Renal cell carcinoma (RCC) is notorious for the lack of sensitivity to chemotherapy (8), but the underlying mechanisms are not elucidated. Nitidine chloride (NC) holds promise as a drug treatment against RCC as it was shown to have a pro-apoptotic effect on RCC and inhibit tumor growth by suppressing the ERK signaling pathway in a xenograft model (9, 10). However, in human lung adenocarcinoma cells, NC efficacy was inversely correlated with the expression level of the drug transporter ABCA1: cancer cells with downregulated ABCA1 were more sensitive to NC treatment (11). Therefore, we wondered whether ABCA1 could also confer NC resistance in RCC. We treated five different RCC cell lines with a combination of NC and an ABCA1 inhibitor cyclosporin A (CsA). Anti-proliferative effect of NC was significantly enhanced in the presence of CsA for three lines among five, Caki-1, 786-O and A498 (Figure 1A). These cells consistently expressed higher levels of ABCA1 compared to the non-responsive cells (Figure 1B and C), suggesting the acquisition of ABCA1-mediated drug resistance. Notably, ABCA1 expression was positively correlated with expression of the EMT-TF Snail in the RCC cell lines (Figure 1D and E). To investigate clinical relevance of ABCA1 upregulation in cancers, we analyzed ABCA1 expression in cancer using the UCSC Xena platform. Comparative ABCA1 expression data of both normal tissue and primary tumors were available for 24 cancer subtypes in the GDC TCGA database. In addition to glioblastoma and head and neck cancer, all three types of renal cancers (clear cell carcinoma (ccRCC), chromophobe carcinoma and papillary cell carcinoma) exhibited significant ABCA1 upregulation in tumor (Figure 1C and Figure 1—figure supplement 1A and B), indicating that ABCA1 upregulation frequently occurs in clinical renal cancers.

To examine whether the enrichment of ABCA1 protein in malignant cancer cells is detectable in clinical cases, we conducted immunohistochemical analysis on surgical specimens taken from patients with primary ccRCC. The findings showed that ABCA1 was upregulated at the lesion site of high-grade ccRCC (Figure 1G). This clinical data aligns with our cellular experimental results, as Snail expression is known to be upregulated in high-grade ccRCC. Upon detailed comparison among grades, ABCA1 signal was more prominent in the patients with the high-grade classification (Figure 1H and Figure 1—figure supplement 2), suggesting that ABCA1 could serve as a marker for high-grade ccRCC. Xena analysis also revealed significantly higher expression of Snail in ccRCC, also supporting the idea that ABCA1 upregulation is correlated with exogenous Snail expression (Figure 1—figure supplement 1C).

Our results suggest the correlation between chemoresistance and Snail expression level. In order to clarify whether Snail expression alone can confer resistance to anti-cancer drugs in epithelial cells, Snail was ectopically expressed in normal epithelial cells and we investigated changes in resistance to anti-cancer drugs. Overexpression of Snail in a mouse mammary gland epithelial cell line EpH4 abolishes transcription of various genes involved in cell-cell adhesion including E-cadherin (Figure 1I) (12–14). EpH4-Snail cells exhibited coexistence of the mesenchyme-specific intermediate filament Vimentin and the epithelium-specific Cytokeratin-18 (Figure 1J), which is a typical feature of a hybrid E/M state of cancer cells. The hybrid E/M EpH4-Snail cells were significantly more resistant to NC than parental EpH4 cells (Figure 1K). Consistent with the results of kidney cancer cell lines, this resistance was cancelled by CsA (Figure 1K), and upregulation of ABCA1 expression was also observed in EpH4-Snail cells (Figure 1L and M). Consistent with the resistance to the antitumor agent, ABCA1 mainly localized plasma membrane in EpH4-Snail cells (Figure 1M). We also examined another model with a different cell line. In MDCK II, a canine kidney epithelial cell line, TGF-β treatment induced EMT under the expression of the oncogenic KRAS-G12V mutant (15). The upregulation of ABCA1 expression was also observed in this transient hybrid E/M model (Figure 1N), suggesting that similar mechanisms to those in EpH4-Snail are also at play in kidney cell EMT. Notably, induction of ABCA1 expression requires a longer period of treatment with TGF-β (day 8) than Snail induction (day 3), indicating that ABCA1 is upregulated in response to cellular alteration by Snail but not induced directly by TGF-β signaling. Furthermore, in comparison between human esophageal cancer cell lines (21), Snail-positive TE-8 cells exhibited higher expression of ABCA1 than Snail-negative TE-15 (Figure 1O). These results suggest that the induction of ABCA1 by ectopic expression of Snail occurs regardless of cellular background.

Then how does Snail induce ABCA1 expression, since as a transcriptional repressor it is incapable of doing so directly? Upregulation of ABCA1 was not observed in either E-cadherin or α-catenin KO cells (Figure 1L), indicating that ABCA1 induction is driven as a specific response to a fundamental change in cellular state induced by Snail overexpression, not an ancillary effect of disrupting either cell-cell adhesion or apical-basal polarity. Generally, ABCA1 is a transporter for the efflux of cholesterol out of the cell when there is an excess of cholesterol in the cell and ABCA1 upregulation in response to excess loading of cholesterol is mediated by sterol-activated nuclear receptors, LXRs (16). Aside from the cholesterol-LXRs regulatory axis, it was recently reported that ABCA1 expression in epithelial cells is regulated via FOXO3a and c-Myc (17). Activation of FAK/PI3K/Akt signaling depending on cell crowding state downregulates ABCA1 expression via phosphorylation of FOXO3a, a positive transcriptional regulator of ABCA1 (17). We found that phosphorylation level of FOXO3a at S453 tended to be lower in EpH4-Snail cells than in wild-type EpH4 cells (Figure 2—figure supplement 1A). However, since downregulation of Akt signaling by treatment with inhibitors of its activators GSK2334470 (PDK1), LY294002 (PI3K) and Wortmannin (PI3K) did not induce ABCA1 expression in wild-type EpH4 cells (Figure 2—figure supplement 1A), FOXO3a would not be responsible for ABCA1 expression in EpH4-Snail cells. Although ABCA1 expression is also downregulated by c-Myc in breast cancer, no remarkable difference of c-Myc expression was observed between EpH4 and EpH4-Snail cells, indicating that c-Myc is not likely involved in ABCA1 expression in EpH4-Snail cells (Figure 2—figure supplement 1B). Therefore, we next pursued in detail the possibility that LXRs induce ABCA1 in EpH4-Snail cells. Immunofluorescence microscopy revealed that LXRs are highly accumulated in the nuclei of EpH4-Snail cells, suggesting that ABCA1 upregulation could be mediated by LXRs (Figure 2A). Consistently, treatment with an LXR inhibitor GSK2033 abolished ABCA1 expression to an undetectable level in EpH4-Snail cells (Figure 2B). GSK 2033 treatment also canceled NC resistance (Figure 2C), suggesting that cholesterol efflux system activated by LXR is important for acquisition of chemoresistance. However, EpH4-snail cells did not show enhanced sensitivity to NC treatment even when ABCA1 was depleted by knockout (Figure 2—figure supplement 2). Given that CsA inhibits other isoforms of ABC transporters (18), other LXR-regulated efflux pumps may also contribute to chemoresistance.

Since transcriptional activity of LXRs are regulated by cellular cholesterol level, we examined whether ABCA1 is expressed in response to high cholesterol accumulation in the cells. Inhibition of cholesterol biosynthesis by simvastatin, an inhibitor of the rate-limiting enzyme of cholesterol biosynthesis 3-hydroxy-3-methylglutaryl-coenzyme A reductase, remarkably decreased ABCA1 expression in EpH4-Snail cells but residual ABCA1 was still detected (Figure 2D, normal FBS). In contrast, treatment with simvastatin in the medium supplemented with lipid-free FBS completely abolished ABCA1 expression (Figure 2D, lipid-free FBS). Together with the finding that incubation in lipid-free FBS medium alone did not markedly decrease ABCA1 expression, these results suggest that cholesterol biosynthesis is the primary source of excess accumulation of cholesterol in EpH4-Snail cells, but cholesterol uptake from media also significantly contribute to the accumulation as a secondary source.

These findings imply dysregulation of cholesterol metabolism in EpH4-Snail. Filipin staining revealed that cholesterol was prominently accumulated in intracellular structures in EpH4-Snail cells, whereas it localized primarily in the plasma membrane of wild-type EpH4 cells (Figure 2E). Since excess cholesterol can be stored in lipid droplets, we stained cells with Lipi-Green to visualize lipid droplets and found that lipid droplets were significantly enlarged in EpH4-Snail (Figure 2F and G). Consistently, TopFluor cholesterol, a fluorescent cholesterol analogue, was localized to lipid droplets in EpH4-Snail cells (Figure 2H). On the other hand, the intracellular filipin signals did not co-localize with the lipid droplet protein ADRP (perilipin-2) but co-localize with the lysosomal protein LAMP1 instead (Figure 2I). Given that filipin stains unesterified cholesterol, internalized cholesterol accumulates in lysosomes in its unesterified form and in lipid droplets in its esterified form in EpH4-Snail cells.

We next compared the total cholesterol levels in EpH4 cells and EpH4-Snail cells; the cellular amount of cholesterol slightly increased in EpH4-Snail cells, but the difference was modest and not significant (Figure 2J). Therefore, the internalization of cholesterol in EpH4-Snail cells is not simply due to an increase in the amount of cholesterol, but would be due to an abnormality in the subcellular distribution of cholesterol. A large proportion (30-50%) of cellular cholesterol is associated with sphingomyelin, and depletion of sphingomyelin induces internalization of cholesterol (19). To investigate contents of cholesterol and sphingomyelin in the plasma membrane, we utilized the lipid-binding protein probes domain 4 of perfringolysin O (cholesterol, D4) and lysenin (sphingomyelin). Intriguingly, cholesterol content in the plasma membrane was not affected by EMT, but sphingomyelin content decreased in EpH4-Snail cells (Figure 2K and L). Collectively, the increase in a sphingomyelin-unbound form of cholesterol in the plasma membrane results in the internalization of cholesterol and activation of LXR to induce ABCA1 expression in hybrid E/M cells.

The strong interaction between sphingolipids and cholesterol contributes to the formation of tightly packed and less fluid plasma membrane (20). Alteration of this physical property of plasma membrane frequently occurs in cancer cells and is thought to affect malignancy (21). Therefore, we next addressed the impact of sphingomyelin decrease on the lipid packing of plasma membrane using FpCM-SO₃, a recently developed plasma membrane-specific solvatochromic probe (22). Solvatochromic dyes respond to the polarity of their environment, exhibiting a red shift in more polar solvents. In lipid bilayers, these dyes similarly sense differences in lipid packing (order) and display a red shift in more fluid, disordered membranes (Figure 2—figure supplement 3A). Thus, variations in lipid order can be visualized as changes in the ratio of signal intensities of the red to blue channels (Figure 2—figure supplement 3B). The negatively charged and alkylated modifications enable FpCM-SO₃ to stably reside in the plasma membrane of living cells. Ratiometric imaging using FpCM-SO₃ revealed that the plasma membrane of EpH4-Snail cells was more disordered (Figure 2M–O). While plasma membrane fluidity is usually attributed to the cholesterol content (23), our results suggest that ectopic expression of Snail induces fluidization of plasma membrane, not by altering the surface cholesterol level but by decreasing sphingomyelin content.

The present findings suggest that the cause of chemoresistance may trace back to alterations of the balance of sphingomyelin and cholesterol. Therefore, we next investigate how sphingomyelin metabolism is affected by ectopic expression of Snail. We found that the total sphingomyelin content relative to total phospholipids decreased in EpH4-Snail cells (Figure 3A). Consequently, the ratio of cholesterol to SM (Chol/SM ratio) in the cells significantly increased in EpH4-Snail cells (Figure 3B). To investigate the SM profile in detail, we next analyzed the fatty acid composition of SM using LC-MS. In EpH4-Snail cells, the fractions of VLCFA-SMs (22:1, 22:0 and 24:1) were significantly decreased whereas those of LCFA-SMs (14:0, 16:1 and 16:0) were rather increased (Figure 3C), resulting in a prominent decrease in the total VLCFA-SM fraction (Figure 3D). These results suggest that decrease in VLCFA-SMs are responsible for decrease in total SM content in EpH4-Snail cells. On the other hand, the decrease in the cell surface content (Figure 2L) was more prominent than that in total SM (Figure 3A). Given that VLCFA-SMs are suggested to be differently trafficked in recycling from endosome to plasma membrane (24), their reduction may lead to decreased plasma membrane sphingomyelin content by altering its intracellular distribution.

Fatty acid composition of SM is mainly modulated by substrate specificity of two families of enzymes, Elovls and CerSs, that respectively catalyze the rate-limiting step of the fatty acid elongation cycle and the N-acylation of sphingoid bases to produce ceramides (Figure 3E) (25, 26). Therefore, we hypothesized that transcriptional repression of genes from these families causes the decrease in VLCFA-SM content in EpH4-Snail cells. RT-PCR analyses identified ELOVL7 and CERS3 as genes which are repressed specifically in EpH4-Snail cells (Figure 3F and G). Consistent with our hypothesis, Elovl7 and CerS3 control VLCFA-SM production (Figure 3E). Collectively, ectopic expression of Snail represses transcription of VLCFA-SM-producing enzymes and results in a decrease in the amount of VLCFA-SM. This downregulation of biosynthesis pathway is responsible for the induction of ABCA1 expression, because supplementation of VLCFA ceramide decreased the ABCA1 expression in EpH4-Snail cells (Figure 3H). Together with the results of the cholesterol depletion experiment (Figure 2D), the high Chol/SM ratio due to downregulation of VLCFA-SM biosynthesis accounts for the elevated ABCA1 expression in EpH4-Snail cells.

Since ABCA1 expression is not aligned with either the loss of epithelial state (E-cadherin and α-catenin KO; Figure 1L) or a non-epithelial basal state (fibroblasts), upregulation of ABCA1 is characteristic to the hybrid E/M cells expressing ectopic Snail (Figure 3I). To investigate the mechanism underlying this difference in ABCA1 expression, we again compared Chol/SM ratio in EpH4-Snail and normal fibroblast cell lines L-929 and NIH3T3. Chol/SM ratio of these fibroblasts were slightly but not significantly higher than that of EpH4 (Figure 3J). The cholesterol content in NIH-3T3 cells is significantly higher than in EpH4 cells (Figure3—figure supplement 1). Consequently, normal fibroblast would synthesize more SMs to counteract the cholesterol load imposed by the decrease in VLCFA-SMs and stabilize the Chol/SM ratio. By contrast, hybrid E/M cells are unable to respond appropriately to the harmful imbalance between cholesterol and SM due to the incomplete activation of the mesenchymal transcriptome.

To examine whether the above findings apply to human cancer cells, we investigate the Chol/SM ratio of TE-15 and TE-8 (see also Figure 1O) (27). Consistent with our findings in the EpH4-Snail system, Chol/SM ratio was significantly higher in TE-8 cells than in TE-15 cells (Figure 3K). Together with the results of mouse cell lines, normal epithelial and fibroblast cells maintain Chol/SM ratio around 1.0-1.5, and ABCA1 expression as a response to the excess Chol is induced when Chol/SM ratio exceed 1.5 (Figure 3L). Notably, the difference in either cholesterol or SM contents between TE-15 and TE-8 cells, and cholesterol content between EpH4 and EpH4-Snail is not significant when each are compared alone (Figure 2D and Figure 3—figure supplement 1), indicating the importance of considering their functional cooperativity, i.e. to analyze the Chol/SM ratio. Collectively, the cause of chemoresistance can be traced back to the downregulation of VLCFA-SM biosynthesis (Figure 3M). A decrease in sphingomyelin but not in cholesterol induces plasma membrane fluidization, which leads to internalization of cholesterol. The internalized, SM-unbound cholesterol activates LXRs, which in turn elevate ABCA1 expression and thereby confer chemoresistance to cells.

Finally, we examined the possibility of targeting the imbalance of Chol/SM ratio in Snail-positive cancer cells to selectively inhibit their growth. As discussed above, hybrid E/M cells of high Chol/SM ratio must either export cholesterol out of cells or isolate it within cells in lipid droplets as cholesteryl esters to avoid the cytotoxic effects of free cholesterol. Thus, inhibiting the means of clearing excess cholesterol could specifically target cells in the hybrid E/M state for self-extermination. Cholesterol efflux from cells is mainly dependent on ABCA1, and esterification of cholesterol is catalyzed by acyl-CoA:cholesterol acyl transferases (ACATs) (Figure 4A) (28). Among the two ACAT isoforms in humans, SOAT1 is widely expressed, including in the kidney (29). Indeed, all RCC cell lines tested express SOAT1 (Figure 4B), although the expression levels did not correlate with those of Snail in the respective cell lines (Figure 1B). Therefore, we examined the effect of ABCA1 inhibitor CsA and ACAT inhibitor TMP-153 on the EpH4-Snail model and renal carcinoma cells. While CsA non-selectively inhibited growth of all cell lines tested (Figure 4C and E), TMP-153 selectively inhibited growth of Snail-positive cells (EpH4-Snail, 786-O, A498 and Caki-1; Figure 4D and 4F). Thus, sequestration of cholesterol via esterification by ACATs is the primary mechanism for handling excess cholesterol to avoid cell death in these cells. Notably, TMP-153 treatment alone did not provide this selectivity between esophageal cancer cell lines, but combination of TMP-153 and CsA selectively inhibited growth of the Snail-positive TE-8 cells (Figure 4—figure supplement 1 A-C). Collectively, these results suggest that inhibition of cholesterol efflux and cholesterol esterification selectively inhibit growth of cells stably in a Snail-induced hybrid E/M state.

We also investigated the potential of ACAT inhibitors as anti-tumor agents in vivo using xenograft models. We successfully transplanted the 786-O line into nude mice among Snail-positive ccRCC cell lines. Using this system, we found that TMP-153 treatment significantly reduced 786-O tumor volume in vivo (Figure 4G-I). Moreover, the combination of TMP-153 and CsA also inhibited the growth of TE-8 tumor xenografts (Figure 4—figure supplement 1D-F). Together with the in vitro assays (Figure 4C-F and Figure 4—figure supplement 1 A-C), we propose that cholesterol clearance systems, particularly ACATs, can be used as molecular targets to selectively inhibit the growth of Snail-positive cancers.

Cells were grown in DMEM supplemented with 10% FBS under 5% CO₂ at 37°C. Mouse Snail-expressing EpH4 cells (EpH4-Snail)(14), E-cadherin KO and α-catenin KO EpH4 cells (37) were generated previously. EpH4-Snail cells were maintained in medium supplemented with 500 μg/ml G418 (Wako Pure Chemical). EpH4 cells were kindly provided by Dr. Reichmann (Institut Suisse de Recherches, Lausanne, Switzerland), and amplification of mouse-specific genomic sequences was confirmed. HEK293, 786-O, and A498 cells were obtained from the American Type Culture Collection (ATCC); Caki-1 and KMRC-3 cells were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank; TE-8 and TE-15 cells were obtained from the Tohoku University Cell Resource Center for Biomedical Research; and TUHR14TKB cells were obtained from the RIKEN BioResource Research Center (BRC). Human cell lines were authenticated by short tandem repeat (STR) profiling using the GenePrint10 System (Promega). All cell lines were routinely tested and confirmed to be negative for mycoplasma contamination.

pLVET-HA-K-RasG12V-IRES-GFP (Addgene #107140), LAMP1-mRFP-FLAG (#34611), pLentiPGK DEST H2B-iRFP670 (#90237) were transduced using the lentivirus produced in HEK293 cells, and fluorescence-positive cells were collected using a cell sorter Sony SH-800 as needed. pEGFP-C1-ADRP (Addgene #78161) was transfected to cells using Lipofectamine 3000 (Thermo Fischer). Nitidine chloride was purchased from Selleck Biotech, cyclosporin A from Wako, and TMP-153 from Cayman Chemical, filipin complex from Streptomyces filipinensis and GSK2033 from Sigma-Aldrich, Lipi-Green from Dojindo Laboratories. Ceramide (d18:1/22:0; C22:0 ceramide) from Avanti Polar Lipids.

The following primary antibodies were used for immunoblotting (IB), immunofluorescence microscopy (IF) and immunohistochemistry (IHC): Rabbit anti-ABCA1 mAb (96292S, IB, IF), rabbit anti-Snail mAb (3879S, IB), rabbit anti-Akt mAb (4691S, IB), rabbit anti-phospho-Akt (T308) mAb (2965S, IB), rabbit anti-phospho-FoxO3a (S253) pAb (9466S, IB) from Cell Signaling Technology; Rabbit anti-ABCA1 pAb (NB400-105, IHC) from Novus Biologicals; Rat anti-E-cadherin mAb (ECCD-2, IB, IF) from TaKaRa; Mouse anti-cytokeratin 18 mAb (LS-C84878, IF) from LS Bio; Goat anti-LXR alpha + LXR beta pAb (ab24362, IF) from Abcam; Mouse anti-α-tubulin mAb (12G10, IB) produced in house.

The following secondary antibodies were used: HRP-conjugated anti-rat IgG (HAF005) and anti-mouse IgG (HAF007) from R&D Systems; HRP-conjugated anti-rabbit IgG (4030-05) from Southern Biotech; Cy3-conjugated anti-goat IgG (705-165-147) from Jackson Immunoresearch Laboratories.

Total RNAs were prepared with RNeasy Mini Kit (Qiagen) and the retrotranscribed with Superscript III First-Strand Synthesis System (Invitrogen). RT-PCR was then performed using KOD FX (TOYOBO) following manufacturer’s instructions. Primer sequences used are shown in Table S1.

Total lipids were extracted from cultured cells by Bligh-Dyer’s method, dissolved in methanol/chloroform = 1/1 (v/v). Phospholipid content was determined by phosphate assay. SM and Chol content were determined using Sphingomyelin assay kit STA-601 (Cell Biolabs) and Amplex Red Cholesterol Assay Kit (Invitrogen), respectively, according to the manufacturer’s instructions. Fatty acid profile of SM was determined with LC-MS as described previously (39). Briefly, electrospray ionization mass spectrometry analysis was performed on a 6420 triple-quadrupole liquid chromatography–mass spectrometer (Agilent Technologies) equipped with an HPLC system and an auto sampler (Infinity 1260; Agilent Technologies). The extracted lipids were directly subjected to electrospray ionization mass spectrometry analysis. The mobile phase composition was acetonitrile/methanol/water = 18:11:1 (0.1% ammonium formate). The flow rate was 4 μl/min. The mass range of the instrument was set at 650–950 m/z. m/z profiles of SM were extracted according to total ion chromatogram patterns, and all peaks corresponding to [M+H]⁺ ions of d18:1-even chain length fatty acid-SMs were subjected to analyses.

To visualize subcellular localization of cholesterol, cells cultured on glass-bottom dishes were fixed with 4% paraformaldehyde for 15 min at room temperature and stained with 50 μg/ml filipin prepared in PBS for 30 min. To visualize lipid droplets, live cells were washed once with HBSS and stained with Lipi-Green (Dojindo) for 20 min at 37°C. Cells were washed twice with HBSS and observed on a heating stage at 37°C. For immunofluorescence microscopy, cells cultured on coverslips were fixed with 4% paraformaldehyde or 2% formalin prepared in PBS for 15 min at RT and permeabilized with 0.1% Triton X-100 prepared in PBS. Fixed cells were blocked with 1% BSA prepared in PBS for 1 h at RT. Cells were incubated with primary antibodies for 1 h at RT and secondary antibodies for 1 h at RT. Antibodies were prepared in the blocking solution. For detection of ABCA1, cells were fixed with MeOH for 20 min at -30°C and antibodies were prepared in Can Get Signal immunostain Immunoreaction Enhancer Solution B (TOYOBO). Fixed cells were observed at RT. For imaging of plasma membrane cholesterol and sphingomyelin, cells cultured on glass-bottom dishes were washed with HBSS three times, incubated with 1x working solution of CellMask Green plasma membrane stain (Molecular Probes) in HBSS, subsequently incubated with 5 mg recombinant scarlet-tagged D4 or 10 mg RFP-tagged lysenin in 100 ml HBSS for 30 min, washed three times with HBSS and observed at 37°C. For ratiometric imaging using FpCM-SO₃ (22), cells cultured on glass-bottom dishes were incubated with 1 mM FpCM-SO₃ in HBSS for 10 min at 37°C and observed at 37°C without washout. The dye was excited with a 405 nm laser, and 400–500 nm (Blue) and 500–700 nm (Red) signals were split with variable dichroic mirror and simultaneously detected with two photomultipliers. The raw images were gaussian blurred, ratio and mean intensity of two channels was calculated, and then data are expressed as a hue (ratio) – brightness (intensity) image (see Figure 2—figure supplement 3). All observation was performed with a confocal microscope (Carl Zeiss LSM900) equipped with Plan-APO (63×/1.40 NA, oil immersion) objective. Images were acquired using Carl Zeiss Zen 3.4 software. Images were analyzed using ImageJ/Fiji.

Whole cells were lysed with SDS sample buffer and samples were resolved by SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% skim-milk prepared in TBS-T, membranes were incubated with primary antibody for 1 h at RT or overnight at 4°C. Membranes were then washed and incubated with HRP-conjugated secondary antibody for 1 h at RT. Chemiluminescence signal was detected using a LAS4000mini imaging system (Fujifilm) and images were analyzed using ImageJ/Fiji. Protein levels were calculated relative to the signal intensity of a-tubulin as a loading control.

C22:0 ceramide was supplemented to culture medium as described previously (40). The ceramide was dissolved in dodecane/ethanol = 2/98 solution at 0.63 mM (stock solution). The stock solution (or vehicle control) was added to DMEM+10% FBS medium at a final solvent concentration of 0.4%, resulting in a final ceramide concentration of 2.5 mM. The prepared medium was used immediately. Cells were cultured in this medium for 24 h and then subjected to further analysis.

Cells cultured in 96-well microtiter plates (2 × 10³ cells/well, 48 h) were subjected to treatments with nitidine chloride, GSK2033, CsA, TMP-153 or their combination dissolved in DMSO for 24 h at 37°C. In experiments involving GSK2033, the compound was added 24 h prior to treatment with the other drugs. Final DMSO concentrations were less than 0.25%. After treatment, metabolically active cell number was determined using Cell Counting Kit-8 (Dojindo) according to manufacturer’s instructions. Briefly, 10 μl of CCK-8 solution was directly added to each well containing 100 μl culture medium and cells were incubated 1 h at 37°C. Reaction was stopped by adding 10 μl of 1% sodium dodecyl sulfate prepared in MQ and plates were put at 4°C to avoid additional reaction. Absorbance of solution at 450 nm were measured using a spectrophotometer (Thermo GENESYS 10S UV-Vis). Data were expressed as relative to 0.25% DMSO control.

Formalin-fixed and paraffin-embedded surgical specimens were cut into 3 μm sections. These sections were immersed in antigen retrieval solution (pH 9.0) (Nichirei Bioscience Inc.) and heated in a pressure chamber. After that, the sections were incubated with the primary antibodies (rabbit anti-ABCA1 pAb, NB400-105) and later incubated with HRP-labeled goat anti-rabbit secondary antibodies (Nichirei Bioscience Inc.). Immunoreactivity was visualized by using 3–3 Diaminobenzidine Dab Substrate Kits (Nichirei Bioscience Inc.). Informed consent for experimental use of the samples was obtained from all patients in accordance with the ethical guidelines of Aichi Cancer Center, and the study was approved by the Institutional Review Board (Approval No. 2021-0-037). DAB signals were extracted from RGB images by color deconvolution, inverted, and quantified as the mean intensity of pixels exceeding a defined threshold using ImageJ/Fiji.

Four-week-old female athymic nude mice were purchased from Japan SLC and housed in a specific pathogen-free facility. All animal experiments were conducted in accordance with the Guidelines for Animal Experiments of Aichi Medical University and approved by the Institutional Animal Care and Use Committee (Approval No. 2024-58).

To initiate xenografting of 786-O, 2[×[10⁶ cells in 200[μL DMEM were subcutaneously injected into the back of mice by using a 27[G needle. After 7 days, when the inoculated tumor became visible, the mice were randomly divided into treatment and control groups, and treatment was initiated (day 0). TMP-153 (50[mg/kg body weight) or vehicle (0.5% hydroxypropyl methylcellulose with 0.1% polysorbate 80) was intraperitoneally administered to each mouse on days 0, 4, 7, 11, 14, and 18 to each mouse. The tumor volume was measured every week and calculated using the modified ellipsoid formula (1/2[×[length[×[width²). The xenografting of TE-8 was performed with some modifications. For this, 1[×[10⁷ cells were injected. A combination of TMP-153 and CsA (both at 10[mg/kg body weight) or vehicle was intraperitoneally administered to each mouse on days 0 and 4. Finally, the tumor volume was measured 36 days after the first administration.

Microsoft Excel for Microsoft 365 MSO 2306, GraphPad Prism v8.4.1 and Python version 3.12.7 were used for analyses and displays of quantitative data. Data are expressed as mean with each point or error bars representing standard deviations (SD). Significant difference was indicated as follows: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.