Abstract Hepatocellular carcinoma (HCC) is the most common form of liver cancer, with an estimated incidence of over one million cases by 2025. The advances in vaccines against HBV and HCV have led to a decrease in the number of virus-related cases; however, the obesity epidemic has contributed to a drastic increase in metabolic dysfunction-associated steatohepatitis (MASH) related HCC cases (e.g., obesity and type 2 diabetes induced HCC). Considering this trend, we aim to understand how lifestyle choices, like food composition, in combination with alcohol consumption at a “social drinking level” impacts host metabolism, and subsequently the development of MASH-induced HCC. We hypothesize that the impact of dietary composition on dysbiosis and immune cells, particularly adaptive immune cell metabolic status, will drive the phenotype. We have developed a MASH-induced HCC murine model, with long-term dietary intervention including seven different customized diets: normal chow (NC), ketogenic, solid high fat diet (sHFD), or isocaloric liquid Lieber DeCarli Diets (LDC): high fat/low carb (HF), high fat/low carb with ethanol (HF-EtOH), low fat/high carb (LF), and low fat/high carb with ethanol (LF-EtOH). Mice began special diet in late adolescences and disease progression was monitored over the course of a year, where mice developed HCC. Our model allowed for a comprehensive understanding of MASH-induced HCC where disease initiation could be studied beyond known contributors like obesity or body mass index (BMI). The animal model was analyzed comprehensively with high-parameter flow cytometry (FC), 16S sequencing, bulk RNA-seq, scRNA/ATAC-seq, and WGS. Long-term diet intervention led to macronutrient specific microbiota dysregulation, alterations in systemic metabolism and suppression of adaptive immune response, consequently impacting the progression of HCC. Customized dietary interventions showed a specific phenotype regarding HCC development and progression with distinct mutational genotype and particular response to immunotherapy. T and B cell subpopulation deficiency had a significant impact on MASH-induced HCC development in a macronutrient composition dependent manner. In summary, each dietary intervention showed a distinct pattern and effect on dysbiosis, systemic metabolic reprograming, cellular transcriptomic and epigenetic alteration, and particularly immune cell effector function. Additionally, some compositions supported the immuno-escape mechanism of malignant cells through regulation of their MHC machinery. Collectively, these accumulated data construct an atlas for understanding the underlying molecular mechanisms contributing to lifestyle-induced HCC providing the knowledge needed to advance effective strategies for treatment and prevention. Citation Format: Nicolas T. Ryujin, Jian Huang, Albert Nguyen, Jared Edwards, Yuhe Cheng, Jessica Wen, Ashish Damania, Nadim Ajami, Spencer Rosario, Mark Long, Ludmil Alexandrov, Shabnam Shalapour. Macronutrient composition dictates MASH-associated HCC immunosurveillance through microbiota alteration and metabolic adaptation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3974.

INTRODUCTION:Sternal intraosseous (IO) access has advantages over humeral and tibial access for fluid resuscitation in military medical settings due to superior flow rates and pharmacokinetics. However, the morphology of the young adult manubrial cortical and medullary bone as it relates to IO catheter tips of currently available FDA-approved IO access devices is unclear. MATERIALS AND METHODS:Computed tomography manubrium images of active duty service members (N = 93) aged 18 to 30 were assessed by radiologists, including manubrial height, width, and cortical thickness, as well as total thickness and medullary thickness at the thickest (superior) and thinnest (inferior) parts of the manubrium. Inference regarding medullary or cortical IO catheter tip placement rates were made for FAST1 (5.5 mm catheter depth) and TALON (4.5 mm catheter depth) sternal IO devices. Data were analyzed using ANOVA, non-parametric, and correlational statistics at P < 0.05. RESULTS:As inferred from case-specific CT-derived manubrium dimensions and maximal catheter tip penetration depths, both FAST1 and TALON would be placed in the target medullary space on 100% of opportunities if properly applied in the superior manubrium and on 99% of opportunities if properly applied in the inferior manubrium. Age was not significantly related to IO-relevant manubrial dimensions. CONCLUSIONS:Present findings suggest that both FAST1 and TALON sternal IO catheter tips can be successfully placed into the target medullary bone with high accuracy in male military members aged 18 to 30 who require rapid resuscitation.

BackgroundSarcopenia is prognostic for survival in patients with head and neck cancer (HNC). However, identification of this high-risk feature remains challenging without computed tomography (CT) imaging of the abdomen or thorax. Herein, we establish sarcopenia thresholds at the C3 level and determine if C3 sarcopenia is associated with survival in patients with HNC. MethodsThis retrospective cohort study was conducted in consecutive patients with a squamous cell carcinoma of the head and neck with cross-sectional abdominal or neck imaging within 60 days prior to treatment. Measurement of cross-sectional muscle area at L3 and C3 levels was performed from CT imaging. Primary study outcome was overall survival. ResultsSkeletal muscle area at C3 was strongly correlated with the L3 level in both men (n = 188; r = 0.77; p < 0.001) and women (n = 65; r = 0.80; p < 0.001), and C3 sarcopenia thresholds of 14.0 cm(2)/m(2) (men) and 11.1 cm(2)/m(2) (women) were best predictive of L3 sarcopenia thresholds. Applying these C3 thresholds to a cohort of patients with neck imaging alone revealed that C3 sarcopenia was independently associated with reduced overall survival in men (HR = 2.63; 95% CI, 1.79, 3.85) but not women (HR = 1.18, 95% CI, 0.76, 1.85). ConclusionsThis study identifies sarcopenia thresholds at the C3 level that best predict L3 sarcopenia in men and women. In HNC, C3-defined sarcopenia is associated with poor survival outcomes in men, but not women, suggesting sarcopenia may differentially affect men and women with HNC.

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.

Voluntary wheel running is a valuable metabolic intervention and well-established measure of physical activity in preclinical rodent models. Herein, we describe detailed assembly instructions and provide necessary resources for researchers to build their own running wheels from commercial-off-the-shelf parts and an open-source program at approximately a tenth of the cost of commercially-available options.

Proteins including glycoproteins and lectins play important roles in many biological processes. Therefore, they are vigorously studied in academic, clinical and industrial research. Such research activities often need these proteins in their purest forms. Thus, protein purification constitutes an important step in many scientific projects. Conventional protein purification techniques include affinity chromatography, ion-exchange chromatography and size-exclusion chromatography, and electrophoresis. These techniques have their own limitations. Conventional approaches are generally tedious, multi-step, expensive and time consuming. They generally require elaborate infrastructure and larger starting crude materials. In addition, these techniques sometimes encounter non-specific binding. In order to overcome some of the limitations associated with these conventional methods, our lab developed a protein detection/purification method named “Capture and Release” (CaRe). In this method, a target capturing agent (TCA) captures a specific target (lectin or glycoprotein) in the crude solution and form insoluble complex. The complex is spun down while the other unwanted proteins are washed off. Captured target is released from the TCA by the addition of competitive monovalent ligand, separated by membrane filtration and visualized by gel electrophoresis. We were successful in purifying recombinant human Galectin-3 by CaRe. This method was able to purify glycoproteins as well. CaRe was validated by purifying known lectins and glycoproteins. Compare to conventional techniques, our method is relatively fast, simple, precise and less expensive and it can detect/purify lectins and glycoproteins even from a small volume (~1 ml) of starting material. Thus CaRe can serve as a valuable tool to discover unknown proteins and glycoproteins.

BACKGROUND/OBJECTIVE:Peripheral-nerve blocks (PNBs) using continuous-infusion of local anesthetics are used to provide perioperative analgesia. Yet little research exists to characterize the histopathological effects of continuous long-duration PNBs. Herein we test the hypothesis that continuous peri-neural bupivacaine infusion (3-day vs. 7-day infusion) contributes to histologic injury in a duration-dependent manner using an in vivo model of rat sciatic nerves. METHODS:We placed indwelling catheters in 22 rats for infusion with low-dose (0.5mg/kg/hr) bupivacaine or normal saline proximal to the right sciatic nerves for 3 or 7 consecutive days. Hind-limb analgesia was measured using Von-Frey nociceptive testing. At infusion end, rats were sacrificed, bilateral nerves were sectioned and stained with hematoxylin and eosin and CD68 for evaluation of inflammatory response, and eriochrome to assess damage to myelin. RESULTS:Animals receiving continuous infusion of bupivacaine maintained analgesia as demonstrated by significant decrease (50% on average) in nociceptive response in bupivacaine-infused limbs across time points. Both 7-day saline and bupivacaine-infused sciatic nerves showed significantly-increased inflammation by H&E staining compared to untreated native nerve controls (P = 0.0001, P < 0.0001). Extent of inflammation did not vary significantly based on infusate (7-day saline vs. 7-day bupivacaine P > 0.99) or duration (3-day bupivacaine vs 7-day bupivacaine P > 0.99). No significant change in sciatic nerve myelin was found in bupivacaine-infused animals compared to saline-infused controls, regardless of duration. CONCLUSIONS:Long-duration (7-day) bupivacaine infusion provided durable post-operative analgesia, yet contributed to equivalent neural inflammation as short duration (3-day) infusion of bupivacaine or saline with no evidence of demyelination.

BACKGROUND AND PURPOSE:Sarcopenia is associated with decreased survival in head and neck cancer patients treated with radiotherapy. This study sought to determine whether in-clinic multifrequency bioelectrical impedance analysis (BIA) can identify survival-associated sarcopenia in patients with head and neck cancer. MATERIALS AND METHODS:This prospective observational study enrolled 50 patients with head and neck cancer undergoing radiation therapy. Baseline BIA measures of skeletal muscle (SM) mass, fat-free mass (FFM), and fat mass (FM) were compared to CT-based estimates using linear regression. Sex-specific BIA-derived thresholds for sarcopenia were defined by the maximum Youden Index on receiver operator characteristic (ROC) curves. Patients were stratified by sarcopenia status and OS was compared using the Kaplan-Meier method and log-rank test. RESULTS:Among 48 evaluable patients, BIA measures of body composition were strongly correlated with CT measures: SM mass (r = 0.97; R2 = 0.94; p < 0.0001), FFM (r = 0.97; R2 = 0.94; p < 0.0001) and FM (r = 0.95; R2 = 0.90; p < 0.0001). SM mass index < 9.19 kg/m2 identified sarcopenia men with high sensitivity (91.7%) and specificity (92.9%), whereas in women SM mass index < 6.53 kg/m2 was sensitive for sarcopenia (100%), but not specific. Patients with sarcopenia, defined by either CT or BIA, exhibited decreased OS (HR = not estimable; CT p = 0.009; BIA p = 0.03). CONCLUSION:BIA provides accurate estimates of body composition in head and neck cancer patients. Implementation of BIA in clinical practice may identify patients with sarcopenia at risk for poor survival.

Abstract BACKGROUND The female predominance of meningiomas may reflect hormonal influences on meningioma development, with known presence of estrogen and progesterone receptors. Progestin-associated meningiomas demonstrate a shift in the mutational landscape and are more frequently located at the skull base. Obesity and increased adipocytes increase aromatase and affect male hormone synthesis, thus increases circulating estrogen. OBJECTIVE Report prevalence of obesity in a consecutive series of male patients presenting with meningioma and the interaction with obesity. METHODS A retrospective review (20012019) was performed of male patients diagnosed with meningioma. Body mass index (BMI) >30 kg/m2 was considered obese. Obese male meningioma patients were characterized and compared to nonobese male meningioma patients. RESULTS Of 239 male patients with meningioma, 97 (40.6%) were obese at the time of surgery with overall mean BMI (30.0 kg/m2). This is above the age-adjusted baseline prevalence of obesity for males in Oregon, which was 34.8% between 2015–2016. Mean age at diagnosis for obese males was 55.3 years and 57.0 years for nonobese males. Within the obese group, most lesions were located on the skull base (48.5% n=47) compared to 34.0% convexity (n=33), 15.4% falx (n=16) and 1.0% multiple (n=1). Tumors were WHO grade I (73.6%), grade II (25.6%) or grade III (1.2%). Of the men in our series, patients with skull base meningiomas were more likely to be obese (OR: 1.7, 95% CI: 1.01–2.9, p=0.0309). CONCLUSIONS There is likely a hormonal influence on the pathogenesis of meningioma, and many men diagnosed with this disease are obese. Of 239 male patients with meningioma in our series, 97 (40.6%) were obese which exceeds the 34% age-adjusted baseline prevalence of obesity for males in Oregon. Patients with skull base meningiomas were more likely to be obese, consistent with prior series and suggestive of a hormone-driven association with skull base meningioma.

Stedelin, Brittany; Pangelinan, Kelsey; Edwards, Jared; Cheaney, Barry II BS; Rai, Karan; Raslan, Ahmed M.T; Ernst, Lia Author Information

ImportanceThe negative association of low lean muscle mass (sarcopenia) with survival outcomes in head and neck cancers, including oropharyngeal carcinoma, is established. However, it is not known whether the choice of primary treatment modality (surgery or radiotherapy) is associated with oncologic outcomes of patients with sarcopenia and oropharyngeal squamous cell carcinoma (OPSCC).ObjectiveTo examine whether primary surgical resection or definitive radiotherapy is associated with improved survival for patients with sarcopenia and localized OPSCC.Design, Setting, and ParticipantsA cohort study was conducted of patients with clinically staged T1 to T2, N0 to N2 OPSCC with cross-sectional abdominal imaging within 60 days prior to treatment and treated between January 1, 2005, and December 31, 2017. Skeletal muscle mass was measured at the third lumbar vertebra using previously defined techniques and sarcopenia was defined as less than 52.4 cm2/m2 of muscle for men and less than 38.5 cm2/m2 for women. In addition, associated patient demographic characteristics, cancer data, treatment information, and survival outcomes were assessed. Statistical analysis was performed from December 3, 2018, to August 28, 2019.Main Outcomes and MeasuresPrimary outcomes were overall survival and disease-specific survival.ResultsAmong the 245 patients who met study inclusion criteria, 209 were men (85.3%) and the mean (SD) age was 62.3 (7.8) years. Sarcopenia was detected in 135 patients (55.1%), while normal skeletal muscle mass was detected in 110 patients (44.9%). For the 110 patients without sarcopenia, primary treatment modality was not associated with improved survival. For patients with sarcopenia at diagnosis, primary surgical resection was associated with improved overall survival (hazard ratio [HR], 0.37; 95% CI, 0.17-0.82) and disease-specific survival (HR, 0.22; 95% CI, 0.07-0.68). This association persisted after propensity score matching, as up-front surgery was associated with improved overall survival (HR, 0.33; 95% CI, 0.12-0.91) and disease-specific survival (HR, 0.17; 95% CI, 0.04-0.75) survival.Conclusions and RelevanceThis study suggests that sarcopenia has a negative association with survival for patients with OPSCC. Primary surgery and radiotherapy confer similar survival associations for patients with normal skeletal muscle mass and localized OPSCC. However, up-front surgical resection may be associated with improved survival outcomes for patients with sarcopenia.

A 50-year-old female with a 20-year history of multifocal pulmonary benign metastasizing leiomyoma (BML), and asthma presented with subacute worsening of chronic dyspnea. A contrast-enhanced computerized tomography of the chest showed a single 1.4 × 1.5-cm contrast-enhancing mass in the right lower lobe among numerous non-enhancing bilateral pulmonary BML lesions. Pulmonary angiogram was not performed at that time due to clinical improvement. Four years later, the patient presented with refractory subacute worsening of her chronic dyspnea and was referred for embolization of the pulmonary arteriovenous malformation (PAVM). Two feeder arteries to the PAVM were embolized; each with a 6-mm Amplatzer-IV vascular plug and a 4-mm Nester coil. Follow-up angiograms demonstrated no flow through the PAVM. The patient's dyspnea resolved and she remained asymptomatic at one-year follow-up.

Stedelin, Brittany; Pangelinan, Kelsey; Edwards, Jared; Cheaney, Barry II BS; Raslan, Ahmed M.T; Ernst, Lia Author Information

Hemolysins are natural cytotoxic agents that are capable of destabilizing plasma membrane structures of erythrocytes. We recently identified a plant hemolysin named H e mo l y sin‐ X (HelyX) that showed dual specificity for cholesterol and certain serum glycoproteins, such as asialofetuin and fibrinogen. HelyX was isolated through two different approaches: protein purification protocols as well as organic solvent extraction. Ligand binding properties of HelyX were elucidated by inhibition assays using microtiter plates as well as spectrophotometer. HelyX demonstrated significantly fast kinetics (30–180 seconds) of lysis and ligand binding. Scanning electron microscopic imaging captured distinct pores on the surface of HelyX‐treated erythrocytes. Our results indicate that HelyX disintegrates mammalian red blood cells by forming pores on their surfaces. Support or Funding Information Michigan Technological University This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Structural degradation is detrimental for protein activity and functions. We recently found that the tumor‐associated protein galectin‐3 (Gal‐3) is apparently an exception to this rule. Gal‐3 plays important roles in cancer but its functions are not fully understood. We have found that the affinity purified Gal‐3, when stored with lactose at 4°C for more than six weeks, undergoes complete degradation even in the absence of collagenases. Full‐length Gal‐3 after full degradation produced a single protein band of 17 kDa. This molecular weight is similar to that of the CRD (carbohydrate recognition domain) portion of Gal‐3. Conventionally, the CRD is separated from intact Gal‐3 by enzymatic digestion using collagenases. However, in the present case, Gal‐3 apparently self‐degraded to produce the CRD without the catalytic action of collagenases. Our data indicate that degradation of Gal‐3 significantly alters the protein but the CRD remains intact and functionally active. Like the full‐length Gal‐3, the CRDs interacted with thyroglobulin, chondroitin sulfate A and C. However, the CRDs could not cross‐link these multivalent ligands as revealed by our spectroscopic analysis. Our current observations suggest that Gal‐3 retains it ligand recognition properties even when it is substantially degraded in a challenging cellular environment. Thus the degraded Gal‐3 can still carry out some of the functions of intact Gal‐3. Although the CRDs retain the binding property of its intact precursor (Gal‐3), they lose the ability to cross‐link cellular receptors. As a result, the CRDs generated through degradation may influence cell signaling by competing with intact Gal‐3 for the same receptors in cellular environment.Support or Funding InformationNational Science FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

AbstractImportanceDepleted skeletal muscle mass (sarcopenia) is associated with decreased survival and cancer control in head and neck cancer patients treated with radiotherapy. There is a need for validated measures of body composition that can be implemented in routine clinical workflow.ObjectiveTo validate the use of bioelectrical impedance analysis (BIA) for body composition analysis and diagnosis of sarcopenia in head and neck cancer patients.DesignIn this prospective observational cohort study, baseline 50 patients with head and neck cancer undergoing radiation therapy (RT) were enrolled between February 2016 and March 2017. Baseline BIA measures of skeletal muscle (SM) mass, fat-free mass (FMM), and fat mass (FM) were compared to CT-based estimates of body composition using linear regression. Sex-specific BIA-derived thresholds for sarcopenia were defined by the maximum Youden Index on receiver operator characteristic (ROC) curves of BIA against CT-defined sarcopenia. Changes in body composition across treatment were compared against changes in body weight using linear regression.ParticipantsIn total, 50 patients with pathologically confirmed stage I to IVB non-metastatic head and neck cancer treated with definitive radiation therapy were enrolled.SettingSingle academic referral center.Main Outcome and MeasureThe primary outcome was relative agreement between baseline lean body mass and fat body mass predicted from BIA measurement and CT imaging.ResultsOf the 48 evaluable patients 16 (33.3%) were sarcopenic at baseline based on CT analysis. BIA measures of body composition were strongly correlated with CT measures: SM mass (r = 0.97; R2 = 0.94; p < 0.0001), FFM (r = 0.97; R2 = 0.94; p < 0.0001) and FM (r = 0.95; R2 = 0.90; p < 0.0001). Relationship with normalized indices of SM mass, FFM, and FM was similar between BIA and CT, but not BIA and body mass index (BMI). Patients lost a mean of 5.7 ± 5.8 kg during treatment, of which 1.5 ± 1.9 kg was SM, 2.6 ± 3.3 kg was FFM, and 2.2 ± 2.6 kg was FM. Eight additional patients developed sarcopenia by the end of RT.ConclusionsBIA provides accurate estimates of body composition in head and neck cancer patients. Implementation of BIA in clinical practice may identify patients with sarcopenia.Trial RegistrationClinicalTrials.gov identifier: NCT02615275