Hepatic signal transducer and activator of transcription-3 signalling drives early-stage pancreatic cancer cachexia via suppressed ketogenesis.

BACKGROUND: Patients with pancreatic ductal adenocarcinoma (PDAC) often suffer from cachexia, a wasting syndrome that significantly reduces both quality of life and survival. Although advanced cachexia is associated with inflammatory signalling and elevated muscle catabolism, the early events driving wasting are poorly defined. During periods of nutritional scarcity, the body relies on hepatic ketogenesis to generate ketone bodies, and lipid metabolism via ketogenesis is thought to protect muscle from catabolizing during nutritional scarcity. METHODS: We developed an orthotopic mouse model of early PDAC cachexia in 12-week-old C57BL/6J mice. Murine pancreatic cancer cells (KPC) were orthotopically implanted into the pancreas of wild-type, IL-6-/-, and hepatocyte STAT3-/- male and female mice. Mice were subject to fasting, 50% food restriction, ad libitum feeding or ketogenic diet interventions. We measured longitudinal body composition by EchoMRI, body mass and food intake. At the endpoint, we measured tissue mass, tissue gene expression by quantitative real-time polymerase chain reaction, whole-body calorimetry, circulating hormone levels, faecal protein and lipid content, hepatic lipid content and ketogenic response to medium-chain fatty acid bolus. We assessed muscle atrophy in vivo and C2C12 myotube atrophy in vitro. RESULTS: Pre-cachectic PDAC mice did not preserve gastrocnemius muscle mass during 3-day food restriction (-13.1 ± 7.7% relative to food-restricted sham, P = 0.0117) and displayed impaired fatty acid oxidation during fasting, resulting in a hypoketotic state (ketogenic response to octanoate bolus, -83.0 ± 17.3%, P = 0.0328; Hmgcs2 expression, -28.3 ± 7.6%, P = 0.0004). PDAC human patients display impaired fasting ketones (-46.9 ± 7.1%, P < 0.0001) and elevated circulating interleukin-6 (IL-6) (12.4 ± 16.5-fold increase, P = 0.0001). IL-6-/- PDAC mice had improved muscle mass (+35.0 ± 3.9%, P = 0.0031) and ketogenic response (+129.4 ± 44.4%, P = 0.0033) relative to wild-type PDAC mice. Hepatocyte-specific signal transducer and activator of transcription 3 (STAT3) deletion prevented muscle loss (+9.3 ± 4.0%, P = 0.009) and improved fasting ketone levels (+52.0 ± 43.3%, P = 0.018) in PDAC mice. Without affecting tumour growth, a carbohydrate-free diet improved tibialis anterior myofibre diameter (+16.5 ± 3.5%, P = 0.0089), circulating ketone bodies (+333.0 ± 117.6%, P < 0.0001) and Hmgcs2 expression (+106.5 ± 36.1%, P < 0.0001) in PDAC mice. Ketone supplementation protected muscle against PDAC-induced atrophy in vitro (+111.0 ± 17.6%, P < 0.0001 myofibre diameter). CONCLUSIONS: In early PDAC cachexia, muscle vulnerability to wasting is dependent on inflammation-driven metabolic reprogramming in the liver. PDAC suppresses lipid ß-oxidation and impairs ketogenesis in the liver, which is reversed in genetically modified mouse models deficient in IL-6/STAT3 signalling or through ketogenic diet supplementation. This work establishes a direct link between skeletal muscle homeostasis and hepatic metabolism. Dietary and anti-inflammatory interventions that restore ketogenesis may be a viable preventative approach for pre-cachectic patients with pancreatic cancer.

Identifying trajectories and predictors of chemotherapy-induced peripheral neuropathy symptoms, physical functioning, and falls across treatment and recovery in adults treated with neurotoxic chemotherapy: the PATTERN observational study protocol (NCT05790538).

BACKGROUND: Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating and dose-limiting side effect of systemic cancer therapy. In many cancer survivors, CIPN persists after treatment ends and is associated with functional impairments, abnormal gait patterns, falls, and diminished quality of life. However, little is known regarding which patients are most likely to develop CIPN symptoms that impair mobility and increase fall risk, when this risk develops, or the optimal timing of early intervention efforts to mitigate the impact of CIPN on functioning and fall risk. This study will address these knowledge gaps by (1) characterizing trajectories of symptoms, functioning, and falls before, during, and after treatment in adults prescribed neurotoxic chemotherapy for cancer; and (2) determining the simplest set of predictors for identifying individuals at risk for CIPN-related functional decline and falls. METHODS: We will enroll 200 participants into a prospective, observational study before initiating chemotherapy and up to 1 year after completing chemotherapy. Eligible participants are aged 40-85 years, diagnosed with stage I-III cancer, and scheduled to receive neurotoxic chemotherapy. We perform objective assessments of vibratory and touch sensation (biothesiometry, tuning fork, monofilament tests), standing and dynamic balance (quiet stance, Timed-Up-and-Go tests), and upper and lower extremity strength (handgrip dynamometry, 5-time repeated chair stand test) in the clinic at baseline, every 4-6 weeks during chemotherapy, and quarterly for 1 year post-chemotherapy. Participants wear devices that passively and continuously measure daily gait quality and physical activity for 1 week after each objective assessment and self-report symptoms (CIPN, insomnia, fatigue, dizziness, pain, cognition, anxiety, and depressive symptoms) and falls via weekly electronic surveys. We will use structural equation modeling, including growth mixture modeling, to examine patterns in trajectories of changes in symptoms, functioning, and falls associated with neurotoxic chemotherapy and then search for distinct risk profiles for CIPN. DISCUSSION: Identifying simple, early predictors of functional decline and fall risk in adults with cancer receiving neurotoxic chemotherapy will help identify individuals who would benefit from early and targeted interventions to prevent CIPN-related falls and disability. TRIAL REGISTRATION: This study was retrospectively registered with ClinicalTrials.gov (NCT05790538) on 3/30/2023.

Validation of automated body composition analysis using diagnostic computed tomography imaging in patients with pancreatic cancer.

BACKGROUND: Sarcopenia is associated with complications and inferior oncologic outcomes in solid tumors. Axial computed tomography (CT) scans can be used to evaluate sarcopenia, however manual quantification is laborious. We sought to validate an automated method of quantifying muscle cross-sectional area (CSA) in patients with pancreatic adenocarcinoma (PDAC). METHODS: Mid-L3 CT images from patients with PDAC were analyzed: CSAs of skeletal muscle (SM) were measured using manual segmentation and the software AutoMATiCA, and then compared with linear regression. RESULTS: Five-hundred-twenty-five unique scans were analyzed. There was robust correlation between manual and automated segmentation for L3 CSA (R2 0.94, P < 0.001). Bland-Altman analysis demonstrated a consistent overestimation of muscle CSA by AutoMATiCA with a mean difference of 5.7%. A correction factor of 1.06 was validated using a unique test dataset of 36 patients with non-PDAC peripancreatic malignancies. CONCLUSIONS: Automated muscle CSA measurement with AutoMATiCA is highly efficient and yields results highly correlated with manual measurement. These findings support the potential use of high-throughput sarcopenia analysis with abdominal CT scans for both clinical and research purposes.

Critical changes in hypothalamic gene networks in response to pancreatic cancer as found by single-cell RNA sequencing.

OBJECTIVE: Cancer cachexia is a devastating chronic condition characterized by involuntary weight loss, muscle wasting, abnormal fat metabolism, anorexia, and fatigue. However, the molecular mechanisms underlying this syndrome remain poorly understood. In particular, the hypothalamus may play a central role in cachexia, given that it has direct access to peripheral signals because of its anatomical location and attenuated blood-brain barrier. Furthermore, this region has a critical role in regulating appetite and metabolism. METHODS: To provide a detailed analysis of the hypothalamic response to cachexia, we performed single-cell RNA-seq combined with RNA-seq of the medial basal hypothalamus (MBH) in a mouse model for pancreatic cancer. RESULTS: We found many cell type-specific changes, such as inflamed endothelial cells, stressed oligodendrocyes and both inflammatory and moderating microglia. Lcn2, a newly discovered hunger suppressing hormone, was the highest induced gene. Interestingly, cerebral treatment with LCN2 not only induced many of the observed molecular changes in cachexia but also affected gene expression in food-intake decreasing POMC neurons. In addition, we found that many of the cachexia-induced molecular changes found in the hypothalamus mimic those at the primary tumor site. CONCLUSION: Our data reveal that multiple cell types in the MBH are affected by tumor-derived factors or host factors that are induced by tumor growth, leading to a marked change in the microenvironment of neurons critical for behavioral, metabolic, and neuroendocrine outputs dysregulated during cachexia. The mechanistic insights provided in this study explain many of the clinical features of cachexia and will be useful for future therapeutic development.

Lipocalin 2 mediates appetite suppression during pancreatic cancer cachexia.

Lipocalin 2 (LCN2) was recently identified as an endogenous ligand of the type 4 melanocortin receptor (MC4R), a critical regulator of appetite. However, it remains unknown if this molecule influences appetite during cancer cachexia, a devastating clinical entity characterized by decreased nutrition and progressive wasting. We demonstrate that LCN2 is robustly upregulated in murine models of pancreatic cancer, its expression is associated with reduced food consumption, and Lcn2 deletion is protective from cachexia-anorexia. Consistent with LCN2's proposed MC4R-dependent role in cancer-induced anorexia, pharmacologic MC4R antagonism mitigates cachexia-anorexia, while restoration of Lcn2 expression in the bone marrow is sufficient in restoring the anorexia feature of cachexia. Finally, we observe that LCN2 levels correlate with fat and lean mass wasting and is associated with increased mortality in patients with pancreatic cancer. Taken together, these findings implicate LCN2 as a pathologic mediator of appetite suppression during pancreatic cancer cachexia.

Establishment and characterization of a novel murine model of pancreatic cancer cachexia.

BACKGROUND: Cachexia is a complex metabolic and behavioural syndrome lacking effective therapies. Pancreatic ductal adenocarcinoma (PDAC) is one of the most important conditions associated with cachexia, with >80% of PDAC patients suffering from the condition. To establish the cardinal features of a murine model of PDAC-associated cachexia, we characterized the effects of implanting a pancreatic tumour cell line from a syngeneic C57BL/6 KRASG12D P53R172H Pdx-Cre+/+ (KPC) mouse. METHODS: Male and female C57BL/6 mice were inoculated subcutaneously, intraperitoneally, or orthotopically with KPC tumour cells. We performed rigorous phenotypic, metabolic, and behavioural analysis of animals over the course of tumour development. RESULTS: All routes of administration produced rapidly growing tumours histologically consistent with moderate to poorly differentiated PDAC. The phenotype of this model was dependent on route of administration, with orthotopic and intraperitoneal implantation inducing more severe cachexia than subcutaneous implantation. KPC tumour growth decreased food intake, decreased adiposity and lean body mass, and decreased locomotor activity. Muscle catabolism was observed in both skeletal and cardiac muscles, but the dominant catabolic pathway differed between these tissues. The wasting syndrome in this model was accompanied by hypothalamic inflammation, progressively decreasing brown and white adipose tissue uncoupling protein 1 (Ucp1) expression, and increased peripheral inflammation. Haematological and endocrine abnormalities included neutrophil-dominant leukocytosis and anaemia, and decreased serum testosterone. CONCLUSIONS: Syngeneic KPC allografts are a robust model for studying cachexia, which recapitulate key features of the PDAC disease process and induce a wide array of cachexia manifestations. This model is therefore ideally suited for future studies exploring the physiological systems involved in cachexia and for preclinical studies of novel therapies.

Dexamethasone Chemotherapy Does Not Disrupt Orexin Signaling.

BACKGROUND: Steroid-induced sleep disturbance is a common and highly distressing morbidity for children receiving steroid chemotherapy for the treatment of pediatric acute lymphoblastic leukemia (ALL). Sleep disturbance can negatively impact overall quality of life, neurodevelopment, memory consolidation, and wound healing. Hypothalamic orexin neurons are influential wake-promoting neurons, and disturbances in orexin signaling leads to abnormal sleep behavior. A new class of drug, the orexin receptor antagonists, could be an intriguing option for sleep disorders caused by increased orexinergic output. Our aim was to examine the impact of ALL treatment doses of corticosteroids on the orexin system in rodents and in children undergoing treatment for childhood ALL. METHODS: We administered repeated injections of dexamethasone to rodents and measured responsive orexin neural activity compared to controls. In children with newly diagnosed standard risk B-cell ALL receiving dexamethasone therapy per Children's Oncology Group (COG) induction therapy from 2014-2016, we collected pre- and during-steroids matched CSF samples and measured the impact of steroids on CSF orexin concentration. RESULTS: In both rodents, all markers orexin signaling, including orexin neural output and orexin receptor expression, were preserved in the setting of dexamethasone. Additionally, we did not detect a difference in pre- and during-dexamethasone CSF orexin concentrations in children receiving dexamethasone. CONCLUSIONS: Our results demonstrate that rodent and human orexin physiology is largely preserved in the setting of high dose dexamethasone. The data obtained in our experimental model fail to demonstrate a causative role for disruption of the orexin pathway in steroid-induced sleep disturbance.

Maternal high-fat diet and obesity compromise fetal hematopoiesis.

OBJECTIVE: Recent evidence indicates that the adult hematopoietic system is susceptible to diet-induced lineage skewing. It is not known whether the developing hematopoietic system is subject to metabolic programming via in utero high-fat diet (HFD) exposure, an established mechanism of adult disease in several organ systems. We previously reported substantial losses in offspring liver size with prenatal HFD. As the liver is the main hematopoietic organ in the fetus, we asked whether the developmental expansion of the hematopoietic stem and progenitor cell (HSPC) pool is compromised by prenatal HFD and/or maternal obesity. METHODS: We used quantitative assays, progenitor colony formation, flow cytometry, transplantation, and gene expression assays with a series of dietary manipulations to test the effects of gestational high-fat diet and maternal obesity on the day 14.5 fetal liver hematopoietic system. RESULTS: Maternal obesity, particularly when paired with gestational HFD, restricts physiological expansion of fetal HSPCs while promoting the opposing cell fate of differentiation. Importantly, these effects are only partially ameliorated by gestational dietary adjustments for obese dams. Competitive transplantation reveals compromised repopulation and myeloid-biased differentiation of HFD-programmed HSPCs to be a niche-dependent defect, apparent in HFD-conditioned male recipients. Fetal HSPC deficiencies coincide with perturbations in genes regulating metabolism, immune and inflammatory processes, and stress response, along with downregulation of genes critical for hematopoietic stem cell self-renewal and activation of pathways regulating cell migration. CONCLUSIONS: Our data reveal a previously unrecognized susceptibility to nutritional and metabolic developmental programming in the fetal HSPC compartment, which is a partially reversible and microenvironment-dependent defect perturbing stem and progenitor cell expansion and hematopoietic lineage commitment.

Cancer- and endotoxin-induced cachexia require intact glucocorticoid signaling in skeletal muscle.

Cachexia is a wasting condition defined by skeletal muscle atrophy in the setting of systemic inflammation. To explore the site at which inflammatory mediators act to produce atrophy in vivo, we utilized mice with a conditional deletion of the inflammatory adaptor protein myeloid differentiation factor 88 (MyD88). Although whole-body MyD88-knockout (wbMyD88KO) mice resist skeletal muscle atrophy in response to LPS, muscle-specific deletion of MyD88 is not protective. Furthermore, selective reexpression of MyD88 in the muscle of wbMyD88KO mice via electroporation fails to restore atrophy gene induction by LPS. To evaluate the role of glucocorticoids as the inflammation-induced mediator of atrophy in vivo, we generated mice with targeted deletion of the glucocorticoid receptor in muscle (mGRKO mice). Muscle-specific deletion of the glucocorticoid receptor affords a 71% protection against LPS-induced atrophy compared to control animals. Furthermore, mGRKO mice exhibit 77% less skeletal muscle atrophy than control animals in response to tumor growth. These data demonstrate that glucocorticoids are a major determinant of inflammation-induced atrophy in vivo and play a critical role in the pathogenesis of endotoxemic and cancer cachexia.

Neuropeptides in the pathophysiology and treatment of cachexia.

PURPOSE OF REVIEW: Cachexia occurs in various inflammatory diseases and is characterized by weight loss and muscle wasting. Pro-inflammatory cytokines modulate the activity of neuropeptides and hormones that control energy homeostasis and/or illness behaviors. This review summarizes recent (published within the past 18 months) literature regarding neuropeptides and hormones that have been implicated in the pathophysiology of cachexia, and that are likely to have therapeutic potential for preventing or reversing cachexia in various disease states. RECENT FINDINGS: Hypothalamic pro-opiomelanocortin (POMC) and agouti-related protein (AgRP) neurons are downstream targets for pro-inflammatory cytokines. Genetic or pharmacological blockade of melanocortin receptor signaling preserves lean body mass and attenuates anorexia in experimental models of cachexia. Orally available melanocortin receptor antagonists have been developed and tested in cachectic animals with favorable results. Ghrelin and ghrelin mimetics increase appetite and preserve lean body mass in cachectic patients with diverse underlying diseases. Additional neuropeptide-expressing neurons in the hypothalamus (e.g., orexin neurons) might play a role in cachexia-associated lethargy. SUMMARY: Promising outcomes from recent preclinical studies and/or early clinical trials with melanocortin receptor antagonists and ghrelin mimetics raise hopes that safe and effective anti-cachexia drugs will soon become available for widespread clinical use.

Neuroendocrine profiles in galanin-overexpressing and knockout mice.

The peptide galanin has been implicated in the neuroendocrine regulation of reproduction and energy balance. To gain more insight into the functional significance of galanin in these processes, we studied the phenotype of mice that either overexpress galanin in the brain under the control of the dopamine beta-hydroxylase promoter (GALTG) or have a complete absence of galanin expression (GALKO). Both GALTGs and GALKOs had body weights and feeding patterns that were indistinguishable from wild-type (WT) control animals, and both genotypes were reproductively competent. Serum levels of follicle-stimulating hormone were significantly higher in GALKOs and slightly lower in GALTGs than in their respective WT controls. Both GALTGs and GALKOs showed a normal response to fasting, but when GALKO mice were treated with leptin during fasting, levels of corticosterone and testosterone were altered compared to WT mice. In addition, GALKOs were more sensitive than WT controls to the effects of chronic leptin treatment on body weight and fat pad mass, whereas GALTGs showed responses to this metabolic challenge that were indistinguishable from their controls. When galanin was administered centrally, GALKOs had lower testosterone and corticosterone levels than did WT mice. These results suggest that the complete loss of galanin leads to significant alterations in neuroendocrine homeostasis, whereas targeted overexpression of galanin in the brain does not interfere with normal neuroendocrine function.