Unraveling the impact of cancer-associated fibroblasts on hypovascular pancreatic neuroendocrine tumors.

BACKGROUND: Pancreatic neuroendocrine tumors (PNETs) with low microvessel density and fibrosis often exhibit clinical aggressiveness. Given the contribution of cancer-associated fibroblasts (CAFs) to the hypovascular fibrotic stroma in pancreatic ductal adenocarcinoma, investigating whether CAFs play a similar role in PNETs becomes imperative. In this study, we investigated the involvement of CAFs in PNETs and their effects on clinical outcomes. METHODS: We examined 79 clinical PNET specimens to evaluate the number and spatial distribution of α-smooth muscle actin (SMA)-positive cells, which are indicative of CAFs. Then, the findings were correlated with clinical outcomes. In vitro and in vivo experiments were conducted to assess the effects of CAFs (isolated from clinical specimens) on PNET metastasis and growth. Additionally, the role of the stromal-cell-derived factor 1 (SDF1)-AGR2 axis in mediating communication between CAFs and PNET cells was investigated. RESULTS: αSMA-positive and platelet-derived growth factor-α-positive CAFs were detected in the hypovascular stroma of PNET specimens. A higher abundance of α-SMA-positive CAFs within the PNET stroma was significantly associated with a higher level of clinical aggressiveness. Notably, conditioned medium from PNET cells induced an inflammatory phenotype in isolated CAFs. These CAFs promoted PNET growth and metastasis. Mechanistically, PNET cells secreted interleukin-1, which induced the secretion of SDF1 from CAFs. This cascade subsequently elevated AGR2 expression in PNETs, thereby promoting tumor growth and metastasis. The downregulation of AGR2 in PNET cells effectively suppressed the CAF-mediated promotion of PNET growth and metastasis. CONCLUSION: CAFs drive the growth and metastasis of aggressive PNETs. The CXCR4-SDF1 axis may be a target for antistromal therapy in the treatment of PNET. This study clarifies mechanisms underlying PNET aggressiveness and may guide future therapeutic interventions targeting the tumor microenvironment.

Multimodal 3-D/2-D human islet and duct imaging in exocrine and endocrine lesion environment: associated pancreas tissue remodeling.

Pancreatic intraepithelial neoplasia (PanIN) and islet cell microadenoma are exocrine and endocrine neoplasms of human pancreas that have been linked to pancreatic ductal adenocarcinoma (PDAC) and neuroendocrine tumor, respectively. However, in health and at the surgical margin of pancreatic cancer, it remains unresolved how to simultaneously characterize duct and islet remodeling to investigate the exocrine-endocrine association in the lesion microenvironment. Here, we develop a new vibratome-based approach to detect, confirm, and analyze the two types of pancreas remodeling via stereo/three-dimensional (3-D) and classic/two-dimensional (2-D) histology. Surgical margins of PDAC (n = 10, distal) and cadaveric donor pancreases (n = 10, consecutive cases) were fixed, sectioned by vibratome (350 µm), and surveyed for PanIN and microadenoma via stereomicroscopy. After lesion detection, PanIN and microadenoma were analyzed with 3-D fluorescence imaging and clinical microtome-based histology for confirmation and assessment of microenvironment. Multimodal imaging of PDAC surgical margins and cadaveric donor pancreases detected the peri-PanIN islet aggregation with duct-islet cell clusters. Organ-wide survey of cadaveric donor pancreases shows a marked 2.3-fold increase in the lesion size with the PanIN-islet association vs. without the association. In the survey, we unexpectedly detected the islet cell microadenoma adjacent to (<2 mm) PanIN. Overall, among the 53 early lesions in the cadaveric donor pancreases (PanINs and microadenomas), 81% are featured with the associated exocrine-endocrine tissue remodeling. Multimodal 3-D/2-D tissue imaging reveals local and simultaneous duct and islet remodeling in the cancer surgical margin and cadaveric donor pancreas. In the cadaveric donor pancreas, the peri-PanIN islet aggregation and PanIN-microadenoma association are two major features of pancreas remodeling in the early lesion microenvironment.NEW & NOTEWORTHY We develop a new multimodal 3-D/2-D imaging approach (matched stereomicroscopic, fluorescence, and H&E signals) to examine human duct-islet association in the PDAC surgical margin and cadaveric donor pancreas. In both conditions, peri-PanIN islet aggregation with duct-islet cell clusters was identified. The PanIN-islet cell microadenoma association was unexpectedly detected in the donor pancreas. Our work provides the technical and morphological foundations to simultaneously characterize human islets and ducts to study their association in health and disease.

Cooperative recruitment of Arl4A and Pak1 to the plasma membrane contributes to sustained Pak1 activation for cell migration.

Cell migration requires the coordination of multiple signaling pathways involved in membrane dynamics and cytoskeletal rearrangement. The Arf-like small GTPase Arl4A has been shown to modulate actin cytoskeleton remodeling. However, evidence of the function of Arl4A in cell migration is insufficient. Here, we report that Arl4A acts with the serine/threonine protein kinase Pak1 to modulate cell migration through their cooperative recruitment to the plasma membrane. We first observed that Arl4A and its isoform Arl4D interact with Pak1 and Pak2 and showed that Arl4A recruits Pak1 and Pak2 to the plasma membrane. The fibronectin-induced Pak1 localization at the plasma membrane is reduced in Arl4A-depleted cells. Unexpectedly, we found that Pak1, but not Arl4A-binding-defective Pak1, can recruit a cytoplasmic myristoylation-deficient Arl4A-G2A mutant to the plasma membrane. Furthermore, we found that the Arl4A-Pak1 interaction, which is independent of Rac1 binding to Pak1, is required for Arl4A-induced cell migration. Thus, we infer that there is feedback regulation between Arl4A and Pak1, in which they mutually recruit each other to the plasma membrane for Pak1 activation, thereby modulating cell migration through direct interaction.

The role of S100A9 in the interaction between pancreatic ductal adenocarcinoma cells and stromal cells.

BACKGROUND: A major feature of the microenvironment in pancreatic ductal adenocarcinoma (PDAC) is the significant amount of extracellular matrix produced by pancreatic stellate cells (PSCs), which have been reported to enhance the invasiveness of pancreatic cancer cells and negatively impact the prognosis. METHODS: We analyzed the data from two publicly available microarray datasets deposited in the Gene Expression Omnibus and found candidate genes that were differentially expressed in PDAC cells with metastatic potential and PDAC cells cocultured with PSCs. We studied the interaction between PDAC cells and PSCs in vitro and verified our finding with the survival data of patients with PDAC from the website of The Human Protein Atlas. RESULTS: We found that PSCs stimulated PDAC cells to secrete S100A9, which attracted circulatory monocytes into cancer tissue and enhanced the expression of programmed death-ligand 1 (PD-L1) on macrophages. When analyzing the correlation of S100A9 and PD-L1 expression with the clinical outcomes of patients with PDAC, we ascertained that high expression of S100A9 and PD-L1 was associated with poor survival in patients with PDAC. CONCLUSIONS: PSCs stimulated PDAC cells to secrete S100A9, which acts as a chemoattractant to attract circulatory monocytes into cancer microenvironment and induces expression of PD-L1 on macrophages. High expression of S100A9 and PD-L1 was associated with worse overall survival in a cohort of patients with PDAC.

Therapeutic Ultrasound Halts Progression of Chronic Kidney Disease In Vivo via the Regulation of Markers Associated with Renal Epithelial-Mesenchymal Transition and Senescence.

Low-intensity pulsed ultrasound (LIPUS), a therapeutic type of ultrasound, is known to enhance bone fracture repair processes and help some tissues to heal. Here, we investigated the therapeutic potential of LIPUS for the treatment of chronic kidney disease (CKD) in two CKD mouse models. CKD mice were induced using both unilateral renal ischemia/reperfusion injury (IRI) with nephrectomy and adenine administration. The left kidneys of the CKD mice were treated using LIPUS with the parameters of 3 MHz, 100 mW/cm2, and 20 min/day, based on the preliminary experiments. The mice were euthanized 14 days after IRI or 28 days after the end of adenine administration. LIPUS treatment effectively alleviated the decreases in the body weight and albumin/globulin ratio and the increases in the serum renal functional markers, fibroblast growth factor-23, renal pathological changes, and renal fibrosis in the CKD mice. The parameters for epithelial-mesenchymal transition (EMT), senescence-related signal induction, and the inhibition of α-Klotho and endogenous antioxidant enzyme protein expression in the kidneys of the CKD mice were also significantly alleviated by LIPUS. These results suggest that LIPUS treatment reduces CKD progression through the inhibition of EMT and senescence-related signals. The application of LIPUS may be an alternative non-invasive therapeutic intervention for CKD therapy.

Local islet remodelling associated with duct lesion-islet complex in adult human pancreas.

AIMS/HYPOTHESIS: Islets are thought to be stably present in the adult human pancreas to maintain glucose homeostasis. However, identification of the pancreatic intraepithelial neoplasia (PanIN)-islet complex in mice and the presence of PanIN lesions in adult humans suggest that similar remodelling of islet structure and environment may occur in the human pancreas. To identify islet remodelling in a clinically related setting, we examine human donor pancreases with 3D histology to detect and characterise the human PanIN-islet complex. METHODS: Cadaveric donor pancreases (26-65 years old, n = 10) were fixed and sectioned (350 µm) for tissue labelling, clearing and microscopy to detect local islet remodelling for 3D analysis of the microenvironment. The remodelled microenvironment was subsequently examined via microtome-based histology for clinical assessment. RESULTS: In nine pancreases, we identified the unique peri-lobular islet aggregation associated with the PanIN lesion (16 lesion-islet complexes detected; size: 3.18 ± 1.34 mm). Important features of the lesion-islet microenvironment include: (1) formation of intra-islet ducts, (2) acinar atrophy, (3) adipocyte association, (4) inflammation (CD45+), (5) stromal accumulation (α-SMA+), (6) increase in Ki-67 proliferation index but absence of Ki-67+ alpha/beta cells and (7) in-depth and continuous duct-islet cell contacts, forming a cluster. The duct-islet cell cluster and intra-islet ducts suggest likely islet cell neogenesis but not replication. CONCLUSIONS/INTERPRETATION: We identify local islet remodelling associated with PanIN-islet complex in the adult human pancreas. The tissue remodelling and the evidence of inflammation and stromal accumulation suggest that the PanIN-islet complex is derived from tissue repair after a local injury.

Human pancreatic afferent and efferent nerves: mapping and 3-D illustration of exocrine, endocrine, and adipose innervation.

The pancreas consists of both the exocrine (acini and ducts) and endocrine (islets) compartments to participate in and regulate the body's digestive and metabolic activities. These activities are subjected to neural modulation, but characterization of the human pancreatic afferent and efferent nerves remains difficult because of the lack of three-dimensional (3-D) image data. Here we prepare transparent human donor pancreases for 3-D histology to reveal the pancreatic microstructure, vasculature, and innervation in a global and integrated fashion. The pancreatic neural network consists of the substance P (SP)-positive sensory (afferent) nerves, the vesicular acetylcholine transporter (VAChT)-positive parasympathetic (efferent) nerves, and the tyrosine hydroxylase (TH)-positive sympathetic (efferent) nerves. The SP+ afferent nerves were found residing along the basal domain of the interlobular ducts. The VAChT+ and TH+ efferent nerves were identified at the peri-acinar and perivascular spaces, which follow the blood vessels to the islets. In the intrapancreatic ganglia, the SP+ (scattered minority, ~7%) and VAChT+ neurons co-localize, suggesting a local afferent-efferent interaction. Compared with the mouse pancreas, the human pancreas differs in 1) the lack of SP+ afferent nerves in the islet, 2) the lower ganglionic density, and 3) the obvious presence of VAChT+ and TH+ nerves around the intralobular adipocytes. The latter implicates the neural influence on the pancreatic steatosis. Overall, our 3-D image data reveal the human pancreatic afferent and efferent innervation patterns and provide the anatomical foundation for future high-definition analyses of neural remodeling in human pancreatic diseases.NEW & NOTEWORTHY Modern three-dimensional (3-D) histology with multiplex optical signals identifies the afferent and efferent innervation patterns of human pancreas, which otherwise cannot be defined with standard histology. Our 3-D image data reveal the unexpected association of sensory and parasympathetic nerves/neurons in the intrapancreatic ganglia and identify the sympathetic and parasympathetic nerve contacts with the infiltrated adipocytes. The multiplex approach offers a new way to characterize the human pancreas in remodeling (e.g., fatty infiltration and duct lesion progression).

ARL4A acts with GCC185 to modulate Golgi complex organization.

ADP-ribosylation factor-like protein 4A (ARL4A) is a developmentally regulated member of the ARF/ARL GTPase family. The primary structure of ARL4A is very similar to that of other ARF/ARL molecules, but its function remains unclear. The trans-Golgi network golgin GCC185 is required for maintenance of Golgi structure and distinct endosome-to-Golgi transport. We show here that GCC185 acts as a new effector for ARL4 to modulate Golgi organization. ARL4A directly interacts with GCC185 in a GTP-dependent manner. Sub-coiled-coil regions of the CC2 domain of GCC185 are required for the interaction between GCC185 and ARL4A. Depletion of ARL4A reproduces the GCC185-depleted phenotype, causing fragmentation of the Golgi compartment and defects in endosome-to-Golgi transport. GCC185 and ARL4A localize to the Golgi independently of each other. Deletion of the ARL4A-interacting region of GCC185 results in inability to maintain Golgi structure. Depletion of ARL4A impairs the interaction between GCC185 and cytoplasmic linker-associated proteins 1 and 2 (CLASP1 and CLASP2, hereafter CLASPs) in vivo, and abolishes the GCC185-mediated Golgi recruitment of these CLASPs, which is crucial for the maintenance of Golgi structure. In summary, we suggest that ARL4A alters the integrity of the Golgi structure by facilitating the interaction of GCC185 with CLASPs.

The Arf family GTPase Arl4A complexes with ELMO proteins to promote actin cytoskeleton remodeling and reveals a versatile Ras-binding domain in the ELMO proteins family.

The prototypical DOCK protein, DOCK180, is an evolutionarily conserved Rac regulator and is indispensable during processes such as cell migration and myoblast fusion. The biological activity of DOCK180 is tightly linked to its binding partner ELMO. We previously reported that autoinhibited ELMO proteins regulate signaling from this pathway. One mechanism to activate the ELMO-DOCK180 complex appears to be the recruitment of this complex to the membrane via the Ras-binding domain (RBD) of ELMO. In the present study, we aimed to identify novel ELMO-interacting proteins to further define the molecular events capable of controlling ELMO recruitment to the membrane. To do so, we performed two independent interaction screens: one specifically interrogated an active GTPase library while the other probed a brain cDNA library. Both methods converged on Arl4A, an Arf-related GTPase, as a specific ELMO interactor. Biochemically, Arl4A is constitutively GTP-loaded, and our binding assays confirm that both wild-type and constitutively active forms of the GTPase associate with ELMO. Mechanistically, we report that Arl4A binds the ELMO RBD and acts as a membrane localization signal for ELMO. In addition, we report that membrane targeting of ELMO via Arl4A promotes cytoskeletal reorganization including membrane ruffling and stress fiber disassembly via an ELMO-DOCK1800-Rac signaling pathway. We conclude that ELMO is capable of interacting with GTPases from Rho and Arf families, leading to the conclusion that ELMO contains a versatile RBD. Furthermore, via binding of an Arf family GTPase, the ELMO-DOCK180 is uniquely positioned at the membrane to activate Rac signaling and remodel the actin cytoskeleton.

Immune cell shuttle for precise delivery of nanotherapeutics for heart disease and cancer.

The delivery of therapeutics through the circulatory system is one of the least arduous and less invasive interventions; however, this approach is hampered by low vascular density or permeability. In this study, by exploiting the ability of monocytes to actively penetrate into diseased sites, we designed aptamer-based lipid nanovectors that actively bind onto the surface of monocytes and are released upon reaching the diseased sites. Our method was thoroughly assessed through treating two of the top causes of death in the world, cardiac ischemia-reperfusion injury and pancreatic ductal adenocarcinoma with or without liver metastasis, and showed a significant increase in survival and healing with no toxicity to the liver and kidneys in either case, indicating the success and ubiquity of our platform. We believe that this system provides a new therapeutic method, which can potentially be adapted to treat a myriad of diseases that involve monocyte recruitment in their pathophysiology.

ARL4D recruits cytohesin-2/ARNO to modulate actin remodeling.

ARL4D is a developmentally regulated member of the ADP-ribosylation factor/ARF-like protein (ARF/ARL) family of Ras-related GTPases. Although the primary structure of ARL4D is very similar to that of other ARF/ARL molecules, its function remains unclear. Cytohesin-2/ARF nucleotide-binding-site opener (ARNO) is a guanine nucleotide-exchange factor (GEF) for ARF, and, at the plasma membrane, it can activate ARF6 to regulate actin reorganization and membrane ruffling. We show here that ARL4D interacts with the C-terminal pleckstrin homology (PH) and polybasic c domains of cytohesin-2/ARNO in a GTP-dependent manner. Localization of ARL4D at the plasma membrane is GTP- and N-terminal myristoylation-dependent. ARL4D(Q80L), a putative active form of ARL4D, induced accumulation of cytohesin-2/ARNO at the plasma membrane. Consistent with a known action of cytohesin-2/ARNO, ARL4D(Q80L) increased GTP-bound ARF6 and induced disassembly of actin stress fibers. Expression of inactive cytohesin-2/ARNO(E156K) or small interfering RNA knockdown of cytohesin-2/ARNO blocked ARL4D-mediated disassembly of actin stress fibers. Similar to the results with cytohesin-2/ARNO or ARF6, reduction of ARL4D suppressed cell migration activity. Furthermore, ARL4D-induced translocation of cytohesin-2/ARNO did not require phosphoinositide 3-kinase activation. Together, these data demonstrate that ARL4D acts as a novel upstream regulator of cytohesin-2/ARNO to promote ARF6 activation and modulate actin remodeling.

Lymphatic vessel remodeling and invasion in pancreatic cancer progression.

BACKGROUND: The lymphatic system is involved in metastasis in pancreatic cancer progression. In cancer staging, lymphatic spread has been used to assess the invasiveness of tumor cells. However, from the endothelium's perspective, the analysis downplays the peri-lesional activities of lymphatic vessels. This unintended bias is largely due to the lack of 3-dimensional (3-D) tissue information to depict the lesion microstructure and vasculature in a global and integrated fashion. METHODS: We targeted the pancreas as the model organ to investigate lymphatic vessel remodeling in cancer lesion progression. Transparent pancreases were prepared by tissue clearing to facilitate deep-tissue, tile-scanning microscopy for 3-D lymphatic network imaging. FINDINGS: In human pancreatic ductal adenocarcinoma, we identify the close association between the pancreatic intraepithelial neoplasia (PanIN) lesions and the lymphatic network. In mouse models of PanIN (elastase-CreER;LSL-KrasG12D and elastase-CreER;LSL-KrasG12D;p53+/-), the 3-D image data reveal the peri-lesional lymphangiogenesis, endothelial invagination, formation of the bridge/valve-like luminal tubules, vasodilation, and luminal invasion. In the orthotopic mouse model of pancreatic cancer, we identify the localized, graft-induced lymphangiogenesis and the peri- and intra-tumoral lymphatic vessel invasion. INTERPRETATION: The integrated view of duct lesions and vascular remodeling suggests an active role, rather than a passive target, of lymphatic vessels in the metastasis of pancreatic cancer. Our 3-D image data provide insights into the pancreatic cancer microenvironment and establish the technical and morphological foundation for systematic detection and 3-D analysis of lymphatic vessel invasion. FUND: Taiwan Academia Sinica (AS-107-TP-L15 and AS-105-TP-B15), Ministry of Science and Technology (MOST 106-2321-B-001-048, 106-0210-01-15-02, 106-2321-B-002-034, and 106-2314-B-007-004-MY2), and Taiwan National Health Research Institutes (NHRI EX107-10524EI).