Mesenchymal niche remodeling impairs hematopoiesis via stanniocalcin 1 in acute myeloid leukemia.

Acute myeloid leukemia (AML) disrupts the generation of normal blood cells, predisposing patients to hemorrhage, anemia, and infections. Differentiation and proliferation of residual normal hematopoietic stem and progenitor cells (HSPCs) are impeded in AML-infiltrated bone marrow (BM). The underlying mechanisms and interactions of residual hematopoietic stem cells (HSCs) within the leukemic niche are poorly understood, especially in the human context. To mimic AML infiltration and dissect the cellular crosstalk in human BM, we established humanized ex vivo and in vivo niche models comprising AML cells, normal HSPCs, and mesenchymal stromal cells (MSCs). Both models replicated the suppression of phenotypically defined HSPC differentiation without affecting their viability. As occurs in AML patients, the majority of HSPCs were quiescent and showed enrichment of functional HSCs. HSPC suppression was largely dependent on secreted factors produced by transcriptionally remodeled MSCs. Secretome analysis and functional validation revealed MSC-derived stanniocalcin 1 (STC1) and its transcriptional regulator HIF-1α as limiting factors for HSPC proliferation. Abrogation of either STC1 or HIF-1α alleviated HSPC suppression by AML. This study provides a humanized model to study the crosstalk among HSPCs, leukemia, and their MSC niche, and a molecular mechanism whereby AML impairs normal hematopoiesis by remodeling the mesenchymal niche.

Total leukemic cell count normalized to PBS control. (E) Normal BM CD34 + co-cultured on MSCs and either PBS or rSTC1 was added for 5 days. Secondary CFUs per CD34 + in BM CD34 + .

MSC
MSCs were seeded at 2.5x10 4 /cm2 in a 12-well dish in 400 µL of media at a multiplicity of infection (MOI) of 10 and incubated at 37° C. After 16h the virus is removed and the MSCs are trypsinized and seeded. All co-cultured and scaffolds were started 4 days after MSC transduction. Transduction efficiency was generally >80 %.

Generation of Lentivirus vectors
The generation and validation of lentivirus vector against HIF-1a (Sh-HIF1) is described in our previous publication (20). Briefly, H1-shRNA sequences were subcloned in the lentivirus (pTripΔU3Ef1α-EGFPMCSΔU3) that contains the enhanced GFP (eGFP) gene under the control of the EF1α promoter. An shRNA directed against the dsRed fluorescent protein (RFP) was used as a control (shCTL).
Lentivirus vectors against STC1 were purchased from vectorbuilder based on U6-shRNA sequences with eGFP selection marker. As control an shRNA directed against LacZ was used. shRNA sequences are listed in Table S2.

Generation of AML expressing inducible caspase 9 fragment
A plasmid with the MSCV.IRES.GFP retrovirus backbone expressing a product of FKBP1A fused with a Ser-Gly-Gly-Gly-Ser linker to the small and large subunit of the caspase 9 gene (originally deposited by David Spencer) was obtained from Addgene.
Retrovirus production was carried out identically to the lentivirus production with the exception that retrovirus packaging constructs pHIT60 (expressing gag-pol) and pVSV-g (expressing VSV-G) were used (both a generous gift from Jonathan Stoye).
AML cell lines were transduced at MOI 10 and sorted four days after based on GFP + expression (named AML iCasp9).

Colony forming unit assay
To perform colony forming unit assays, CD34 + cells were plated in the methylcellulose media MethoCult H4434 (StemCell Technologies, Vancouver, Canada). Colonies were scored according to their morphology 14 days following plating using an inverted microscope. Colonies were harvested by washing with PBS and re-plated in secondary assays for an additional 14 days.

Long term colony initiation cell -Assay (LTC-IC)
As a feeder-layer MS-5 were seeded at 2x10 4 /cm 2 on day 0 in a 12-well dish. On day 2 the plate was irradiated at 7.5 grey. 4,000 CD34 + HSPCs are suspended in 1 ml of Myelocult H5100 (Stem cell technologies, Vancouver, Canada). The media from the irradiated feeder-layer plate is removed and replaced with 1 ml of the HSPC suspension per well. 500 µl of media was replaced per week. After 5 /6 weeks, all cells are collected and FACS sorted using a surface marker staining for human CD45 and murine SCA-1 to discriminate MS-5 (Sca-1 + CD45 -) and hematopoietic cells (Sca-1 -CD45 + ). 2.2x10 4 hematopoietic cells were resuspended in 1.1 ml of methylcellulose (Methocult H4434 Stem Cell Technologies, Vancouver, Canada) containing SCF, GM-CSF, IL3 and EPO at manufacturer's concentration. One ml of the suspension was transferred to one of the 6 inner wells of a 12-well dish. The outer wells were filled with dH2O to prevent the methylcellulose from drying. Colonies are scored after 14 days.
The number of colonies from 2x10 4 cells was used to calculate the total number of colonies from the progeny of 4,000 HSPCs. The total colony number was then divided by the average colony output per LTC-IC which determines the number of LTC-IC per 4,000 HSPCs.

Flow cytometry analysis and cell sorting
Flow cytometry analysis was performed using a LSRII flow cytometer and LSRFortessa (BD Biosciences, Oxford, UK). Transduced cells were identified based on their GFP expression or via HLA.A2 (mismatched between UCB and primary patient samples sued). Mouse Human CD73 and CD90 was used to label primary MSC. Sorting was performed on either MoFlo XDP (Beckman Coulter), BD FACS Aria or BD Influx sorters using a 100 μm nozzle. Collection tubes contained 2% FCS/PBS and purity checks were performed to check sort quality. Human grafts in mice were assessed using hCD45, hCD3, hCD19, hCD33, hCD34, hCD38, hCD45RA, hCD90, hCD49f and mouse CD45 antibodies (see table supplementary S2).

Reverse Transcriptase and Real Time Quantitative PCR (RT-QPCR)
The direct-zol RNA purification kit (Zymo Research) was used for samples <1x10 5 cells and the RNeasy Mini kit (Qiagen RNeas, Crawley, UK) for samples between 1x10 5 -1x10 7 cells according to the manufacturer's instructions. mRNA was reverse transcribed by Superscript III Reverse Transcriptase (Invitrogen) with an oligoDT primer (Sigma-Aldrich, Gillingham, Dorset, UK). RT-QPCR was performed on a QuantStudio™ 5/7 (Applied Biosystems) using the standard manufacturer's settings and mRNA expression was quantified with the Comparative CT Method and Ribosomal Protein L18A (RPL18A) (MSC) were used as housekeeping gene controls.
The CT values used were the result of triplicates. The primers used are described in were mixed with 4x Nupage Loading buffer (ThermoFisher Scientific, UK) and boiled at 96° C for 5 min. The samples were run on a denaturing precast 8-12 % bis-tris Nupage gel (ThermoFisher Scientific, UK) and transferred by wet transfer to nitrocellulose membranes. Primary antibodies were incubated ON at 4° C and secondary antibodies 1h at RT. Protein bands were visualized using an enhanced chemiluminescence visualization system (ECL Plus, Amersham Life Sciences).