Effects of Dedifferentiated Fat Cells on Neurogenic Differentiation and Cell Proliferation in Neuroblastoma Cells

Recent reports demonstrated that mesenchymal stem cells (MSCs) can induce differentiation of neuroblastoma (NB) cells. Dedifferentiated fat cells (DFAT) and MSCs have similar properties. The present study investigated whether DFAT can induce NB cell differentiation and suppress cell proliferation. DFAT was obtained from mature adipocytes isolated from adipose tissue from a ceiling culture. NB cells were cultured in a medium with/without DFAT, and subsequently in a DFAT-conditioned medium (CM) with/without phosphatidylinositol 3 kinase (PI3K) inhibitor. Length of neurites was measured, and the mRNA expression levels of the neurofilament ( NF ) and tubulin beta III ( TUB β 3 ) were assessed using quantitative real-time reverse transcription polymerase chain reaction. Cell viability was assessed by the water-soluble tetrazolium salt-1 assay. NB cells cultured with DFAT elongated the neurites and upregulated the expression of NF and Tub β 3 compared with the control. However, NB cells cultured in DFAT-CM demonstrated increased cell viability compared with the control. NB cells cultured with DFAT-CM and PI3K inhibitor suppressed cell viability and demonstrated increased neurite length and expression and upregulation of Tub β 3 . Therefore, the combined use of DFAT-CM and PI3K inhibitors suppresses the proliferation of NB cells and induces their differentiation. DFAT may offer new insights into therapeutic approaches in NB.


INTRODUCTION
Neuroblastoma is an embryonal tumor derived from undifferentiated neural crest cells and is the most common extracranial solid tumor during childhood. Tumors may be derived from anywhere in the sympathetic nervous system. Primary tumors are mainly concentrated in the abdomen (65%), half of which occur in the adrenal medulla [1]. Most neuroblastomas that develop during infancy spontaneously dedifferentiate without treatment and have favorable prognosis. However, neuroblastomas that occurring 12 months after birth may have poor outcomes. Clinical features, including age at onset and molecular biological markers of tumor tissues, are closely related to the prognosis. MYCN amplification, found in 20%-30% of patients with neuroblastoma, is a strong indicator of poor prognosis. Risk stratification based on these indicators are used to make treatment decisions [1][2][3].
The standard treatment for high-risk neuroblastoma is a multimodal approach, comprising chemotherapy and radiation therapy, and autologous hematopoietic stem-cell transplantation. Over the last decade, high-dose chemotherapy combined with autologous hematopoietic stem cell transplantation has been shown to improve event-free survival compared with conventional chemotherapy [4][5]. However, despite clinical remission with such a treatment, the 3-year event-free survival rate remains low at 31%-47% because of recurrence from residual lesions [4][5][6].
Mesenchymal stem cells (MSCs) are fibroblast-like cells, isolated from the bone marrow, adipose tissue, and fetal appendage tissues (such as the umbilical cord, umbilical cord blood, and placenta). MSCs are multipotent cells that differentiate into various cell types, such as osteoblasts, adipocytes, and chondrocytes [7][8]. MSCs are not only multipotent but also promote regeneration and supply immunoregulatory signals and are expected to be therapeutic cells with immunotherapeutic potential.

MSCs induce differentiation of neuroblastoma cells by secreting neurotrophic factors and growth
factors such as nerve growth factor (NGF), glial cell-derived neurotrophic factor, and brain-derived neurotrophic factor (BDNF) [9][10]. In a previous study, intertumoral injection of MSCs in a neuroblastoma mouse model inhibited tumor growth by inducing apoptosis. This resulted in longer survival in the MSC-treated mice [11]. Although MSCs have such high potential, clinical problems also have been reported. Harvest of bone marrow-derived MSCs is highly invasive. Additionally, geriatric patients with low bone density or those with osteoporosis do not have the required number of MSCs. Adipose tissue-derived stem cells (ASCs) could be an alternative to bone marrow-derived MSCs because they can be easily isolated without painful procedures or donor site injury [12,13].
However, similar to bone marrow MSCs, ASCs also comprise a heterogeneous cell population in the stromal tissue. Additionally, ASCs at early passage contain endothelial cells, smooth muscle cells, and pericytes. Additionally, ASCs cultured with serial passages continue to express various endothelial cell markers [12]. Therefore, other stem cell sources are required.
Harvesting of cells irrespective of age with minimal invasiveness is desirable. Additionally, the harvested cells must have high proliferative ability and homogeneity [14,15]. As a cell source that solves these problems, Matsumoto et al. [16] demonstrated that dedifferentiated fat cells (DFAT), obtained from ceiling culture of mature adipocytes, isolated from the adipose tissue, can actively proliferate and have an ability to differentiate similar to MSCs. DFAT are recognized as cells that have acquired a trait similar to that of MSCs, because the cell surface antigen profile of DFAT is similar to that of bone marrow-derived MSCs and ASCs. Cytokines released from DFAT were also found similar to those released from bone marrow-derived MSCs and ASCs. Cytokines have been reported to contain neurotrophic factors, such as BDNF [17]. Therefore, DFAT may also induce differentiation of neuroblastoma cells like MSCs.
Various neurotrophic and growth factors secreted by MSCs may exert therapeutic effects by promoting the differentiation of neuroblastoma cells [9][10]. However, whether DFAT have a differentiation-inducing effect on neuroblastoma cells remains unclear. In the present study, we aimed to determine the effect of DFAT on neuronal differentiation and cell viability in a human neuroblastoma cell line.

Human neuroblastoma cell lines
NB9 cells were acquired from the RIKEN Cell Bank (Tsukuba, Japan) and were cultured in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) with 15% fetal bovine serum (FBS) (Sigma Aldrich, St. Louis, MO) and 1% penicillin-streptomycin-glutamine (Thermo Fisher Scientific) at 37 °C in a 5% CO 2 incubator. Cells were subcultured to a fresh medium when the growth reached 90% confluence.
SK-N-SH cells were acquired from the American Type Culture Collection (ATCC Manassas, VA, USA). SK-N-SH cells were cultured as monolayers in Dulbecco's modified Eagle medium (Thermo Fisher Scientific) with 10% FBS, 1% Minimal Essential Medium non-essential amino acids (Thermo Fisher Scientific), 1% sodium pyruvate (Thermo Fisher Scientific), and 1% penicillin-streptomycin-glutamine, at 37 °C in a 5% CO 2 incubator. Cells were subcultured to a fresh medium when the growth reached 90% confluence.

Isolation and cell culture
Approximately 1-2 g of subcutaneous adipose tissue obtained from patients undergoing surgery was processed for preparing DFAT based on a previous report. The adipose tissue was cut into small pieces and treated with 0.1% (w/v) collagenase solution (Sigma Aldrich) at 37 °C for 30 min under agitation. After agitation, the mixture was passed through a filter of pore size 250 µm to remove undigested tissue. The filtered liquid was centrifuged at 135 g for 3 min, and the floating top layer containing the mature adipocytes was collected. After washing with phosphate-buffered saline

DFAT conditioned medium
The sixth-passage DFAT were placed in 100 mm dishes and allowed to grow at 37 °C in 5% CO 2 until the cells reached 70% confluence. After washing with PBS, the medium was replaced with 5 mL of DMEM containing 5% FBS. The DFAT were incubated for 72 h, and the supernatant was collected and centrifuged at 1,500 g for 10 min. The supernatant was passed through a Millex-HV 0.45 µm filter (Merck Millipore, Burlington, MA). The collected DFAT-CM were frozen at -80 °C and thawed on the day of the experiments.

Co-culture of neuroblastoma cell lines and DFAT
NB9 cells (5 × 10 4 ) or SK-N-SH (1 × 10 5 ) were seeded in six-well plates. The neuroblastoma cells were incubated for 7 days in the absence (control group) or presence (co-culture group) of DFAT following 24 hours of seeding. In the co-culture group, DFAT (1 × 10 5 ) were seeded in the cell culture insert 0.4 µm PET membrane (BD Falcon). The cells were incubated in DMEM supplemented with 10% FBS. Seven days after incubation, the neurite length and the expression level of neural differentiation markers in NB cells were evaluated to assess the differentiation potential.

Cell proliferation
SK-N-SH or NB9 cells (5 × 10 3 ) were seeded in 96-well plates. Twenty-four hours after seeding, neuroblastoma cells were incubated for 3 days in the absence (control group) or presence of DFAT-CM (DFAT-CM group). In the DFAT-CM group, 100 µL of DFAT-CM was added to 100 µL of basal medium (DMEM containing 20% FBS). In the control group, 100 µL of DMEM was added to 100 µL of the basal medium. After culturing the neuroblastoma cells for 3 days under these conditions, the proliferation ability was evaluated using the WST-1 assay.

Combined effect of DFAT-CM and phosphatidylinositol 3 kinase (PI3K) inhibitor on neuroblastoma cells
The pharmacological inhibitor of PI3K, LY294002, was obtained from Tocris Bioscience (Bristol, England) and reconstituted according to the manufacturer's specifications. NB9 cells (5 × 10 4 ) were seeded into six-well plates. Twenty-four hours after seeding, neuroblastoma cells were incubated for 3 days in the absence (control group) or presence of DFAT-CM (DFAT-CM group), in combination with DFAT-CM and PI3K inhibitor (DFAT-CM + PI3K inhibition). In the LY294002 and DFAT-CM + LY294002 groups, the concentration of LY294002 was adjusted to 10 µM. After culturing the neuroblastoma cells for 3 days under these conditions, the differentiation potential of the neuroblastoma cells was evaluated. NB9 cells (5 × 10 3 cells/well) were seeded in 96-well plates. Twenty-four hours after seeding, the neuroblastoma cells were incubated under the above conditions for 3 days, and their proliferation ability was evaluated.

Neurite outgrowth assessment
For neurite outgrowth assessment in neuroblastoma cells, at least 15 random fields were photographed using a BZ-X710 phase-contrast light microscope (KEYENCE, Osaka, Japan).
Neurite lengths were measured manually using the National Institutes of Health ImageJ software (Rasband, W. S., ImageJ, U. S. National Institutes of Health, Bethesda, ML, https://imagej.nih.gov/ij/, 1997-2018) [18][19]. Fifty cells were observed and counted to have neurites. A neurite was defined as a process outgrowth that was longer than twice the diameter of the cell body.

Statistical Analysis
The Mann-Whitney U test was used for statistical comparisons between the two groups. Statistical comparisons between multiple groups were performed using one-way analysis of variance followed by Tukey's multiple comparison test for post hoc analysis. The analysis was performed using EZR software (Saitama Medical Center, Jichi Medical University, Saitama, Japan) [20]. Statistical significance was set at p<0.05.

Effects of DFAT on neuroblastoma cells by co-culture
When the cell morphology of NB9 cells was observed with a phase-contrast microscope, the co-culture group had more cells with neurites than the control group (Fig. 1a). Based on the quantitative analysis of neurite lengths, the percentage of cells with neurite outgrowth was significantly higher in the co-culture group than in the control group (Fig. 1b). SK-N-SH cells had similar results in that neurite outgrowth was observed in the co-culture group (Figs. 1c and d).
Subsequent to the neurite outgrowth assay to confirm NB9 cell differentiation, mRNA expression of neuronal specific markers (NF and TUBβ3) was assessed by real-time RT-PCR. The mRNA levels of NF and TUBβ3 were significantly higher in the NB9 cells of the co-culture group than in those of the control group (p<0.05) (Fig. 1e). SK-N-SH cells showed similar results; the mRNA levels of NF and TUBβ3 were significantly increased in the co-culture group when compared to the control group (p<0.05) (Fig. 1f).

Effects of DFAT-CM on neuroblastoma cells
To determine whether DFAT-CM can affect the viability of human neuroblastoma cells, the viability of NB9 and SK-N-SH cells was assessed by WST-1 assay. The cell viability of NB9 cells cultured in DFAT-CM was significantly higher compared with that cultured in the control medium (p<0.05) (Fig. 2a). Similar results were observed in SK-N-SH cells (Fig. 2b).
From the aforementioned results, it can be stated that DFAT-CM can induce neuronal differentiation in neuroblastoma cells. However, DFAT-CM was demonstrated to promote the proliferation of neuroblastoma cells.

Combined effect of DFAT-CM and PI3K inhibitor on neuroblastoma cells
According to literature, cell proliferation signaling pathways in neuroblastoma cells are activated by PI3K/AKT that exists an intermediate signaling pathway [21]. To determine whether inhibition of PI3K activity affects proliferative potential and the differentiation of neuroblastoma cells, we treated NB9 cells with LY294002 to inhibit PI3K and subsequently, incubated with DFAT-CM or control medium. First, the concentration of LY294002 required to suppress the proliferative potential of NB9 cells was determined. Twenty-four hours after seeding the NB9 cells, DFAT-CM and LY294002 were added and incubated. On the third day of culture, cell proliferation was evaluated using the WST-1 assay. LY294002, a selective inhibitor of PI3K, suppressed NB9 cell proliferation at 10µM (Fig. 3a). In addition, LY294002 significantly suppressed the cell proliferation ability promoted by DFAT-CM on NB9 cells at 10µM (Fig. 3a). These data indicate that the PI3K inhibitor has a suppressive effect on the cell growth-promoting action of DFAT-CM.
After NB9 cells were incubated for 3 days in combination with DFAT-CM and LY294002, the neurite outgrowth of neuroblastoma cells was analyzed. From the quantitative analysis of neurite length, neurite outgrowth cells were significantly higher in the DFAT-CM group than in the control group. Treatment of NB9 cells with LY294002 did not significantly increase the percentage of neurite outgrowth cells (Figs. 3b and c). The expression of TUBβ3 was significantly increased in both the DFAT-CM and LY294002 groups compared with that in the control group (Fig. 3d). In the DFAT-CM+LY294002 group, the expression of TUBβ3 was significantly increased not only in the control group but also in the DFAT-CM and LY294002 groups (Fig. 3d). These results suggest that PI3K inhibitors increase the mRNA level of TUBβ3, and this effect is enhanced by combination with DFAT-CM.
We investigated the changes in AKT phosphorylation in NB9 cells following the addition of PI3K inhibitors. We confirmed that the addition of DFAT-CM led to the phosphorylation of AKT in NB9 cells (Fig. 4). The phosphorylation of AKT was inhibited in the presence of the PI3K inhibitor LY294002 at a concentration of 10 µM (Fig. 4).

DISCUSSION
In this study, we demonstrated that DFAT can induce differentiation in neuroblastoma cells. Our data demonstrated that co-culturing neuroblastoma cells with DFAT promoted neurite formation.

Neurite outgrowth suggests differentiation of neuroblastoma into nerve cells and is a phenomenon
observed when differentiation is induced in the neural progenitor cells by factors such as retinoic acid and NGF [22]. The neurite outgrowth of neuroblastoma cells indirectly co-cultured with DFAT via cell culture inserts suggests that the factors secreted from DFAT induced the differentiation of neuroblastoma cells. We also found that the expression of neuronal specific markers, including NF and TUBβ3, from neuroblastoma cells was enhanced by indirect co-culture with DFAT cells. This suggests that DFAT may induce the differentiation of neuroblastoma cells, even at the mRNA level. Our study had several limitations. The effect of DFAT on neuroblastoma mouse models is yet to be known. Additionally, we did not compare DFAT with the other types of cells, such as MSCs.
Further studies are required to evaluate the effect of the combined administration of DFAT-CM with a PI3K inhibitor in improving neuroblastoma.         combination with a PI3K inhibitor on NB cell differentiation. NB9 cells were cultured in the presence or absence of DFAT-CM, in combination with a PI3K inhibitor for three days. NB9 cells were photographed using a phase-contrast light microscope after culture (scale bar, 100 μm). (c) Neurite outgrowth of NB9 cells was enhanced by DFAT-CM, but the PI3K inhibitor did not affect neurite outgrowth. (d) Total RNA was extracted, and TUBβ3 was quanti ed using real-time reverse transcription polymerase chain reaction.
Relative expression was analyzed using the comparative CT method with GAPDH as the internal control. Expression of TUBβ3 was increased by DFAT-CM and was further increased by the combined use of a PI3K inhibitor. Bar: mean ± SE. Data shown for triplicate wells, *p<0.05.

Figure 4
Image showing the results of Western blotting analysis for phospho-AKT expression in NB9 cells. NB9 cells were cultured in the presence or absence of dedifferentiated fat cells-conditioned medium (DFAT-CM), in combination with a phosphatidylinositol 3-kinase (PI3K) inhibitor for 24 h. The cell protein extracts were then extracted and Western blotting analysis for phospho-AKT expression in NB9 cells was performed. DFAT-CM led phosphorylation of AKT in NB9 cells. The phosphorylation of AKT was inhibited in the presence of LY294002.