Adipogenesis induces growth inhibition of dedifferentiated liposarcoma

2.1 Cell lines and reagents

LIPO-246 and LIPO-863B, LP6, and MLS-402 and MLS-1765 cell lines were kindly provided by Dr Dina Lev, Dr Jonathan A. Fletcher, and Dr Pierre Aman, respectively. The cells were cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% FBS (Gibco) and 1% antibiotic-antimycotic (Gibco) at 37°C and in a 5% CO₂ incubator. Cell lines were validated for human cell line authentication (STR DNA profiling, Figure S1) using an AmpFLSTR Identifiler PCR Amplification Kit (Thermo Fisher Scientific). Mycoplasma contamination was not detected in any cells.

2.2 Adipogenic differentiation assay

Cells were seeded into a 6-well plate in DMEM medium, which was then replaced with an adipogenic differentiation medium (StemPro Adipogenic Differentiation Kit; Invitrogen, Carlsbad, CA, USA), all four components, or the indicated combination (Figure 1C) of inducing adipogenic differentiation reagents with dexamethasone, IBMX, indomethacin, or insulin (Sigma, St Louis, MO, USA) in complete DMEM medium every 3–4 days. After 21 days, the cells were stained with an oil Red O staining kit (Lifeline Cell Technology, Carlsbad, Ca, USA) according to the manufacturer's instructions.

A more detailed version of materials and methods is included in Data S1.

3.1 Growth inhibition of human WDLPS/DDLPS cells by inducing adipogenesis in vitro

To examine whether adipogenic stimulation induces adipogenesis in human LPS cells, we carried out oil Red O staining after the cells had been cultured in commercial adipogenic induction medium. LIPO-863B (WDLPS) and LP6 (DDLPS) cells showed numerous lipid droplets compared to the corresponding control cells (cells not treated with adipogenic induction medium) (Figure 1A). Next, we reviewed the literature to identify methods of pharmacologically inducing adipogenic differentiation in human cells. Many research groups have reported that the four compounds, dexamethasone, IBMX, indomethacin, and insulin, can induce adipogenic differentiation of human bone marrow stem cells.19 Currently, dexamethasone, indomethacin, and insulin are used to treat immune disorders and attenuate uncontrolled blood sugar levels, whereas IBMX is considered a potential drug for treating inflammation. Based on this information, we examined whether these four compounds could induce adipogenic differentiation of WDLPS/DDLPS cells. WDLPS (LIPO-863B) and DDLPS (LIPO-246 and LP6) cells showed an increased level of oil Red O staining positivity, whereas myxoid LPS cells (MLS-402 and MLS-1765) did not (Figure 1B). Interestingly, treatment with these compounds inhibited the growth of WDLPS/DDLPS cells but not myxoid LPS cells (Figure 1B). These results indicate that adipogenic differentiation can inhibit the growth of WDLPS/DDLPS but not myxoid LPS cells.

To determine which of these compounds induces adipogenic differentiation and growth inhibition, WDLPS/DDLPS cells were treated with combinations of one, two, three, or four of the compounds (data not shown). We found that some of the two-compound combinations showed induction of adipogenic differentiation and inhibition of growth similar to those shown by the four-compound combination. Therefore, we compared oil Red O staining positivity and growth inhibition for each two-compound combination (Figure 1C). Compared to that in the corresponding control cells (Figure 1B, No-Adipogenesis), several combinations showed increased oil Red O staining positivity and reduced growth in WDLPS/DDLPS cells. LIPO-246, LIPO-863B, and LP6 cells showed the greatest response to treatment combinations 4, 1, and 4, and were also more responsive to treatments 5, 4, and 1, respectively (Figure 1D).

We monitored the expression level of NANOG, OCT-4, and SOX2, genes involved in maintaining stemness (Figure 2A). LIPO-246 cells treated with combination 4 showed upregulated expression of all three genes compared to those treated with combination 5, and showed a higher level of OCT-4 expression than cells treated with all four compounds. LIPO-863B cells treated with combination 1 showed upregulated OCT-4 expression compared to those treated with combination 4, whereas their NANOG and SOX2 expression levels were similar to those of cells treated with all four compounds. LP6 cells treated with combination 4 showed upregulated NANOG expression compared to those treated with combination 1; however, their OCT-4 and SOX2 expression levels were similar to those of cells treated with all four compounds.

We also compared the expression level of genes serially induced during the transcriptional regulation of adipogenesis: SREBP1 (all stages), C/EBPβ (early stage), C/EBPα and PPAR-γ (middle to late stages), and ADIPSIN and LPL (late stage), for each of the treatment combinations (Figure 2B).20 LIPO-246 cells treated with combination 4 showed upregulated expression of all genes except ADIPSIN compared to those treated with combination 5, and the expression levels of all genes except PPAR-γ were similar to those in the cells treated with all four compounds. Expression of C/EBPβ was higher after treatment with all four compounds. LIPO-863B cells treated with combination 1 showed higher expression levels of all genes except C/EBPβ compared to those treated with combination 4, and their levels of PPAR-γ, ADIPSIN, and LPL expression were similar to those following treatment with all four compounds. LP6 cells treated with combination 4 showed higher SREBP1, PPAR-γ, and ADIPSIN expression levels compared to those treated with combination 1, whereas their levels of C/EBPβ, PPAR-γ, and LPL expression were similar to those following treatment with all four compounds.

Next, we examined the translational upregulation of these genes during induction with the selected combinations for 7, 14, and 21 days (Figure 2C). LIPO-863B cells treated with all four compounds showed higher expression levels of all proteins except SREBP1 compared to those treated with combination 1, and their level of C/EBPβ expression was similar to that following treatment with combination 1. LP6 cells treated with combination 4 showed similar expression levels of all proteins compared to those following treatment with all four compounds. Taken together, these results suggest that treatment with specific combinations (combination 4 for LP6 and LIPO-246, and combination 1 for LIPO-863B) induces adipogenic potency by transcriptional and translational upregulation of genes related to stemness and early-late adipogenic differentiation.

3.2 Inhibition of human DDLPS growth by inducing adipogenesis in vivo

To evaluate the antitumorigenic effect of treatment with adipogenesis-inducing compounds in vivo, LIPO-246, LIPO-863B, and LP6 cells were s.c. inoculated into nude mice and randomly treated with vehicle, two-compound combinations, or all four compounds. In the pilot trial, LIPO-863B cells formed tumors with a high penetrance (3/3); however, their volume reached only approximately 200 mm³ after monitoring for 32 days post-injection (Figure S2). Therefore, a full-scale trial could not be carried out using LIPO-863B xenografts even though reduced tumor growth (ie, weight and volume) was observed following treatment with all four compounds and combination 1 (Figure S2). LIPO-246 xenografts showed reduced tumor growth following treatment with all four compounds, but not combination 4 (Figure 3A). Tumor growth was reduced in the LP6 xenografts treated with combination 4 and all four compounds in a method dependent on the number of compounds (Figure 3B). We then carried out histological analysis on the xenograft model-derived tumors (Figure S3). Cells from the compound-derived tumors showed fewer proliferative features and had low cellularity compared to those from the vehicle-derived tumors (Figure S3). Tumors treated with all four compounds showed increased oil Red O staining positivity compared to those treated with combination 4 (Figure 3C).

In parallel with our in vitro experiments, we examined whether treatment with the compounds enhanced the transcriptional and translational regulation of the genes involved in stemness and adipogenesis in vivo (Figure 3D-F). LP6 xenografts treated with combination 4 showed upregulated expression of all genes except OCT-4 compared to those treated with all four compounds (Figure 3D, left panel). Treatment with all four compounds caused the expression of all genes except SREBP1 and ADIPSIN to be upregulated compared to treatment with combination 4 (Figure 3D, middle and right panels). However, the expression levels following treatment with combination 4 were considerably higher than in those without treatment (Veh). LP6 xenografts treated with all four compounds showed upregulated expression of all proteins except NANOG compared to that by treatment with combination 4 (Figure 3E). Tumors treated with all four compounds showed increased expression and nuclear localization of SREBP1 than those treated with combination 4. (Figure 3F). These findings indicate that the induction of adipogenesis and stemness inhibits the potency of DDLPS tumors in vivo.