Introduction
Giant cells with polyploidy and multiple nuclei have been observed during normal growth and development as well as in pathologic states, such as cancer initiation, progression, and therapy resistance [
1‐
7]. The polyploid giant cells observed in solid tumors were termed as PGCCs (polyploid giant cancer cells), but other terms including poly‐aneuploid cancer cells (PACC) [
8], multinucleated giant cells [
9], osteoclast-like giant cells [
10], and cancer-associated macrophage-like cells (CAML) [
11] are also in use describing multinucleated giant-sized cells. Cancers with proven viral etiology are characterized by PGCC [
12]. Functional studies of PGCCs provided evidence that these cells arise from normal diploid cells under stress, show stem cell-like properties, and give rise to tumors [
7]. PGCCs in solid tumors are well documented but their existence in liquid tumors, such as acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS,) is not well documented.
MDS is a heterogeneous group of hematopoietic stem cell disorders characterized by cytopenias due to ineffective hematopoiesis and an increased propensity for transformation to AML in a third of the patients [
13]. Recurrent cytogenetic abnormalities are characteristic of both MDS and AML [
14]. In MDS, almost 50% of the patients show an abnormal karyotype recurrently involving chromosomes 3, 5, 7, 8, 11, 12, 17, 20, and Y but polyploidy is rare [
15]. Similarly, in AML, be it de novo or secondary (arising from MDS), aneuploidy is common, but polyploidy is rare [
16,
17]. Several cases with near tetraploid to tetraploid cells in both MDS and AML patients have been reported using descriptive terms like “giant” and “bizarre” cells, but the significance of these polyploidal giant cells remains unclear [
18‐
21].
Apart from the cytogenetic abnormalities, somatic mutations in over 40 frequently mutated genes with functions in diverse cellular processes such as DNA methylation, chromatin modification, splicing, and transcription are reported in MDS [
22]. Along with DNA methylation, RNA splicing genes are the most frequently mutated in MDS [
22]. Particularly, mutations in
SF3B1 have been described in up to 85% patients with a subtype of MDS characterized by ring sideroblasts [
22‐
24].
Previously, our group and others have shown that heterozygous point mutations in
SF3B1 affect pre-mRNA splicing, owing to its role in facilitating the interaction of snRNP spliceosomal complex with branch site, resulting in retention of 10 to 25 nucleotides long-intron upstream of 3’ acceptor splice sites in over 1000 genes [
25‐
27]. This widespread defect in pre-mRNA splicing of many genes results in cellular stress. A subset of the affected genes is involved in a variety of cellular functions, including iron metabolism, erythropoiesis, and mitochondrial activities [
28‐
31].
In this study, we report our observations that introduction of the most frequent hotspot mutation in SF3B1 gene in K562 cells results in an increased frequency of polyploid multinucleated giant cells that are resistant to serum starvation conditions and commonly used chemotherapeutic agent, azacytidine, approved for the treatment of both MDS and AML. We show that PGCCs are distinct from the occasional megakaryocyte observed in K562 cells. We also show, using cell proliferation markers, cytogenetics, and time-lapse videography, that PGCC are capable of cell division. Finally, the mutant cells show an increase in mitochondrial biomass.
These observations provide the first reported evidence of the appearance of PGCC in liquid cancers and may have implications in the initiation and progression of MDS and AML as well as resistance to chemotherapy.
Materials and methods
Cells and cell culture method
The wild-type and
SF3B1 K700E mutation bearing K562 cells were described previously [
26,
32]. A synonymous change or mutation (c.2098A > G) was introduced using CRISPR/Cas9 genome engineering method. The wild-type and the mutant K562 cells were cultured in IMDM media (Thermo) supplemented with 10% fetal bovine serum (GE system) and 1% Penicillin–streptomycin (Gibco).
Flow cytometry
Flow cytometry analysis was carried out in Fortessa II (BD Biosciences) using CellQuest software (BD Biosciences, San Jose CA, USA). Scatter plots and cell quantification analysis were carried out using FlowJo software (version 10.1) (BD Biosciences) or FCS Express 7 (De Novo Software).
Propidium Iodide DNA staining
The genome content of the wild-type and mutant K562 cells was measured using Propidium Iodide (PI). 4 WT and 4 mutant (K700E) K562 clones were used for the assay. A total of 1 million cells for each experiment were fixed in cold 70% ethanol for 1 h. The cells were washed with 1xPBS twice. The supernatant was discarded and the pellet was resuspended in 1 mL of mixture of Ribonuclease A (50 µL from 100 µg/mL stock) and PI (200 µL from 50 µg/mL stock) in 1xPBS. The cells were stained for 20 min at room temperature in dark. The cells were washed with 1xPBS twice, resuspended in 500 uL 1xPBS, and analyzed using Flow cytometry.
Nuclear and mitochondrial staining
WT and mutant (K700E) K562 cells were washed with 1xPBS twice. The cells were finally resuspended at a density of 1 million/mL in 1xPBS to which Mitotracker Rhodamine 123 solution (Sigma) was added to a concentration of 50 µM from a 25 mM stock. The cells were incubated in dark for 1 h at room temperature. After 1 h, 10 µg/µL Hoechst 3342 (Thermo) solution was added for nuclear staining and CellBrite Fix 555 (Biotium) for plasma membrane staining to the cells and incubated in dark for 10 min at room temperature. The cells were then washed twice with 1xPBS and resuspended in 100 µL of 1xPBS. Out of which 30 µL was loaded on 35-mm glass bottom plates (MatTeK Inc, USA) and observed under confocal microscope.
Treatment with 5-azacytidine
Three clones each of WT and K700E mutants were seeded in 6-well plate at a concentration of 3 × 105 cells /well. The cells were treated with 25 µM 5-Azacytidine (SellekChem) (stock 10 mM) or DMSO as control for 72 h in IMDM media supplemented with 10% FBS. For IC50 calculation cells were treated with different concentrations of 5-Azacytidine from 0 to 100 µM for 48 h and 72 h. The cytotoxicity was analyzed using AnnexinV /PI staining and measured through Flow Cytometry.
Microscopy
Confocal microscopy was carried out using Nikon Ti Eclipse-inverted microscope and analyzed using Nikon NIS-AR Elements software (Nikon Inc, USA). High-resolution images were taken using STORM (Stochastic Optical Reconstruction Microscopy) 100x/1.49 objective lens. Light microscopy and imaging were done using Olympus Ix51 microscope attached with Olympus DP72 camera. Images were further analyzed using Fiji Software (
https://imagej.net/Fiji).
CD61 staining
The CD61 expression analysis in WT and mutant cells was carried out using Flow cytometry and microscopy. Three clones each of WT and K700E mutants were seeded in 6-well plate at a density of 3 × 105 cells per well in 3 sets. One set was treated with 25 µM 5-Azacytidine (SellekChem), one set with 5 nM PMA (Sigma), and one set with 300uM CoCl2 (Sigma). All the treatments were done in IMDM media with 10% FBS for 72 h. For CD61 expression using flow cytometry, cells were washed with 1xPBS twice and stained with 5 uL of CD61 (BioLegend) in 500 uL 1xPBS. Staining was carried out for 20 min at room temperature in dark. The CD61 expression was analyzed using Fortessa II (BD Biosciences).
For microscopy, the CD61-stained cells were further stained with Hoechst (Thermo) for 10 min. The stained cells were washed with 1xPBS, mounted on glass slide, and visualized under Olympus microscope at 20X magnification. The images were captured using Olympus DP74 camera attached to the microscope.
Time-lapse microscopy
The WT and mutant K562 cells were resuspended in StemCell MethoCult (H4034, StemCell, Inc) media at a concentration of 1 × 105 cells/mL. Cells were then seeded in 96-well glass-bottomed plates (Greiner Inc) at a density of 104 cells/well and monitored for time lapsed for 24 h. Images were captured using BioTek Cytation 5 plate reader with BioSpa 8 automated incubator (BioTek) at a frequency of every 30 min and analyzed using Gen 5 (BioTek) and Fiji (Image J) softwares.
Actively growing WT and mutant K562 cells in duplicate were processed for metaphase preparations using standard methods [
33]. Briefly, cells were treated with hypotonic solution, fixed in methanol:acetic acid fixative and slides were prepared. FISH analysis was performed using a tri-color combination of fluorescent-labeled centromere probes for chromosomes 3 (orange), 7 (green), and chromosome 17 (aqua) obtained from Abbott Molecular (IL, USA).
Ki-67 staining
For Ki-67 immunofluorescence experiments, overnight cultures of WT and K700E cells (`2 × 106) were first washed with 1xPBS and fixed in 4% paraformaldehyde in PBS for 10 min at room temperature. Cells were then gently washed with ice-cold PBS and permeabilized with 0.5% Triton TX-100/PBS on ice for 10 min. Cells were then washed twice with room-temperature PBS and then incubated with Ki-67 antibody (1:200 dilution in 1xPBS-2.5 ug/mL-Biolegend 350,501) for 1 h at room temperature in dark. The cells were then washed with 1xPBS twice at room temperature. After washing, cells were incubated with secondary anti-mouse Rabbit antibody (1:500 in 1xPBS) labeled with Alexa Fluor 488 (Thermo Life Technology) for 30 min at room temperature. The cells were washed twice with 1xPBS. The nuclei were counterstained with Hoechst as described above and plated in 35-mm glass bottom plates at a concentration of 1 × 104 cells/mL. The images were captured using confocal microscope with 40X objective lens.
Statistical analysis
All the statistical analysis were carried out using GraphPad Prism 6. P values were calculated using unpaired Student’s t test (Welch’s Corrected) between 3 to 5 WT and mutant (K700E) clones. The difference was found significant at P < 0.05.
Discussion
PGCCs are rare cells that are observed both in vivo (biopsies from patients) and in vitro (primary cell cultures and cell lines) in studies dating back to over two centuries, but their significance has been appreciated more in the last decade as functional studies mainly in solid tumors implicated their role in tumorigenesis, metastasis, and therapy resistance [
1‐
4,
6,
8,
12,
37‐
39]. Multinucleated giant Reed–Stenberg cells [
40] are a hallmark of Hodgkin’s lymphoma, but evidence of PGCCs in liquid cancers is sparse consisting of case reports of tetraploid cells with giant cell morphology [
18‐
21]. Monocyte-derived macrophages were shown to form multinucleated giant cells and act as nurse cells that de novo generate other cell types in vitro [
41]. Also, multinucleated giant cells are observed around new bone material after implantation [
42]. Taken together, it appears that multinucleated giant cells are formed in response to stress and play important roles in a variety of human diseases, including cancer. The exact origin of these cells is not clear, but evidence, in a few cases, supports the role of monocyte macrophages that fuse to generate these cells. Finally, the genes and pathways involved in the formation of multinucleated giant cells are not known.
In our attempt to study the molecular and cellular defects due to
SF3B1 mutation, we observed that introduction of the hotspot mutation in K562 cells, a continuously growing cell line derived from chronic myelogenous leukemia in terminal blast crises widely used as a model for myeloid malignancies, resulted in an increased frequency of giant cells in multiple clones derived from mutant cells but not in wild-type clones. The presence of increased number of giant cells in mutants was confirmed both by microscopic observations and flow cytometry using forward (FSC) and side (SSC) scatter. Typically, cells with large size have increased FSC. The increase in giant cells in the mutant, and not wild-type, is unrelated to the CRISPR/Cas9-mediated genomic alterations since the wild-type cells were similarly manipulated by introducing a synonymous change (Fig.
1A) next to the mutant nucleotides. Also, the giant cell formation was found to be specific to K700E mutation in
SF3B1 gene as introduction of another well-established mutation (P95H) in
SRSF2 gene did not result in increased giant cell formation in K562 cells (Supplementary Figure S1). It is interesting to note that macrocytic anemia which is characterized by giant cells is frequently present in MDS subtype with increased ring sideroblasts and thrombocytosis with SF3B1 mutations [
25,
43]. Also, mice or zebra fish carrying SF3B1 K700E mutations show macrocytic anemia suggesting this mutation is associated with increase cell size in certain cell types. Since PGCCs are characterized by polyploidy and multiple nuclei, DNA content was calculated using several independent measures that include propidium iodide staining, metaphase and FISH analysis, and Hoechst staining followed by microscopy. In multiple independent clones, PI staining showed that giant cells were polyploid with > 6 N DNA content (K562 is a near triploid). This polyploidal nature of giant cells was confirmed using FISH analyses on metaphases, where greater than 3 N chromosomes and multiple copies of FISH probes asymmetrically distributed among multiple nuclei were documented. Interestingly, FISH analysis also found evidence of “mitotic catastrophe,” which, in the context of giant cells, is believed to be due to abnormal or incomplete mitoses frequently observed in PGCC, resulting in aneuploid or nearly diploid daughter cells [
44‐
49].
PGCCs in solid tumors are observed after radiation or chemotherapeutic interventions and have been well documented both in patient biopsies and in vitro studies [
3,
5,
8,
9,
11,
35,
38,
50‐
52]. PGCC formation is believed to be a natural response to stress [
4,
5,
53]. Azacytidine, a DNA methylation inhibitor, is approved for use in MDS and AML [
54]. Similar to PGCCs in solid tumors [
53], we found PGCC in this liquid cancer to be resistant to Azacytidine at a dose that killed half the K562 cells as evidenced by an enrichment of PGCCs. In response to Azacytidine, we showed a higher percentage of death in WT cells and a higher percentage of surviving fraction of giant cells in mutants (K700E) indicating these cells are resistant to the anti-cancer drug (Fig.
3). We observed similar resistance of giant cells to stress induced by serum starvation. Short-term serum starvation results in cell cycle arrest and is commonly used in cell synchronization methods, but long-term starvation results in cellular apoptosis and death. The giant cells persisted after 18 days of starvation with mutants showing higher survival in culture compared to WT cells (Supplementary Figure S5).
One concern regarding the use of K562 cells is that they can differentiate toward erythroid or megakaryocyte lineages under appropriate conditions [
55]. Specifically, hemin treatment results in increased differentiation of these cells to erythrocyte lineage [
56] and PMA treatment to megakaryocyte lineage [
36]. To rule out that these giant cells are not spontaneously differentiated megakaryocyte, we stained the cells with CD61, a marker for megakaryocyte differentiation (Fig.
4B and C). We also stained cells that were treated with either azacytidine or CoCl
2 that is known to create hypoxic conditions and increase PGCC formation [
7] and PMA that is well established to differentiate K562 into mature megakaryocytes. Unlike PMA treatment, where there was a robust increase in CD61 uniformly in all cells, we noticed a modest increase in CD61 expression with Azacytidine or CoCl
2 treatment, likely due to deregulation of gene expression (Fig.
4B). Most importantly, the giant cell morphology and nuclear morphology (which is typically multilobulated in megakaryocytes) observed in PMA-treated cells are distinct from that observed with 5-Azacytidine or CoCl
2 treatment suggesting the PGCCs are not megakaryocytes but a distinct subset of cells (Fig.
4A and C).
The multinucleated state of PGCCs is achieved by cell fusion and/or nuclear division with failed cytokinesis [
5]. Also, PGCCs are known to divide giving birth to normal-sized cells with near normal ploidy by asymmetrically dividing their genome content [
44,
48]. Time-lapse microscopy combined with Ki-67 proliferation marker data suggested that the giant cells observed in mutants are capable of cell division to give rise to daughter cells (Fig.
5). Additionally, we observed various morphological states of giant cells in culture with appearance of budding or bursting as documented in PGCCs from solid tumors (Supplementary Figure S6). We also observed cell fusion happening in culture when giant cells are in the vicinity of smaller cells (Supplementary File S7). Our observations indicate that these giant cells are capable of both endoreduplication as well as cell fusion and can give rise to small size progeny as reported previously for solid tumors [
5].
As noted earlier, mutations in
SF3B1 gene are associated with the appearance of ring sideroblasts; erythroblasts characterized by iron laden mitochondria surrounding the nuclei of erythroblasts. We observed a 3–4fold increase in mitochondrial biomass in mutant cells compared to wild-type but the significance of this observation in the formation and functions of PGCC is not clear (Fig.
6).
The significance of our observations is that we document PGCCs in a cell line derived from a hematological cancer for the first time. We show that the increased frequency of PGCC formation is linked to a specific genetic event, that is, introduction of a point mutation in a splicing factor, suggesting possible distinct molecular mechanisms for PGCC formation.
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