Metabolic reprogramming by mutant GNAS creates an actionable dependency in intraductal papillary mucinous neoplasms of the pancreas.

BACKGROUND: Oncogenic '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. OBJECTIVE: We aim to unveil the consequences of mutant Gnas R201C expression on phenotype, transcriptomic profile and genomic dependencies. DESIGN: We 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. RESULTS: Induction 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. CONCLUSION: Multiple 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.

Blockade of histamine receptor H1 augments immune checkpoint therapy by enhancing MHC-I expression in pancreatic cancer cells.

BACKGROUND: Although immune checkpoint blockade (ICB) therapy has proven to be extremely effective at managing certain cancers, its efficacy in treating pancreatic ductal adenocarcinoma (PDAC) has been limited. Therefore, enhancing the effect of ICB could improve the prognosis of PDAC. In this study, we focused on the histamine receptor H1 (HRH1) and investigated its impact on ICB therapy for PDAC. METHODS: We assessed HRH1 expression in pancreatic cancer cell (PCC) specimens from PDAC patients through public data analysis and immunohistochemical (IHC) staining. The impact of HRH1 in PCCs was evaluated using HRH1 antagonists and small hairpin RNA (shRNA). Techniques including Western blot, flow cytometry, quantitative reverse transcription polymerase chain reaction (RT-PCR), and microarray analyses were performed to identify the relationships between HRH1 and major histocompatibility complex class I (MHC-I) expression in cancer cells. We combined HRH1 antagonism or knockdown with anti-programmed death receptor 1 (αPD-1) therapy in orthotopic models, employing IHC, immunofluorescence, and hematoxylin and eosin staining for assessment. RESULTS: HRH1 expression in cancer cells was negatively correlated with HLA-ABC expression, CD8+ T cells, and cytotoxic CD8+ T cells. Our findings indicate that HRH1 blockade upregulates MHC-I expression in PCCs via cholesterol biosynthesis signaling. In the orthotopic model, the combined inhibition of HRH1 and αPD-1 blockade enhanced cytotoxic CD8+ T cell penetration and efficacy, overcoming resistance to ICB therapy. CONCLUSIONS: HRH1 plays an immunosuppressive role in cancer cells. Consequently, HRH1 intervention may be a promising method to amplify the responsiveness of PDAC to immunotherapy.

ERAP2 is a novel target involved in autophagy and activation of pancreatic stellate cells via UPR signaling pathway.

BACKGROUND/OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) is characterized by excessive desmoplasia and autophagy-dependent tumorigenic growth. Pancreatic stellate cells (PSCs) as a predominant stromal cell type play a critical role in PDAC biology. We have previously reported that autophagy facilitates PSC activation, however, the mechanism remains unknown. We investigated the mechanism of autophagy in PSC activation. METHODS: We compared gene expression profiles between patient-derived PSCs from pancreatic cancer and chronic pancreatitis using a microarray. The stromal expression of target gene in specimen of PDAC patients (n = 63) was analyzed. The effect of target gene on autophagy and activation of PSCs was investigated by small interfering RNAs transfection, and the relationship between autophagy and ER stress was investigated. We analyzed the growth and fibrosis of xenografted tumor by orthotopic models. RESULTS: In analysis of gene expression microarray, endoplasmic reticulum aminopeptidase 2 (ERAP2) upregulated in cancer-associated PSCs was identified as the target gene. High stromal ERAP2 expression is associated with a poor prognosis of PDAC patients. Knockdown of ERAP2 inhibited unfolded protein response mediated autophagy, and led to inactivation of PSCs, thereby attenuating tumor-stromal interactions by inhibiting production of IL-6 and fibronectin. In vivo, the promoting effect of PSCs on xenografted tumor growth and fibrosis was inhibited by ERAP2 knockdown. CONCLUSIONS: Our findings demonstrate a novel mechanism of PSCs activation regulated by autophagy. ERAP2 as a promising therapeutic target may provide a novel strategy for the treatment of PDAC.

Impact of early mobilization on discharge disposition and functional status in patients with subarachnoid hemorrhage: A retrospective cohort study.

ABSTRACT: Whereas early rehabilitation improves the patients' physical function in patients with cerebral infarction and hemorrhage, complications in the early stage are the main barriers in patients with subarachnoid hemorrhage (SAH). Therefore, the clinical impact of early rehabilitation in patients with SAH is not well documented. We sought to investigate whether early mobilization is associated with favorable discharge disposition and functional status in patients with SAH.Hospitalization data of 35 patients (65.7 ±â€Š13.7 years, 37.1% men) were retrospectively reviewed. The early and delayed mobilization groups were defined as those who had and had not participated in walking rehabilitation on day 14, respectively. We investigated whether patients were discharged or transferred to another hospital and assessed their functional status using the Functional Ambulation Categories, Ambulation Index, Glasgow Outcome Scale, and modified Rankin Scale scores.Nine patients (69.2%) in the early mobilization group and one patient (4.5%) in the delayed mobilization group were discharged home directly (P < .001). In multivariate logistic regression analysis, early mobilization was independently associated with home discharge after adjustment using the World Federation of Neurosurgical Societies grade (adjusted odds ratio = 30.20, 95% CI = 2.77-329.00, P < .01). Early mobilization was associated with favorable functional status at discharge through multivariate linear regression analysis (standardized beta = 0.64 with P < .001 for the Functional Ambulation Category and beta = -0.62 with P < .001 for the modified Rankin Scale, respectively).Early mobilization was associated with home discharge and favorable functional status at discharge. Larger prospective studies are warranted.

Repositioning of duloxetine to target pancreatic stellate cells.

Pancreatic cancer cells (PCCs) are surrounded by an abundant stroma, which is produced by pancreatic stellate cells (PSCs). PSCs promote tumor cell proliferation and invasion. The objective of the current study was to identify compounds that suppress PSC activation. Gene expression profiles of cancer-derived fibroblasts and normal fibroblasts were used, and the pathway analysis suggested altered pathways that were chosen for validation. It was found that the 'neuroactive ligand-receptor interaction' pathway from the Kyoto Encyclopedia of Genes and Genomes pathway analysis was one of the altered pathways. Several compounds related with this pathway were chosen, and changes in PSC activity were investigated using fluorescence staining of lipid droplets, reverse transcription-quantitative PCR, western blotting, and invasion and migration assays. Among these candidates, duloxetine, a serotonin-noradrenaline reuptake inhibitor, was found to suppress PSC activation and disrupt tumor-stromal interaction. Thus, duloxetine may be a potential drug for suppressing PSC activation and pancreatic cancer growth.

Efficient pre-treatment for pancreatic cancer using chloroquine-loaded nanoparticles targeting pancreatic stellate cells.

Pancreatic stellate cells (PSCs) play a key role in desmoplastic stroma, which is a characteristic of pancreatic ductal adenocarcinoma (PDAC), and they also enhance the malignancy of pancreatic cancer cells. Our previous study reported chloroquine's mitigating effects on PSC activation; however, the drug is known to induce adverse effects in clinical practice. The present study aimed to reduce chloroquine doses and develop a useful pre-treatment that targets PSCs using nanoparticles. Poly lactic-co-glycolic acid (PLGA) nanoparticles were used as carriers and loaded with indocyanine green (Nano-ICG) or chloroquine (Nano-CQ). Tumor accumulation of Nano-ICG was evaluated using an in vivo imaging system. The effects of chloroquine, Nano-CQ and/or chemotherapy drug gemcitabine were investigated in an orthotopic xenograft mouse model. Nano-ICG selectively accumulated in pancreatic tumors and persisted therein for over 7 days after administration. Additionally, Nano-ICG accumulated in the peritoneal metastasized regions, but not in the liver, kidney and normal pancreatic tissues. Nano-CQ reduced the density of activated PSCs at lower chloroquine doses and significantly restrained tumor progression in combination with gemcitabine. In conclusion, the PLGA nanosystem successfully delivered the drug to pancreatic tumors. Nano-CQ efficiently reduced PSC activation and may be a promising novel pre-treatment strategy for PDAC.

New high-throughput screening detects compounds that suppress pancreatic stellate cell activation and attenuate pancreatic cancer growth.

BACKGROUND/OBJECTIVES: Pancreatic stellate cells (PSCs) are involved in abundant desmoplasia, which promotes cancer cell aggressiveness and resistance to anti-cancer drugs. Therefore, PSCs are suggested to be a promising therapeutic target by attenuating PSC activation to inhibit tumor-stromal interactions with pancreatic cancer cells. Here, we developed a screen to identify compounds that reduce the activity of PSCs and investigated the effect of candidates on pancreatic cancer. METHODS: Lipid droplet accumulation in PSCs was used to observe differences in PSC activity and a new high-throughput screening platform that quantified lipid droplets in PSCs was established. A library of 3398 Food and Drug Administration-approved drugs was screened by this platform. Validation assays were performed in vitro and in vivo. RESULTS: Thirty-two compounds were finally selected as candidate compounds by screening. These compounds decreased α-smooth muscle actin expression and inhibited autophagic flux in PSCs in vitro. Among the candidates, three drugs selected for validation assays inhibited the proliferation and migration of PSCs and invasion of cancer cells by disrupting tumor-stromal interactions. Production of extracellular matrix molecules was also decreased significantly by this treatment. In vivo testing in xenograft models showed that dopamine antagonist zuclopenthixol suppressed tumor growth; this suppression was significantly increased when combined with gemcitabine. CONCLUSIONS: A new screening platform that focused on the morphological features of PSCs was developed. Candidate drugs from this screening suppressed PSC activation and tumor growth. This screening system may be useful to discover new compounds that attenuate PSC activation.

Bone marrow-derived macrophages converted into cancer-associated fibroblast-like cells promote pancreatic cancer progression.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a desmoplastic reaction caused by cancer-associated fibroblasts (CAFs), which provokes treatment resistance. CAFs are newly proposed to be heterogeneous populations with different functions within the PDAC microenvironment. The most direct sources of CAFs are resident tissue fibroblasts and mesenchymal stem cells, however, the origins and functions of CAF subtypes remain unclear. Here, we established allogeneic bone marrow (BM) transplantation models using spontaneous PDAC mice, and then investigated what subtype cells derived from BM modulate the tumor microenvironment and affect the behavior of pancreatic cancer cells (PCCs). BM-derived multilineage hematopoietic cells were engrafted in recipient pancreas, and accumulated at the invasive front and central lesion of PDAC. We identified BM macrophages-derived CAFs in tumors. BM-derived macrophages treated with PCC-conditioned media expressed CAF markers. BM-derived macrophages led the local invasion of PCCs in vitro and enhanced the tumor invasive growth in vivo. Our data suggest that BM-derived cells are recruited to the pancreas during carcinogenesis and that the specific subpopulation of BM-derived macrophages partially converted into CAF-like cells, acted as leading cells, and facilitated pancreatic cancer progression. The control of the conversion of BM-derived macrophages into CAF-like cells may be a novel therapeutic strategy to suppress tumor growth.

Neutrophil extracellular traps promote liver micrometastasis in pancreatic ductal adenocarcinoma via the activation of cancer­associated fibroblasts.

Cancer­associated fibroblasts (CAFs) promote the progression of pancreatic ductal adenocarcinoma (PDAC) via tumor­stromal interactions. Neutrophil extracellular traps (NETs) are extracellular DNA meshworks released from neutrophils together with proteolytic enzymes against foreign pathogens. Emerging studies suggest their contribution to liver metastasis in several types of cancer. Herein, in order to investigate the role of NETs in liver metastasis in PDAC, the effects of NET inhibitors on spontaneous PDAC mouse models were evaluated. It was demonstrated that DNase I, a NET inhibitor, suppressed liver metastasis. For further investigation, further attention was paid to liver micrometastasis and an experimental liver metastasis mouse model was used that was generated by intrasplenic tumor injection. Furthermore, DNase I also suppressed liver micrometastasis and notably, CAFs accumulated in metastatic foci were significantly decreased in number. In vitro experiments revealed that pancreatic cancer cells induced NET formation and consequently NETs enhanced the migration of hepatic stellate cells, which was the possible origin of CAFs in liver metastasis. On the whole, these results suggest that NETs promote liver micrometastasis in PDAC via the activation of CAFs.

S100P regulates the collective invasion of pancreatic cancer cells into the lymphatic endothelial monolayer.

Lymph node metastasis is an independent prognostic factor in pancreatic cancer. However, the mechanisms of lymph node colonization are unknown. As a mechanism of lymphatic metastasis, it has been reported for other types of cancer that spheroids from tumor cells cause circular chemorepellent­induced defects (CCIDs) in lymphatic endothelial monolayers. In pancreatic cancer, such mechanisms of metastasis have not been elucidated. The present study evaluated the involvement of this new mechanism of metastasis in pancreatic cancer and investigated the associated factors. In human pancreatic cancer tissue, it was observed that clusters of cancer cells penetrated the wall of lymphatic ducts around the primary tumor. An in vitro co­culture system was then used to analyze the mechanisms of tumor cell­mediated disruption of lymphatic vessels. Time­lapse microscopic imaging revealed that spheroids from pancreatic cancer cells caused circular defects in lymphatic endothelial monolayers. CCID formation ability differed depending on the cell line. Neither aggregation of spheroids nor adhesion to lymphatic endothelial cells (LECs) exhibited a significant correlation with this phenomenon. The addition of supernatant from cultured cancer cells enhanced CCID formation. Microarray analysis revealed that the expression of S100 calcium binding protein P (S100P) was significantly increased when LECs were treated with supernatant from cultured cancer cells. Addition of a S100P antagonist significantly suppressed the migration of LECs and CCID formation. The present findings demonstrated that spheroids from pancreatic cancer cells caused circular defects in lymphatic endothelial monolayers. These CCIDs in pancreatic cancer were partly regulated by S100P, suggesting that S100P may be a promising target to inhibit lymph node metastasis.

Prediction of Negative Outcomes in Non-Surgical Treatment for Appendiceal Abscess in Adults.

OBJECTIVES: Non-surgical treatment is an acceptable approach for managing appendiceal abscess in adults. However, it is only applicable for selected patients, and conversion to surgery is mandatory for failed conservative treatment. This study aimed to determine the predictive factors for unsuccessful outcomes. METHODS: Of 594 patients with acute appendicitis, 34 (5.7%) diagnosed with appendiceal abscess were initially treated conservatively. Patients were divided into two groups: the conservative group, which was successfully treated with antibiotics and percutaneous abscess drainage, and the conversion group, which comprised patients who had surgical conversion despite conservative treatment. Risk factors for the conversion group were investigated by comparing clinical and radiological parameters between the two groups. RESULTS: Eight (23.4%) patients were converted to surgical management at an average of 5.5 days of non-surgical treatment. An abscess size greater than 40 mm and a lower rate of improvement in the white blood cell (WBC) count were significant factors for predicting conversion in multivariate analysis. The conversion group had a long operative time and high morbidity and operative conversion rates (change of proposed initial operation). Early conversion to operation group, i.e., less than 5 days of treatment, contributed to a significantly shorter hospital stay, lower hospital cost, and relatively shorter operative time (p = 0.02, p = 0.04, and p = 0.11, respectively). CONCLUSIONS: Contributing factors in predicting unsuccessful outcomes for non-surgical treatment include an abscess size greater than 40 mm and a low rate of improvement in WBC count on the first day of antibiotic treatment.

Autophagy inhibition enhances antiproliferative effect of salinomycin in pancreatic cancer cells.

BACKGROUND: Salinomycin has cytotoxic effects on various types of malignancy and induces autophagy. However, it has not been clarified whether autophagy induced by salinomycin treatment has a protective or cytotoxic role. We investigated whether salinomycin affects autophagy in pancreatic cancer cells and whether autophagy induced by salinomycin treatment has a protective or cytotoxic role in these cells. METHODS: We investigated the effect of salinomycin using three pancreatic cancer cell lines. We investigated effect on proliferation and the CD133 positive fraction using flow cytometry. In addition, we monitored the change in autophagic activity after salinomycin treatment using fluorescent immunostaining, western blotting, and flow cytometry. Finally, knockdown of ATG5 or ATG7 by siRNA was used to investigate the impact of autophagy inhibition on sensitivity to salinomycin. RESULTS: Salinomycin suppressed the proliferation of pancreatic cancer cells in a concentration dependent manner, and reduced the CD133 positive fraction. Salinomycin enhanced autophagy activity in these cells in a concentration dependent manner. Autophagy inhibition made pancreatic cancer cells more sensitive to salinomycin. CONCLUSIONS: Our data provide the first evidence indicating that autophagy induced by salinomycin have a protective role in pancreatic cancer cells. A new therapeutic strategy of combining salinomycin, autophagy inhibitors, and anticancer drugs could hold promise for pancreatic cancer treatment.

Task-specific brain reorganization in motor recovery induced by a hybrid-rehabilitation combining training with brain stimulation after stroke.

Recently, we have developed a new hybrid-rehabilitation combining 5Hz repetitive transcranial magnetic stimulation and extensor motor training of the paretic upper-limb for stroke patients with flexor hypertonia. We previously showed that the extensor-specific plastic change in M1 was associated with beneficial effects of our protocol (Koganemaru et al., 2010). Here, we investigated whether extensor-specific multiregional brain reorganization occurred after the hybrid-rehabilitation using functional magnetic resonance imaging. Eleven chronic stroke patients were scanned while performing upper-limb extensor movements. Untrained flexor movements were used as a control condition. The scanning and clinical assessments were done before, immediately and 2 weeks after the hybrid-rehabilitation. As a result, during the trained extensor movements, the imaging analysis showed a significant reduction of brain activity in the ipsilesional sensorimotor cortex, the contralesional cingulate motor cortex and the contralesional premotor cortex in association with functional improvements of the paretic hands. The activation change was not found for the control condition. Our results suggested that use-dependent plasticity induced by repetitive motor training with brain stimulation might be related to task-specific multi-regional brain reorganization. It provides a key to understand why repetitive training of the target action is one of the most powerful rehabilitation strategies to help patients.

Tuning of electronic properties of single-walled carbon nanotubes under homogenous conditions.

Reversible and non-bonding interaction between SWNTs and ODCB is observed from the analyses of visible near-infrared absorption data and Raman spectroscopies (see spectra). The solvent effect on SWNTs effectively controls the electronic structure of SWNTs under homogeneous conditions.The dispersion of single-walled carbon nanotubes (SWNTs) in o-dichlorobenzene (ODCB) is studied. Reversible and non-bonding interaction between SWNTs and ODCB is observed from the analyses of visible near-infrared (Vis-NIR) absorption data and Raman spectroscopy. This interaction should be one of the important factors for stable dispersion of SWNTs in ODCB. The solvent effect on SWNTs effectively controls the electronic structure of SWNTs under homogeneous conditions.