ABSTRACT
Cachexia is a complex syndrome that is often associated with cancer. Chemotherapy, one of the maincancer treatments, worsens weight loss in cancer-induced cachexia. In this context, it is thought that fat loss precedes muscle loss, and that alterations in adipose tissue are associated with tumours. However, the effect of cancer treatment on adipose tissue is not well understood. This study aimed to evaluate the impact of chemotherapy alone on mature 3T3-L1 adipocytes to identify the mechanisms contributing to adipose tissue alteration. The murine cell line 3T3-L1, a model of mature adipocytes, was used in this study. After differentiation, cells were treated for 48 h with a chemotherapy cocktail called FLOT composed of 5-fluorouracil, leucovorin, oxaliplatin and docetaxel at two concentrations (FLOT 1X and 0.1X). The control group was treated with the vehicle of the chemotherapy cocktail. Viability, mitochondrial function and dynamics, lipid metabolism, and cellular stress were also evaluated. FLOT 1X chemotherapy significantly reduced viability of mature 3T3-L1 cells and inhibited lipid accumulation. Interestingly, while FLOT 1X treatment downregulated lipogenesis markers, FLOT 0.1X treatment upregulated some of them. Although, the treatment showed no effect on mitochondrial respiration or density, it significantly increased expression of oxidative stress and inflammation markers in adipocytes.This in vitro study provides the first evidence of FLOT chemotherapy’s direct effects on healthy mature adipocytes. The results demonstrate significant treatment-induced reductions in cell viability along with dysregulation of both lipogenic and lipolytic pathways. These findings elucidate previously unrecognized mechanisms underlying adipose tissue dysfunction in cancer cachexia.
KEYWORDS: adipocytes, mouse, FLOT, lipid metabolism, high resolution respirometry, reactive oxygen species
GRAPHICAL ABSTRACT
Introduction
Cachexia is a complex metabolic syndrome affecting approximately 50–80% of patients with cancer [1,2]. It is characterized by muscle loss, with or without fat loss, and accounts for up to 20% of all cancer-related deaths. This condition is particularly prevalent in pancreatic and oesophageal cancers and can be exacerbated by treatments, such as platin-based therapies [3,4]. Treatments for resectable oesophageal adenocarcinomas, such as the FLOT regimen (oxaliplatin, 5-fluorouracil (5-FU), docetaxel, and leucovorin), are highly toxic [5,6]. Patients are prone to unintentional weight loss due to anorexia, dysphagia, and nutrient malabsorption [7]. While muscle loss is associated with poor outcomes and increased mortality, loss of adipose tissue (AT) is associated with an impaired response to anti-cancer treatments [8]. Moreover, fat loss may precede muscle wasting during cancer progression, and prevention of this fat depletion could have a protective role, as observed in a murine model of skin cancer [9].
The mass and function of AT are disrupted by cancer cachexia, but little is known about the effects of chemotherapy. Most chemotherapeutic drugs are not target-specific and cannot distinguish between tumour cells and normal non-cancerous cells [10,11]. Cisplatin has been extensively studied and is known to induce cell death in adipocyte-like cells [12], whereas mesenchymal stem cells are resistant to cytotoxic effects [13]. Few studies have focused on the effects of chemotherapeutic agents on mature adipocytes [10,14]. Indeed, the authors showed that doxorubicin decreased adipocyte viability and decreased adipogenesis in mesenchymal stem cells.
Previous studies have demonstrated that chemotherapy can induce significant metabolic dysfunction in cancer patients [15,16]. For instance, cisplatin and doxorubicin have been shown to impair adipocyte differentiation, decrease lipid storage capacity, and increase lipolysis, resulting in fat loss and metabolic dysregulation [17]. These alterations are often accompanied by heightened oxidative stress and mitochondrial dysfunction, which further contribute to AT wasting. The latter is linked to poor prognosis, decreased chemotherapy tolerance, and lower survival rates [18], impairing patient quality of life [19,20]. However, most studies have focused on individual chemotherapeutic agents, rather than combination therapies like FLOT, commonly used in gastrointestinal cancers [5,21]. Given the distinct mechanisms of action of each FLOT component (5-fluorouracil, leucovorin, oxaliplatin, and docetaxel), the combinatorial regimen likely induces adipocyte metabolic alterations that may differ qualitatively and quantitatively from single-agent chemotherapy effects.
Understanding how chemotherapy-induced metabolic dysregulation in adipose tissue contributes to dysfunctions cited above is essential for developing targeted interventions to mitigate cachexia and enhance therapeutic responses. This study aimed to investigate the direct effects of FLOT chemotherapy on metabolic function, mitochondrial activity, and oxidative stress in mature 3T3-L1 adipocytes (a murine fibroblast-derived cell line), addressing a critical knowledge gap regarding combination chemotherapy-induced AT dysregulation. As FLOT is a first-line treatment for oesophageal adenocarcinoma, understanding its specific impact on adipocyte biology is clinically relevant. It was hypothesized that 48-hour FLOT exposure would induce significant metabolic adaptations in white adipocytes, potentially mirroring the adipose tissue alterations observed in chemotherapy-treated patients.
Results
1X FLOT chemotherapy treatment decreased mature 3T3-L1 viability
Cell viability was significantly decreased (p < 0.05) by the 1X FLOT cocktail compared with that observed in the control group (Figure 1). At this concentration, an average of 80% of cells remained for subsequent experiments. At lower concentrations, FLOT treatment did not significantly reduce the cell viability. According to this result, FLOT 1X and 0.1X concentrations only were then tested.
Figure 1.
Effect of FLOT chemotherapy treatment on mature 3T3-L1 viability. Mature 3T3-L1 cells were treated for 48 h with the FLOT treatment at different concentrations of FLOT and H2O2 as a negative control of toxicity. Four independent experiments were performed with 2 to 6 technical replicates: ctrl (n = 19), H2O2 (n = 8), FLOT 0.01X (n = 10), FLOT 0.1X (n = 21), and FLOT 1X (n = 21). The results are presented as the mean percentage of the control ± S.E.M. Kruskal-Wallis test and Dunn test post-hoc, ***p < 0.001, *p < 0.05 versus control.
Ctrl: control; FLOT: 5-fluorouracil, leucovorin, oxaliplatin and docetaxel; H2O2: hydrogen peroxide.
1X FLOT chemotherapy treatment decreased lipid metabolism of mature 3T3-L1
Lipid metabolism is a critical point to explore because lipid storage is the main function of mature adipocytes. Neutral lipids accumulation in lipid droplets was evaluated using the oil red O (ORO) staining (Figure 2). The absorbance measured was significantly lower with the FLOT 1X treatment than with the control, but not with the FLOT 0.1X.
Figure 2.
Effect of FLOT chemotherapy treatment on lipid droplets accumulation in mature 3T3-L1. Mature 3T3-L1 were treated for 48 h with different FLOT treatment concentrations. Lipid droplets were stained with oil red O. Absorbance of the red oil O was measured at 500 nm. One independent experiment was performed with 12 technical replicates: ctrl (n = 12), FLOT 0.1X (n = 12), FLOT 1X (n = 12). The results are presented as the mean percentage of the control ± S.E.M. Kruskal-Wallis test and Dunn test post-hoc, ***p < 0.05 versus control.
Ctrl: control; FLOT: 5-fluorouracil, leucovorin, oxaliplatin and docetaxel.
Lipid metabolism in mature 3T3-L1 cells after a 48 h FLOT chemotherapy treatment at 1X or 0.1X, was investigated through both protein levels and gene expression. Representative blots of the protein levels are presented in Figure 3a. The patatin like phospholipase domain containing 2 (PNPLA2) also called adipose triglyceride lipase (ATGL), which is the first enzyme involved in the breakdown of triglycerides, showed similar protein levels under all conditions (Figure 3b). Protein levels of perilipin 1 (PLIN1), a key regulator of lipolysis and lipid storage, were decreased (p < 0.05) in the FLOT 1X condition compared to both the control and FLOT 0.1X (Figure 3c).
Figure 3.
Effect of FLOT chemotherapy treatment on lipid metabolism protein levels in mature 3T3-L1. Mature 3T3-L1 were treated for 48 h with different FLOT treatment concentrations. Four independent experiments were performed with 3 technical replicates (n = 12). (a) representative images of blots for PNPLA2 and PLIN1. Protein accumulations of PNPLA2 (b) and PLIN1 (c) normalized by ‘Ponceau’ red staining are presented according to treatment. The results are presented as the mean ± S.E.M., and each individual replicate is represented as a white dot. Kruskal-Wallis test and Dunn test post-hoc, ***p < 0.05 between groups.
AU: arbitrary unit; Ctrl: control; FLOT: 5-fluorouracil, leucovorin, oxaliplatin and docetaxel; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; PLIN1: perilipin 1; PNPLA2: patatin like phospholipase domain containing 2.
The relative gene expression of fatty acid synthase (Fasn) was significantly lower (p < 0.05) in the FLOT 1X treatment compared to both the control and FLOT 0.1X (Figure 4a). Relative gene expression of diacylglycerol O-acyltransferase 1 (Dgat1) (Figure 4b) was also significantly lower in the FLOT 1X treatment group than in the 2 other groups. The Dgat2 relative gene expression (Figure 4c) was higher with the FLOT 0.1X treatment compared to both the control and FLOT 1X.
Figure 4.
Effect of FLOT chemotherapy treatment on lipid metabolism gene expression in mature 3T3-L1.Mature 3T3-L1 cells were treated for 48 h with different FLOT treatment concentrations. Five independent experiments were performed, with 3 technical replicates (n = 15). Gene expressions of Fasn (a), Dgat1 (b), Dgat2 (c), Cpt1a (d), and Cd36 (e) were normalized by Rplp0 gene expression. The results are presented as the mean ± S.E.M. and each individual replicate is represented as a white dot. Kruskal-Wallis test and Dunn test post-hoc, ***p < 0.05 between groups.
AU: arbitrary unit; Cd36: cluster of differentiation 36; Cpt1a: carnitine palmitoyltransferase 1α; Ctrl: control; Dgat: diacylglycerol O-acyltransferase; Fasn: fatty acid synthase; FLOT: 5-fluorouracil, leucovorin, oxaliplatin and docetaxel; Rplp0: ribosomal protein lateral stalk subunit P0.
Finally, carnitine palmitoyltransferase 1 alpha (Cpt1a), responsible for acyl carnitine formation and lipid entry into the mitochondria for their oxidation, was not differentially expressed between the groups (Figure 4d), whereas the cluster of differentiation 36 (Cd36) relative gene expression was significantly decreased with FLOT 1X treatment compared to the control. At the same time, its expression increased with FLOT 0.1X compared to the FLOT 1X (Figure 4e).
1X FLOT chemotherapy treatment had no effect on mitochondrial density but decreased mitochondrial dynamics in mature 3T3-L1
Because mitochondria play a major role in metabolic adaptation and energy production from glucose and lipids, their metabolism was investigated. Mitochondrial density was assessed using the ratio of mitochondrial DNA to nuclear DNA (mtDNA-to-nDNA). FLOT 1X treatment increased significantly this ratio compared to control (Figure 5a). Mitochondrial biogenesis was examined by measuring peroxisome proliferator-activated receptor gamma coactivator 1 alpha (Ppargc1a) relative gene expression. It was lower in the FLOT 1X group than in the control and FLOT 0.1X groups (Figure 5b). However, similar PPARGC1A protein levels were observed between the groups (Figure 5c).
Figure 5.
Effect of FLOT chemotherapy treatment on mitochondrial biogenesis, density and dynamics in mature 3T3-L1. Mature 3T3-L1 cells were treated for 48 h with different FLOT treatment concentrations. Four independent experiments were performed with 2 to 3 technical replicates (n = 11–15). Mitochondrial density was evaluated using the mitochondrial (mt-nd6) DNA per nuclear (Ndufb6) DNA ratio (a). Protein accumulation of PPARGC1A (c) was normalized by ‘Ponceau’ red staining and presented with a representative image of the blot. Gene expressions of Ppargc1a (b), Dnm1l (d), and Mfn1 (e) were normalized by Rplp0 gene expression. The results are presented as the mean ± S.E.M. and each individual replicate is represented as a white dot. Kruskal-Wallis test and Dunn test post-hoc, ***p < 0.05 between groups.
AU: arbitrary unit; ctrl: control; Dnm1l: dynamin-related protein 1; FLOT: 5-fluorouracil, leucovorin, oxaliplatin and docetaxel; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; Mfn1: mitofusin 1; Mt-nd6: mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 6; Ndufb6: NADH:ubiquinone oxidoreductase subunit B6; Ppargc1a: peroxisome proliferator-activated receptor gamma coactivator 1-alpha; Rplp0: ribosomal protein lateral stalk subunit P0.
The mitochondrial fusion and fission pathways were also explored. Relative gene expression of dynamin 1 like (Dnm1l) (i.e. fission, Figure 5d) and mitofusin 1 (Mfn1) (i.e. fusion, Figure 5e) were both significantly decreased with FLOT 1X compared to both the control and FLOT 0.1X conditions.
Mitochondrial activity was evaluated by measuring oxygen (O2) consumption using high-resolution respirometry. Mitochondrial respiration was not different between the conditions for all respiratory states measured (Figure 6).
Figure 6.
Effect of FLOT chemotherapy on mitochondrial respiration in mature 3T3-L1. Measurements were performed in presence of glutamate (G), malate (M), ADP (D), octanoyl-carnitine (O), and succinate (S) in mature 3T3-L1 after a 48 h treatment with FLOT 1X. Three independent experiments were performed with 1 to 3 technical replicates (n = 5 to 7). The results are presented as the mean ± S.E.M., and each individual replicate is represented as a white dot. Differences between the treated and control groups were assessed using the t-test, NS.
ADP: adenosine diphosphate; ce: cells; ctrl: control; FLOT: 5-fluorouracil, leucovorin, oxaliplatin and docetaxel.
1X FLOT chemotherapy treatment increased antioxidant defences in mature 3T3-L1
Hydrogen peroxide (H2O2) flux was measured (Figure 7a) with the OROBOROS O2k-Fluorometer along with mitochondrial respiration (Figure 7b), allowing calculation of the free radical leak (FRL) (Figure 7c). None of these parameters were significantly different following FLOT 1X treatment compared with the control (Figure 7a,b,c). Mitochondrial antioxidant defences were then determined by evaluating nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) and superoxide dismutase 2 (SOD2) protein levels (representative blots in Figure 8a), and Sod2, Nfe2l2 and catalase (Cat) relative gene expression. SOD2 protein levels did not differ between the groups (Figure 8b), but Sod2 relative expression was higher in the FLOT 0.1X group than in the control and FLOT 1X groups (Figure 8c), whereas Cat relative expression was higher in the FLOT 1X group than in the other groups (Figure 8d). NFE2L2 protein levels were higher with both concentrations of FLOT than with the control (Figure 8e), but Nfe2L2 relative gene expression was not different between the groups (Figure 8f).
Figure 7.
Effect of FLOT chemotherapy on mitochondrial ROS production and FRL in mature 3T3-L1. Measurements were performed in the presence of glutamate (G), malate (M), succinate (S), rotenone (Rot), ADP (D), and antimycin a (Ama). Three independent experiments were performed with 2 to 3 technical replicates (n = 6–7). Both H2O2 (a) and oxygen (b) fluxes are required for the free radical leak (FRL) calculation (c). The formula used is the following: H2O2 fluxes divided by twice O2 consumption with the result multiplied by 100. The results are presented as the mean ± S.E.M., and each individual replicate is represented as a white dot. Differences between the treated and control groups were assessed using the Wilcoxon rank-sum exact test, *p < 0.05.
ADP: adenosine diphosphate; ce: cells; ctrl: control; FLOT: 5-fluorouracil, leucovorin, oxaliplatin and docetaxel; FRL: Free Radical Leak; H2O2: hydrogen peroxide.
Figure 8.
Effect of FLOT chemotherapy treatment on mitochondrial antioxidant defense in mature 3T3-L1. Mature 3T3-L1 cells were treated for 48 h with different FLOT treatment concentrations. Four (B, E) to five (C, D, F) independent experiments were performed with 2 to 3 technical replicates (n = 11–15). (a) representative images of the blots for both SOD2 and NFE2L2. Protein levels of SOD2 (b) and NFE2L2 (e) were normalized by ‘Ponceau’ red staining. Gene expression of Sod2 (c), cat (d) and Nfe2l2 (f) were normalized by Rplp0 gene expression. Results are presented as the mean ± S.E.M., and each individual replicate is represented as a white dot. Kruskal-Wallis test and Dunn test post-hoc, ***p < 0.05 between groups.
AU: arbitrary unit; Cat: catalase; ctrl: control; FLOT: 5-fluorouracil, leucovorin, oxaliplatin and docetaxel; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; NFE2L2: nuclear factor (erythroid-derived 2)-like 2; SOD2: superoxide dismutase 2; Rplp0: ribosomal protein lateral stalk subunit P0.
1X FLOT chemotherapy increased expression of inflammation markers in mature 3T3-L1
As antioxidant defences increased with the treatment, cell death was first examined using caspase 3 (CASP3), an apoptosis marker. Representative blots of CASP3 protein levels are presented in Figure 9a. Protein levels remained unchanged between the conditions (Figure 9b), and no cleaved CASP3 was observed. Nevertheless, its relative gene expression was significantly lower with the 1X concentration compared to both control and FLOT 0.1X (Figure 9c). As CASP3 May be activated by interleukin 6 (IL6), a pro-inflammatory cytokine, its relative gene expression was measured (Figure 9d). The latter was significantly increased with FLOT 1X treatment compared with the control. Relative gene expression of the necrosis pathway gene toll like receptor 4 (Tlr4), which is also activated by inflammation, was significantly decreased in the FLOT 1X group but increased (p = 0.082) in the FLOT 0.1X group compared to the control (Figure 9e). Reactive oxygen species (ROS)-activated phosphorylation of mitogen-activated protein kinase (MAPK14/MAPK1) was unchanged regardless of the treatment (Figure 9f).
Figure 9.
Effect of FLOT chemotherapy treatment on inflammation and cell death in mature 3T3-L1. Mature 3T3-L1 cells were treated for 48 h with different FLOT treatment concentrations. Four (B, f) to five (C, D, E) independent experiments were performed with 2 to 3 technical replicates (n = 7–15). (a) representative images of the blots for CASP3 and total MAPK1/MAPK14. Protein levels of CASP3 (b) and MAPK1/MAPK14 (f) was normalized by ‘Ponceau’ red staining. Gene expressions of Casp3 (c), Il6 (d) and Tlr4 (e) were normalized by Rplp0 gene expression. The results are presented as the mean ± S.E.M., and each individual replicate is represented as a white dot. Kruskal-Wallis test and Dunn test post-hoc, ***p < 0.05 between groups.
AU: arbitrary unit; CASP3: caspase 3; ctrl: control; FLOT: 5-fluorouracil, leucovorin, oxaliplatin and docetaxel; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; Il6: interleukin 6; MAPK: mitogen activated protein kinase; Rplp0: ribosomal protein lateral stalk subunit P0; Tlr4: toll like receptor 4.
Discussion
This in vitro study is the first to evaluate the effect of FLOT chemotherapy on mature murine adipocytes. The treatment decreased mature adipocyte viability and gene expression of markers involved in mitochondrial dynamics and lipogenesis. Moreover, FLOT treatment increases cellular stress and inflammation.
FLOT treatment for 48 h significantly reduced the viability of mature 3T3-L1 cells only at a 1X concentration. To date, this cocktail has not been tested in this cell type. Previous studies on cisplatin and doxorubicin have shown severe mitochondrial dysfunction in adipocytes. Doxorubicin is a chemotherapeutic agent that is known to have many adverse effects. It induced mature 3T3-L1 cell death after 96 h of incubation, showing only about 15% viable cells at the highest concentrations (i.e. 1 µM) [10]. In contrast, a 3 h-treatment of undifferentiated 3T3-L1 cells, with adriamycin (300 nM) showed no toxicity but impaired differentiation [14]. Our study differs from previous work in both the type of chemotherapeutic agents used and their concentrations. Specifically, we tested a combination regimen containing oxaliplatin at 25.2 µM (FLOT 1X), a substantially higher than reported in earlier studies. Despite this, only mild toxicity was observed, with approximately 80% of the cells remaining metabolically active. The closest studies on chemotherapy molecules were in vitro studies using adipose-derived mesenchymal stem cells. After 48 h of incubation with 10 µg·mL−1 (33 µM) cisplatin, proliferation was inhibited [22]. Another study on adipose-derived stem cells showed a decreased viability with 5-FU at 0.1 mg·mL−1 (0.77 mm) after a 48 h-treatment [23]. As concentrations, molecules, and incubation times were different between all the previous studies, it is difficult to conclude even if most of the studies showed a decreased viability of the cells after incubation with chemotherapeutic molecules. Further research is needed to determine whether specific components of FLOT (e.g., oxaliplatin, 5-FU) drive distinct mitochondrial stress responses compared to single-agent chemotherapy treatments.
FLOT 1X 48 h-treatment decreased lipid metabolism in mature 3T3-L1 cells, as evaluated by Fasn, Dgat1, Dgat2 and Cd36 relative gene expression and PLIN1 protein levels. Surprisingly, FLOT 0.1X increased Dgat2 and Cd36 relative gene expression compared to than in both the control and FLOT 1X groups. Lower concentrations may induce differential adaptive mechanisms compared to higher concentrations. In mouse adipocytes, chemotherapy has been shown to decrease total AT weight after two cycles of combined irinotecan and 5-FU chemotherapy [24]. Furthermore, Dgat2 and Fasn gene expressions were decreased in mice after treatment, which is concordant with the observations of the present study. Inhibition of Dgat1 and Dgat2 has been shown to decrease lipid droplet accumulation [25]. These results are concordant with the decreased lipid accumulation observed with FLOT 1X and the decrease in fat mass observed following platin-based chemotherapy in mice and humans [4,26]. In addition to the decrease in lipogenesis, lipolysis was downregulated as well. Indeed, PLIN1 protein levels were lower after treatment, but not PNPLA2 protein levels. Lipolytic activity is known to be induced by cancer, and induces cancer cachexia via AT depletion [9,27]. Chemotherapy-induced inhibition may be protective against AT loss and cachexia. Platin-based chemotherapy has also been shown to induce cachexia [4]. Indeed, it has been shown that inhibition of the lipolysis pathway with a chemical agent in tumour-bearing mice attenuates cancer cachexia [28]. The observed reduction in lipid accumulation following FLOT treatment suggests significant dysregulation of adipocyte lipid metabolism. This metabolic disruption may stem from multiple mechanisms: (1) impaired mitochondrial function leading to compromised adenosine tri-phosphate (ATP) production and consequent metabolic stress, and (2) transcriptional reprogramming evidenced by downregulation of key lipid metabolism regulators (Pparg, Srebf1, Cd36). Further investigation should focus on upstream pathways such as Prkaa2 and Mtor to elucidate the precise mechanisms governing FLOT-induced lipid dysregulation. However, one possible explanation is that FLOT-induced lipid metabolism impairment may be partially compensated by mitochondrial adaptations, such as increased fatty acid oxidation or alternative metabolic pathways.
A 48 h-FLOT treatment increased mitochondrial density, but Ppargc1a gene expression which is involved in the mitochondrial biogenesis pathway, was decreased. In healthy mice treated with cisplatin, twice, for 4 days, white AT Ppargc1a gene expression was not different from the control group [29]. However, PPARGC1A protein levels were increased after cisplatin treatment. Conversely, in another pre-clinical model, mice bearing C26-colon tumours treated with oxaliplatin and 5-FU, showed decreased muscle PPARGC1A protein levels, but not gene expression, compared to the control group [30]. Further investigations are required to determine the effects of platin-based treatments on mitochondrial biogenesis in adipocytes. Mitochondrial dynamics have also been studied, as they regulate mitochondrial density. Both fusion and fission with the gene expression of Mfn1 and Dnm1l respectively, decreased after 48 h chemotherapy FLOT treatment. In a cancer cell line, 20 nM docetaxel alone induced mitochondrial fission measured by fluorescence microscopy [31]. Since FLOT is a combination of docetaxel with other molecules, and the concentration used was higher (30.9 µM) but with the same incubation time (48 h), this may explain why the present results are not concordant with the literature.
In addition to the alteration of mitochondrial dynamics, mitochondrial antioxidant defences increased in adipocytes after treatment. However, directly measured ROS production did not increase. Oxaliplatin has been shown to increase ROS production in peripheral nerve axons [32]. As mitochondria are the major contributors to ROS production, especially through complexes I and III of the respiratory chain [33], an increase in oxidative stress may be due to an alteration in the mitochondrial respiratory chain without an effect on mitochondrial respiration. The measurement of ATP production may provide a better understanding of this dysfunction. Nfe2l2 has been shown to increase mitochondrial antioxidant responses mediated by Sod2 and Cat [34] which increased after FLOT treatment. In colorectal cancer cells, oxaliplatin alone reduced the Nfe2l2 signalling pathway, leading to an increase in ROS production [35]. The effects of oxaliplatin appear to be inconsistent between cancerous and non-cancerous cells. A mouse model of cachexia showed fat depletion after a combination of 5-FU, leucovorin, and oxaliplatin (i.e. FOLFOX), a combination close to the FLOT one, for five consecutive weeks [26]. This could be linked to the present in vitro model with a decrease in cell viability and the cachectic effect observed in clinical practice such as adipose wasting [4]. Interestingly, while FLOT treatment significantly reduced cell viability, it did not lead to a marked increase in cleaved caspase levels, suggesting that apoptosis may not be the primary mechanism of cell death under these conditions. This apparent discrepancy could be explained by the involvement of caspase-independent pathways, such as necroptosis, autophagy-associated cell death, or other non-canonical forms of cell death. Further investigations will be required to delineate the exact mechanisms by which FLOT exerts its cytotoxic effects. However, our findings show a strong correlation between FLOT treatment and metabolic dysfunction, additional functional validation is needed to confirm whether these effects are directly caused by mitochondrial impairment or are secondary to broader transcriptional changes.
Limitations and future directions
This study provides novel insights into the metabolic effects of FLOT chemotherapy on adipocytes. However, it is essential to recognize the limitations of in vitro models, which do not account for systemic influences such as inflammatory cytokines, hormonal fluctuations, and interactions with other tissues that occur in vivo. Furthermore, adipose tissue remodelling in cancer patients may result from chronic metabolic stress rather than the acute 48-hour chemotherapy exposure used in this study.
Given these considerations, future research should incorporate longer-term adipocyte culture models, co-culture systems with immune cells, or in vivo murine models to better assess whether chemotherapy-induced changes in lipid metabolism persist and contribute to the progression of cachexia. While this is the first study to investigate the effects of FLOT on in vitro mature adipocytes, it is important to note that the model was designed to mimic clinical treatment in terms of molecular ratio, but it was delivered over a 48-hour period, unlike the cyclical chemotherapy approach typically used in clinical practice.
To more closely simulate the clinical scenario, future studies should explore: (1) extended exposure durations to model cumulative effects, and (2) post-treatment recovery periods to identify any persistent metabolic adaptations. Despite the short duration of exposure, the use of varying concentrations allowed us to study the effects at doses that did not compromise cell viability, yet still altered key parameters, such as lipid metabolism.
Future studies should include lipidomic profiling to investigate whether FLOT affects fatty acid oxidation and triglyceride synthesis, as well as pharmacological rescue experiments using mitochondrial stabilizers (e.g., resveratrol or coenzyme Q10) to assess whether reversing mitochondrial dysfunction can mitigate lipid metabolism impairment. These approaches would provide a more mechanistic understanding of how chemotherapy disrupts adipocyte function and help identify potential therapeutic targets for alleviating cachexia-related adipose tissue wasting. In contrast to previous studies on cisplatin and doxorubicin, where mitochondrial respiration was severely impaired due to electron transport chain disruption and excessive ROS production, the metabolic effects of FLOT appear to be more moderately preserved, with mitochondrial respiration showing partial resistance. This suggests that while FLOT impacts adipocyte metabolism, its mitochondrial toxicity may differ from that of individual chemotherapeutic agents. Further comparative studies are required to determine whether specific components of the FLOT regimen drive this differential response or if a compensatory metabolic adaptation occurs in adipocytes after treatment.
A dose effect was not observed, and even an opposite effect of different concentrations has been shown for some parameters. The lower concentration (FLOT 0.1X), as it was not toxic, may allow adaptation mechanisms that were not possible at the higher concentration (FLOT 1X). The 3T3-L1 cell line is a murine immortalized cell line that does not reflect AT complexity in vivo because it is a tissue not exclusively composed of mature adipocytes. In this study, we focused on the effect on mature adipocytes; however, chemotherapy may also have effects during differentiation.
The present study is the first to explore the effects of FLOT on healthy mature adipocytes. The results demonstrated that the treatment decreased cell viability and impaired lipid metabolism, which is in line with what has been observed in chemotherapy-induced cachexia in clinical settings. These results support the hypothesis that AT plays an important role in the tumour microenvironment and metabolic response to cancer. Moreover, through cytokine secretion and regulation of metabolic enzymes, adipocytes exhibited high inflammatory and cellular stress levels after FLOT treatment, which may have an impact on cancer cell proliferation, metastasis, and chemoresistance, as it has been reported for other chemotherapeutic treatments. Further studies are needed to evaluate the effects of FLOT chemotherapy on adipocytes, especially in humans.
The findings of this study highlight the clinical relevance of managing chemotherapy-induced cachexia, particularly in the context of FLOT chemotherapy, which disrupts lipid metabolism and mitochondrial function in adipocytes. Preserving adipose tissue function may slow cachexia progression, with potential therapies including mitochondria-targeted antioxidants (e.g., Coenzyme Q10, resveratrol), PPARG agonists to restore lipid metabolism, and metabolic modulators like metformin to improve mitochondrial efficiency. Future clinical trials should assess whether these interventions reduce fat loss and improve treatment tolerance in chemotherapy patients. Future research should also examine FLOT chemotherapy in vivo to assess the impact of systemic inflammation, hormonal changes, and tumour burden on adipose dysfunction. Ultimately, targeted metabolic interventions could help mitigate chemotherapy-induced adipose dysfunction and cachexia, contributing to improved cancer treatment outcomes.
Material and methods
3T3-L1 cell culture and treatments
3T3-L1 cells from ATCC (LGC Standards, Molsheim, France) were cultured in 12-well plates, as described by Pinel et al. [36]. Briefly, cells were cultured up to confluence (day 0). To initiate differentiation 0.5 mm 3-isobutyl-1-methylxanthine (IBMX), 1 µM dexamethasone and 10 µg/mL insulin were added to the medium. After 48 h (day 2), cells were maintained in the medium with insulin only to maintain differentiation. Insulin was removed at day 4 and cells were maintained until day 8. On day 8, cells were incubated for 48 h with a chemotherapy cocktail of four molecules (FLOT): 5-FU, Oxaliplatin and Docetaxel in the following ratio (52:1.7:1) and excess leucovorin, which is representative of the ratio used in humans. This mixture was prepared in a solution of 5% glucose and 10% (v/v) ethanol to improve the solubilization of the molecules. The cocktail was diluted 1:50 (1X), 1:500 (0.1X) and 1:5000 (0.01X) in proliferation medium. The 1X final concentrations were 1.9 mm 5-Fluorouracil (Sigma-Aldrich® St. Louis, MO, USA), 210.0 µM Leucovorin (Merck Millipore®, Burlington, VT, USA), 25.2 µM Oxaliplatin (Sigma-Aldrich® St. Louis, MO, USA) and 30.9 µM Docetaxel (Sigma-Aldrich® St. Louis, MO, USA). The solution used to solubilize the cocktail was used as a control and was diluted 1:50 in the proliferation medium. For all the experiments, media changes were standardized to every 48 hours and experiments were conducted at the same time of day to account for circadian metabolic variations. The 48-hour treatment duration was selected based on previous studies examining chemotherapy-induced cell death and pilot experiments [11,12]. Representative pictures of cultured cells before and after differentiation are presented in Figure 10.
Figure 10.
Differentiation of 3T3-L1 pre-adipocytes in mature adipocytes Pre-adipocytes at day 0 (a) and mature adipocytes with lipid droplets at day 8 of differentiation (b).
Cell viability
After 48 h FLOT treatment, the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to measure cellular metabolic activity, an indicator of cell viability and proliferation. Briefly, cells were incubated with a 0.5 mg·mL−1 solution of MTT (Sigma-Aldrich® St. Louis, MO, USA) diluted in the proliferation medium for 2 h at 37°C. The tetrazolium crystals were dissolved in dimethyl sulphoxide (DMSO), and the optical density (OD) was measured at 570 nm according to the manufacturer’s instructions on a plate reader (Epoch, BioTek, Colmar, France). Cells were treated with 8.8 mm H2O2, as a positive control for toxicity, under the same conditions as the treated cells.
Oil red O lipid staining
ORO staining (Sigma-Aldrich® St. Louis, MO, USA) was used to stain and detect neutral lipids in the lipid droplets and quantify lipid accumulation. After FLOT treatment, medium was carefully removed and cells were fixed in 10% formalin (Sigma-Aldrich® St. Louis, MO, USA) for 1 h and stained for 10 min with a 0.73 M ORO solution (Sigma-Aldrich® St. Louis, MO, USA) diluted in 60% isopropanol (Sigma-Aldrich® St. Louis, MO, USA) in water. The medium was discarded, and the cells were washed with water until the total excess stain was eliminated. Then a 10 min incubation with 100% isopropanol was performed to dissolve the stain lipid droplets. After homogenization, the absorbance was measured at 500 nm using a plate reader (Epoch, BioTek, Colmar, France). Blank was assessed using 100% isopropanol. To overcome the variability between plates, the results were expressed as a percentage of the control for each plate.
Mitochondrial respiration
To measure mitochondrial oxidative phosphorylation respiration, high-resolution respirometry was performed using an Oxygraph-2k (Oroboros Instruments, Innsbruck, Austria). Briefly, cells were cultured as previously described (3T3-L1 cell culture and treatments section) on a 100 mm petri dish. After treatment, the cells were harvested with a non-enzymatic dissociation solution (Sigma-Aldrich® St. Louis, MO, USA). The cells were then centrifuged at 350 g at room temperature (RT) for 5 min. The resulting pellet was resuspended in 500 µL of respiration medium (MIR05) at 37°C. Thirty-five microlitres of the cell suspension were injected into the respirometric chambers, where measurements were performed in duplicate. All measurements were conducted at oxygen concentrations above 110 mmol·mL−1 at 37°C. After performing respirometric testing of the optimal digitonin concentration, it appeared that the dissociation agent sufficiently permeabilized the cells [37]. Table 1 reports the reagents and their concentrations in the chamber after addition as part of the substrate-uncoupler-inhibitor titration (SUIT) protocol. The detailed protocol is illustrated in Figure 11a. Data were recorded and analysed using DatLab software (v7.4.0.4, Oroboros Instruments, Innsbruck, Austria). Results were normalized to protein concentration, which was determined using a Rapid Gold BCA kit (Thermo Fischer ScientificTM, Waltham, MA, USA) according to the manufacturer’s instructions.
Table 1.
Solutions used for respirometry and fluorimetry measurements.
| Solution | [chamber] | Solvent |
|---|---|---|
| Glutamate | 10 mm | H2O |
| Malate | 2 mm | H2O |
| ADP | 5 mm | H2O |
| Octanoyl-carnitine | 1.5 mm | DMSO |
| Cytochrome C | 0.01 mm | H2O |
| Succinate | 10 mm | H2O |
| Rotenone | 0.5 µM | EtOH |
| Antimycin A | 2.5 µM | EtOH |
| SOD2 | 5 U/mL | H2O |
| HRP | 1 U/mL | MIR05 |
| AmplexRed | 0.01 mm | DMSO |
| H2O2 | 0.0001 mm | HCl |
ADP: adenosine diphosphate; DMSO: dimethylsulfoxide; EtOH; ethanol; H2O: ultrapure water; H202: hydrogen peroxide; HCl: chlorohydric acid; HRP: horseradish peroxidase; MIR05: respiration medium; SOD2: superoxide dismutase 2.
Figure 11.
SUIT protocol for respirometry and fluorimetry analysis. The SUIT protocol is designed to explore mitochondrial respiration globally.The cells were permeabilized following detachment from the culture vials. After cell addition (ce), glutamate (G) and malate (M) were added to measure the CI-linked LEAK respiration. Both serve as donors of NADH,H+, a substrate of the respiratory chain complex I.A: To obtain the OXPHOS respiratory state, ADP (D) was added and converted to ATP, by ATP synthase. OXPHOS status was also measured in the presence of an additional substrate, octanoyl-carnitine (O), which is a carnitine-dependent β-oxidation substrate. To obtain maximal coupled oxidative capacity, succinate (S), a FADH2-based substrate was added to activate complex II. Finally, cytochrome c (c) was used to evaluate mitochondrial membrane integrity. B: This protocol allows for the evaluation of mitochondrial ROS production by converting the superoxide anion produced in H2O2 in the presence of SOD2. To ensure that every superoxide anion was converted to H2O2, SOD2 was added at the beginning of the experiment. The AmplexRed reaction with H2O2 is catalysed by HRP and produces the stable fluorescent compound; resorufin. After cell addition (ce), glutamate (G) and malate (M) were added to measure the CI-linked LEAK respiration and H2O2 production. Succinate (S) was added to measure the CI-CII-linked LEAK respiration, also called non-phosphorylating resting state. It also increases the membrane potential, provides redox power in the Q-junction, and is responsible for ROS production. The addition of rotenone (Rot) inhibits complex I, which blocks backward electron flow and allows measurement of the succinate pathway control state. ADP (D) was added to initiate OXPHOS. Antimycin A (Ama) was added to inhibit complex III, induce residual oxygen consumption, and also increase ROS production.
ADP: adenosine diphosphate; ATP: adenosine triphosphate; FADH2: flavin adenine dinucleotide; H2O2: hydrogen peroxide; HRP: Horseradish peroxidase; NADH,H+: nicotinamide adenine dinucleotide; OXPHOS: oxidative phosphorylation; ROS: reactive oxygen species; SOD2: superoxide dismutase 2; SUIT: substrate uncoupler inhibition titration.
H2O2 fluxes measurement
Simultaneous measurements of H2O2 flux and mitochondrial respiration were performed using an Oxygraph-2k. An O2k-Fluo LED2-Module equipped with Fluo-Sensors Green (Oroboros Instruments; excitation 525 nm, emission ~600 nm; Oroboros Instruments, Innsbruck, Austria) was plugged into the device for H2O2 detection using the fluorophore Aldrich® UltraRed. Sample preparation was similar to the high-resolution respirometry experiment. Thirty-five microlitres of the cell suspension were injected into the respirometric chambers in duplicate. Table 1 reports the reagents and their final concentrations in the chamber as part of the SUIT protocol, which is detailed in Figure 11b. All experiments were conducted at 37 °C under continuous agitation (750 rpm) and hyperoxic conditions (240 µM > [O2] > 450 µM).
This protocol allows the calculation of the fraction of electrons that reduce O2 to free radicals rather than reaching complex IV to be reduced to water. Because two electrons are needed to reduce one mole of O2 to H2O2 whereas four electrons are needed to reduce one mole of O2 to water, the percent FRL was calculated as the rate of free radical production divided by twice the rate of O2 consumption, with the result multiplied by 100 [33,38].
RNA isolation, reverse transcription and qPCR
To co-extract DNA and RNA, cells were harvested in 1 mL TRIzol reagent (Thermo Fischer ScientificTM, Waltham, MA). Cells were vortexed in order to complete cell lysis before addition of 200 µL of chloroform isoamyl alcohol (Sigma-Aldrich® St. Louis, MO). After a 2 min incubation at RT, samples were centrifuged (16 000 g, 15 min, 4°C). The red layer contained DNA and the translucent layer contained RNA.
The RNA phase was transferred to a tube containing 500 µL cold isopropanol. Samples were incubated for 30 min at 4°C and centrifuged (16 000 g, 15 min, 4°C). RNA pellets were washed twice with 1 mL ethanol. After drying, the pellet was incubated 10 min at 60°C with 20 µL of ultrapure and sterile water.
The DNA fraction was added with 500 µL back extraction buffer (guanidine thiocyanate 4 M; sodium citrate 50 mm and Tris base 1 M) and shaken (270 rpm, 10 min, RT). Samples were then centrifuged (16 000 g, 30 min, 4°C). The upper fraction was collected and added to 400 µL isopropanol at 4°C. The mixture was incubated (30 min, 4°C) and centrifuged (16 000 g, 15 min, 4°C). After drying, the pellet was incubated (10 min, 60°C) with 20 µL of ultrapure sterile water.
RNA and DNA concentrations and purity were assessed by spectrophotometry using OD at 260 and 280 nm (NanoDropLite; Thermo Fischer ScientificTM, Waltham, MA, USA). Quality was judged good if the OD260-to-OD280 ratio was between 1.8 and 2.
Mitochondrial density was quantified using the mtDNA (mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1, Mt-nd6)-to-nDNA (NADH:ubiquinone oxidoreductase subunit B6, Ndufb6) ratio. PCR was performed from 0.1 ng of DNA with ONEGreen Fast qPCR Premix (Ozyme), following the manufacturer’s instructions. The primers used are listed in Table 2. Data analysis was performed using methods described by Quiros et al. [39].
Table 2.
Primers used for qPCR.
| Gene | Gene Accession N° | Sequence (5’ − 3’) |
|---|---|---|
| Casp3 (Forward) | ENSMUSG00000031628 | GAG GCT GAC TTC CTG TAT GCT |
| Casp3 (Reverse) | AAC CAC GAC CCG TCC TTT | |
| Cat (Forward) | ENSMUSG00000027187 | CCT TCA AGT TGG TTA ATG CAG A |
| Cat (Reverse) | CAA GTT TTT GAT GCC CTG GT | |
| Cd36 (Forward) | ENSMUSG00000002944 | TTG TAC CTA TAC TGT GGC TAA ATG AGA |
| Cd36 (Reverse) | CTT GTG TTT TGA ACA TTT CTG CTT | |
| Cpt1a (Forward) | ENSMUSG00000024900 | GAT GAC GGC TAT GGT GTT TCC TAC |
| Cpt1a (Reverse) | TCC CAA AGC GGT GTG AGT CTG | |
| Dgat1 (Forward) | ENSMUSG00000022555 | TTC CGC CTC TGG GCA TT |
| Dgat1 (Reverse) | AGA ATC GGC CCA CAA TCC A | |
| Dgat2 (Forward) | ENSMUSG00000030747 | ACT CTG GAG GTT GGC ACC AT |
| Dgat2 (Reverse) | GGG TGT GGC TCA GGA GGA T | |
| Dnm1l (Forward) | ENSMUSG00000022789 | TGC CTC AGA TCG TCG TAG TG |
| Dnm1l (Reverse) | TGA CCA CAC CAG TTC CTC TG | |
| Fasn (Forward) | ENSMUSG00000025153 | GAC ACT GCT GCG TGC CAA G |
| Fasn (Reverse) | CAC AGA CAC CTT CCC GTC AC | |
| Il6 (Forward) | ENSMUSG00000025746 | GCT ACC AAA CTG GAT ATA ATC |
| Il6 (Reverse) | CCA GGT AGC TAT GGT ACT CCA | |
| Mfn1 (Forward) | ENSMUSG00000027668 | TTG CCA CAA GCT GTG TTC GG |
| Mfn1 (Reverse) | TCT AGG GAC CTG AAA GAT GGG C | |
| Mt-nd6 (Forward) | ENSMUSG00000064368 | TAC CCG CAA ACA AAG ATC ACC CAG C |
| Mt-nd6 (Reverse) | AGG AGG GAT TGG GGT AGC GGC | |
| Ndufb6 (Forward) | ENSMUSG00000071014 | TGG AGC GAT TCT GGG ATA AC |
| Ndufb6 (Reverse) | GCC TTG ATG GAA CTG AGA GG | |
| Nfe2l2 (Forward) | ENSMUSG00000015839 | GGG GAA CAG AAC AGG AAA CA |
| Nfe2l2 (Reverse) | CCG TAA TGC ACG GCT AAG TT | |
| Ppargc1a (Forward) | ENSMUSG00000029167 | GAA GTG GTG TAG CGA CCA ATC |
| Ppargc1a (Reverse) | AAT GAG GGC AAT CCG TCT TCA | |
| Rplp0 (Forward) | ENSMUSG00000067274 | ACT GGT CTA GGA CCC GAG AAG |
| Rplp0 (Reverse) | TCC CAC CTT GTC TCC AGT CT | |
| Sod2 (Forward) | ENSMUSG00000006818 | GAC CCA TTG CAA GGA ACA A |
| Sod2 (Reverse) | GTA GTA AGC GTG CTC CCA CAC | |
| Tlr4 (Forward) | ENSMUSG00000039005 | CAA GAA CAT AGA TCT GAG CTT CAA CCC |
| Tlr4 (Reverse) | GCT GTC CAA TAG GGA AGC TTT CTA GAG |
Casp3: caspase 3; Cat: catalase; Cd36: cluster of differentiation 36; Cpt1a: carnitine palmitoyltransferase 1 alpha; Dgat: diacylglycerol o-acyltransferase; Dnm1l: dynamin 1 like; Fasn: fatty acid synthase; Il6: interleukin-6; Mfn1: mitofusin 1; Mt-nd6: mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 6; Ndufb6: NADH:ubiquinone oxidoreductase subunit B6; Nfe2l2: nuclear factor (erythroid-derived 2)-like 2; Ppargc1a: peroxisome proliferator-activated receptor gamma coactivator 1α; Rplp0: ribosomal protein lateral stalk subunit P0; Sod2: superoxide dismutase 2; Tlr4: toll like receptor 4.
To quantify Fasn, Dgat1, Dgat2, Cpt1a, Cd36, Ppargc1a, Dnml1, Mfn1, Il6, Sod2, Nfe2l2, Casp3 and Tlr4 gene expression, RNA was reverse-transcribed using the Applied Biosystem kit (Thermo Fischer ScientificTM, Waltham, MA, USA). Briefly, 2 µg of RNA were retro-transcribed according to the manufacturer’s instructions. Complementary DNA (cDNA) was diluted 1:10 and used as a template for Real-time PCR performed using ONEGreen Fast qPCR Premix (Ozyme, Saint-Cyr-l’Ecole, France), according to the manufacturer’s instructions. The ΔΔCt method was used for data analysis as described by Livak et al. [40]. Ribosomal protein lateral stalk subunit P0 (Rplp0) was used as a housekeeping gene and the mean of the controls for normalization. The results are expressed as relative gene expression. The primers used are listed in Table 2.
Western blot
Chemicals were purchased from Sigma-Aldrich® (St. Louis, MO, USA), unless otherwise indicated.
3T3-L1 adipocytes were harvested in lysis buffer (HEPES 50 mm; NaCl 150 mm (Merck Millipore®, Burlington, VT, USA); 10 mm EDTA; 10 mm NaPPi; 25 mm β-glycero-phosphate; 100 mm NaF; glycerol 10%; 2 mm sodium orthovanadate; proteases inhibitor cocktail 0.50%.). After homogenization, samples were centrifuged (12 000 g, 15 min, 4°C). Protein quantification was performed using the Rapid Gold BCA kit (Thermo Fischer ScientificTM, Waltham, MA, USA) according to the manufacturer’s instructions.
Briefly, 15 µg of protein were loaded onto 10% acrylamide gels with loading buffer. A molecular ladder (OZYC006, Ozyme, Saint-Cyr-l’Ecole, France) was also added onto each gel. Proteins bands were transferred onto 0.2 µm PVDF membranes and stained with Ponceau Red solution. The membranes were then incubated in 5% fat-free dry milk in Tris Buffer Saline (TBST; (Euromedex, Souffelweyersheim, France) and 0.1% Tween 20 (Euromedex, Souffelweyersheim, France)) for 1 h. The membranes were then incubated overnight at 4°C with the primary antibodies listed in Table 3. Secondary antibodies were used based on the primary antibody source, i.e. anti-rabbit (Thermo Fischer ScientificTM, Waltham, MA, USA) and anti-mouse (Thermo Fischer ScientificTM, Waltham, MA, USA), and incubated for 1 h at RT. Chemiluminescence acquisition was performed using Fusion Solo S (Vilber Lourmat, Le Mans, France) after incubation with HRP substrates (Immobilion®, Western HRP substrate; Merck Millipore®, Burlington, VT, USA). Protein bands were quantified by densitometry using MultiGauge V3.2 software (Fujifilm, Saint Quentin en Yvelines, France). The relative fold-change was calculated using membrane images obtained after Ponceau Red staining for normalization.
Table 3.
Primary antibodies used for Western blot.
| Protein | Dilution | Reference |
|---|---|---|
| CASP3 | 1:1000 | Cell Signaling Technology #9662 |
| GAPDH | 1:5000 | Sigma-Aldrich #G9545 |
| MAPK1 | 1:1000 | Sigma-Aldrich #M0800 |
| MAPK14 | 1:500 | Sigma-Aldrich #M8177 |
| NFE2L2 | 1:1000 | GeneTex #103322 |
| PPARGC1A | 1:500 | Abcam #ab191838 |
| PLIN1 | 1:1000 | Cell Signaling Technology #D1D8 |
| PNPLA2 | 1:1000 | Abcam #207799 |
| SOD2 | 1:1000 | Abcam #ab137037 |
CASP3: caspase 3; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MAPK: mitogen-activated protein kinase; NFE2L2: nuclear factor (erythroid-derived 2)-like 2; PPARGC1A: peroxisome proliferator-activated receptor gamma coactivator 1 alpha; PLIN1: perilipin 1; PNPLA2: patatin like phospholipase domain containing 2; SOD2: superoxide dismutase 2.
Statistical analysis
All data are presented as mean ± standard error of the mean (S.E.M.). For statistical analyses, the normality of the distribution was checked using the Shapiro-Wilk test. The assumption of homoscedasticity was tested using the Bartlett’s test. Comparisons between treatment conditions were performed using analysis of variance (ANOVA) or a non-parametric Kruskal-Wallis test when the assumptions of ANOVA were not met. When appropriate (omnibus p-value less than 0.05), a post-hoc test was applied for pairwise multiple comparisons, i.e. a Tukey-Kramer test after ANOVA and a Dunn test after a Kruskal-Wallis test. To compare only two conditions, a paired t-test or Wilcoxon rank-sum exact test was used. All analyses were performed using R Statistical Software (v4.4.1; R Core Team 2024) and statistical significance was set at p < 0.05.
Acknowledgments
The authors utilized an AI-based language assistance tool (ChatGPT, GPT-4, OpenAI) to enhance the clarity and readability of the English language in this manuscript. The authors are solely responsible for the scientific content and integrity of the work.
All authors have read and approved the final version of the manuscript.
Correction Statement
This article has been republished with minor changes. These changes do not impact the academic content of the article.
Funding Statement
This work was supported by the “Ligue Contre le Cancer région Auvergne-Rhône-Alpes et Saône-et-Loire.”.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Abbreviations
- 3T3-L1
murine preadipocyte cell line
- 5-FU
5-fluorouracil
- AT
adipose tissue
- BIOPS
biopsy preservation solution
- FLOT
5-fluorouracil, leucovorin, oxaliplatin, docetaxel
- MIR05
respiration medium
- ORO
Oil Red O
- SUIT
substrate-uncoupler-inhibition titration
Data availability statement
The data that support the findings of this study are openly available in Mendeley Data at http://doi.org/10.17632/vvtc3s8p6w.1.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that support the findings of this study are openly available in Mendeley Data at http://doi.org/10.17632/vvtc3s8p6w.1.