and 1 DC cluster. Comparing CD8 T cells present in blood versus tumor revealed that the most differentially expressed genes (DEGs) found in blood were characteristic of naïve T cells, while genes present in the TME were consistent with activated T cell phenotypes. In addition, sub-clustering revealed 3 distinct CD8 T cell types that included two activated-like populations, with one of these populations also expressing an activated/exhausted program typified by expression of PDCD1, LAG3, and GZMA. Naïve-like T cells were most highly represented in the blood, while activated and activated/exhausted-like genes were more present in the TME.
Inhibiting the actions of human VEGF-A blocks the enhanced tumor growth in autologously engrafted mice. Notably, IPA Upstream Regulator Analysis identified VEGFA, a central player in tumor growth and vascularization, as a key upstream inducer of genes in the TME (FDR p= 5.65 × 10−13). Indeed, expression VEGFA itself was nearly absent in blood but induced in the TME, especially in macrophages and VEGFA targets were highly represented among the DEGs between tumor and blood.To test the relevance of VEGF-A in the TME, we selectively blocked human VEGF-A by treating autologous mice humanized from Mel2 with the anti-hVEGF-A antibody bevacizumab that has high affinity for human VEGF-A yet low affinity for mouse VEGF-A. PDXs grown in untreated autologously engrafted MISTRG6 mice grew significantly larger than those in non-engrafted littermate control hosts (p&lt;0.05). When treated with bevacizumab, the enhanced tumor growth was significantly abrogated, with bevacizumab-treated mice bearing significantly smaller tumors compared with controls (p&lt;0.001).
Future Directions: Thus, these in silico and in vivo results suggest that human VEGF-A production in the autologous TME enhances tumor growth in MISTRG6 PDX models and underscores the utility of the MISTRG6 system for pre-clinical testing of drugs that act on human immune components of the TME. By engrafting mice with bone marrow derived stem cells followed by implantation of tumor derived from the same donor, we have demonstrated that autologous MISTRG6 models recapitulate important features of the human TME, including sufficient immunosuppression to prevent tumor clearance, presence of activated/exhausted T cells, and innate immune cells including DCs, monocytes, NK cells, and macrophages, the latter especially relevant to the production of VEGF-A.
Citation Format: Michael Chiorazzi, Jan Martinek, Bradley Krasnick, Yunjiang Zheng, Keenan Robbins, Rihao Qu, Gabriel Kaufmann, Zachary Skidmore, Laura Henze, Frederic Brösecke, Adam Adonyi, Jun Zhao, Liang Shan, Esen Sefik, Jacqueline Mudd, Ye Bi, S Peter Goedegebuure, Malachi Griffith, Obi Griffith, Abimbola Oyedeji, Sofia Fertuzinhos, Roland Garcia-Milian, Daniel Boffa, Frank Detterbeck, Andrew Dhanasopon, Justin Blasberg, Benjamin Judson, Scott Gettinger, Katerina Politi, Yuval Kluger, A Karolina Palucka, Ryan Fields, Richard A. Flavell. Autologous humanized PDX modeling for immuno-oncology recapitulates the human tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr NG11.