Patient-Derived Bone Marrow Spheroids Reveal Leukemia-Initiating Cells Supported by Mesenchymal Hypoxic Niches in Pediatric B-ALL

Abstract

B-cell acute lymphoblastic leukemia (B-ALL) results from the expansion of malignant lymphoid precursors within the bone marrow (BM), where hematopoietic niches and microenvironmental signals provide leukemia-initiating cells (LICs) the conditions to survive, proliferate, initiate disease, and relapse. Normal and malignant lymphopoiesis are highly dependent on the BM microenvironment, particularly on CXCL12-abundant Reticular (CAR) cells, which provide a niche for maintenance of primitive cells. During B-ALL, leukemic cells hijack BM niches, creating a proinflammatory milieu incompetent to support normal hematopoiesis but favoring leukemic proliferation. Although the lack of a phenotypic stem cell hierarchy is apparent in B-ALL, LICs are a rare and quiescent population potentially responsible for chemoresistance and relapse. Here, we developed novel patient-derived leukemia spheroids (PDLS), an ex vivo avatar model, from mesenchymal stromal cells (MSCs) and primary B-ALL cells, to mimic specialized niche structures and cell-to-cell intercommunication promoting normal and malignant hematopoiesis in pediatric B-ALL. 3D MSC spheroids can recapitulate CAR niche-like hypoxic structures that produce high levels of CXCL10 and CXCL11. We found that PDLS were preferentially enriched with leukemia cells displaying functional properties of LICs, such as quiescence, low reactive oxygen species, drug resistance, high engraftment in immunodeficient mice, and long-term leukemogenesis. Moreover, the combination of PDLS and patient-derived xenografts confirmed a microenvironment-driven hierarchy in their leukemic potential. Importantly, transcriptional profiles of MSC derived from primary patient samples revealed two unique signatures (1), a CXCL12low inflammatory and leukemia expansion (ILE)-like niche, that likely supports leukemic burden, and (2) a CXCL11hi immune-suppressive and leukemia-initiating cell (SLIC)-like niche, where LICs are likely sustained. Interestingly, the CXCL11+ hypoxic zones were recapitulated within the PDLS that are capable of supporting LIC functions. Taken together, we have implemented a novel PDLS system that enriches and supports leukemia cells with stem cell features driven by CXCL11+ MSCs within hypoxic microenvironments capable of recapitulating key features, such as tumor reemergence after exposure to chemotherapy and tumor initiation. This system represents a unique opportunity for designing ex vivo personalized avatars for B-ALL patients to evaluate their own LIC pathobiology and drug sensitivity in the context of the tumor microenvironment.

Acute lymphoblastic leukemia-secreted miRNAs induce a proinflammatory microenvironment and promote the activation of hematopoietic progenitors

“…The same was suggested for MSC when RNAseq data were analyzed from our previous work available in E-MTAB-10838 https:// www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-10838/ and when TLR8 expression was investigated by multiparametric flow cytometry in CD45 − CD73 + CD105 + CD90 + primary stromal cells (Figure 8(C), left and right panels) 17 Together, miR-146a-5p, miR-181b-5p, and miR-199b-3p are secreted by ALL cells inside exosomes into the BM microenvironment with apparently direct effect on both, residual normal hematopoietic cells, including innate immune cells, and MSC.…”

“…Our previous work has shown that ALL mesenchymal stromal cells (MSCs) display a proinflammatory profile with nuclear NF-κB translocation and contribute to an aberrant microenvironment through an increased secretion of cytokines including IL-1α, IL-6, IL-12p70, and TNFα. 15,17,22,23 Here, we report 3 miRNAs, which are up-regulated in ALL and contain the GU motifs between 18th and 21th positions, determinant for binding to the TLR8. The revealed ALL-miRNAs function as agonist TLR8 ligands, promoting inductive signals for a proinflammatory connection between tumor and normal early hematopoiesis in ALL cells.…”

“…15 Considering the underestimated roles of proinflammatory mechanisms in childhood cancer progression and relapse, we aimed to investigate if miRNAs could participate as mediators in the establishment of inducive feedback loops between tumor cells and their microenvironment in acute lymphoblastic leukemia (ALL), as transferable and dual-functioning miRNAs may play detrimental roles for normal hematopoiesis while supporting the formation of tumor niches and cooperating to expression of genetic aberrations within the leukemic BM. 16,17 ALL is the most common type of pediatric cancer in the world and, despite effectiveness of current treatment protocols reaching 85% long-term survival rates and low numbers of failure on remission induction in some highly developed countries, 18 abundant questions on the mechanisms involved in resistance to therapy, maintenance, progression, and high-risk prognosis are to be addressed. 19 The creation of proinflammatory microenvironments in the BM has emerged as a potential promoter of leukemic cells proliferation and survival, concomitant to a role in the replacement of normal hematopoiesis.…”

In Vitro and In Vivo Modeling of Normal and Leukemic Bone Marrow Niches: Cellular Senescence Contribution to Leukemia Induction and Progression

“…Hence, several protocols for bioengineering 3D HSC niches have been established for mouse and human HSC and LSC expansion [4,[103][104][105][106][107][108][109][110][111]. A well-designed artificial niche must be adaptable and scalable, facilitate selective incorporation of a variety of biochemical signals and allow the use of high-throughput or single-cell techniques, in addition to overcoming biomechanical and biotransport restrictions [68].…”

“…A well-designed artificial niche must be adaptable and scalable, facilitate selective incorporation of a variety of biochemical signals and allow the use of high-throughput or single-cell techniques, in addition to overcoming biomechanical and biotransport restrictions [68]. A big step towards the modeling of the leukemic niche was the possibility to grow leukemic cells as spheroids [107,112,113], or their implantation into polymer matrixes [37,109,112] or inside porous scaffolds [103,105,111,114]. The use of bioreactors or microfluidic devices further improved nutrient supply and increased the number of parameters involved on the niche building [115][116][117][118][119].…”