Ewing sarcomas developed in the hu-CD34+ model are biologically and transcriptionally distinct from tumors developed in an immunodeficient model. A, Orthotopic Ewing sarcomas are successfully established in a humanized (hu-CD34+) mouse. B, Representative CD99 and NKX2.2 IHC staining of Ewing sarcomas developed in a hu-CD34+ model, shown with a magnification of 200×. C, Representative CD99 and NKX2.2 IHC staining of lung metastases that developed in the hu-CD34+ Ewing sarcoma model, shown with a magnification of 200×. D, Immunophenotyping performed on peripheral blood mononuclear cells obtained from mice bearing TC32 Ewing sarcoma tumors at euthanasia following tumor growth demonstrates persistence of engrafted human immune cells. HuCD45 represents % of all peripheral lymphocytes that are huCD45 positive. The remaining cells that are hu-CD45 negative represent (defective) peripheral blood mononuclear cells of mouse origin. The percent of hu-CD45 cells that express CD20, CD3, and CD33 (representing B cells, T cells, and myeloid cells, respectively) is shown. n = 36 mice. Error bars represent SD. E, Representative flow cytometry plot demonstrates low (1.24% of all cells in the tumor) baseline huCD45+ infiltration in Ewing sarcomas developed in hu-CD34+ mice. F, Schematic depicting hu-CD34+ vs. NSG mouse models used in these experiments. G, Bilateral lungs were harvested from hu-CD34+ or NSG mice harboring TC32 Ewing sarcoma tumors, and the presence of metastatic foci per section was determined by an independent pathologist. Each bar represents average number of metastatic lesions per slide in individual mouse. ***, P t test was performed. n = 9 humanized mice and n = 6 NSG mice. H, Volcano plot of DEGs in TC32 tumors established in hu-CD34+ mice vs. NSG mice. I, Tumors were established (TC32 cell line) in hu-CD34+ or NSG mice. Time from tumor injection to survival was determined using pre-specified end points for survival (see “Materials and Methods”). Kaplan–Meier estimate was used for analysis. N = 7 and N = 9 for Hu-CD34+ and NSG, respectively. ns = P value ≥ 0.05. F, Created in BioRender. Bailey, K. (2025) https://BioRender.com/4jtlo4v.
Daley et al. (Wed,) studied this question.