Co-culture with leukemia cells decreases proliferation and increases chemoprotective capacity of normal mesenchymal stromal cells


 Background Bone marrow mesenchymal stromal cells (BM-MSCs) are essential structural and functional components of the BM microenvironment and play an important role in acute myeloid leukemia (AML) pathogenesis. BM-MSCs isolated from AML patients (AML-MSCs) show distinct signatures from normal BM-MSCs. However, the exact abnormalities of AML-MSCs and the origin of these abnormalities are still unknown.Methods In this study, we evaluated the proliferative activity of AML-MSCs, and the influence of leukemia cells (LCs) on BM-MSCs. These two cell types were co-cultured using an in vitro co-culture system, and the biological functions of AML-MSCs, healthy donor derived MSCs (HD-MSCs), and LC-treated HD-MSCs (LCtrHD-MSCs) were compared by flow cytometry, and CCK-8 and chemotaxis assays. Student t-test (between two groups) and one-way ANOVA (more than 2 groups) were used to compare differences. Pearson correlation coefficients were used to assess correlations between two factors.Results AML-MSCs display a significant proliferative deficiency, which correlates with primary leukemic blast cell counts but not with patients’ age. Inhibition of BM-MSC proliferation could be induced by leukemia cells through direct contact. Co-cultured leukemia cells also increase expression of several inflammatory cytokines, and chemokines in BM-MSCs. Furthermore, LCtrHD-MSCs reduced apoptosis, and increased migration and chemoresistance in co-cultured AML cells, comparable with AML-MSCs.Conclusions Our results showed that leukemia cells can induce healthy donor derived BM-MSCs to exhibit AML-MSC-like characteristics and indicated that AML-MSC abnormalities may be partly induced by leukemia cells.

and accumulation of immature myeloid blasts. Following advances in the understanding of the molecular and genetic alterations associated with AML and the development of chemotherapeutic agents, the majority of patients achieve complete remission (Rm) at initial treatment. However, patients eventually develop recurrence, which is very common and is partly due to the development of chemoresistance 1 . Multiple factors, for example, the existence of leukemic stem cells (LSCs) and the protective bone marrow (BM) niche have been found to be involved in the chemoresistance 2,3 . Although treatments targeting LSCs as well as the leukemia BM microenvironment have gained certain clinical effects, the interactions between leukemia cells (LCs) and their BM niche need further investigation.
BM derived mesenchymal stromal cells (BM-MSCs), also named as mesenchymal stem cells, are essential structural and functional components of the BM microenvironment and play an important role in hematopoiesis by regulating hematopoietic stem and progenitor cell (HSPC) quiescence, self-renewal, proliferation and differentiation 4,5 . In the past decades, many studies have demonstrated that BM-MSCs are important in the development of hematological malignancies and that they contribute to chemoresistance through the release of specific soluble cytokines 6,7 or through their interaction with LCs 8 . Therefore, despite advancements in our understanding of the molecular and genetic alterations in AML, the abnormalities in BM-MSCs derived from AML patients (AML- MSCs) have not been fully elucidated. AML-MSCs overexpress several chemokines and cytokines, such as stromal cell-derived factor-1 (SDF-1, also known as CXCL12) 9 , prostaglandins (PGs) 10 , galectin 3 11 , apoptosis repressor with caspase recruitment domain (ARC) protein 12 , and IL-10 13 and were more prefer to support LC survival compared with normal MSCs.
Proteomic profiling has also revealed a different protein expression profile of AML-MSCs, and certain MSC protein expression signatures may be related to patient survival and remission 11 . Several studies have shown that abnormalities in AML-MSCs may contribute to AML pathogenesis 9, 11, 12 , but the origin of these abnormalities is still unclear.
Cytogenetic analysis of tumor-specific genetic alterations has provided controversial results. Huang et al. have found that part of the AML patients carry mutations in their BM-MSCs, some of which overlap with and some that are distinct from their corresponding leukemic blasts 13 . However, another study performed on a larger cohort did not identify any molecular abnormalities in AML-MSCs 14 . Recent studies have demonstrated that LCs modify the normal hematopoietic niche into a "leukemia niche" to selectively support LC survival and induce a distinct cross-talk between LCs and MSCs 15,16 . However, whether the abnormalities in AML-MSCs originate from the influence of LCs or are due to mutations is still unclear. To evaluate the influence of LCs on MSCs, we cultured the two cell types using an in vitro co-culture system and compared the gene expression profile as well as the biological functions of the different MSCs.  Table 1.

MSC isolation, culture and characterization
Mononuclear cells were isolated from BM by Ficoll (specific gravity 1.077; Sigma-Aldrich, St. Louis, MO, USA) density gradient centrifugation. Cells were washed twice with PBS, resuspended at a concentration of 1.0 × 10 6 cells/mL with DMEM/F12 containing 10% FBS and 1% antibiotic mixture (penicillin, 100 U/mL and streptomycin sulphate, 100 μg/mL) and then incubated at 37°C in T25 culture flasks in a humidified atmosphere supplemented with 5% CO 2 . The non-adherent cells were removed after 48 h and the culture medium was changed every 3 days. Adherent MSCs were passaged at 80% confluency. Cell surface markers, such as CD105, CD29, CD44, CD45, CD34, and human leukocyte antigen (HLA)-DR (Becton Dickinson and Company, NY, USA) were detected using flow cytometry (Beckman Coulter, Inc., CA, USA). The MSCs were digested at passage 3 and reseeded in 24-well culture plates at a density of 1.0 × 10 5 cells/mL. Coculture with primary LCs or LC lines were applied when MSCs reached 70% confluency. The AML patient derived (AML-), healthy donor derived (HD-), and LC treated HD (LCtrHD-) MSCs were used for comparison of their biological functions.

Co-culture of LCs with MSCs
Primary LCs from BM were separated using Ficoll gradient and seeded on a MSC layer in

Detection of aldehyde dehydrogenase positive (ALDH + ) LSCs
To detect ALDH + leukemic stem cells, AML cell lines including NB4, U937, Dami, and Kasumi-1 were labeled with activated ALDEFLUOR™ Reagent (ALDEFLUOR TM Kit, StemCell, Vancouver, Canada) before and after co-culture with different MSCs. After incubation, flow cytometry was performed to detect the percent of ALDH + cells.

RNA sequencing
The MSCs cultured alone or together with the AML cell line THP-1 were digested following washed thoroughly using PBS and 0.25%trypsin (if need) to remove THP-1 and collected for transcriptome sequencing. Briefly, total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA) and treated with DNase and a Ribo-Zero Magnetic Kit to digest doublestranded and single-stranded DNA and deplete rRNA, respectively. Purified mRNA was fragmented into small pieces and used to construct a cDNA library. Sequencing was performed on a BGISEQ500 platform (BGI, Shenzhen, China). The rRNA, low quality samples, linker contamination, and unknown base N content were filtered out from raw data. The clean reads were then mapped to the genome database and Bowtie2 were used to align the clean reads to the reference sequence. RSEM was used to calculate the number of reads mapping to genes. Differentially expressed genes between the 2 groups were screened using the criteria that log2 fold change must be ≥ 1 or ≤ -1, p value must be ≤ 0.001, and the false discovery rate (FDR) must be ≤ 0.001.

Data analysis
Data are presented as mean ± standard error of the mean (SEM). SPSS ** software (Math Soft, Inc., Seattle, WA, USA) was used to perform statistical analyses. Student t-test (between two groups) and one-way ANOVA (more than 2 groups) were used to compare differences. The Pearson correlation was used to evaluate correlations between two factors.

AML-MSCs showed reduced proliferative capacity
We expanded AML-MSCs (n = 6) and HD-MSCs (n = 6) and evaluated the phenotypic and biological characteristics of both cell groups. Both AML-MSCs and HD-MSCs displayed similar spindle-shaped fibroblast like morphologies (Fig. 1a), expressed typical MSC markers (CD105 + CD29 + CD44 + ) and were negative for hematopoietic markers (CD45 − CD34 − HLA-DR − ) in a comparable way ( Supplementary Fig. 1). During MSC expansion, we found that AML-MSCs showed decreased proliferative activity. The adherent cells in primary AML-MSC cultures were significantly less than those in HD-MSCs cultures at the same culture time (Fig. 1a). AML-MSCs needed 22-37 days to reach 80% confluency, while HD-MSCs needed 12-20 days (average = 29.17 ± 6.11 vs. 16.00 ± 3.63, p < 0.01, Fig. 1b). This difference declined with prolonging the culture time in vitro. When cultured at passage 3, no significant differences were found between the two groups (7.83 ± 1.33 vs. 6.83 ± 0.75, p > 0.05, Fig. 1b). The proliferative activity of MSCs may be influenced by various factors such as patients' age, leukemic blast cell counts etc. Thus, we carried out a correlative analysis between these factors and the confluency time of primary culture.
As shown in Fig. 1c and 1d, there was a significant positive correlation between the primary leukemic blast cell count and confluency time (Pearson r = 0.8494, p = 0.0323, Fig. 1c). In contrast, no correlation was found with patients' age (Pearson r = 0.5022, p = 0.0962, Fig. 1d). These results indicate that the decreased proliferative activity may be induced by AML cells but is not related to patients' age.

LCs suppress MSC proliferation via direct cell contact
To further evaluate the influence of LCs on MSC proliferation, we co-cultured AML cell lines profile between the two cell groups (Fig. 3b). Sequencing data were deposited in the China National Gene Bank (CNGB, CNP0000695). There were totally 2808 differently expressed genes (2182 up regulated and 626 down regulated) between the two cell groups (Fig. 3c).

BM-MSCs co-culture did not increase the number of ALDH + LSCs
ALDH is increasingly regarded as a leukemia-initiating cell (LIC) or LSC marker 22 (Fig. 4). After co-culture, neither HD-MSCs nor AML-MSCs increased the percentage of ALDH1 + LCs (Fig. 4, p > 0.05). This result suggests that, at least in AML LCs, co-culture with MSCs is unable to enhance the number of ALDH1 + LSCs.

LC-treated MSCs support survival and promote migration of AML cells
The BM niche supports both normal CD34 + HSPC and AML cell survival and proliferation, and the AML-MSCs have been reported to support LC survival and proliferation 15 − 17, 19 .
To study the influence of different MSCs on AML cell viability, apoptosis, migration, and drug resistance, we co-cultured THP-1 cells together with HD-MSCs, AML-MSCs, or LCtrHD-MSCs. We showed a significant difference in THP-1 apoptosis, migration, and drug resistance between the different cultures. All the three kinds of MSCs supported cell viability similarly in vitro, although the AML-and LCtrHD-MSCs slightly increased the viability of co-cultured THP-1 cells, but the difference was not significant (115.77 ± 13.26% and 110.82 ± 33.04% respectively vs. 100%, p > 0.05. Table 2 and Fig. 5a). All the three kinds of MSCs reduced the apoptosis rate of co-cultured THP-1 cells. Both AML-and LCtrHD-MSCs showed stronger anti-apoptosis ability than HD-MSCs (Table 2 and Fig. 5b,c), but the difference between AML-and LCtrHD-MSCs was not significant (3.35 ± 1.11% vs.
4.32 ± 1.42%, p > 0.05). Regarding chemotaxis, both AML-and LCtrHD-MSCs attracted more LCs than HD-MSCs (2.76 ± 0.47 × 10 5 and 1.92 ± 0.26 × 10 5 respectively vs. 0.66 ± 0.25 × 10 5 , p < 0.05. Table 2 Figure 5e shows the influence of different MSCs on THP-1 chemoresistance. Compared to THP-1 cells cultured alone, HD-MSCs did not improve chemoresistance (34.99 ± 3.61% vs. 45.64 ± 6.82%, p > 0.05. Table 2), while the AML-and LCtrHD-MSCs significantly increased the percent of Arc-a resistant THP-1 cells (67.41 ± 5.82% and 59.49 ± 11.00% respectively vs. 45.64 ± 6.82%, p < 0.05. Table 2 In this study, we found that AML-MSCs showed decreased proliferative potential in primary culture, which was related with the leukemic blast cell counts but not the age of patients.  18 . Furthermore, reduced proliferation capacity and increased adipogenic potential of AML-MSCs has also reported by other researchers 28,29 . When human LCs were transplanted into immuno-deficient mice, the BM microenvironments (including vascular structures and osteoblastic-lining cells) were damaged along with the leukemic dissemination, and subsequent chemotherapy could induce the formation of a protective niche around the residual LCs 30 . This research suggested that LCs can also inhibit proliferation and function of niche cells in vivo. In addition, AML-MSCs and MDS-MSCs have been shown to exhibit a senescence-associated phenotype and function, including a large flat morphology, increased β-galactosidase activity, and increased secretion of pro-inflammatory cytokines as well as chemokines 31,32 . We hypothesized that, both the decreased proliferative capacity and the senescenceassociated phenotype are probably induced by leukemic blasts.
Studies using leukemic mice models have reported that LCs transplanted into a mouse model can "hijack" the normal BM niche into a leukemic niche which impairs normal hematopoiesis, favors leukemic stem cell survival and expansion 15,33 . It has been shown that AML-MSCs show special characteristics including over expression of a series of chemokines and cytokines 11 − 13, 31, 32 , increased adipogenic and osteoblastic potential, and improved the ability to support leukemia progenitor cells survival 29,34

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Publication of this manuscript has been approved by all co-authors.
Availability of data and materials: We will make data and materials described in this manuscript freely available to scientists wishing to use them for non-commercial purposes.
Competing interests: All authors declare no conflicts of interest.

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