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Background Cancer cachexia is a metabolic disorder characterized by the progressive loss of fat and lean mass that results in significant wasting, ultimately leading to reduced quality of life and increased mortality. Effective therapies for cachexia are lacking, potentially owing to the mismatch in clinically relevant models of cachexia. Specifically, cachexia observed in a clinical setting is commonly associated with advanced or late‐stage cancers that are metastatic, yet pre‐clinical metastatic models of cachexia are limited. Furthermore, the prevalence of cachexia in head and neck cancer patients is high, yet few pre‐clinical models of head and neck cancer cachexia exist. In addition to these shortcomings, cachexia is also heterogeneous among any given cancer, whereas patients with similar disease burden may experience significantly different degrees of cachexia symptoms. In order to address these issues, we characterize a metastatic model of human papilloma virus (HPV) positive head and neck squamous cell carcinoma that recapitulates the cardinal clinical and molecular features of cancer cachexia. Methods Male and female C57BL/6 mice were implanted subcutaneously with oropharyngeal squamous cell carcinoma cells stably transformed with HPV16 E6 and E7 together with hRas and luciferase (mEERL) that metastasizes to the lungs (MLM). We then robustly characterize the physiologic, behavioural, and molecular signatures during tumour development in two MLM subclones. Results Mice injected with MLM tumour cells rapidly developed primary tumours and eventual metastatic lesions to the lungs. MLM3, but not MLM5, engrafted mice progressively lost fat and lean mass during tumour development despite the absence of anorexia (P

Lipocalin 2 (LCN2) is a pleiotropic molecule that is induced in the central nervous system (CNS) in several acute and chronic pathologies. The acute induction of LCN2 evolved as a beneficial process, aimed at combating bacterial infection through the sequestration of iron from pathogens, while the role of LCN2 during chronic, non-infectious disease remains unclear, and recent studies suggest that LCN2 is neurotoxic. However, whether LCN2 is sufficient to induce behavioral and cognitive alterations remains unclear. In this paper, we sought to address the role of cerebral LCN2 on cognition in both acute and chronic settings. We demonstrate that LCN2 is robustly induced in the CNS during both acute and chronic inflammatory conditions, including LPS-based sepsis and cancer cachexia. In vivo, LPS challenge results in a global induction of LCN2 in the central nervous system, while cancer cachexia results in a distribution specific to the vasculature. Similar to these in vivo observations, in vitro modeling demonstrated that both glia and cerebral endothelium produce and secrete LCN2 when challenged with LPS, while only cerebral endothelium secrete LCN2 when challenged with cancer-conditioned medium. Chronic, but not short-term, cerebral LCN2 exposure resulted in reduced hippocampal neuron staining intensity, an increase in newborn neurons, microglial activation, and increased CNS immune cell infiltration, while gene set analyses suggested these effects were mediated through melanocortin-4 receptor independent mechanisms. RNA sequencing analyses of primary hippocampal neurons revealed a distinct transcriptome associated with prolonged LCN2 exposure, and ontology analysis was suggestive of altered neurite growth and abnormal spatial learning. Indeed, LCN2-treated hippocampal neurons display blunted neurite processes, and mice exposed to prolonged cerebral LCN2 levels experienced a reduction in spatial reference memory as indicated by Y-maze assessment. These findings implicate LCN2 as a pathologic mediator of cognitive decline in the setting of chronic disease.

This study presents the first messenger RNA (mRNA) therapy for metastatic ovarian cancer and cachexia-induced muscle wasting based on lipid nanoparticles that deliver follistatin (FST) mRNA predominantly to cancer clusters following intraperitoneal administration. The secreted FST protein, endogenously synthesized from delivered mRNA, efficiently reduces elevated activin A levels associated with aggressive ovarian cancer and associated cachexia. By altering the cancer cell phenotype, mRNA treatment prevents malignant ascites, delays cancer progression, induces the formation of solid tumors, and preserves muscle mass in cancer-bearing mice by inhibiting negative regulators of muscle mass. Finally, mRNA therapy provides synergistic effects in combination with cisplatin, increasing the survival of mice and counteracting muscle atrophy induced by chemotherapy and cancer-associated cachexia. The treated mice develop few nonadherent tumors that are easily resected from the peritoneum. Clinically, this nanomedicine-based mRNA therapy can facilitate complete cytoreduction, target resistance, improve resilience during aggressive chemotherapy, and improve survival in advanced ovarian cancer.

Microglia in the mediobasal hypothalamus (MBH) respond to inflammatory stimuli and metabolic perturbations to mediate body composition. This concept is well studied in the context of high fat diet induced obesity (HFDO), yet has not been investigated in the context of cachexia, a devastating metabolic syndrome characterized by anorexia, fatigue, and muscle catabolism. We show that microglia accumulate specifically in the MBH early in pancreatic ductal adenocarcinoma (PDAC)‐associated cachexia and assume an activated morphology. Furthermore, we observe astrogliosis in the MBH and hippocampus concurrent with cachexia initiation. We next show that circulating immune cells resembling macrophages infiltrate the MBH. PDAC‐derived factors induced microglia to express a transcriptional profile in vitro that was distinct from that induced by lipopolysaccharide (LPS). Microglia depletion through CSF1‐R antagonism resulted in accelerated cachexia onset and increased anorexia, fatigue, and muscle catabolism during PDAC. This corresponded with increased hypothalamic–pituitary–adrenal (HPA) axis activation. CSF1‐R antagonism had little effect on inflammatory response in the circulation, liver, or tumor. These findings demonstrate that microglia are protective against PDAC cachexia and provide mechanistic insight into this function., • Microgliosis occurs in the mediobasal hypothalamus early in pancreatic cancer.• Microglia respond to pancreatic cancer‐derived factors and produce arginase‐1.• Microglia depletion worsens cachexia during pancreatic cancer.

A priority in cancer research is to innovate therapies that are not only effective against tumor progression but also address comorbidities such as cachexia that limit quality and quantity of life. We demonstrate that TLR7/8 agonist R848 induces anti-tumor responses and attenuates cachexia in murine models of pancreatic ductal adenocarcinoma (PDAC). In vivo, tumors from two of three cell lines were R848-sensitive, resulting in smaller tumor mass, increased immune complexity, increased CD8+ T-cell infiltration and activity, and decreased Treg frequency. R848-treated mice demonstrated improvements in behavioral and molecular cachexia manifestations, resulting in a near-doubling of survival duration. Knockout mouse studies revealed that stromal, not neoplastic, TLR7 is requisite for R848-mediated responses. In patient samples, we found Tlr7 is ubiquitously expressed in stroma across all stages of pancreatic neoplasia, but epithelial Tlr7 expression is relatively uncommon. These studies indicate immune-enhancing approaches including R848 may be useful in PDAC and cancer-associated cachexia., In the treatment of pancreatic ductal adenocarcinoma (PDAC), comorbidities such as cachexia limit quality of life and survival. Here, the authors show TLR7/8 agonist R848 remodels host and tumour immune responses, promoting survival and attenuating cachexia in murine models of PDAC.

Weight loss and anorexia are common symptoms in cancer patients that occur prior to initiation of cancer therapy. Inflammation in the brain is a driver of these symptoms, yet cellular sources of neuroinflammation during malignancy are unknown. In a mouse model of pancreatic ductal adenocarcinoma (PDAC), we observed early and robust myeloid cell infiltration into the brain. Infiltrating immune cells were predominately neutrophils, which accumulated at a unique central nervous system entry portal called the velum interpositum, where they expressed CCR2. Pharmacologic CCR2 blockade and genetic deletion of Ccr2 both resulted in significantly decreased brain-infiltrating myeloid cells as well as attenuated cachexia during PDAC. Lastly, intracerebroventricular blockade of the purinergic receptor P2RX7 during PDAC abolished immune cell recruitment to the brain and attenuated anorexia. Our data demonstrate a novel function for the CCR2/CCL2 axis in recruiting neutrophils to the brain, which drives anorexia and muscle catabolism., eLife digest Weight loss, decreased appetite and fatigue are symptoms of a wasting disorder known as cachexia, which is common in several serious diseases such as AIDS, chronic lung disease and heart failure. Up to 80 percent of people with advanced cancer also develop cachexia, and there are no effective treatments. It is not known how cachexia develops, but symptoms like appetite loss and fatigue are controlled by the brain. One theory is that the brain may be responding to a malfunctioning immune response that causes inflammation. While the brain was thought to be protected from this, new research has shown that it is possible for cells from the immune system to reach the brain in some conditions. To find out if this also happens in cancer, Burfeind et al. studied mice that had been implanted with pancreatic cancer cells and were showing signs of cachexia. Samples from the mice’s brains showed that immune cells known as neutrophils were present and active. A protein known as CCR2 was found in higher levels in the brains of these mice. This protein is involved in the movement of neutrophil cells through the body. To see what effect this protein had, Burfeind et al. gave the mice a drug that blocks CCR2. This prevented the neutrophils from entering the brain and reduced the symptoms of cachexia in the mice. To further confirm the role of CCR2, the mice were genetically modified so that they could not produce the protein. This reduced the number of neutrophils seen in the brain but not in the rest of the body. This suggests that a drug targeting CCR2 could help to reduce the symptoms of cachexia, without disrupting the normal immune response away from the brain. This approach would still need to be tested in clinical trials before it is possible to know how effective it might be in humans.

Cachexia, a devastating wasting syndrome characterized by severe weight loss with specific losses of muscle and adipose tissue, is driven by reduced food intake, increased energy expenditure, excess catabolism, and inflammation. Cachexia is associated with poor prognosis and high mortality and frequently occurs in patients with cancer, chronic kidney disease, infection, and many other illnesses. There is no effective treatment for this condition. Hypothalamic melanocortins have a potent and long-lasting inhibitory effect on feeding and anabolism, and pathophysiological processes increase melanocortin signaling tone, leading to anorexia, metabolic changes, and eventual cachexia. We used 3 rat models of anorexia and cachexia (LPS, methylcholanthrene sarcoma, and 5/6 subtotal nephrectomy) to evaluate efficacy of TCMCB07, a synthetic antagonist of the melanocortin-4 receptor. Our data show that peripheral treatment using TCMCB07 with intraperitoneal, subcutaneous, and oral administration increased food intake and body weight and preserved fat mass and lean mass during cachexia and LPS-induced anorexia. Furthermore, administration of TCMCB07 diminished hypothalamic inflammatory gene expression in cancer cachexia. These results suggest that peripheral TCMCB07 treatment effectively inhibits central melanocortin signaling and therefore stimulates appetite and enhances anabolism, indicating that TCMCB07 is a promising drug candidate for treating 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.

A priority in cancer research is to innovate therapies that are not only effective against tumor progression but also address comorbidities such as cachexia that limit quality and quantity of life. We demonstrate that TLR7/8 agonist R848 induces anti-tumor responses and attenuates cachexia in murine models of pancreatic ductal adenocarcinoma (PDAC). In vivo, tumors from two of three cell lines were R848-sensitive, resulting in smaller tumor mass, increased immune complexity, increased CD8

Hypothalamic inflammation is a key component of acute sickness behavior and cachexia, yet mechanisms of inflammatory signaling in the central nervous system remain unclear. Previous work from our lab and others showed that while MyD88 is an important inflammatory signaling pathway for sickness behavior, MyD88 knockout (MyD88KO) mice still experience sickness behavior after inflammatory stimuli challenge. We found that after systemic lipopolysaccharide (LPS) challenge, MyD88KO mice showed elevated expression of several cytokine and chemokine genes in the hypothalamus. We therefore assessed the role of an additional inflammatory signaling pathway, TRIF, in acute inflammation (LPS challenge) and in a chronic inflammatory state (cancer cachexia). TRIFKO mice resisted anorexia and weight loss after peripheral (intraperitoneal, IP) or central (intracerebroventricular, ICV) LPS challenge and in a model of pancreatic cancer cachexia. Compared to WT mice, TRIFKO mice showed attenuated upregulation of Il6, Ccl2, Ccl5, Cxcl1, Cxcl2, and Cxcl10 in the hypothalamus after IP LPS treatment, as well as attenuated microglial activation and neutrophil infiltration into the brain after ICV LPS treatment. Lastly, we found that TRIF was required for Ccl2 upregulation in the hypothalamus and induction of the catabolic genes, Mafbx, Murf1, and Foxo1 in gastrocnemius during pancreatic cancer. In summary, our results show that TRIF is an important inflammatory signaling mediator of sickness behavior and cachexia and presents a novel therapeutic target for these conditions.

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.

Purpose/Objectives: The disease-associated wasting condition cachexia is a common complication of pancreatic ductal adenocarcinoma (PDAC) that impacts quality of life and portends poor survival. Undernutrition is a major driver of wasting in PDAC, yet cachexia remains refractory to nutritional supplementation. By modifying nutritional challenges at different stages of cachexia development, we sought to understand the relative contributions of undernutrition and metabolic reprogramming on adipose and skeletal muscle wasting. Materials/Methods: Adult mice received orthotopic PDAC tumor injections (KrasG12D; p53R172H/+; Pdx1-cre) or sham injections. Mice were fasted or food restricted at different times during tumor growth in a series of 2x2 factorial studies. Anthropometrics, voluntary wheel running activity, and body composition were measured throughout study. Blood glucose and ketones were measured by glucometer and ketometer. Ketogenic potential was assessed using octanoate challenge after fasting. Liver mRNA levels were measured using qPCR. Hepatic IL-6 signaling was assessed using qPCR and ELISA for plasma IL-6. Results: PDAC mice maintained food intake and wheel running for 8 days after tumor injection, before progressive decline. Loss of adipose mass in PDAC mice occurred only in the context of decreased nutrition, whereas loss of lean mass preceded decreases in food intake. Overnight fasting mitigated differences in fat mass between groups, whereas the effect of fasting on skeletal muscle loss was similar in PDAC and control mice. Fasting blood glucose and ketogenic potential were lower in PDAC mice, suggesting impaired hepatic response to metabolic stress. In the liver, glycolytic genes were increased, whereas gluconeogenic and ketogenic genes were decreased in PDAC mice. Wasting and hepatic reprogramming occurred independently of hepatic IL-6 signaling. Conclusions: Undernutrition drives PDAC-associated adipose wasting, yet muscle wasting occurs prior to changes in food intake and in the absence of systemic inflammation. Hepatic macronutrient partitioning and metabolic gene expression are altered in PDAC, implicating impaired adaptive responses to negative energy balance in PDAC-associated cachexia. Citation Format: Heike Mendez, Xinxia Zhu, Brennan Olsen, Daniel L. Marks, Aaron J. Grossberg. Impaired adaptation to negative energy balance in pancreatic cancer-associated wasting [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C19.

PURPOSE: With recent advances in immunotherapy, many novel cancer treatments are rapidly entering the clinical arena. However, immunotherapies may differ from traditional chemotherapies in their effects on cachexia and treatment-associated sickness. Cachexia is a common comorbidity of cancer that limits therapeutic options, decreases quality of life, and increases mortality risk. Many chemotherapy agents induce or worsen cachexia by independently causing anorexia, weight loss, muscle wasting, and fatigue. Acute systemic inflammation, an effect of many immunotherapies, results in sickness responses that are typically self-limited in healthy individuals. Whether immunotherapy-associated sickness is self-limited or chronic in the context of cancer, and how cachexia is impacted by immunotherapy, remains unknown. METHODS: We recently found that the TLR7/8 agonist R848 reduces tumor size in mice implanted with epithelial cells from a syngeneic KRASG12D/+ P53R172H/+ Pdx-Cre (KPC) pancreatic tumor. To assess cachexia outcomes and R848-induced sickness, mice were orthotopically implanted with KPC cells or saline, and 2 days later were randomized to daily IP R848 (10µg) or vehicle until sacrifice (n=5-7/group/experiment, 3 experiments). Mice were tracked for weight, food intake, body composition, and locomotor activity (LMA), with end-stage analysis of tissue mass and gene expression. RESULTS: Initially, KPC-bearing mice treated with R848 developed significant weight gain and ascites, but had a similar degree of anorexia and decreased LMA as vehicle-treated KPC-bearing mice. However, ongoing R848 resulted in subsequent tumor regression, decreased ascites, increased appetite, and increased LMA. At necropsy, KPC-bearing mice treated with R848 had a 50-70% reduction in tumor mass, histologically characterized by lymphocytic infiltrate and germinal centers. Furthermore, R848-treated KPC-bearing mice had improved total lean mass and heart mass; decreased expression of genes related to skeletal and cardiac muscle catabolism (Mafbx, Murf1, Foxo1, Bnip3, Gabarapl, Ctsl) and hepatic acute phase reactants (Orm1, Apcs); a trend toward decreased CNS inflammatory gene expression (Selp, Il1r1); and unchanged brown adipose tissue thermogenic gene expression (Ucp1). In sham-operated mice, R848 resulted in self-limited anorexia and weight loss, without muscle wasting or decreased LMA. Current work is underway to elucidate tumor intrinsic and tumor extrinsic mechanisms of R848 in this model. SUMMARY: These studies show that R848 does not independently cause sustained sickness, and in a murine model of pancreatic cancer, can induce antitumor responses and improve cachexia outcomes. This represents a key difference from many cytotoxic chemotherapies and suggests immunotherapy approaches may be useful in the treatment of cachexia-associated malignancies. Citation Format: Katherine A. Michaelis, Mason A. Norgard, Xinxia Zhu, Peter R. Levasseur, Katherine R. Pelz, Kevin G. Burfeind, Terry K. Morgan, Daniel L. Marks. The TLR7/8 agonist R848 induces antitumor responses and attenuates cachexia in a murine model of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3779.

The central melanocortin system plays a key role in the regulation of food intake and energy homeostasis. We investigated whether genetic or pharmacologic blockade of central melanocortin signaling attenuates cardiac cachexia in mice and rats with heart failure. Permanent ligation of the left coronary artery (myocardial infarction (MI)) or sham operation was performed in wild-type (WT) or melanocortin-4 receptor (MC4R) knockout mice. Eight weeks after surgery, WT-Sham mice had significant increases in lean body mass (LBM; PPPPPPP

Chronic kidney disease (CKD) is associated with an increase in inflammatory cytokines and can result in cachexia with loss of muscle and fat stores. We previously demonstrated the efficacy of treating a model of cancer cachexia with ghrelin and a ghrelin receptor agonist. Currently, we examine a surgical model of CKD in rats, resulting in uremia and decreased accrual of lean body mass. Treatment with ghrelin and two ghrelin receptor agonists (BIM-28125 and BIM-28131) resulted in increased food intake and an improvement in lean body mass accrual that was related in part to a decrease in muscle protein degradation as assessed by muscle levels of the 14-kDa actin fragment resulting from cleaved actomyosin. Additionally, there was a decrease in circulating inflammatory cytokines in nephrectomized animals treated with ghrelin relative to saline treatment. Ghrelin-treated animals also had a decrease in the expression of IL-1 receptor in the brainstem and a decrease in expression of prohormone convertase-2, an enzyme involved in the processing of proopiomelanocortin to the anorexigenic peptide α-MSH. We conclude that ghrelin treatment in uremia results in improved lean mass accrual in part due to suppressed muscle proteolysis and possibly related to antiinflammatory effects.

Cancer cachexia is a syndrome of weight loss that results from the selective depletion of skeletal muscle mass and contributes significantly to cancer morbidity and mortality. The driver of skeletal muscle atrophy in cancer cachexia is systemic inflammation arising from both the cancer and cancer treatment. While the importance of tumor derived inflammation is well described, the mechanism by which cytotoxic chemotherapy contributes to cancer cachexia is relatively unexplored. We found that the administration of chemotherapy to mice produces a rapid inflammatory response. This drives activation of the hypothalamic-pituitary-adrenal axis, which increases the circulating level of corticosterone, the predominant endogenous glucocorticoid in rodents. Additionally, chemotherapy administration results in a significant loss of skeletal muscle mass 18 hours after administration with a concurrent induction of genes involved with the ubiquitin proteasome and autophagy lysosome systems. However, in mice lacking glucocorticoid receptor expression in skeletal muscle, chemotherapy-induced muscle atrophy is completely blocked. This demonstrates that cytotoxic chemotherapy elicits significant muscle atrophy driven by the production of endogenous glucocorticoids. Further, it argues that pharmacotherapy targeting the glucocorticoid receptor, given in concert with chemotherapy, is a viable therapeutic strategy in the treatment of cancer cachexia.