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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. 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. 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. 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.

Supplemental Materials Supplemental Figures Suppl. Fig. S1 (related to Fig. 1). Multimodal imaging of islet aggregation with duct-islet cell cluster in PDAC surgical margin. (A-I) and (J-R) are two examples of the coexistence of islet aggregation, PanIN, acinar atrophy, and fibrosis in the surgical margin distal to PDAC (case No. 1628870 and 4442070). In G, H, P, Q, varying amounts of mucin were observed in the duct lesions. In I and R, the high-resolution images identify the duct (CK7+) -islet (insulin+ or glucagon+) cell cluster. Green, CK7; blue, insulin; magenta, glucagon; white, nuclei. Suppl. Fig. S2 (related to Fig. 2). Detection and confirmation of duct lesion in human donor pancreas (gallery display). Suppl. Fig. S3-1 (related to Fig. 4S). Detection and characterization of an endocrine-exocrine lesion mixture in human donor pancreas (CgA+, PGP9.5+, glucagon+ microadenoma with CK7+ epithelium in a stroma-rich environment). (A-E) Detection of glucagon+ microadenoma. A-C show microadenoma (arrow) in vibratome section. Fluorescence tissue map (D) identifies the intra-microadenoma ducts (CK7+, green; enlarged in inset i and ii, asterisk). Magenta, glucagon; blue, insulin; white, nuclei. PGP9.5 staining (E, cyan) confirms the islet/neuroendocrine lesion. Red, CD31. (F-M) Confirmation of microadenoma-duct lesion mixture with multiplex CgA, H&E, CD45, and Ki-67 signals. The endocrine-exocrine lesion mixture is associated with stromal accumulation (F-I; asterisk, enlarged area), leukocyte infiltration (J, K; asterisk/arrow, enlarged area), and prominent cell proliferation (L, M; box, enlarged area; blue arrows, Ki-67+ nuclei). An adjacent PanIN (within 2 mm) is presented in the next page. Black arrows in F, G, J, L indicate the same microenvironment. Suppl. Fig. S3-2 (related to Fig. 5). Detection of a PanIN lesion adjacent to the microadenoma in panel A-M (Suppl. Fig. S3-1). (N-P) Microadenoma and its microenvironment visualized via stereomicroscopy. N-P and A-M examine the same microenvironment (arrow). Vibratome section P is further processed for H&E histology to confirm the PanIN lesion. (Q, R) Gross view and enlarged H&E images of PanIN. The gold-standard H&E histology (cyan arrows from P to Q to R, same environment) confirms the low-grade PanIN lesion and peri-lesional stroma. Box in Q is magnified in R to specify the lesion. Suppl. Fig. S4 (related to Fig. 4O-R). False positive result in fluorescence imaging of islet cell microadenoma in human pancreas. (A-C) Gross view and side-by-side display of fluorescence and transmitted light signals of microadenoma. The three images were derived from an optically cleared microadenoma (refractive index: 1.52). A: microadenoma alongside pancreatic lobules (red, blood vessels, CD31; green, lymphatic vessels, D2-40; white, nuclei, DAPI). B: enlarged fluorescence image of vasculature (blood and lymphatic vessels). C: overlay of transmitted light and nuclear signals. Adipocytes and blood clots (black spots) are prominently seen in C via in-depth transmitted light signals. Number 1-6 indicate the adipocytes (#1-2) and blood clots (#3-6) shown in both B and C. Alphabet a-f indicate the adipocytes and blood clots appear only in C. CD31 signals of B is presented in D. B-D examine the same microenvironment. (D, E) False positive result revealed by analysis of CD31 fluorescence signals of microadenoma. Relative signals intensity along the central line of D (0-2,000 pixels) is presented in E. Both CD31-labeled blood vessels and the blood clot-induced autofluorescence create local increases in the signal intensity (E). Number 4 in E indicates the autofluorescence caused by the blood clot #4 in panel B-D. =============================================================================================== Supplemental Tables Suppl. Table S1(Related to Materials and Methods).Clinical data of pancreatic ductal adenocarcinoma cases. Suppl. Table S2(Related to Materials and Methods).Pancreas organ donor information and lesion summary. Suppl. Table S3(Related to Materials and Methods).Summary of primary antibodies used in illustrations. Suppl. Table S4(Related to Materials and Methods).Summary of color codes presented in illustrations. =============================================================================================== Supplemental Video Suppl. Video S1 (related to Fig. 3L-N). Tile scan and in-depth recording of PanIN microenvironment in human pancreas. This video shows a survey of PanIN microenvironment to identify the associated exocrine-endocrine tissue remodeling. Duct lesion and islet a- and b-cells form a complex in the peri-lobular space. Green, CK7; magenta, glucagon; blue, insulin; white, nuclei. Overlay of fluorescence and transmitted light (gray) signals is used to identify the boundaries of pancreatic lobules and CK7+ epithelium.

Supplemental Materials Supplemental Figures Suppl. Fig. S1 (related to Fig. 1). Detection and confirmation of duct lesion in human donor pancreas (gallery display). Suppl. Fig. S2 (related to Fig. 3). False positive result in fluorescence imaging of pancreatic microadenoma. (A-C) Gross view and side-by-side display of fluorescence and transmitted light signals of microadenoma. The three images were derived from an optically cleared microadenoma (refractive index: 1.52). A: microadenoma alongside pancreatic lobules (red, blood vessels, CD31; green, lymphatic vessels, D2-40; white, nuclei, DAPI). B: enlarged fluorescence image of vasculature (blood and lymphatic vessels). C: overlay of transmitted light and nuclear signals. Adipocytes and blood clots (black spots) are prominently seen in C via in-depth transmitted light signals. Number 1-6 indicate the adipocytes (#1-2) and blood clots (#3-6) shown in both B and C. Alphabet a-f indicate the adipocytes and blood clots appear only in C. CD31 signals of B is presented in D. B-D examine the same microenvironment. (D, E) False positive result revealed by analysis of CD31 fluorescence signals of microadenoma. Relative signals intensity along the central line of D (0-2,000 pixels) is presented in E. Both CD31-labeled blood vessels and the blood clot-induced autofluorescence create local increases in the signal intensity (E). Number 4 in E indicates the autofluorescence caused by the blood clot #4 in panel B-D. Suppl. Fig. S3 (related to Fig. 4). Detection of PanIN adjacent to microadenoma. (A-C) Microadenoma and its microenvironment visualized via stereomicroscopy. A-C and Fig. 4 examine the same microenvironment (arrow). Vibratome section C is further processed for H&E histology to confirm the PanIN lesion. (D, E) Gross view and enlarged H&E images of PanIN. The gold-standard H&E histology (cyan arrows from C to D to E, same environment) confirms the low-grade PanIN lesion and peri-lesional stroma. Box in D is magnified in E to specify the lesion. =============================================================================================== Supplemental Tables Suppl. Table S1(Related to Materials and Methods).Summary of primary antibodies used in illustrations. Suppl. Table S2(Related to Materials and Methods).Summary of color codes presented in illustrations. =============================================================================================== Supplemental Video Suppl. Video S1 (related to Fig. 2). Tile scan and in-depth recording of PanIN microenvironment in human pancreas. This video shows a survey of PanIN microenvironment to identify the associated exocrine-endocrine tissue remodeling. Duct lesion and islet a- and b-cells form a complex in the peri-lobular space. Green, CK7; magenta, glucagon; blue, insulin; white, nuclei. Overlay of fluorescence and transmitted light (gray) signals is used to identify the boundaries of pancreatic lobules and CK7+ epithelium.

Graphic Abstract Local alpha-cell hyperplasia in human pancreas Supplementary Tables Supplementary Table 1 (Related to Materials and Methods). Summary of primary antibodies used in illustrations. Supplementary Table 2 (Related to Materials and Methods). Summary of color codes presented in illustrations.

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).

Aptamer-based nanovectors that actively bind to circulating monocytes enhance drug delivery to the heart and to 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.

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.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.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 (CD45We 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.

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.

Supplementary figure legends Supplementary Fig. 1 (Related to Fig. 1). Gallery of pancreatic tissue maps showing peri-lobular islet aggregation and duct lesion (PanIN) -islet complex in human donor pancreases. Magenta: glucagon. Green: CK7. White: nuclei. Tile scanning and overlay of fluorescence and transmitted light signals are used to detect and identify the lesion-islet complex. Supplementary Fig. 2 (Related to Fig. 2). Intra-islet ducts in human pancreas. This unique structure is identified in the PanIN-islet complex via quadruple staining of glucagon (magenta, a-cells), insulin (blue, b-cells), CK7 (green, ductal epithelium), and nuclei (white). Supplementary tables Supplementary Table 1 (Related to Materials and Methods).Summary of primary antibodies used in illustrations. Supplementary Table 2 (Related to Materials and Methods).Summary of color codes presented in illustrations. Supplementary video legends Supplementary Video 1 (Related to Fig. 1). High-definition presentation of PanIN in PanIN-islet complex. This video uses a side-by-side illustration to compare the lesion in the H&E micrograph (left) and the 3-D image stack (right, in-depth recording of fluorescence and transmitted light signals). The H&E micrograph provides the ground-truth tissue information to identify this PanIN-1B lesion. Its feature is truthfully presented in the recording (right), confirming our 3-D imaging approach. Magenta: glucagon. Green: CK7. White: nuclei. Supplementary Video 2 (Related to Fig. 2).In-depth recording of islet aggregation and intra-islet duct in PanIN-islet complex. Left: PanIN-1A lesion and its islet association. Right: islet aggregation and formation of intra-islet duct (00:05-00:12). Blue: insulin. Magenta: glucagon. Green: CK7. White: nuclei. Supplementary Video 3 (Related to Fig. 3).In-depth recording of stromal accumulation in PanIN-islet complex. Left: map of pancreas and projection of lesion-islet complex (arrow, zoom-in area). Right: zoom-in examination of stromal accumulation in lesion-islet complex. Blue: insulin. Yellow: a-SMA. Green: CK7. White: nuclei. Supplementary Video 4 (Related to Fig. 4).Lymphangiogenesis associated with PanIN-islet complex. Left: in-depth recording of lymphatic vessels (green, D2-40) around duct lesions and aggregated islets (magenta, glucagon). Right: 3-D projection of lymphatic vessels in the microenvironment. White: nuclei. Supplementary Video 5 (Related to Fig. 5).Peri- and intra-islet cell proliferation in PanIN-islet microenvironment. Left: gross view of aggregated islets (3-D projection and 2-D image). Right: zoom-in examination of Ki-67+ nuclei. The Ki-67+ nuclei are identified inside and around the islet but not enclosed by the glucagon (magenta) or insulin (blue) signals. Blue: insulin. Magenta: glucagon. Green: nuclei. Yellow: Ki-67. In the first half of the video (00:00-00:10), the overlap of yellow (Ki-67) and green (nuclear staining) signals is used to detect and confirm the cell proliferation. In the second half (00:11-00:21), the same microenvironment is displayed without the green signals to emphasize the locations of Ki-67+ nuclei. Supplementary Video 6 (Related to Fig. 5).Second example of cell proliferation in PanIN-islet microenvironment. Upper panel: in-depth recording of Ki-67+ nuclei. Lower panel: projection of Ki-67+ signals to register their locations. Blue: insulin. Magenta: glucagon. Green: nuclei. Yellow: Ki-67. Sixteen Ki-67+ nuclei are identified in this view. The numbers at the lower panel indicate their order of appearance in the video. In particular, number 1, 2, 4, 5, 6, 13, and 14 are adjacent to the glucagon+ (magenta) or insulin+ (blue) cells but not enclosed by the two endocrine markers.

Supplementary figure legends Supplementary Fig. 1 (Related to Fig. 1). Gallery of pancreatic tissue maps showing peri-lobular islet aggregation and duct lesion (PanIN)-islet complex in human donor pancreases. Magenta: glucagon. Green: CK7. White: nuclei. Tile scanning and overlay of fluorescence and transmitted light signals are used to detect and identify the alterations of pancreatic tissues. Supplementary Fig. 2 (Related to Fig. 2). Intra-islet ducts in human pancreas. This unique structure is identified in the area of lesion-islet complex with quadruple staining of glucagon (magenta, a-cells), insulin (blue, b-cells), CK7 (green, ductal epithelium), and nuclei (white). Supplementary tables Supplementary Table 1 (Related to Materials and Methods).Summary of primary antibodies used in illustrations. Supplementary Table 2 (Related to Materials and Methods).Summary of color codes presented in illustrations. Supplementary video legends Supplementary Video 1 (Related to Fig. 1). High-definition presentation of early PanIN lesion in lesion-islet complex. This video uses a side-by-side illustration to compare the lesion in the H&E micrograph (left) and the 3-D image stack (right, in-depth recording of fluorescence and transmitted light signals). The H&E micrograph provides the ground-truth tissue information to define this PanIN-1B lesion. Its feature is truthfully presented in the recording (right), confirming our 3-D imaging approach. Magenta: glucagon. Green: CK7. White: nuclei. Supplementary Video 2 (Related to Fig. 2).In-depth recording of islet aggregation and intra-islet duct in lesion-islet complex. Left: PanIN-1A lesion and its islet association. Right: islet aggregation and formation of intra-islet duct (00:05-00:12). Blue: insulin. Magenta: glucagon. Green: CK7. White: nuclei. Supplementary Video 3 (Related to Fig. 3).In-depth recording of stromal accumulation in lesion-islet complex. Left: map of pancreas and projection of lesion-islet complex (arrow, zoom-in area). Right: zoom-in examination of stromal accumulation in lesion-islet complex. Blue: insulin. Yellow: a-SMA. Green: CK7. White: nuclei. Supplementary Video 4 (Related to Fig. 4).Lymphangiogenesis associated with lesion-islet complex. Left: in-depth recording of lymphatic vessels (green, D2-40) around duct lesions and aggregated islets (magenta, glucagon). Right: 3-D projection of lymphatic vessels in the microenvironment. White: nuclei. Supplementary Video 5 (Related to Fig. 5).Peri- and intra-islet cell proliferation in lesion-islet microenvironment. Left: gross view of aggregated islets in 3-D projection and 2-D image. Right: zoom-in examination of Ki-67+ nuclei. The Ki-67+ nuclei are identified inside and around the islet but not enclosed by the glucagon (magenta) or insulin (blue) signals. Blue: insulin. Magenta: glucagon. Green: nuclei. Yellow: Ki-67. In the first half of the video (00:00-00:10), the overlap of yellow (Ki-67) and green (nuclear staining) signals is used to detect and confirm the cell proliferation. In the second half (00:11-00:21), the same microenvironment is displayed without the green signals to emphasize the locations of Ki-67+ nuclei. Supplementary Video 6 (Related to Fig. 5).Second example of cell proliferation in lesion-islet microenvironment. Upper panel: in-depth recording of Ki-67+ nuclei. Lower panel: projection of Ki-67+ signals to register their locations. Blue: insulin. Magenta: glucagon. Green: nuclei. Yellow: Ki-67. Sixteen Ki-67+ nuclei are identified in this view. The numbers at the lower panel indicate their order of appearance in the video. In particular, number 1, 2, 4, 5, 6, 13, and 14 are adjacent to the glucagon+ (magenta) or insulin+ (blue) cells but not enclosed by the two endocrine markers.

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 critical 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 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).

Background: The link of pancreatic cancer to diabetes (type 3c) suggests that islets are influenced by the cancer lesion progression. However, at an early stage, i.e., when the pancreas appears to be normal, how the endocrine pancreas is affected by the initial duct lesion formation remains unclear. Aims. To apply panoramic 3-D histology to detect early duct lesions in human cadaveric donor pancreases and examine the peri-lesional islet environment. Methods: Tissue clearing was applied to the freshly perfused donor pancreases (target) and cancer surgical biopsies (malignant control) for 3-D microscopy. Results: Large-scale tissue scanning detects early duct lesions (PanIN 1A/B) in three donor pancreases (3/5; nondiabetic; age (yr)/BMI: 40/24, 53/28, and 51/20). High-definition 3-D fluorescence and 2-D transmitted-light and H&E images reveal: 1) peri-lesional islet aggregation, 2) epithelium-islet complex (e.g., intra-islet duct), 3) adipocyte association, 4) inflammation, 5) stromal activation, and 6) Ki-67+ ductal and stromal cells but not the β- or α-cells. Conclusions: We systematically developed a 3-D/2-D integrative imaging approach to detect and characterize the islet microenvironment around early duct lesions. Our data indicate a proliferative setting, but the islet aggregation appears not to arise from β- or α-cell division; instead, it may be due to lobular atrophy and/or evolve from duct-to-islet differentiation. Disclosure H. Chien: None. T. Chiang: None. S. Peng: None. M. Chung: None. C. Lee: None. Y. Tien: None. S. Tang: None.

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.

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 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.

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.