Bone marrow specimens, collected according to international and institutional guidelines, were obtained through BM aspiration from 26 pediatric patients referred to the “IMIEM” Children’s Hospital (Toluca, Mexico) and to the “Moctezuma” Children’s Hospital (Mexico City, Mexico) and diagnosed with ALL, before any treatment (Table S1 in Supplementary Material). Control BM specimens were obtained from six healthy children undergoing minor orthopedic surgery. Mononuclear cells (MNC) were obtained by Ficoll-Paque (GE Healthcare) density gradient centrifugation and then enriched in the CD34⁺ fraction using the human CD34 MicroBead Kit UltraPure human (MiltenyiBiotec) according to the manufacturer’s instructions. Cell purity and yield were measured by flow cytometry using a BD FASCanto II. All procedures were approved by the Ethics, Research, and Biosafety Committees at the participant hospitals and by the National Committee of Scientific Research at the Mexican Institute for Social Security (CIEICE-007-01-13, R-2012-3602-29, and R-2015-785-120). All the samples were collected after written informed consent from the parents.

Mononuclear cells (2–4 × 10⁶) from normal or leukemic BM were placed in culture with low glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 100 U/ml of penicillin/streptomycin (Gibco). MSC were then isolated by their plastic adherence properties according to the International Society for Cellular Therapy (23). Upon confluence, cell monolayers were trypsinized and reseeded for their expansion and biological characterization for at least one more week. All experiments were performed with harvested cells from the third or fourth passage. Immunophenotype of MSC was confirmed by flow cytometry as CD45⁻CD34⁻HLA-DR⁻CD90⁺CD73⁺CD105⁺.

To evaluate the chondrogenic differentiation potential of normal and ALL-MSC, they were cultured with chondrogenic medium [DMEM-alpha glucose, supplemented with 0.1µM dexamethasone, 50 µg/ml ascorbic acid, 110 µg/ml sodium pyruvate, 40 µg/ml prolin, 10 ng/ml TGF-β1, 50 mg/ml ITS+ premix (Becton Dickinson), 6.25 µg/ml insulin, 6.25 µg/ml transferrin, 6.25 ng/ml selenic acid, 1.25 mg/ml bovine serum albumin, and 5.35 mg/ml linoleic acid] followed by Alcian blue staining after 14 days. For adipogenic differentiation, MSC were grown in medium supplemented with 0.5mM isobutil-metilxantin, 1µM dexametasone, 10µM insulin, and 200µM indomethacin, before red oil staining. Osteogenic differentiation was investigated using low glucose DMEM medium supplemented with 15% of osteogenic supplement, 10⁻⁷M dexamethasone, and 0.2 mM ascorbic acid. Cells were grown during 3 weeks and then stained with von Kossa dye.

Mesenchymal stromal cells’ progenitor frequencies were recorded by fibroblast colony formation assay. Crescent numbers of MNC were plated in DMEM supplemented with 10% of FBS and 14 days later Wright–Giemsa staining was performed to determine the CFU-F content.

A carboxifluorescein (CFSE) dilution assay was conducted using 10mM CFSE per 5 × 10⁵ MSC and dye dilution evaluated by flow cytometry at 24 and 48 h.

Transcription of differentiation-related genes was evaluated by RT-PCR using the following primers: PPARγ2, Runx2, Sox9, CXCL12, HIF-1α, and IL7, with the 18s ribosomal RNA as housekeeping control. cDNAs were subject of amplification by RT-PCR (95°C initial denaturalization for 5 min, 35 amplification cycles of 5 s at 95°C, and aligning temperature in function of every single gene during 30 s and 72°C during 10 min) in a thermal cycler T100 ™BIORAD. AlphaView software (FluorChem System) was used for analysis. Primer sequences: Runx2: F: 5′-CACCATGTCAGCAAAACTTCTT-3′; R: 5′-TCACGTCGCTCATTTTGC-3′. Sox9: F: 5′-GTACCCGCACTTGCACAAC-3′; R: 5′-TCGCTCTCGTTCAGAAGTCTC-3′. PPARG2: F: 5′-TCCATGCTGTTATGGGTGAA-3′; R: 5′-TGTGTCAACCATGGTCATTTC-3′. CXCL12: F: 5′-CCAAACTGTGCCCTTCAGAT-3′; R: 5′-CTTTAGCTTCGGGTCAATGC-3′. 18s: F: 5′-AAATCCAGCGCAGTACAAGATCCCA-3′; R: 5′-TTTCTTCTTGGACACACCCACGGT-3′.

Around 1,000 lymphoid progenitors were seeded (input value) in alpha-MEM 10% FBS supplemented with 100 ng/ml rhSCF, 100 ng/ml rhFlt3-L, 20 ng/ml rhIL-7, and 10 ng/ml rhIL-15. Cocultures were maintained during 3–6 weeks, and B precursor cells production was evaluated by multiparametric flow cytometry (FACSCalibur, BD). Results were recorded as yield per input (absolute number of B cell precursors that are produced per each initial progenitor).

Most primary and secondary antibodies came from Abcam: mouse monoclonal CXCL12 (MM0211-9N26), rabbit recombinant monoclonal SCF (EP665Y), rabbit polyclonal HIF-1α (AB103063), or mouse monoclonal connexin-43 (Cx-43) (4E6.2). Goat Anti-Mouse Alexa Fluor^® 488 (IgG H&L) (ab150113), Goat Anti-Rabbit Alexa Fluor^® 405 (IgG H&L) (ab175652), and Donkey Anti-Mouse Alexa Fluor^® 405 (IgG H&L) (ab175658). Rabbit Anti-Human phospho-NF-kB p65 (ser536) (93H1) came from cell signaling. Source for immunophenotyping antibodies: CD34-APC (Clone 581, BioLegend), CD19-FITC (Clone HIB19, BioLegend), CD45-PECy5 (Clone HI30, BioLegend), HLA-DR-PE (clone L243, BioLegend), CD90-APC (5E10, BD), CD73-PE (AD2, BD), and CD105-FITC (266, BD).

Supernatants were removed from normal and ALL-MSC cultures and the secreted factors quantified by multiplex cytokine detection assay (Milliplex Map, Millipore) according to the manufacturer’s instructions.

Mesenchymal stromal cells were treated with 100µM cobalt chloride II (CoCl₂) during 24 h. Cytoplasmic stabilization of HIF-1α was measured by flow cytometry and its nuclear translocation by indirect immunofluorescence microscopy.

Mesenchymal stromal cells–hematopoietic cell cocultures were supplemented with BrdU during 24 h and leukemic cells harvested, fixed, and permeabilized. DNAse treatment was performed for 1 h at 37°C before cell incubation with anti-BrdU. Proliferation rates were analyzed by multiparametric flow cytometry.

Mesenchymal stromal cells were fixed with paraformaldehyde 4% and permeabilized with PBS–Triton 0.01%. Fc receptors were blocked with 3% PBS–FBS, and cells were incubated overnight with anti-CXCL12, anti-SCF, anti-HIF-1α, anti-IL-7, anti-Cx-43, or anti-phospho-p65 monoclonal antibodies. AlexaFluor^®488 and AlexaFluor^®405 conjugated antibodies were further added. Nuclei were stained with propide iodide before cell preparations were analyzed in a confocal microscope (NIKON). Images were analyzed using the ImageJ 1.51h software (Wayne Rasband, NIH, USA).

Mesenchymal stromal cells were induced to form spheroids in non-adherent wells and leukemic precursor cells cocultured with each spheroid. Normal or leukemic cells were labeled with fluorescent dyes CellTraceViolet^® or CellTrace Far Red^® (Life Technologies) to distinguish each other by confocal microscopy throughout competence assays. Immunophenotype of leukemic cells were performed by flow cytometry after spheroid enzymatic digestion. CXCL12 expression was concomitantly evaluated by confocal microscopy. Briefly, spheroids were washed several times with PBS followed by fixing and permeabilization for 30 min. Spheroids were incubated with antibodies as described before.

Prism V3.02 (GraphPad) software was used to perform statistical data analysis. Yield per input results were showed as media values ± SD. Differences within groups were established by non-parametric test Mann–Whitney, considering significant probability values <0.05. For three or more groups, the Kruskal–Wallis test has been applied. Comparative analyses are reported as Mean ± SEM.

Bone marrow MSC from childhood ALL patients were characterized according to their morphological, phenotypical, and functional properties established as the minimal criteria by the International Society for Cellular Therapy (32). No differences in morphological aspects between cells from both sources were apparent. However, ALL-MSC showed intercellular disorganization when monolayer structures were observed (Figure 1A). Remarkably, although CFU-F capacity and cell proliferation were reduced (Figures 1B,C), increasing proliferation activity was recorded when 7-day cultures were performed, suggesting that once the ALL primary MSC are adapted to in vitro culture conditions, autocrine growth factors may induce their high extent proliferation. Nevertheless, simple population dynamics assays by cell counting suggest ALL-MSC have no ability to reach normal rates (Figure S1 in Supplementary Material). In contrast, neither their differentiation potential to chondrogenic, adipogenic, and osteogenic lineages nor their phenotypes were truly compromised (Figures 1D,E). As known, although specific MSC markers are still under investigation, currently the most accepted phenotype is CD34⁻CD45⁻CD14/CD11⁻CD90⁺CD73⁺CD105⁺ with variable expression of MHC II, CD29 (integrin β1), CD49b (integrin α2), CD44 (HCAM), CD54 (ICAM-1), CD58 (LFA-3), and CD166 (ALCAM). Here, our data indicate a correspondence in the phenotype of the MSC from ALL and NBM, showing minor differences in expression of HLA-ABC, CD13, CD73, CD140b, CD44, CD54, CD49b, and CD166, which may relate to potential abnormalities in the capacity of cell–cell intercommunication with leukemic precursors within the BM. Interestingly, as previously reported for angiogenic-stimulated abnormal MSC in hematological malignancies (33), CD90 was reduced in a subpopulation of ALL (Figure 1E; Figure S1 in Supplementary Material). Whether this molecule may discriminate more primitive stages of mesenchymal linage, as it does in the hematopoietic system, remains elusive. Differentiation gene transcripts, including Runx2 (osteogenesis), Sox9 (chondrogenesis), and PPARG2 (adipogenesis) revealed a substantial reduction in Sox9 expression by ALL cells, suggesting altered lymphopoiesis associated to defects in osteochondrogenesis (Figure 1F) as reported (34). Despite the fact that PPARG2 expression was not obviously altered, a number of ALL BM samples displayed augmented ability to differentiate toward adipocytes (Figure 1D).

Notably, the hematopoietic support capability of MSC from ALL BM was increased when compared to their normal counterparts (Figure 2A), though it is uncertain the normal or leukemic origins of such supported cells. When normal primitive hematopoietic cells are placed onto ALL-MSC monolayers to support cell differentiation, the production of CD19⁺ B cells and CD56⁺ NK cells is critically impaired (evaluated as cell frequencies and as yield per input progenitor). In contrast, near 30 and 15% of the produced cells are CD19⁺ B-lineage or CD56⁺ NK cells, respectively, in normal to normal conditions (Figure S2 in Supplementary Material). Together, the data may strengthen the notion that abnormal MSC from ALL BM constitute special niches convenient for leukemic cell proliferation and/or differentiation but unable to sustain normal hematopoietic development (Figure S3 in Supplementary Material). Moreover, we extended these studies to explore the ALL-MSC ability of intercommunicating to HSPC by evaluating the display of the major adhesion VLA-4/VCAM-1 axis and found a substantial reduction of VLA-4 expression by ALL hematopoietic MNC, as well as decreased VCAM-1 expression by ALL-MSC. Thus, suboptimal adhesion and interplay between hematopoietic and mesenchymal components of the ALL BM may contribute to malignant cell maintenance at the expense of normal hematopoiesis (Figure S2 in Supplementary Material).

At least 16 soluble factors relevant to lymphoid development were aberrantly secreted, as supernatant contents indicated high levels of pro-inflammatory cytokines IL-1α, IL-6, IL-12p70, and TNFα, as well as interferon type I and type II and early growth factors like Flt3, G-CSF, and IL-7 (Figure 2B). Remarkably, CXCL12 (stromal derived factor-1) and SCF (stem cell factor), two components of the hematopoietic microenvironment and main regulators of stem cell maintenance that have shown to be key players in the early lymphopoiesis pathway, were critically reduced. Accordingly, ALL-MSC-conditioned medium contained high amounts of G-CSF, which has shown the ability of mobilizing hematopoietic cells from BM through a CXCL12/CXCR4 axis breaking mechanism. The observed pro-inflammatory profile was supported by the nuclear NF-κB translocation in ALL-MSC (Figure 2C). Our findings suggest a pro-inflammatory microenvironment contributed by activated MSC that may impact normal and leukemic developmental dynamics.

A crucial regulatory axis of the cross talk between lymphoid progenitors and the hematopoietic microenvironment is CXCL12/CXCR4. MSC producing high amounts of CXCL12 and SCF constitute the primary niche for B-lymphoid progenitor cells (20). Concurring with our previous supernatants observation, confocal microscopy revealed the per-cell-basis reduced production of both factors (Figures 3A–D). In contrast, IL-7 lymphoid niche appears unaltered in ALL (Figure 3E). To explore the biological mechanism associated to CXCL12 loss, we investigated the gene transcription extent and found it heterogeneously impaired among patient samples (Figure 4A). In addition, the intercommunicating stromal cell Gap-junction molecule, Cx-43, which drives secretion of CXCL12 appeared frankly downregulated in ALL-derived MSC (Figure 4B). As it is believed that in BM, MSC maintain hematopoietic cells attached to them in a semi-quiescence state through the dynamic production of CXCL12, lack of Cx-43 impacting the release of CXCL12 (35) may have important implications in architecture and function of BM niches. Moreover, homing to the endosteal niche might relate to Cx-43, due to its participation in the bidirectional traffic from BM to the periphery and vice versa during ALL (36).

HIF-1α is a major transcriptional regulator of CXCL12 (37). Here, a critical reduction of its expression with a cytoplasmic localization instead of the typical nuclear translocation seen in their normal counterpart was recorded in ALL-MSC (Figure 5A). Furthermore, experimental hypoxia was not able to increase HIF-1α expression to normal levels. Chemical imitation of hypoxia by Chloride Cobalt (CoCl₂) has been reported to create a hypoxic-like condition by disturbing the constant proteasomal degradation of HIF-1α and allowing its cytoplasmic accumulation and further nuclear translocation even during normoxic settings. Although expression of CXCL12 showed a slight boost, the end balance was never as high as from NBM (Figure 5B; Figure S4 in Supplementary Material).

Thus, in ALL, the CXCL12-dependent lymphoid niche might be intrinsically altered due at least to an impaired transcriptional regulation by HIF-1α and to an abnormal functional activity of Cx-43.

To investigate the biological relevance of loss of CXCL12 in leukemic progression, we conducted an in vitro cell proliferation assay by incorporation of BrdU under distinct CXCL12 scenarios. Primary leukemic cells were treated either with CoCl₂ to increase CXCL12 expression or with carbenoxolone (CBX), a Cx-43 inhibitor, to indirectly reduce CXCL12, and their proliferative status was analyzed (Figure 5C). Of interest, malignant proliferation extent was inversely proportional to CXCL12 expression in MSC. Cell treatment with CBX did not increase the proliferation of ALL cells over the initial mock settings, suggesting, as expected, that CXCL12 regulation may result from multiple addition elements. Since the role of CXCL12 in early lymphopoiesis has been vastly studied (18, 19), our findings suggest that CXCL12 lymphoid niche may have an additional function in normality by limiting proliferation of malignant precursor cells.

Because in vitro maintenance of primary ALL cells in conventional culture conditions and the 3D in vivo BM structure is critical to generate specialized hematopoietic niches, we explored the possibility of improving the microenvironmental conditions and increasing yield per input values by mimicking the structural BM properties through 3D cocultures in stromal spheroids. We have learned from this novel system that primitive normal and ALL CD34⁺ cells get the ability of colonizing them with high efficiency (Figures 6A–C) (Balandran et al., manuscript in preparation).

Population dynamics was studied by a competence assay between purified CD34⁺ cell populations from ALL BM or healthy donors mobilized peripheral blood that were previously stained with red and blue commercial fluorescent dyes, respectively.

As expected, CXCL12 was induced in 3D cultures, possibly due to reconnection of MSC and the putative hypoxia condition within the system (Figure 6D). Notably, leukemic MSC were less colonized by normal progenitors compared to the NBM counterparts at the first 24 h (Figure 6E). However, 120 h later, the normal niches were colonized by leukemic cells simultaneously to an apparent decrease of CXCL12 and displacement of normal progenitors (Figure 6F; Figure S5 in Supplementary Material).