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

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.

Histone H3K9 and H3K14 acetylation at the promoter of the LGALS9 gene is associated with mRNA levels in cervical cancer cells.

Galectin-9 levels have been reported to be altered in several cancer types, but the mechanism that regulates the expression of Galectin-9 has not been clarified. Galectin-9 is encoded by the LGALS9 gene, which gives rise to eight mRNA variants. The aims of this study were: (a) to identify the mRNA variants of LGALS9, (b) to characterize CpG methylation and H3K9 and H3K14 histone acetylation at the promoter of the LGALS9 gene, and (c) to characterize the relationship between these modifications and LGALS9 expression level in cervical cancer cells. All mRNA variants were detected in HaCaT (nontumoural keratinocytes) and SiHa cells, and seven were observed in HeLa cells. The promoter region of LGALS9 contains eight CpG dinucleotides. No hypermethylation pattern related to low LGALS9 expression was identified in tumour cells. Chromatin immunoprecipitation analysis demonstrated higher acetylation of H3K9ac and H3K14ac in HaCaT cells, which was related to higher mRNA levels. The presence of the mRNA variants suggests that alternative splicing may regulate the expression of galectin-9 isoforms. The results of this study suggest that histone acetylation, but not promoter CpG methylation, may be involved in the transcriptional regulation of the LGALS9 gene.