BM-MSCs display altered gene expression profiles in B-cell acute lymphoblastic leukemia niches and exert pro-proliferative effects via overexpression of IFI6

Human ALL (Nalm-6 and RS4;11) cells, which were acquired from the Laboratory of Hematopoietic Stem Cell Transplantation Center of Guizhou Province (Guiyang, China), were cultured in a RPMI-1640 medium involving FBS (10%), streptomycin (100 mg/mL) and penicillin (100 units/mL) at 37 °C with 5% CO₂.

For the acquisition of BM-MSCs, we separated BM-MSCs from the bone marrow aspirates of B-ALL patients (n = 37) with their consent. The aspirates were subjected to ficoll gradient centrifugation, and subsequently cultured in the complete medium of adult BM-MSCs (Saiye, Shanghai, China). The phenotypic traits and differentiation capacities of BM-MSCs have been confirmed by us priorly [18].

For the construction of co-culture system, the culture plate was initially seeded with BM-MSCs at 1 × 10⁵/ml for 24 h, and then added with leukemia cells at a 4:1 ratio. For the establishment of the leukemia niche, MSCs were subjected to a 72-h co-cultivation with leukemia cells with reference to previously published literature [13]. In each independent repeated experiment, separately cultured MSCs from the same patient as control. For the collection of suspended leukemia cells in the co-culture system, Nalm-6/RS4;11 cells were pipetted cautiously from the monolayer MSCs. For some leukemia cells adhering to MSCs surface, PBS-EDTA 1 was used to wash the co-culture extensively to remove all the leukemia cells.

Vincristine sulfate, which was product of Taoshu Biotechnology (Shanghai, China), was prepared in phosphate buffer solution (PBS). AMD3100 and PD98059 from MedChemExpress (Shanghai, China) were formulated separately in dimethyl sulfoxide (DMSO) and anhydrous ethanol. The AKT, ERK, phospho-AKT and phospho-ERK antibodies were procured from Cell Signaling Technology (Danvers, MA, USA)., while the CXCR4 and IFI6 antibodies were procured separately from Solibao Biotechnology (Beijing, China) and ImmunoWay Biotechnology (Plano, TX, USA). Proteintech Group (Wuhan, China) was the provider of the secondary antibody used herein for Western blot.

MSCs (500 ul) were seeded in a 24-well culture plate at 5 × 10⁴/ml. After the MSCs adhered overnight, the MSCs culture medium was aspirated, and 500 ul of Nalm-6/RS4;11 cells (1-3 × 10⁵/ml) were inoculated on MSCs. The mono-cultured Nalm-6/RS4;11 cells were set as a control, where RPMI 1640 medium was used as a culture medium. The leukemia cells in each well were collected after incubation for 24, 48, 72 and 96 h, respectively. Then, the leukemia cells were quantified by direct counting using a cell counter, and the cell growth curves were drawn according to the results.

After harvesting and PBS-washing, the cells were subjected to Annexin-V/propidium iodide (PI) staining to assay the apoptotic ratio as per the advised protocol (7Sea Pharmatech, Shanghai, China). To assess the cell cycle, RNase A and PI (7Sea Pharmatech, Shanghai, China) were utilized to treat the gathered MSCs, followed by flow cytometry (BD Biosciences, San Jose, USA).

Matrigel-coated and uncoated Transwell chambers were used for invasion and migration experiments, respectively. The lower chamber was added with 650 ul of MSCs (1 × 10⁵/mL) and incubated overnight. Next, the upper chamber (pore size: 8.0 um, Corning Incorporated, Costar) was added with 100 ul of Nalm-6/RS4; 11 cells (4 × 10⁵/mL) and subjected to a 24-h incubation. An inverted microscope was utilized to surveil the leukemia cell migration and invasion in the lower chamber, followed by photographing under 40 × magnification. Quantification of migrated and invaded cells was accomplished by direct counting using a cell counter.

For adherent BM-MSCs, after washing with PBS in a 6-well plate, the procedure for β-galactosidase staining was implemented as per the specific guidelines. then followed by observation of the proportion of cells stained blue (senescent cells) under a microscope and photographing (200 ×). The proportion of senescent cells in each group was evaluated with 5 random fields of view, and then the senescence of cells in each group was assessed by value averaging.

Human IFI6-silencing RNA (si-IFI6) and IFI6-overexpressing clone lentiviral particles (LV-IFI6) were the Genechem (Shanghai, China) products. Transfection of si-IFI6/LV-IFI6 was accomplished as per the manufacturer protocol. Controls used were the empty vector (EV)-transfected BM-MSCs.

Trizol reagent (Qiagen, Hilden, Germany) was utilized to extract the total RNA of cells, the FastKinggDNA Dispelling RT SuperMix (Qiagen, Hilden, Germany) was utilized to reversely transcribe the RNA extract to cDNA. Then, cDNA was analyzed by quantitative real-time PCR (qRT-PCR) in accordance with the protocols of primers and Talent qPCR PreMix (SYBR Green) (Qiagen, Hilden, Germany). For the target gene, their relative expression levels were estimated by the comparative CT (2^−△CT) approach following normalization to β-actin. Table 1 details the human primers (Generay Bioteach, Shanghai, China) used.

Cells were collected and lysed with Radio ImmunoprecipitationAssay (RIPA) lysis buffer (Beyotime, Shanghai, China) involving phenylmethylsulfonyl fluoride (PMSF; 1%). Protein (10–30 μg) was isolated on SDS-PAGE and then shifted onto the PVDF membrane. A 1–2 h blockade of the membrane proceeded using skimmed milk (5%) at room temperature, followed by an overnight incubation using primary antibodies at 4 ℃. Thereafter, an extra 1-h incubation of the membranes was accomplished using secondary antibody at ambient temperature, and then the protein expression was assayed with electrochemiluminescence reagent. Gray value analysis was performed via the Image J software against the internal β-actin reference.

For transcriptome sequencing analysis, the processed MSCs and leukemia cells were collected respectively. After cells were lysed by trizol reagent, the samples were sent to Shanghai Liebing Information Technology and Hangzhou Lianchuan Biotechnology for transcriptome sequencing analyses. Meanwhile, the gene expression profile dataset GSE101454 related to MSCs derived from patients with B-Cell Precursor ALL (BCP-ALL) in GEO database was selected. This dataset provided microarray analysis data of mono-cultured BM-MSCs and BCP-ALL cells- co-cultured BM-MSCs (for 40 h).

The DEGs in the datasets were analyzed and screened, where the screening conditions were: |log2FC|> 1, Flase Discovery Rate (FDR) < 0.05, P < 0.05. During the experimentation, Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were performed on the DEGs via the Database For Annotation Visualization and Integrated Discovery (DAVID) software, and the biological functions and signaling pathway changes enriched by the differential genes were screened out. Gene set enrichment analysis (GSEA) 4.2.3 was utilized to assess the enrichment of gene sets.

Xenograft assays in mice were approved by the Guizhou Medical University's Animal Care Welfare Committee. 4–6 weeks-old female nonobese diabetes/severe combined immunodeficiency (NOD/SCID) mice were chosen, each of which was given subcutaneous injection of RS4;11/Nalm-6 cells, a RS4;11/Nalm-6-MSC mixture, a RS4;11/Nalm-6-MSCs-EV mixture, and a RS4;11/Nalm-6-MSCs-LV-IFI6 mixture (1 × 10⁶ MSCs mixed with 4 × 10⁶ RS4;11/Nalm-6 cells) into the left chest. Growth of tumors was surveilled every 3 d through length (L) and width (W) determination, and the computational formula for tumor volume was: 0.5 LW². After injection for 25d/34d, the tumor tissues were extracted from mice and embedded in paraffin for further study. The procedure for immunohistochemistry (IHC) staining was implemented as per the specific guidelines. The primary and secondary antibodies used were 1:100 dilutions.

SPSS 20.0 was utilized to assess the data. Independent student's t-test was employed for making two groups comparison, while one-way ANOVA was adopted for homogeneity of variance assessment among multiple groups. Non-normal data were subjected to the Kruskal–Wallis non-parametric test. P-values were indicated as follows: *P < 0.05;**P < 0.01; ***P < 0.001.

To explore the possible differences between MSCs in the leukemia niche (MSCs co-culture with leukemia cells for 72 h) and MSCs cultured alone, we compared these two MSC groups regarding cell stemness, self-renewal, senescence, adipogenic and osteogenic differentiation, cell cycle, as well as cell apoptosis. In terms of cell stemness-associated indicators (POU5F1, SOX2, NANOG), POU5F1 was found to be significantly increased in the MSC–Nalm-6 co-culture system, but no obvious changes were seen in the expressions of SOX2 and NANOG (Fig. 1A). For the self-renewal-associated indicators (RUNX1, HOXB4), the markers of adipogenic (ADIPOQ, PPAR-γ) and osteogenic differentiation (RUNX2, BGLAP), the changes between the two groups were not significant (Fig. 1B, C and D). However, when MSCs co-cultured for 72 h with Nalm-6/RS4;11 were subjected to adipogenic and osteogenic differentiation experiments, the differentiation abilities of co-cultured MSCs were weakened compared to the mono-cultured MSCs (Fig. 1E).

Regarding the cell senescence-related markers, compared to the mono-cultured MSCs, the expressions of p53, p21 and p16 in the co-cultured MSCs presented upward trends, and the change in p21 was more obvious (Fig. 1F, G). On the basis of the foregoing results, the changes of cell senescence between the two groups were further observed by senescence-associated β-galactosidase staining, which found markedly increased senescence degree of co-cultured MSCs compared to that of mono-cultured ones (Fig. 1H). Suggestively, the MSCs in the leukemia niche might have varying degrees of weakened differentiation abilities and senescence changes.

Next, we further explored the possible changes in the apoptosis and cell cycle of co-cultured MSCs. Through testing, we found that in the MSC–Nalm-6 co-culture system, MSCs underwent a certain degree of apoptosis (Fig. 1I, J), and exhibited pronouncedly more S phase cells and declined proportion of G0/G1 phase cells, but no similar changes were observed in MSCs in the RS4;11 co-culture system (Fig. 1K). Based on the above results, we speculated that the biological characteristics of MSCs in the leukemia niche might have changed to varying degrees.

In the following experiments, we further compared the molecular biological changes between the two groups by transcriptome sequencing analysis. Given the more obvious changes of Nalm-6-co-cultured MSCs in the previous detection, we selected the MSCs co-cultured with Nalm-6 for 72 h for sequencing analysis. Figure 2A, B and C separately illustrate the distributions of gene expressions in the tested samples (Fig. 2A), the correlations between samples (Fig. 2B), as well as the clustering features of gene expression patterns (Fig. 2C). Through differential gene analysis, it was found that 5,543 of 20,097 gene expression profiles detected were expressed differentially in MSCs in the co-culture system (|log₂FC|> 1, FDR < 0.05), with 2,874 up-regulated genes and 2,669 down-regulated genes (Fig. 2D, E). As revealed by further GO analysis, the DEGs in the co-cultured MSCs were implicated in biofunctions like cell division, cell cycle and DNA replication, and were also involved in several tumor-promoting biofunctions such as protein phosphorylation, cell migration and angiogenesis (Fig. 2F). Through a concurrent GSEA, we discovered the collective enrichment of particular datasets, such as positive cell cycle regulation, B cell receptor signaling pathway and DNA replication, in the co-culture group (Fig. 2G). Contrastively, representative enrichment of subsets like ribosome, drug metabolism cytochrome P450 and metabolism of xenobiotics by cytochrome P450 was noted in MSCs mono-culture group (Fig. 2H). These results suggested that MSCs in leukemia niches have different gene expression profiles and biological functions, including several functions that promote tumor progression.

In the experiments below, further emphasis was placed on the DEGs in MSCs in the leukemia niche. We searched the Gene Expression Omnibus (GEO) database and screened a dataset (GSE101454) with a certain degree of similarity to our study. Through DEG analysis on this dataset, 11 DEGs were screened out (|log₂FC|> 1, P < 0.05), all of which were up-regulated genes in co-culture system (Fig. 3A). Subsequently, the data from gene expression profiling in the present work were assessed by the same condition setting (Fig. 3B). We performed an intersection analysis on the DEGs in the two gene expression profiling datas and drew a Venn diagram (http://​bioinformatics.​psb.​ugent.​be/​webtools/​Venn/​) (Fig. 3C), founding that a total of 7 genes generated intersections, namely MX1, IFITM1, IFIT3, ISG15, IFI6, IFI44L and IFIT1. Based on these results, the expression of these 7 genes in the mono-cultured and co-cultured MSCs were further examined via qRT-PCR, finding varying degrees of elevations in the 7 genes in the co-culture system (Figs. 3D, E). By comparing the up-regulated folds, it was found that the IFI6 level in the co-cultured MSCs had the highest fold increase (Table 2).

According to the above results, we further verified the difference in the IFI6 expression in mono-cultured and co-cultured MSCs via western blot. Prominently higher IFI6 level was noted in the co-cultured MSCs (Fig. 4A). To explore whether the increased expression of IFI6 has some biological effect, we conducted preliminary exploration through the GEPIA database (http://​gepia.​cancer-pku.​cn/​detail.​php) to assess the normal and tumor tissue levels of IFI6, discovering obviously increased IFI6 levels in several tumors (Fig. 4B). As revealed by survival analysis, high IFI6 level was linked to lower rate of overall survival (Fig. 4C). Thus, we further explored the potential impact of IFI6 in MSCs on the biological function of B-ALL cells. Initially, the expression of IFI6 in MSCs was up-regulated by lentiviral transfection reagent. The fluorescence microscopy (Additional file 1: Figure S1A), qRT-PCR and western blot was employed to validate the transfection efficiency (Fig. 4D, E). By functional testing, results showed that the high expression of IFI6 in MSCs had a slight effect on the changes of sensitivity of leukemic cells to vincristine (Fig. 4F), migration and invasiveness of leukemia cells (Fig. 4G, H), but exerted a pro-proliferative effect on the leukemia cells (Fig. 4I, J). Further down-regulation of IFI6 in MSCs (Additional file 1: Figure S1B, Additional file 2: Figure S2A, B) led to the opposite results in cell proliferation (Additional file 2: Figure S2C, D). As implied by these findings, the elevated IFI6 in MSCs might promote the proliferation of leukemia cells.

In the following experiments, we further applied mice with NOD/SCID to investigate IFI6's role in the growth and proliferation of leukemia cells. We subcutaneously injected RS4;11 (n = 3) and a suspension of RS4;11 and MSCs/MSCs-EV/MSCs-LV-IFI6 (4:1) (n = 3) into the left chest of mice, respectively, and then observed the growth of tumor tissues (Fig. 5A). According to the results, the volume and mass of tumor tissues in the RS4;11 + MSCs-LV-IFI6 group were obviously increased in contrast to the RS4;11, RS4;11 + MSCs and RS4;11 + MSCs-EV groups (Fig. 5B, C and D). In addition, in vivo experimental validation of Nalm-6 cell lines yielded similar results (Additional file 3: Figure S3A, B, C and D). Figure 5E, F displayed the hematoxylin–eosin (HE) staining results and the expressions of IFI6 and Ki-67 in various groups, more IFI6 and Ki-67 expression was observed in tumor tissues of the RS4;11 + MSCs-LV-IFI6 group compared with the RS4;11, RS4;11 + MSCs and RS4;11 + MSCs-EV groups. These datas suggested that high expression of IFI6 in MSCs might promote the leukemia cell growth and multiplication in vivo.

Next, we aimed to explore the underlying mechanism by which IFI6 promotes leukemia cell proliferation. Stromal cell derived factor 1 (SDF-1)/chemokine receptor 4 (CXCR4) is an important signaling axis that mediates the communication between tumor and stromal cells. We speculated whether the increased expression of IFI6 in MSCs could facilitate the leukemia cell multiplication by stimulating the SDF-1/CXCR4 axis. Through the expression comparison for SDF-1 in co-cultured MSCs between the EV, LV-IFI6 and control groups, a prominent elevation of SDF-1 level was noted in the MSCs of the LV-IFI6 group (Fig. 6A). Based on this result, the level of CXCR4, the SDF-1 receptor, in leukemia cells was further examined, finding that in the co-culture system, up-regulation of IFI6 could significantly increase the leukemia cell level of CXCR4 (Fig. 6B), while down-regulation of IFI6 yielded the opposite results (Fig. 6C, D). In subsequent experiments, we further detected the proliferative signaling pathways of AKT and ERK downstream of the SDF-1/CXCR4 axis in leukemia cells. Evidently elevated p-ERK level was noted in the IFI6 up-regulated group, while the levels of p-AKT and AKT both presented upward trends (Fig. 6E, F). However, down-regulation of IFI6 did not change the expression of p-AKT significantly, but obviously decreased the expression level of p-ERK (Fig. 6G, H). Suggesting that the activated SDF-1/CXCR4 axis might further activate the ERK signaling pathway to promote leukemia cell proliferation. To confirm this conjecture, the co-culture system was incorporated with AMD3100, a specific CXCR4 suppressor, and it was found that AMD3100 could effectively reduce the p-ERK level in the up-regulated IFI6 group (Additional file 4: Figure S4). Further, the AMD3100 incorporation into the co-culture system was found capable of weakening the pro-proliferative effect of up-regulated IFI6 (Fig. 6I, J). Also, the addition of the ERK inhibitor PD98059 to the co-culture system of MSCs with up-regulated IFI6 also showed consistent results (Fig. 6K, L). As implied by these findings, the pro-proliferative function of IFI6 for leukemia cells might be exerted through the SDF-1/CXCR4/ERK signal stimulation.

To further unravel IFI6's role in the molecular biological characteristics of leukemia cells, we co-cultured MSCs from EV group and LV-IFI6 group with RS4;11 cells for 72 h, respectively, and then collected leukemia cells for transcriptome sequencing analysis. Figure 7A, B displayed the distributions of gene expression and the gene expression densities of the tested samples. Through differential gene expression analysis (|log₂FC|> 1, P < 0.05), the total number of DEGs was found to be 210 in the LV-IFI6 group, of which 113 were up-regulated and 97 were down-regulated (Fig. 7C). Figures 7D, E showed volcano and cluster plots (Top 100) of DEGs between the two groups. Through GO analysis, it was found that the biological functions enriched by differential genes included signal transduction, protein phosphorylation and positive regulation of cell population proliferation et al. (Fig. 7F). KEGG analysis revealed that the enriched signaling pathways included changes in MAPK and cytokine-cytokine receptor signaling pathway (Fig. 7G). Further, Over-Representation Analysis (ORA) (http://​webgestalt.​org/​) showed that the enriched biological functions included cell conmmunication and cell proliferation (Fig. 7H). These data seemed to be consistent with the above experimental results in this study. Taken together, we speculated that the IFI6 level elevation in the co-cultured MSCs produced a certain effect on the gene expression profile and proliferation of leukemia cells.