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H.R. Lee et al.
kinases16 or interaction with the extracellular matrix. However, despite these protective signals, a role for the stroma in the clonal development of leukemic blasts for the acquisition of chemoresistance has not been demonstrated.
Here, we show that subsets of leukemic cells in stromal contact undergo reversible changes associated with a stem cell-like phenotype and drug-resistant state. These changes are stochastic, and distinct from changes induced by other mechanisms of chemoresistance, thus representing a new class of drug-resistant cells developed in the leukemic microenvironment.
Methods
Human sample collection
Primary leukemic blasts were collected from newly diagnosed AML patients without prior treatment history. Part of the BM samples are from AML patients who had complete medical records during 5 years of follow-up in clinical courses. Human mesenchymal stromal cells (MSC) were separated from the BM of normal donors under informed consent. This study was approved by the Institutional Review Boards of St. Mary’s Hospital and Catholic University of Korea.
Animals
C57/BL6 mice were obtained from the Jackson Laboratories (Bar Harbor, ME). Bis+/+, Bis+/-, Bis-/- mice17 were provided by Dr. Jeong-Hwa Lee (Catholic University of Korea). Mice with dis- ruption of interleukin-4 (IL-4) receptor18 were provided by Dr. Chang Yul Kang (Seoul National University).
Mouse and human acute myeloid leukemia cells and mesenchymal stromal cells
Fresh murine or human MSC were analyzed in the BM using flowcytometry. Cultured MSC (passage five to eight) were obtained by serial plating of BM cells in the DMEM containing 10% fetal bovine serum as described.19,20 For generation of murine AML cells, fluorouracil (5-FU) treated BM cells were transduced with MN-1 or Meis1/HoxA9 through retroviral infection as described.21,22 For co-culture, MSC were irradiated (15 Gy) 18-24 hours prior to use and leukemic cells were seeded on the MSC for co-culture. For co-culture with transwell, MSC were seeded into the upper chamber (6-well type, polyethylene terephthalate [PET] membrane with 0.4 mm pores; BD Bioscience, San Diego, USA) and leukemic cells were seeded into the lower well.
Flow cytometry of leukemic cells and mesenchymal stromal cells
Murine leukemic cells were analyzed by flow cytometry using the following antibodies: CD45.1-APC (BD PharMingen, USA), Lineage cocktail (StemCell Technologies Inc, Canada), Sca-1-PE- Cy7 and c-kit-PE (BD PharMingen). For human leukemic cells, CD45-APC, CD34-BV421, CD90-FITC (BD PharMingen) anti- bodies were used. For MSC, anti-CD106 (VCAM-1)-biotin, CD51-PE (eBioscience, CA, USA.), CD140a (PDGFRa)-APC, Sca- 1-PE-Cy7 (BD PharMingen) were used.
Treatment of antibody and cytotoxic drug
Leukemic cells seeded on irradiated MSC were treated with anti-IL-4 antibody (R&D Systems Inc., USA), anti-CD106 (VCAM-1) (R&D Systems Inc.) for 3 days. For in vivo antibody injections, mice received intraperitoneal injection of anti-IL-4 Ab (1 mg/kg) (R&D Systems Inc.) or intravenous injection of anti- VCAM-1 antbody (10 mg/kg) (Bio X cell, USA) along with
immunoglobulin G (IgG) from rat serum (Bio X cell). Cytotoxicity of in vivo leukemic cells was examined by treat- ment with 100 mg/kg of Ara-C (Sigma-Aldrich, MO, USA) and 3 mg/kg of doxorubicin hydrochloride (Sigma).
Gene expression analysis
Sequencing libraries of two subjects were prepared according to the TruSeq Stranded Total RNA Sample Preparation guide. Aligned reads were quantified using HTSeq-count.23 Differentially expressed genes, fold ratio, P-value, and false dis- covery rate were identified by edgeR algorithm24 for each sub- ject. Enriched KEGG pathways were identified by GSEA-P.25
Statistical analysis
In order to compare the generation of CD90+ subsets from individual primary human leukemia patients’ samples, or the responses of individual patients’ leukemia cells to chemothera- py, we used Mann-Whitney test. In order to compare the differ- ences of means in specific experimental settings, we used a stan- dard unpaired, two-tailed student t-test. The frequencies of leukemia-initiating cells in limiting dilution analysis were calcu- lated by applying Poisson statistics with 95% Confidence Interval (CI) representing ±2 standard error of the mean (SEM).
Results
A subset of leukemic cells acquires a stem cell-like phenotype by contact with mesenchymal stroma
In order to investigate the influence of stromal cells on the function of leukemic cells, we employed an in vitro co- culture model of murine leukemic cells in contact with BM-derived MSC. Murine AML cells were generated by transducing BM mononuclear cells (MNC) with menin- gioma-1 (MN1)21 or HoxA9-Meis1 (H9M1)22 (Figure 1A). When co-cultured with MSC a subset of MN1 leukemic cells acquired a Sca-1(+) phenotype (Lin-c-kit+sca-1+; LSK) mimicking normal hematopoietic progenitors, while the majority remained Sca-1(-) (Lin-c-kit+sca-1-) (Figure 1B). The acquisition of Sca-1(+) phenotypes was similarly observed in other types of leukemic cells (H9M1) or leukemia cell line (C1498) independent of irradiation (Online Supplementary Figure S1A and B). The emergence of the Sca- 1(+) subset was dependent on direct contact with the mes- enchymal stroma (Online Supplementary Figure S1C), as these cells were not observed in stroma-free conditions or in stromal co-culture with a transwell filter (Figure 1B).
In order to determine if acquisition of the Sca-1(+) phe- notype occurs in vivo, MN1 leukemogenic cells (Lin-c- kit+sca-1-) were transplanted into mice. Consistent with the in vitro results, a subset of leukemic cells (GFP+) in recipient mice acquired a Sca-1(+) (Lin-c-kit+sca-1+) pheno- type (Figure 1C).
In order to determine whether acquisition of the Sca- 1(+) phenotype in leukemic cells originated from their fusion with stromal cells, as implicated previously,26 we co-cultured MN1 leukemic cells (GFP+) with MSC trans- duced with YFP. None of the GFP+ leukemic cells co- expressed YFP (Figure 1D). Moreover, there was no differ- ence in cell size between Sca-1(+) and Sca-1(-) cells, as determined by identical forward scatter in flow cytome- try, and no increase in tetraploidy in the Sca-1(+) cells (Figure 1E). Similarly, there was no evidence of cell fusion in this in vivo generated Sca-1(+) subset (Online Supplementary Figure S1D).
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haematologica | 2022; 107(2)