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The ability to cryopreserve natural killer (NK) cells has a significant potential in modern cancer immunotherapy. Current cryopreservation protocols cause deterioration in NK cell viability and functionality. This work reports the preservation of human cytokine‐activated NK cell viability and function following cryopreservation using a cocktail of biocompatible bioinspired cryoprotectants (i.e., dextran and carboxylated ε‐poly‐L‐lysine). Results demonstrate that the recovered NK cells after cryopreservation and rewarming maintain their viability immediately after thawing at a comparable level to control (dimethyl sulfoxide‐based cryopreservation). Although, their viability drops in the first day in culture compared to controls, the cells grow back to a comparable level to controls after 1 week in culture. In addition, the anti‐tumor functional activity of recovered NK cells demonstrates higher cytotoxic potency against leukemia cells compared to control. This approach presents a new direction for NK cell preservation, focusing on function and potentially enabling storage and distribution for cancer immunotherapy. Cryopreservation of natural killer (NK) cells has a potential to create great impact on modern cancer immunotherapy. Current cryopreservation methods utilize toxic cryoprotectants (e.g., dimethyl sulfoxide) causing deterioration in NK cell viability and functionality. An innovative approach to preserve human NK cell viability and functionality following cooling and rewarming is developed using a cocktail of biocompatible bioinspired cryoprotectants.

Advances in precision molecular imaging promise to transform our ability to detect, diagnose and treat disease. Here, we describe the engineering and validation of a new cystine knot peptide (knottin) that selectively recognizes human integrin αvβ 6 with single-digit nanomolar affinity. We solve its 3D structure by NMR and x-ray crystallography and validate leads with 3 different radiolabels in pre-clinical models of cancer. We evaluate the lead tracer’s safety, biodistribution and pharmacokinetics in healthy human volunteers, and show its ability to detect multiple cancers (pancreatic, cervical and lung) in patients at two study locations. Additionally, we demonstrate that the knottin PET tracers can also detect fibrotic lung disease in idiopathic pulmonary fibrosis patients. Our results indicate that these cystine knot PET tracers may have potential utility in multiple disease states that are associated with upregulation of integrin α v β 6 . Knottin is a cystine knot peptide. Here, the authors develop a knottin-based tracer for positron emission tomography and demonstrate its ability to detect cancer and idiopathic pulmonary fibrosis through selective binding to integrin αvβ 6 .

BackgroundOncogenic ‘hotspot’ mutations of KRAS and GNAS are two major driver alterations in intraductal papillary mucinous neoplasms (IPMNs), which are bona fide precursors to pancreatic ductal adenocarcinoma. We previously reported that pancreas-specific Kras G12D and Gnas R201C co-expression in p48Cre; KrasLSL-G12D; Rosa26LSL-rtTA; Tg (TetO-GnasR201C) mice (‘Kras;Gnas’ mice) caused development of cystic lesions recapitulating IPMNs.ObjectiveWe aim to unveil the consequences of mutant Gnas R201C expression on phenotype, transcriptomic profile and genomic dependencies.DesignWe performed multimodal transcriptional profiling (bulk RNA sequencing, single-cell RNA sequencing and spatial transcriptomics) in the ‘Kras;Gnas’ autochthonous model and tumour-derived cell lines (Kras;Gnas cells), where Gnas R201C expression is inducible. A genome-wide CRISPR/Cas9 screen was conducted to identify potential vulnerabilities in KrasG12D;GnasR201C co-expressing cells.ResultsInduction of Gnas R201C—and resulting G(s)alpha signalling—leads to the emergence of a gene signature of gastric (pyloric type) metaplasia in pancreatic neoplastic epithelial cells. CRISPR screening identified the synthetic essentiality of glycolysis-related genes Gpi1 and Slc2a1 in Kras G12D;Gnas R201C co-expressing cells. Real-time metabolic analyses in Kras;Gnas cells and autochthonous Kras;Gnas model confirmed enhanced glycolysis on Gnas R201C induction. Induction of Gnas R201C made Kras G12D expressing cells more dependent on glycolysis for their survival. Protein kinase A-dependent phosphorylation of the glycolytic intermediate enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) was a driver of increased glycolysis on Gnas R201C induction.ConclusionMultiple orthogonal approaches demonstrate that Kras G12D and Gnas R201C co-expression results in a gene signature of gastric pyloric metaplasia and glycolytic dependency during IPMN pathogenesis. The observed metabolic reprogramming may provide a potential target for therapeutics and interception of IPMNs.

Abstract Advances in precision molecular imaging promise to transform our ability to detect, diagnose and treat disease. Here, we describe the engineering and validation of a new cystine knot peptide (knottin) that selectively recognizes human integrin αvβ 6 with single-digit nanomolar affinity. We solve its 3D structure by NMR and x-ray crystallography and validate leads with 3 different radiolabels in pre-clinical models of cancer. We evaluate the lead tracer’s safety, biodistribution and pharmacokinetics in healthy human volunteers, and show its ability to detect multiple cancers (pancreatic, cervical and lung) in patients at two study locations. Additionally, we demonstrate that the knottin PET tracers can also detect fibrotic lung disease in idiopathic pulmonary fibrosis patients. Our results indicate that these cystine knot PET tracers may have potential utility in multiple disease states that are associated with upregulation of integrin α v β 6 .

Objectives To compare mesoscopic epi-fluorescence tomography (MEFT) and EPRI-illumination reflectance imaging (EPRI) for quantitative tumour size assessment in mice. Methods Tumour xenografts of green/red fluorescent protein (GFP/RFP)-expressing colon cancer cells were measured using MEFT, EPRI, ultrasound (US) and micro computed tomography (μCT) at day 14 post-injection ( n  = 6). Results from MEFT and EPRI were correlated with each other and with US and μCT (reference methods). Tumour volumes were measured ex vivo by GFP and RFP fluorescence imaging on cryoslices and compared with the in vivo measurements. Results High correlation and congruency were observed between MEFT, US and μCT (MEFT/US: GFP: r 2  = 0.96; RFP: r 2  = 0.97, both P  < 0.05; MEFT/μCT: GFP: r 2  = 0.93; RFP: r 2  = 0.90; both P  < 0.05). Additionally, in vivo MEFT data were highly correlated and congruent with ex vivo cryoslice imaging results (GFP: r 2  = 0.96; RFP: r 2  = 0.99; both P  < 0.05). In comparison, EPRI significantly overestimated tumour volumes ( P  < 0.05), although there was a significant correlation with US and μCT (EPRI/US: GFP: r 2  = 0.95; RFP: r 2  = 0.94; both P  < 0.05; EPRI/μCT GFP: r 2  = 0.86; RFP: r 2  = 0.86; both P  < 0.05). Conclusions Fluorescence distribution reconstruction using MEFT affords highly accurate three-dimensional (3D) tumour volume data showing superior accuracy compared to EPRI. Thus, MEFT is a very suitable technique for quantitatively assessing fluorescence distribution in superficial tumours at high spatial resolution. Key Points • Mesoscopic epi-fluorescence tomography (MEFT) is an important new molecular imaging technique . • MEFT allows accurate size determination of superficial tumours with high resolution. • MEFT is a suitable technique for longitudinal assessment of tumour growth. • MEFT allows 3D reconstruction and quantification of fluorescence distributions.

Advances in precision molecular imaging promise to transform our ability to detect, diagnose and treat disease. Here, we describe the engineering and validation of a new cystine knot peptide (knottin) that selectively recognizes human integrin αvβ with single-digit nanomolar affinity. We solve its 3D structure by NMR and x-ray crystallography and validate leads with 3 different radiolabels in pre-clinical models of cancer. We evaluate the lead tracer's safety, biodistribution and pharmacokinetics in healthy human volunteers, and show its ability to detect multiple cancers (pancreatic, cervical and lung) in patients at two study locations. Additionally, we demonstrate that the knottin PET tracers can also detect fibrotic lung disease in idiopathic pulmonary fibrosis patients. Our results indicate that these cystine knot PET tracers may have potential utility in multiple disease states that are associated with upregulation of integrin α β .

Abstract RNF43 is an E3 ubiquitin ligase that is recurrently mutated in pancreatic ductal adenocarcinoma (PDAC) and precursor cystic neoplasms of the pancreas. The impact of RNF43 mutations on PDAC is poorly understood and autochthonous models have not been sufficiently characterized. In this study we describe a genetically engineered mouse model (GEMM) of PDAC with conditional expression of oncogenic Kras and deletion of the catalytic domain of Rnf43 (KRC) in exocrine cells. We demonstrate that Rnf43 loss results in an increased incidence of high-grade cystic lesions of the pancreas and PDAC. Importantly, KRC mice have a significantly decreased survival compared to mice containing only an oncogenic Kras mutation. By use of single cell RNA sequencing we demonstrated that KRC tumor progression is accompanied by a decrease in macrophages, as well as an increase in T and B lymphocytes with evidence of increased immune checkpoint molecule expression and affinity maturation, respectively. This was in stark contrast to the tumor immune microenvironment observed in the Kras/Tp53 driven PDAC GEMM. Furthermore, expression of the chemokine, CXCL5, was found to be specifically decreased in KRC cancer cells by means of epigenetic regulation and emerged as a putative candidate for mediating the unique KRC immune landscape. This GEMM establishes RNF43 as a bona fide tumor suppressor gene in PDAC and puts forth a rationale for an immunotherapy approach in this subset of PDAC cases. Competing Interest Statement The authors have declared no competing interest. Footnotes * Disclosures A.M. receives royalties for a pancreatic cancer biomarker test from Cosmos Wisdom Biotechnology, and this financial relationship is managed and monitored by the UTMDACC Conflict of Interest Committee. A.M. is also listed as an inventor on a patent that has been licensed by Johns Hopkins University to ThriveEarlier Detection. All other authors are free of conflicts of interest and have nothing to disclose. * Funding Sources AM is supported by NIH grants R01 CA220236 and R01 CA218004. AM is also funded by the Sheikh Khalifa Bin Zayed Foundation