Acute myeloid leukemia shapes the bone marrow stromal niche in vivo.

Emerging evidence suggests that acute myeloid leukemia (AML) remodels the bone marrow (BM) niche into a leukemia-permissive microenvironment, while suppressing normal hematopoiesis. The influence of AML on bone tissue architecture and osteogenic cell differentiation has been documented in murine models, however, the impact of patient-derived AML cells on human BM stromal cells (BMSC) has only been investigated using conventional in vitro approaches. We assessed the differentiation potential of AML-derived BMSC using two in vivo models that recapitulate the complex organization of the human hematopoietic niche. We found that BMSC derived from pediatric AML patients: i) exhibit a reduced mature bone formation, ii) develop an osteoprogenitor-rich niche, iii) generate bone/BM organoids with a higher adipocytic differentiation, and iiii) support the formation of osteoclasts in a similar proportion to normal donor controls. All these aspects may contribute to the inhibition of normal hematopoietic stem and progenitor cell development and propagate selective blast cell survival and expansion. AML is a heterogeneous disorder characterized by the clonal proliferation of blasts in the BM. Leukemic cells compete with normal hematopoietic stem cells for niche occupation and this results in alterations of the BM microenvironment and the generation of a “leukemic niche” that selectively supports the malignant clone. AML-induced changes in the BM microenvironment have been confirmed in multiple in vitro and in vivo studies. Murine AML models have shown several alterations in the BM niche components (e.g., osteo-progenitors and osteoblasts) positively correlated with leukemogenesis. Similarly, a decrease in osteoblast number has been observed in the BM of AML patients together with reduced osteocalcin serum levels. Moreover, studies have also reported that BMSC, one of the main cellular components of the hematopoietic niche, derived from AML patients exhibit a number of molecular and functional alterations, such as translocations, gene expression modifications, reduced clonogenic potential, decreased proliferation, higher senescence, impaired in vitro adipogenic and osteogenic differentiation, increased support of leukemia growth, and imbalanced regulation of endogenous hematopoiesis. By contrast, other studies have reported that AML-BMSC display normal morphology and differentiation properties. The humanized models are currently the only experimental system to reproduce an in vivo three-dimensional structure of the human BM niche, despite the limitations related to the potential interference of other components of recipient origins. Several in vivo models have been described using normal or genetically modified human BMSC to generate a humanized BM niche that enables robust AML cell engraftment and allows evaluation of factors critical for the development/progression of leukemic cells within the niche. The aim of our work was to evaluate if AML-BMSC have undergone significant changes in their capability to form bone and a BM niche after exposure to patient leukemia in the BM. We isolated and expanded in vitro BMSC from the BM of newly diagnosed pediatric AML patients (AML-BMSC) and healthy donors (HD-BMSC). Patients and healthy controls were age-matched. BMSC donor characteristics are summarized in the Online Supplementary Table S1. BMSC from both sources displayed a spindle-shaped, elongated morphology and formed discrete fibroblast colony-forming units with no differences in colony-forming efficiency (CFE) and in the number of cumulative population doublings (CPD) (Figure 1A-C). Similarly, BMSC derived from both sources revealed an identical in vitro immunophenotype consistent with standard criteria (Figure 1D). As a few studies have described impaired hematopoietic support capacities of AML-derived BMSC, we investigated several cell-bound as well as secreted factors governing the hematopoiesis within the niche. We found significantly diminished mRNA levels of Kit-ligand (KITLG), while other hematopoiesis regulatory molecules such as VCAM1, Angiopoietin-1, CXCL12, and Jagged1 were unaffected (Figure 1E). We then evaluated the in vitro skeletogenic potential of AML-BMSC versus HD-BMSC by performing quantitative gene expression of known osteogenic and chondrogenic genes at baseline in monolayer cultures without the addition of any inducing factors. We found a similar expression in both AML-BMSC and HD-BMSC, except for SP7/Osterix levels which were significantly reduced in AML-BMSC (Figure 1F). In vitro adipogenic, osteogenic, and chondrogenic differentiation assays showed normal tri-lineage differentiation potential for AML-BMSC population as proven by morphology, cytochemical staining, and upregulation of mRNA levels of tissue-specific markers (Figure 1G). The previously published data on in vitro AML-BMSC functional properties, mainly conducted using adult patient cohort samples, are contradictory. One of the reasons for such heterogeneity could be related to the age of patients. AML in young and adult patients should be considered differently since the biological and molecular characteristics of leukemic cells are different. In previous studies that included also pediatric cases, the reduction of adipogenic and osteogenic differentiation potential was correlated with AML characteristics at diagnosis and not to the patients’ age. In contrast, as our results confirm, the differential proliferative capacity of AML-BMSC is related to the age of the patients, with older patient samples displaying a reduction in proliferative ability when compared to younger patient samples. The conventional in vitro differentiation assays are partially predictive of the in vivo physiologic functions of BMSC as these cultures leverage artificial inducing differentiation factors that do not necessarily reflect the intrinsic physiological potential of the cells. Therefore, in order to accurately assess the in vivo functional properties of AML-BMSC in a physiologic environment we used two distinct heterotopic transplantation models to assess the osteogenic activity as well as the capacity to establish a complete hematopoietic niche, respectively. The first assay, which allows evaluation of the BMSC differentiation capacity into osteoblasts based on the formation of histologically-provable bone, was performed by implanting AML-BMSC or HD-BMSC loaded on an osteoconductive hydroxyapatite/tricalcium phosphate carrier in subcutaneous tissues of immunocompromised SCID/beige mice. Histological analysis of the transplants harvested at 8 weeks revealed bone deposition in both groups (Figure 2A-B). Immunostaining of sections with an anti-osterix antibody showed the presence in AML-derived ossicles of osterix-expressing osteoprogenitor cells accompanied by osterix-positive osteocytes (Figure 2C, top panels). Immunostaining with an antiosteocalcin antibody, a marker for mature osteoblasts, revealed a virtual absence of positive cells along the bone surfaces in the AML-derived implants and osteocyte immunoreactivity in both AMLand HD-derived