High-fat diet and obesity each increase tumor cell proliferation and muscle wasting in experimental cancer cachexia.

INTRODUCTION
Cachexia, or unintentional weight loss with metabolic derangement and systemic inflammation, is a frequent and devastating complication of cancer (1). In advanced cancer and cancer of the upper gastrointestinal system, cachexia is marked by the progressive loss of skeletal muscle and adipose tissue. Skeletal muscle loss leads to impaired physical function, reduced quality of life, and poor response to therapy; indeed, cachexia is attributed as the cause of death in more than 30% of patients with cancer (2). Cachexia results from the complex interplay between cancer, inflammation, and metabolism, all processes highly influenced by intrinsic and external biological variables. Therefore, designing effective therapies for cachexia requires an understanding of these variables, including the contributions of body condition and lifestyle in the cancer patient.
The obesity paradox has been observed in diverse diseases, such that overweight and obese patients are both predisposed to serious chronic illness yet experience longer survival times compared to normal weight individuals. To this end, a higher body mass index (BMI) correlates with a reduced chance of death and hospitalization in patients with non-cancer cachectic diseases (3, 4). Pre-existing obesity might exert protection simply by providing additional body mass for catabolism prior to reaching a fatal level of wasting. Thus, perhaps individuals with greater body mass are protected compared with those of smaller mass; indeed, we previously reported that obese patients with lung cancer survive longer regardless of stage or sub-type (5). In contrast, patients with breast, colorectal, prostate or pancreatic cancer who had a greater BMI experienced increased mortality and a greater risk for relapse (6). Importantly, BMI-based definitions of obesity are inadequate to represent physiological fitness and body composition and many studies support muscle mass and muscle quality as better predictors of survival in cancer than BMI-based classifications of obesity (7). The few preclinical models that have examined this question support worsening of cachexia by obesity; HFD-induced obesity increased Yoshida sarcoma tumor growth and cachexia (8) and increased Lewis lung carcinoma (LLC) tumor growth while exacerbating skeletal muscle mitochondrial dysfunction and damage (9). Given these conflicting results, how obesity alters tumor growth, and its interplay in the development of cachexia is not readily clear.
Adequate nutrition is necessary to maintain body weight, including skeletal muscle and fat mass. Anorexia and malabsorption result in malnourishment and contribute to cancer cachexia, although nutritional repletion is insufficient to halt catabolism. Nevertheless, dietary manipulation can modulate metabolism in cancer cachexia, e.g., through the administration of metabolically active lipids or replenishment of limiting amino acids (10–12). As such, various defined diets and dietary supplements have been tested for their ability to reduce cachexia, both in experimental models and in the clinical setting (13, 14). Given the interest in popular diets and more accurately modelling human dietary habits, there is merit in studying diets of varied composition in the setting of cachexia development and therapy. To this end, a prior study fed mice a “Western”-type diet with most calories derived from dietary fat in an experimental cachexia model. In these mice with MAC16 colon adenocarcinoma, body weight loss and tumor growth were reduced proportionately to dietary fat content (15, 16). Furthermore, high-fat diet (HFD) intake was shown to attenuate cachexia, reduce oxidative stress, and ameliorate tissue inflammation in animal models of chronic kidney disease and pulmonary hypertension (17, 18). A high-fat and low-carbohydrate diet prevented tumor growth and inflammation in the C26 model of cachexia (19). Taken together, these results suggest that high-fat diet at the onset of cancer could be protective.
To begin to understand and address these conflicting observations of tumor growth and cachexia development, we sought to assess and disentangle the effects of obesity and high fat diet separately in the LLC model of experimental cancer cachexia. We defined the effects of HFD and obesity on cachexia development, systemic inflammation, tumor growth, and tissue IL-6 signaling. We report that HFD alone and obesity with HFD each exacerbated cachexia as evidenced by reduced muscle mass, increased inflammation, and enhanced tumor cell proliferation. These results highlight distinct, interacting effects underlying accelerated tumor progression with obesogenic diets and poorer outcomes in obesity associated with low muscle mass. Furthermore, our findings suggest that the overlapping contributions of high fat diet and chronic adiposity could be manipulated to slow cachexia and improve survival in cancer.

MATERIALS AND METHODS

Cell culture
Lewis lung carcinoma (LLC) cells (ATCC) were cultured in high glucose (4.5 g/L) Dulbecco’s Modified Eagle Medium (DMEM) supplied with 10% fetal bovine serum, 1% glutamine, 1% sodium pyruvate, and 1% penicillin/streptomycin. Cells were passaged at low density and were maintained in a 5% CO2, 37°C humidified incubator. For proliferation studies, LLC cells were cultured in a 96-well plate and exposed to complete medium containing 10% plasma collected from either lean or obese mice for up to 6 days (n=6). Cell proliferation was assayed with the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI).

Mice
All animal procedures were approved by the University of Miami Institutional Animal Care and Use Committee (Protocol #05–105) and carried out in accordance with NIH publication No. 86–23. Male 12-week-old C57BL/6J diet-induced obese mice were purchased from Jackson Laboratories and maintained on JAX High Fat Diet D12492 (20% kcal from protein, 20% from carbohydrate, 60% from fat,) for the duration of the experiment (Obese-HFD). Upon arrival to the animal facility, mice were acclimated for 1 week then randomized to LLC or control groups. Normal lean, male 12-week-old C57BL/6J controls were purchased at the same time and randomized into four groups: Lean-HFD with or without LLC or Lean-low-fat diet(LFD) (Control Diet D12450B, 20% kcal from protein, 70% from carbohydrate, 10% from fat) with or without LLC, maintaining HFD or LFD, respectively, for the duration of the study. Mice were injected subcutaneously in the flank with 2×106 LLC cells to seed tumor growth or PBS for controls and were group housed by diet with LLC and PBS mice mixed in the same cage to account for potential cage effects. Mice were given ad libitum access to designated diet and housed in a conventional facility with 12-hr light and dark cycles. All mice were weighed daily. Body composition was measured in conscious non-tumor bearing lean and obese mice by magnetic resonance (EchoMRI). Mice were euthanized 23 days later at the humane endpoint, in the ad libitum state. Under isoflurane anesthesia, blood was collected by cardiac puncture into heparinized tubes, centrifuged, and the platelet-poor plasma was flash frozen and store at −80°C. Thoracotomy and removal of the heart assured death. Tissues were collected, weighed, snap-frozen in liquid nitrogen, and stored at −80°C. Tumors were isolated, weighed, fixed in 4% paraformaldehyde, and embedded in paraffin. We did not control for potential confounders of cage location on the rack. Operators were not blinded because tumor status and diet were obvious. No mice were excluded from the analysis of cachexia phenotypes.

Western blotting analysis
Total protein extract was collected by homogenizing tissues (quadriceps, liver, or spleen) in RIPA buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0) with protease inhibitor cocktail (Roche, Indianapolis, IN). Cell debris was removed by centrifugation (15 min at 14,000 × g), and supernatants were collected and stored at −80°C. BCA protein assay (Thermo Fisher Scientific, Suwanee, GA) was used to measure protein concentration. Protein extracts (30 mg) were electrophoresed in 4–15% gradient SDS gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% w/v milk in 1X TBS, 0.1% Tween-20 (TBST) at room temperature for 1 hour, then incubated overnight with diluted antibody in blocking buffer at 4°C with gentle shaking. After washing with TBST, the membrane was incubated at room temperature for 1 hour with either goat anti-rabbit (Santa Cruz Biotechnology Inc., Santa Cruz, CA) or goat anti-mouse (Bio-Rad Laboratories, Hercules, CA) IgG secondary antibodies conjugated to horseradish peroxidase. Protein bands were visualized by incubation with enhanced chemiluminescence (Pierce Super Signal Pico or Dura, Thermo Fisher Scientific, Suwanee, GA) and exposure to film. Antibodies used were: pSTAT3-Y705 (9145), STAT3 (9132), p-AKT-S473 (4060), AKT (4685), p-Foxo3a-S381/321 (9465), Foxo3a (2497), and GAPDH (2118) from Cell Signaling Technology (Beverly, MA); and IL-6 (MABF41) from EMD Millipore (Billerica, MA). Initial analysis was performed on individual samples obtained from each experimental group. Band intensity was quantified by ImageJ, normalized to GAPDH and the results plotted in the graphs shown. In the blots shown here, samples were obtained by pooling 6–9 independent samples from each experimental group. These results are visually representative of the average signal obtained by individual level analysis.

Immunohistochemistry
Tissue sections (8 μm) were cut from paraffin-embedded tumors and mounted onto glass slides. Slides were deparaffinized by incubation in xylenes, 100% ethanol, 95% ethanol, 70% ethanol, and water. Reference tissues were stained with Hematoxylin & Eosin using standard protocols. For immunohistochemistry, a 10 min incubation in 3% H2O2 quenched endogenous peroxidase activity in tissue sections, and then sections were treated in 10 mM citrate buffer solution for 30 min at 80°C for antigen retrieval. Tissues were blocked in 8% BSA in PBS for 1 h at room temperature, then incubated overnight in a humidified box at 4°C with primary antibody: PCNA (P8825; Sigma Aldrich, St. Louis, MO) or IL-6 (6672; Abcam, Cambridge, MA). Sections were incubated with DAKO EnVision+ System-HRP secondary antibody (DAKO, Carpinteria, CA) for 1 h at room temperature. Signals were visualized with DAKO Liquid DAB+ substrate chromogen according to the manufacturer’s instructions. Slides were counterstained with Harris hematoxylin, dehydrated, and mounted with Cytoseal XYL mounting medium (Thermo Fisher Scientific, Suwanee, GA). Results were analyzed by photographing random fields with a Leica DMI3000B microscope and digital images were recorded using a DFC310FX digital color camera and LAS AF software (Leica, Buffalo Grove, IL). ImageJ (NIH) was used to quantify brown staining of IL-6 and PCNA positive nuclei versus total numbers of nuclei that were counted. Results presented are from 4 tumor samples per group.

Blood analyte profiling
Platelet-poor plasma samples were thawed at room temperature, vortexed, and clarified at 13,000 × g for 5 min. Cytokine levels were quantified with the Inflammatory Cytokine Mouse Multiplex Panel (Life Technologies, Grand Island, NY) on a Luminex Magpix, following the manufacturer’s instructions (Austin, TX). Data was interpreted using Luminex xPONENT software. Unknown values for each analyte measured in the multiplex were determined with respect to an 8-point standard curve.

Transcriptome analysis
Publicly available skeletal muscle transcriptome datasets GSE114820: LLC tumor bearing mice (28 day LLC vs day 0 control) and GSE10785: HFD mice (10 week old C57BL6/J obese vs lean) (20, 21) were compared and pathway analysis performed using Illumina BaseSpace Correlation Engine.

Statistical analysis
Prism (GraphPad) was used for statistical analysis and plotting of the data. For differences in tissue mass endpoints between control and tumor-bearing groups across the three variables (lean-LFD, lean-HFD, obese-HFD), we used a regular 2-way ANOVA (not repeated measures) with Holm-Sidak’s multiple comparisons test for normally distributed data. For non-normal data, we used the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparison test within the LLC condition. Multiplicity adjusted P values are reported. p<0.05 was considered significant.

RESULTS

High fat diet and obesity with HFD lead to greater weight loss in LLC cachexia
To assess the effects of diet and body composition in cancer cachexia, lean and diet-induced obese C57Bl/6J male mice were used. Lean mice were divided into a low-fat diet (lean-LFD) and a high-fat diet (lean-HFD) group, initiated on day 1 when mice were 12 weeks old (Figure 1A). Obese mice were maintained on a high-fat diet (obese-HFD) from 6 weeks of age to the end of the experiment. Obese-HFD mice weighed more on the day of injection than the other groups (Figure 1B). Mice were injected with LLC cells or saline at 12-weeks. In the absence of cancer, lean-LFD mice did not gain body mass (+2.64%, NS) (Figure 1C). In contrast, tumor-free mice switched to HFD on day 1 (lean-HFD) gained 14.93% body weight (P<0.0001 vs. day 1), while obese-HFD mice gained 13.46% (P<0.0001 vs. day 1). No significant difference in weight gain was observed between tumor-free lean-HFD and obese-HFD groups. Weight gain is quantified as the body weight from day 0 (normalized as final body weight/initial body weight); thus obese-HFD mice gained more mass than lean-HFD mice. To validate the obese model, obese non-tumor bearing controls had increased fat tissue percentage compared to lean non-tumor bearing controls (data not shown).
All LLC-injected mice gained weight from days 1 through 19, reflecting tumor growth. Weight loss began at day 20 in both the lean-HFD and obese-HFD cancer groups, in contrast to the lean-LFD tumor-bearing group, which continued to gain weight. By day 23, the obese-HFD group had plummeted to the initial body weight and exhibited sickness and moribund behaviors, prompting euthanasia of all cohorts despite normal functioning in lean-LFD tumor-bearing mice. Changes in tumor-free body weight were determined (Figure 1D). Lean-LFD LLC mice did not meet the definition of “cachectic,” having lost only 3.4% of initial body weight (Figure 1E). Lean-HFD and obese-HFD with cancer both lost more weight than lean-LFD with cancer compared to their diet non-tumor controls. Moreover, weight loss in obese-HFD LLC was greater than in lean-HFD. Thus, the combination of HFD and obesity induced more pronounced cachexia than HFD alone. This enhanced wasting represented ~2.8g more weight loss than lean-HFD mice experienced. These results indicate that lean-HFD and obese-HFD both increased weight loss over the control LFD group.

HFD and obesity each enhance LLC-induced skeletal muscle and fat loss in obese mice
As is typical, LLC tumor growth resulted in the loss of skeletal muscle mass and adipose tissue depots. All muscle groups were examined, including gastrocnemius, quadriceps and tibialis, and showed weight loss in LLC lean-LFD mice compared to non-tumor bearing control (lean-LFD) mice. Among LLC mice, loss of gastrocnemius and tibialis mass was greater with HFD, and even more wasted in the presence of obesity and HFD (Figure 2A, C). Two-way ANOVA showed that tumor status contributed 26.62% of variation (p<0.0001) and diet/body composition 36.4% (p<0.0001) for quadriceps mass variance. Multiple comparisons analysis revealed that muscle mass was significantly different across all groups, such that obese-HFD mice experienced more wasting than lean-HFD, and lean-HFD mice experienced more wasting than lean-LFD mice. Thus, HFD exacerbated muscle wasting in LLC cachexia, and obesity was a further cachexia stimulus.
LLC-induced cardiac wasting was observed only in obese HFD, although cardiac mass tended to be reduced in lean-HFD. Changes in adipose tissue were variable across depots (Figures 2E-H). Intrascapular brown fat and omental white fat were greatly and consistently depleted across groups; however retroperitoneal and gonadal/epididymal white fat were somewhat preserved by HFD, an effect not modulated by obesity.

Diet-induced obesity is associated with reduced skeletal muscle and heart mass
At the time of euthanasia, tumor-free obese-HFD mice showed marked reductions in all limb muscles examined and the heart (Figure 2, Table 1, 3). As anticipated, intrascapular brown, omental, and retroperitoneal adipose depots were increased in lean-HFD compared to lean-LFD and were further increased in obese-HFD mice (Figure 2, Table 1, 3). Furthermore, gonadal adipose tissue was increased in obese-HFD non-tumor bearing mice compared to lean-LFD and lean-HFD groups. Collectively, this low muscle mass in the setting of high body mass index is termed sarcopenic obesity when observed in older patients and myopenic obesity in the non-elderly with muscle wasting conditions. Summary of differences are reported in Table 2. In comparison to tumor-free controls, the differences were greatest in HFD-fed mice.

HFD increased tumor mass, but reduced hepatosplenomegaly
Obese- HFD corresponded with a significant increase in tumor mass in obese versus lean mice 23 days after injection (Figure 3A [lean-LFD 5.6 ± 1.9g; lean-HFD 6.5 ± 1.2g; obese-HFD 8.8 ± 1.4g). In lean-LFD tumor-bearing mice, liver and spleen mass was markedly increased (+38% and +213%, respectively) 23 days after injection (Figure 3B, C). The development of tumor-associated hepato- and splenomegaly was partially prevented in both lean-HFD and obese-HFD mice (Figure 3B, C). Overall, as a fraction of body weight, tumor mass in obese-HFD mice was not different from lean-HFD, but was greater than in lean-LFD (P=0.036 by ANOVA test, P=0.0346 by Tukey’s multiple comparisons test).

HFD and obesity increased inflammatory cytokines and reduced insulin and adiponectin in tumor-bearing mice
To investigate whether modulation of humoral factors, such as growth factors and cytokines, were associated with diet and contributed to obesity, we analyzed serum from LFD lean and obese, and obese-HFD mice by multianalyte profiling (Figure 4). Obese-HFD LLC mice showed significantly elevated levels of pro-inflammatory mediators (Il-6, LIF, and Il-1) compared to lean mice and controls. Insulin and adiponectin are markers of metabolic dysregulation in obesity and inflammation. Tumor-bearing mice also displayed hypoinsulinemia and decreased adiponectin levels in all experimental groups compared to non-tumor bearing mice (Figure 4).

HFD enhanced muscle atrophy associated pathways in obese tumor-bearing mice
Both the IL-6/STAT3 and IGF-1/AKT pathways have been linked to the regulation of muscle size (22–24), therefore, we evaluated these pathways in our mice. The muscles of HFD-fed, LLC-injected mice (both lean and obese) had significantly increased phospho-Stat3 levels (Figure 5A, B), consistent with elevated circulating levels of Il-6 and LIF (Figure 4). Analogously, reduced insulin levels (Figure 4) correspond with HFD-associated reductions of Akt and Foxo3a phosphorylation in the muscle of LLC-injected mice (Figure 5A, B). In both cases, obese mice had exacerbated differences in phospho-Stat3 which are in line with the results for muscle weight (Figure 2A).

LLC tumors represent a major source of IL-6
Taken together, these results suggest that the IL-6/STAT3 pathway plays an important role in muscle wasting associated with our mouse model. To identify the source of IL-6, we measured IL-6 protein levels in the quadriceps muscle, liver, spleen and tumor tissues (Figure 6A, B). Generally, IL-6 was decreased in muscle of tumor-bearing mice, although this reduction was less evident in obese-HFD mice (Figure 6A, B). No relevant differences were measured in livers of LLC-injected mice versus controls. Splenic IL-6 protein levels from LLC-injected mice were decreased compared to controls, apart from unchanged levels in lean-HFD mice (Figure 6A, B). IL-6 was elevated in tumors from lean-HFD mice and, to a greater extent, from obese-HFD mice, as compared to tumors from lean-LFD mice (Figure 7A, B). These data strongly implicate tumors as a major source of IL-6 in our mouse model.

HFD and obesity promoted tumor cell proliferation in vivo and in vitro
HFD in the setting of obesity resulted in increased tumor weight by day 23. To determine whether this was a result of enhanced cell proliferation in the tumor, we assayed the proliferation marker PCNA by immunohistochemistry in tumor tissue sections (Figure 7C-E). PCNA positive cells more than doubled in lean-HFD tumors (+115%, p<0.001 vs. lean-LFD) and, to a greater extent in obese-HFD tumors (+173% vs. lean LFD) (Figure 7D, E). This enhanced proliferation likely explains the increased tumor size.
To determine whether if HFD metabolic have a direct effect on tumor proliferation, we exposed LLC cells to serum from obese-HFD mice (non-tumor bearing). LLC cells exposed to serum from obese-HFD mice proliferated significantly faster than cells exposed to serum from lean mice (+143% after 6 days), suggesting that mediators present in the serum of obese mice fueled cancer cell proliferation (Figure 7F).
Next, we compared transcriptional changes following diet-induced obesity or LLC-induced cachexia using publicly available bulk RNAseq from skeletal muscle. We overlapped differentially expressed genes in the gastrocnemius from LLC tumor bearing mice vs PBS controls (Bioset 1) (20, 21) to differentially expressed genes from obese vs lean male mice (Bioset 2) (25) (Figure 8A). There were 704 overlapping differentially expressed genes with 481 genes changing in the same direction (Figure 8B;Supplemental Table 1). Next, we identified Hallmark pathways up- or down-regulated using the 481 genes (Figure 8B-C). We identified IL-6_JAK_STAT3 signaling, adipogenesis, and inflammatory response were upregulated and are known to promote muscle wasting (Figure 8B). In summary, high-fat diet alone is sufficient to induce muscle wasting, and cancer accelerates high-fat diet-induced muscle wasting (Figure 8D). High-fat diet and obesity in the context of cancer exacerbate muscle wasting and promote tumor growth.

DISCUSSION
Our results show that both HFD and obesity do not prevent cancer-associated muscle and fat depletion and importantly, exacerbate the development of cachexia. This may be due to mechanisms conferring increased tumor proliferation and the development of an elevated inflammatory state. These results are consistent with recent work that showed obesity increased tumor growth and exacerbated skeletal muscle mitochondrial dysfunction and damage in LLC (9). Furthermore, our results are consistent with work from Prado et al. (26) that showed severe muscle depletion might occur irrespective of higher BMI in patients with respiratory or gastrointestinal tract tumors. This condition, named “sarcopenic obesity,” is associated with great variability in body composition of cancer patients and affected functional status, chemotherapy resistance, and survival (26). Sarcopenic obesity is used in the context of aging. Our experiment was conducted on 12wk old mice, not a model of aging; yet in the absence a tumor, normalized muscle mass was lower in HFD-obese mice, demonstrating physiologically relevant obesity with low muscle mass in young animals. CT scan-associated body composition analysis reveals muscle loss as a powerful predictor of mortality in obese cancer patients (7), highlighting the necessity to move beyond BMI alone (27).
One of the main mechanisms underlying cachexia is the establishment of increased systemic inflammation associated with the activation of signaling pathways leading to muscle wasting (22, 28). Similarly, obesity correlates with low-grade chronic inflammation and increased levels of circulating fatty acids that recruit immune cells and, in turn, enhance the inflammatory response (29). Evidence suggests that energy accumulation (such as in HFD intake and obesity) plays a pivotal role in the origin of chronic inflammation (30). Conversely, caloric restriction correlates with low inflammation (31). In the present work, no significant variation was observed between pro-inflammatory cytokine levels from lean and obese control mice; however, we did measure increased pro-inflammatory cytokine levels from tumor-bearing mice. These results are consistent with clinical reports of obese patients with increased circulating levels of TNF, IL-6, and IL-1b that correlate with higher BMI (32).
The primary source of inflammatory cytokines in an organism remains unclear. As previously reported in conditions of elevated adiposity, cytokines might derive from adipocytes themselves or from increased recruitment of inflammatory cells, such as macrophages (33, 34). To identify the primary source of inflammation in our model, we assessed IL-6 levels in several tissues, including, but not limited to muscle and tumor. IL-6 was markedly decreased in the muscles of LLC-injected mice in all experimental groups. The discrepancy of these results with the increased STAT3 phosphorylation in muscle of tumor-injected mice that were either obese or lean on a HFD might suggest the activation of a negative feedback response that attempts to preserve muscle mass in the presence of elevated atrophying stimuli. The in vivo tumor environment was associated with significantly elevated IL-6 levels. While we did not measure the IL-6 levels in other tissues, including adipose depots, it is possible that other tissue contribute to the systemic increase in IL-6. Nonetheless, our data shows that when comparing skeletal muscle, spleen, liver, and tumor, the tumor represents a major source of IL-6. IL-6 is likely not the only contributing factor promoting exacerbated tumor growth. Obesity-associated chronic inflammation can also affect cancer development and growth. Indeed, pharmacologic, and genetic inhibition of IL-6 and TNF can limit the severity of some obesity-related cancers (35, 36). Similarly, fatty acid synthase associated with increased lipid bioavailability represents an excellent milieu for cancer cell initiation and proliferation (37). Our in vitro results show that LLC cells have enhanced proliferation in the presence of serum collected from obese mice, supporting previous studies in humans (38, 39). Moreover, our results aid in developing rationale for why obese-HFD mice have larger LLC tumors.
We also examined the circulating levels of insulin and adiponectin. Insulin is a peptide hormone produced by pancreatic beta cells, and it is released in response to elevated blood glucose. Insulin is related to insulin-like growth factor (IGF-1), whose role in cancer cachexia has been extensively investigated (23, 40). Activation of the insulin receptor (IR) and IGF-1R recruits PI-3K, which, in turn, activates AKT, a known regulator of muscle growth (24). Hyperinsulinemia, hyperglycemia, and insulin resistance are normally associated with obesity (41, 42). Although we were not able to reproduce these conditions in our experimental model, low insulin levels and reduced AKT pathway activation were observed, consistent with previous reports from experimental cachexia models (23, 40).
Adiponectin is a peptide hormone secreted from visceral adipose tissue, and its main functions are related to energy homeostasis, and carbohydrate and lipid metabolism. Adiponectin also has anti-inflammatory properties, possibly functioning by suppressing the migration of monocytes and macrophages (43). Moreover, adiponectin levels are inversely correlated with increased cancer risk, suggesting that adiponectin might protect against the development of several malignancies (44). Altogether, this seems to suggest that low adiponectin levels could be associated with the establishment of inflammation-promoting conditions and tumor growth. Our results showing increased adiponectin levels in obese and HFD-fed control mice and decreased levels in LLC-injected mice support this hypothesis.
This study is not without limitations. Food intake was not measured. However, accurate measurement of food intake requires singly housing mice and weighing the remaining food as well as the ort (shredded crumbs of food produced by mice, which increases with stressors like cachexia). Weighing subtracting remnant chow from starting amounts does not account for ort and artificially inflates food intake, disproportionately for mice with tumors. Given the texture of the high fat diet—crumbly and oily, it is not possible to weigh the remnants accurately and thus we did not conduct such measurements. Also, because we used male mice, follow up experiments are needed to determine whether our findings are applicable in female mice. While the LLC is a validated model of cancer cachexia, we acknowledge that LLC tumors burden was higher than typically encountered in people with cancer. Lastly, while we used a common high fat diet (60% calories from fat), we acknowledge that fat percentage, source of fat (e.g. animal versus plant), and diet compositions can impact cancer cachexia outcomes. Given this was not purpose of our experiment, other work is needed to determine the relationship between fat source and diet composition on cancer cachexia progression.
Overall, our experimental mouse model indicates that neither HFD nor obesity protects against cancer cachexia. Intensified wasting, including both muscle and fat loss, was associated with increased systemic inflammation, possibly due to high pro-inflammatory cytokine levels. This could contribute to increased tissue wasting by promoting the activation of pathways associated with muscle atrophy and fostering tumor proliferation. Based on our results and accumulating evidence indicating obesity as a primary risk factor for cancer development, our findings provide compelling evidence for preventing weight gain to limit accelerated tumor and cachexia progression in the general population.

Supplementary Material
DATA AVAILABILITY
Supplemental Table 1 is available at https://doi.org/10.5281/zenodo.18166640.