Identification of novel vascular projections with cellular trafficking abilities on the microvasculature of pancreatic ductal adenocarcinoma
Pancreatic ductal adenocarcinoma (PDAC) is a nearly lethal neoplasm. It is a remarkably stroma‐rich, vascular‐poor and hypo‐perfused tumour, which prevents efficient drug delivery. Paradoxically, the neoplastic cells have robust glucose uptake, suggesting that the microvasculature has adopted an alt...
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| Published in: | The Journal of pathology Vol. 236; no. 2; pp. 142 - 154 |
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| Main Authors: | , , , , , , , , , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
Chichester, UK
John Wiley & Sons, Ltd
01-06-2015
Wiley Subscription Services, Inc |
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| Online Access: | Get full text |
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| Summary: | Pancreatic ductal adenocarcinoma (PDAC) is a nearly lethal neoplasm. It is a remarkably stroma‐rich, vascular‐poor and hypo‐perfused tumour, which prevents efficient drug delivery. Paradoxically, the neoplastic cells have robust glucose uptake, suggesting that the microvasculature has adopted an alternative method for nutrient uptake and cellular trafficking. Using adapted thick tumour section immunostaining and three‐dimensional (3D) construction imaging in human tissue samples, we identified an undiscovered feature of the mature microvasculature in advanced PDAC tumours; long, hair‐like projections on the basal surface of microvessels that we refer to as 'basal microvilli'. Functionally, these basal microvilli have an actin‐rich cytoskeleton and endocytic and exocytic properties, and contain glucose transporter‐1 (GLUT‐1)‐positive vesicles. Clinically, as demonstrated by PET–CT, the tumour microvasculature with the longest and most abundant basal microvilli correlated with high glucose uptake of the PDAC tumour itself. In addition, these basal microvilli were found in regions of the tumour with low GLUT‐1 expression, suggesting that their presence could be dependent upon the glucose concentration in the tumour milieu. Similar microvasculature features were also observed in a K‐Ras‐driven model of murine PDAC. Altogether, these basal microvilli mark a novel pathological feature of PDAC microvasculature. Because basal microvilli are pathological features with endo‐ and exocytic properties, they may provide a non‐conventional method for cellular trafficking in PDAC tumours. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. |
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| Bibliography: | ark:/67375/WNG-MW9KXKRZ-C Supplemental Information(related to Supplementary materials and methods). Steps to measure microvessel parameters.(related to Supplementary materials and methods). Calibrating the length of basal microvilli.(related to Figure A). Correlation analysis of microvessel parameters and PDAC patient prognosis. (A) Comparison of percentage microvessel coverage and microvessel perimeter in PDAC with those in the normal pancreas and HCC; **p < 0.01, using one-way ANOVA. (B) Branches of microvasculature in pancreas, PDAC and HCC (N, number of samples; n, number of microvessels). (C) Kaplan-Meier survival curves of patients (N = 86) relative to measured microvessel parameters. Survival significances are estimated by log-rank test; values are cut off by median value (n, number of deceased patients). (C) Representative 3D images of microvessels in HCC and PDAC tissues; boxed panel, CD34-positive migrating tip cell and a tip cell connected to the plate body at the end of a microvessel. Of note, patients did not receive any treatment prior to surgery for these tissue samples. Scale bar = 20 µm.(related to Figures and ). Low HIF-1α levels present in PDAC and high HIF-1a levels present in HCC. (A) HIF-1α expression was not detected in 11/12 of PDACs and normal pancreas (2/2) samples by immunoblotting; C, PDAC; N, normal pancreas. Three representative tissue microarrays of HIF-1α and CD34 immunostaining in PDAC patients (17 arrays; n = 200 patients). HIF-1α expression was observed in PDAC microvasculature (A3-B-3; black arrows, microvessels), non-invasive precursor lesions (A1-D-3 and A3-E-7; pink arrows) and 88/197 neoplastic cells (A1-D-7, HIF-1α-positive neoplastic cells). (B) Correlation of microvessel parameters with HIF-1α expression in neoplastic cells of PDAC patients (N, patient number); p values were calculated by Spearman's correlation. (C) VEGFR2 expression in the precancerous lesion (C1, mucinous) and expression in a small fraction of tumour cells (D6). (D) HIF-1α expression pattern in HCC tissue by immunoblotting and IHC; HIF-1α was detected in all 13 HCC samples and highly expressed in three non-neoplastic livers (C, tumour; N, liver). CD34 and HIF-1α immunostaining in tissue microarrays of HCC tissues (N = 158) and representative images of HIF-1α expression patterns in HCC patients and their corresponding microvessel characteristics. A HCC sample with a high level of HIF-1α and dilated microvessels (D-11) and another sample negative for HIF-1α has compressed microvessels (C-3). (E) The correlation of microvessel parameters with HIF-1α expression level of tumour cells in HCC tissues; p values were calculated using Pearson's correlation. (F) Representative images of VEGFR2 expression in HCC patients by immunostaining; VEGFR2 was expressed by liver hepatocytes, tumour cells and endothelial cells (Pt1; pink arrow, endothelial cell) and by granulocytes and endothelial cells (Pt2 and Pt3; pink arrow, endothelial cell). Scale bar = 20 µm.(related to Figures and ). PDAC microvessels are mature with high pericyte coverage, while HCC microvessels are immature with nascent and minimal pericyte coverage. (A) Co-immunostaining of PDAC and HCC microvessels for endothelial marker, CD31 and pericyte marker, SMA; boxed panels show SMA-positive pericytes in PDAC (PDAC: Pt1 and Pt2) and microvessel branches with no SMA-positive pericytes coverage in HCC (HCC: Pt1 and Pt2; white arrows, microvessels without pericytes). (B) Co-immunostaining of PDAC and HCC microvessels with CD34 and Desmin; boxed panels show the pericyte coverage in PDAC and HCC. (C) Co-immunostaining of PDAC and HCC microvessels for CD34 and pericyte marker, PDGFRβ; boxed panels show the PDGFRβ-positive pericyte coverage pattern in HCC and PDAC. (D) Co-immunostaining of PDAC and HCC microvessels for CD34 and pericyte marker, NG2; boxed panels show pericytes at the branch points and completely surrounding PDAC microvessels (PDAC; Pt1, Pt2; yellow arrows, pericytes). Scale bars = 50 µm.(related to Figures and ). PDAC microvessels have a complete basement membrane. (A) Triple fluorescent immunostaining of microvessels (CD31) to image the microvessels and their basement membrane (collagen IV), as well as pericyte coverage (SMA) in PDAC and HCC tumour sections; yellow arrows, basement membrane; white arrows, bare basement membrane; scale bars = 50 µm. (B) TEM image of microvessels in PDAC; white arrows, three consecutive junctional complexes; red arrow, a tight junction; yellow arrow, intermediate junction.(related to Figures and ). Basal microvilli are present in the functional microvessels of advance-stage PDAC tumours. (A) GLUT-1 immunostaining identified travelling RBCs in the lumen of a microvessel with basal microvilli. (B) Co-immunostaining microvessels with CD34 and actin filament marker, phalloidin; pink arrow, basal microvilli; white arrow, cytoplasm terminal web. (C) 3D images of CD34-stained microvessel in non-invasive pancreatic tumour precursor lesions, including PanIN-1, PanIN-2 and PanIN-3. (D) 3D image of a microvessel with long basal microvilli in aggressive PDAC; yellow dotted line, invasive front (see Movie S2). (E) 3D image of microvessels with basal microvilli in proximity to PDAC invasive front; white arrows, 'hairy' microvessels; yellow arrows, non-tumour microvessels in close proximity to invasive tumour cells; pink arrow, GLUT-1-positive invasive cells. (F) 3D image of microvessel with basal microvilli in GLUT-1-postive tumour region; white arrows, 'hairy' microvessels; pink and yellow arrows, GLUT-1-positive metastatic neoplastic cells; yellow arrows, GLUT-1; boxed region indicates magnified area (see Movie S2).(related to Figure ). Basal microvilli in PDAC may enhance glucose transport. (A) Basal microvilli versus SUVmax values in another five PDAC patients. (B) Relationship between SUVmax values of patients and percentage coverage of microvessels in tumours; statistical significance performed using Spearman's correlation. (C) GLUT-1 expression levels in MiaPaCa-2 cells in response to varying glucose concentrations and hypoxic stress; (left panel) harvested after 36 h stress; (right panel) harvested after 24 h culture in DMEM/10% FBS/glucose). For glucose deprivation, cells were maintained in glucose-depleted DMEM without serum. For hypoxia induction, 400 µm CoCl2 was added to the medium and the cells were incubated for 36 h. (D) GLUT-1 and CD34 co-immunostaining in PDAC demonstrate a spatial expression pattern in PDAC neoplastic cells (dotted line in circled region); 3D images of a spatial GLUT-1 expression patterns in PDAC tumour in lower magnification; white arrows, region of low GLUT-1 expression. (E) Spatial GLUT-1 expression in neoplastic cells in close proximity to a microvessel with extensive basal microvilli; 20 µm; 1 µm interval; inner panel displays the position of microvessel to the neoplastic duct. (F) Correlation between GLUT-1 expression in neoplastic cells and the distance to microvessels; significance was measured using Spearman's correlation.Movie S1. (related to Figure ). 3D demonstration of microvasculature with basal microvilli in PDAC tumours (PDAC i-iv) and 3D demonstration of normal pancreas and HCC tumour microvessels.Movie S2. (related to Figure S4). 3D demonstration of Figure S4E, F; DAPI, blue; CD34, red; GLUT-1, green.Movie S3. (related to Figure D). 3D demonstration of Figure D, PDAC-i; DAPI, blue; CD34, red; phospho-VEGFR2Y996, green.Movie S4. (related to Figure D, C). 3D video of Figure C, D, in z axis: (C) DAPI, blue; CD34, red; GLUT-1, green; (D) CD34, blue; phalloidin, red; GLUT-1, green.Movie S5. (related to Figure S6C). 3D demonstration of spatial GLUT-1 expression in PDAC neoplastic cells in close proximity to long basal microvilli; CD34, red; GLUT-1, green.Movie S6. (related to Figure ). 3D demonstration of 'hairy' microvessels in KPC mouse tumour and microvessels in KPC mice near normal pancreatic tissue; CD34, green; DAPI, blue.Correlation of microvascular parameters and clinical pathological characteristics in patientsSummary of pericyte characteristicsBasic clinical characteristics of panNET and HBs patientsList of antibodies and dyes ArticleID:PATH4506 istex:DAA30CC57DC9D449797DCC4A47CF3503CB99D34A ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
| ISSN: | 0022-3417 1096-9896 |
| DOI: | 10.1002/path.4506 |