A robust immunohistochemical (IHC) assay for VEGFR2 was developed to investigate its utility for patient tailoring in clinical trials. The sensitivity, specificity, and selectivity of the IHC assay were established by siRNA knockdown, immunoblotting, mass spectrometry, and pre-absorption experiments. Characterization of the assay included screening a panel of multiple human cancer tissues and an independent cohort of non-small cell lung carcinoma (NSCLC, n = 118) characterized by TTF-1, p63, CK5/6, and CK7 IHC. VEGFR2 immunoreactivity was interpreted qualitatively (VEGFR2 positive/negative) in blood vessels and by semi-quantitative evaluation using H-scores in tumor cells (0–300). Associations were determined among combinations of VEGFR2 expression in blood vessels and tumor cells, and clinico-pathologic characteristics (age, sex, race, histologic subtype, disease stage) and overall survival using Kaplan-Meier analyses and appropriate statistical models. VEGFR2 expression both in blood vessels and in tumor cells in carcinomas of the lung, cervix, larynx, breast, and others was demonstrated. In the validation cohort, 99/118 (83.9%) NSCLC tissues expressed VEGFR2 in the blood vessels and 46/118 (39.0%) showed high tumor cell positivity (H-score ≥10). Vascular and tumor cell expression were inversely correlated (p = 0.0175). High tumor cell expression of VEGFR2 was associated with a 3.7-fold reduction in median overall survival in lung squamous-cell carcinoma (SCC, n = 25, p = 0.0134). The inverse correlation between vascular and tumor cell expression of VEGFR2 and the adverse prognosis associated with high VEGFR2 expression in immunohistochemically characterized pulmonary SCC are new findings with potential therapeutic implications. The robustness of this novel IHC assay will support further evaluation of its utility for patient tailoring in clinical trials of antiangiogenic agents.

We have successfully developed sensitive assays to measure active and total FGF21, which show the majority of total FGF21 in plasma is active FGF21.

Background & Aim: Oxyntomodulin (Oxm) is a proglucagon-derived peptide agonist of both the GLP-1 and glucagon receptors and is a key regulator of gastric acid secretion and energy expenditure. Differential processing from proglucagon hinders assay immunoassay selectivity. Method & results: Antibody engineering was used to develop a sandwich immunoassay that selectively measures endogenous Oxm. The pre- and postprandial levels of Oxm from 19 healthy individuals over the course of 2 h were measured. Postprandial increases in Oxm occurred within minutes and levels significantly correlated with those obtained using previously published mass spectrometry assays. Conclusion: This sandwich immunoassay is appropriately sensitive and selective and is also amenable to high-throughput application for the reliable determination of endogenous levels of intact Oxm from human samples.

Cancer-induced cachexia is a hypermetabolic condition characterized by the unintentional wasting of muscle and adipose tissue, affecting over 80% of patients with pancreatic ductal adenocarcinoma (PDAC). Muscle wasting during cachexia is due to increased skeletal muscle protein degradation via ubiquitin-proteasome and autophagy-lysosome pathways. Autophagy-lysosome degradation requires delivery of cargo to the lysosome for destruction and recycling. Macroautophagy is the most prevalent component of autophagy, encompassing bulk and selective autophagy, and it requires the de novo synthesis of an autophagosome. Bulk autophagy randomly engulfs portions of the cytoplasm. Selective autophagy is mediated through selective autophagy receptors (SAR), which bind and couple cargo to the autophagosome via the general autophagy ligands LC3B and GABARAP. While an increase in the general autophagy machinery is well described in cachectic muscle, much less is known about how complexes and organelles are selectively targeted for degradation. Methods: Here, 12-week-old male C57BL/6J mice were orthotopically implanted with 1x105 KPC cells; controls underwent sham surgery. Half of the tumor-bearing mice were treated with 120 mg/kg gemcitabine and 10 mg/kg nab-paclitaxel (GemNP) at 4 and 10 days. For endpoint analysis, mice were euthanized at 14 days when KPC mice had significant body weight, muscle mass and muscle protein loss compared to SHAM controls. In our in vitro model of PDAC cachexia, KPC-conditioned media (CM) induces C2C12 myotube wasting; thus, myotubes were treated with 50% KPC-CM or control for 48hrs. Results: GemNP reduced end tumor mass by nearly 25% and prevented body weight and muscle loss. mRNAseq of gastrocnemius muscle demonstrated induction of ribosomal component gene expression, while deep proteomics revealed reduction of 30 ribosomal component proteins in KPC mice, consistent with ongoing destruction of ribosomes. Gene expression of general autophagy ligands, LC3B (2.9-fold) and GABARAP (1.6-fold), were increased in KPC mice. Gene expression for SARs associated with ribophagy (NUFIP1, 2.6-fold) and reticulophagy (Fam134b, 11.3-fold) were increased in KPC. FAM134b protein (1.8-fold) was also increased in KPC mice. SARs for lipophagy (PNPLA2, 3.2- and PNPLA8, 1.8-fold) and mitophagy (BNIP3, 4.5-fold) were induced in KPC versus SHAM, while SARs for glycophagy and ferritinophagy were similar. SQSTM1 (3.9-fold) and NBR1 (1.9-fold), general SARs for multiple organelles including aggrephagy, lysophagy, proteaphagy and pexophagy, were increased in KPC. This activation of SARs was due to tumor-induced wasting and not chemotherapy as SAR gene expression in KPC-GemNP mice was similar to SHAM controls. Finally, we also observe markers of SAR-mediated autophagy in our in vitro model of PDAC cachexia. Conclusion: These data indicate that muscle wasting in PDAC cachexia is through activation of selective autophagy of ribosomes, mitochondria, lipid droplets, endoplasmic reticulum, protein aggregates, lysosomes, peroxisomes, and proteasomes. Citation Format: Brittany R. Counts, Ashok Narasimhan, Tara S. Umberger, Emma H. Doud, Amber L. Mosley, Teresa A. Zimmers. Skeletal Muscle Selective Autophagy Receptors are induced PDAC Cachexia [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A062.