Fascin is regulated by slug, promotes progression of pancreatic cancer in mice, and is associated with patient outcomes - PubMed (original) (raw)

. 2014 May;146(5):1386-96.e1-17.

doi: 10.1053/j.gastro.2014.01.046. Epub 2014 Jan 23.

Jennifer P Morton 1, YaFeng Ma 1, Saadia A Karim 1, Yan Zhou 1, William J Faller 1, Emma F Woodham 1, Hayley T Morris 1, Richard P Stevenson 1, Amelie Juin 1, Nigel B Jamieson 2, Colin J MacKay 2, C Ross Carter 2, Hing Y Leung 1, Shigeko Yamashiro 3, Karen Blyth 1, Owen J Sansom 1, Laura M Machesky 4

Affiliations

Fascin is regulated by slug, promotes progression of pancreatic cancer in mice, and is associated with patient outcomes

Ang Li et al. Gastroenterology. 2014 May.

Abstract

Background & aims: Pancreatic ductal adenocarcinoma (PDAC) is often lethal because it is highly invasive and metastasizes rapidly. The actin-bundling protein fascin has been identified as a biomarker of invasive and advanced PDAC and regulates cell migration and invasion in vitro. We investigated fascin expression and its role in PDAC progression in mice.

Methods: We used KRas(G12D) p53(R172H) Pdx1-Cre (KPC) mice to investigate the effects of fascin deficiency on development of pancreatic intraepithelial neoplasia (PanIn), PDAC, and metastasis. We measured levels of fascin in PDAC cell lines and 122 human resected PDAC samples, along with normal ductal and acinar tissues; we associated levels with patient outcomes.

Results: Pancreatic ducts and acini from control mice and early-stage PanINs from KPC mice were negative for fascin, but approximately 6% of PanIN3 and 100% of PDAC expressed fascin. Fascin-deficient KRas(G12D) p53(R172H) Pdx1-Cre mice had longer survival times, delayed onset of PDAC, and a lower PDAC tumor burdens than KPC mice; loss of fascin did not affect invasion of PDAC into bowel or peritoneum in mice. Levels of slug and fascin correlated in PDAC cells; slug was found to regulate transcription of Fascin along with the epithelial-mesenchymal transition. In PDAC cell lines and cells from mice, fascin concentrated in filopodia and was required for their assembly and turnover. Fascin promoted intercalation of filopodia into mesothelial cell layers and cell invasion. Nearly all human PDAC samples expressed fascin, and higher fascin histoscores correlated with poor outcomes, vascular invasion, and time to recurrence.

Conclusions: The actin-bundling protein fascin is regulated by slug and involved in late-stage PanIN and PDAC formation in mice. Fascin appears to promote formation of filopodia and invasive activities of PDAC cells. Its levels in human PDAC correlate with outcomes and time to recurrence, indicating it might be a marker or therapeutic target for pancreatic cancer.

Keywords: Actin Cytoskeleton; EMT; Pancreas; Tumor Progression.

Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1

High fascin histoscore predicts poor survival and recurrence in human PDAC. (A) Representative images of fascin staining in human PDAC. (B) Kaplan-Meier analysis showing cases with high histoscore have poorer outcomes compared with low expression (P = .011 by log-rank test). (C) Boxplot of fascin histoscore vs tumor grade. (D) Boxplot of fascin histoscore vs vascular invasion. (E) Boxplot of fascin histoscore vs time to recur. (F) Representative fascin staining in KPC mice as indicated. Yellow dashes outline the tumor. Insets show high-magnification views of ductal cells. Purple arrows: fascin-positive cells in normal islets. Fascin-positive cells in PanIN3 are indicated by purple arrows. Scale bars = 50 μm for normal and PanINs, 200 μm for early PDAC.

Figure 2

Figure 2

Fascin is required for early PDAC formation. (A) Gene targeting strategy for generating FKPC mice. (B) Kaplan-Meier curves. (C) Top: Western blot analyses of tumor tissue. Bottom: Histology of PDAC H&E (top), immunohistochemistry for fascin (middle) and p53 (bottom). (D) Dot plot of primary tumor-to-body weight ratios at sacrifice (mean ± SEM). (E) Two-hour bromodeoxyuridine (BrdU), Ki67+, phospho-histone H3+ (PHH3), and cleaved caspase 3+ (CC3) cells in PDACs from KPC and FKPC mice. n ≥ 16 fields from n ≥ 4 mice (mean ± SEM). (F) Left: Number of PDAC-positive KPC and FKPC mice at indicated times. ∗P < .05 by χ2 test. Middle: Primary pancreas-to-body weight ratios (mean ± SEM). ∗P < .05; ∗∗P < .01 by Mann Whitney U test. Right: Relative tumor size, lower quartile, median, and upper quartile are shown. ∗P < .05 by Mann Whitney U test. Scale bars in (C) = 100 μm.

Figure 3

Figure 3

Fascin is a target of slug in PDAC. (A) Expression of EMT markers in a representative panel of 10 independent KPC PDAC cell lines. (B) Spearman correlation analysis of fascin and slug protein expression in mouse PDAC cell lines. Fascin and slug expression level in PDAC cell lines was plotted as relative expression to 070669 PDAC cell line, with other cell lines numbered as in (A). (C) Left: Western blot analysis with control, Flag-slug, Flag-snail, and twist expressing 070669 PDAC cells for proteins as indicated. Bar graphs: Relative protein levels of fascin or quantitative polymerase chain reaction analysis mRNA in 070669 PDAC cells expressing EMT Tfs as indicated (mean ± SEM, n = 3). ∗P < .05; ∗∗P <.01, Student t test. (D) Phase and immunofluorescence microscopy of 070669 PDAC cells expressing EMT Tfs as indicated. Low E-cadherin and high fascin expressing cells are indicated by yellow arrows. Scale bars in (D) = 10 μm for immunofluorescence, 50 μm for phase. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) loading control in (A) and (C).

Figure 4

Figure 4

Slug drives fascin expression in KRasG12D p53R172H Pdx1-Cre (KPC)-driven PDAC. (A) Fluorescent images of sections from normal pancreas and PanINs co-stained for E-cadherin (W, white), fascin (green), slug (red), and DNA (4′,6-diamidino-2-phenylindole [DAPI], blue). Insets show high-magnification views of ductal cells. (B) Well (top) and poorly differentiated (bottom) PDACs from KPC mice co-stained for E-cadherin, fascin, and slug. Insets higher magnification. Scale bars in (A) and (B) = 20 μm.

Figure 5

Figure 5

Fascin is a slug target gene. (A) Schematic showing the potential E-box element in intron 1 of the fascin gene and regions targeted by 3 primer pairs (#1−3, red lines). Primer pair #1 targets the putative E-box, and primer pair and #2 and #3 target downstream regions. The number in parentheses indicates distance downstream of transcription start site. (B) Chromatin from 070669 PDAC cells expressing Flag-slug was immunoprecipitated using Flag antibody and polymerase chain reaction was performed on the chromatin Immunoprecipitation product using 3 primer pairs. Primers for an E-cadherin promoter E-box element were used as positive control (mean ± SEM). ∗∗P < .01 by Student t test. (C) Top: the putative E-box on the mouse fascin gene and mutations. Bottom: Relative luciferase activities of 070669 PDAC cells transfected as indicated. n = 3 experiments (mean ± SEM). ∗∗P < .01; ∗P < .05 by Student t test.

Figure 6

Figure 6

Fascin is required for efficient metastasis in KPC mice. (A) H&E staining of (left) bowel and (right) peritoneal invasion by cells from KPC and FKPC tumors. Insets show zoom of invasion area. Black arrows indicate direction of invasion. (B) Table shows incidence of invasion (top) and metastasis (bottom) in KPC and FKPC mice with PDAC (∗P < .05; ∗∗P < .01, χ2 test). (C) Incidence and volume of ascites in PDAC-bearing KPC and FKPC mice (left: ∗∗P < .01, χ2 test; right: mean ± SEM. ∗∗P < .01 by Mann-Whitney U test). (D) Top: Representative pictures of metastatic nodules (red arrows) on intestinal mesentery of KPC and FKPC mice. Bottom: Mesenteric metastasis from KPC mice H&E (left), fascin (middle), and p53 (right). (E) Left: Number of mesenteric metastases; ∗∗P < .01 by Mann-Whitney U test. Right: Survival of mice with liver metastases. Blue and red dotted lines indicate median survival for KPC and FKPC mice, respectively. (F) Liver metastasis from KPC mice for H&E (left), fascin (middle), and p53 (right). Scale bar in (A) and (D) = 100 μm and in (F) = 50 μm.

Figure 7

Figure 7

Fascin mediates peritoneal metastasis via promotion of transmesothelial intercalation. (A) Still image of live GFP-fascin in PDAC cells transmigrating through a red CMTPX CellTracker (CT)-labeled confluent Met5A monolayer. Yellow stars indicate fascin-positive filopodia. (B) Intercalation for individual cells during 10 hours (n = 100 cells, 8 fields, mean ± SEM, n = 3) ∗∗P < .01 by Student t test. (C) Time-lapse video stills of PDAC cells intercalating between MCs. Protrusions are indicated by yellow arrows. (D) PDAC cells as indicated were injected intraperitoneally into nude mice. Yellow arrows indicate tumor nodules. (E) Mesenteric tumor nodules from GFP-fascin−rescued cells express fascin and p53. Insets show high magnification. (F) Number of mesenteric and diaphragm metastases. n = 10 mice per condition. ∗P < .05; ∗∗P < .01 by Mann-Whitney U test. Scale bars in (A), (C), and (D) = 10 μm and in (E) = 100 μm.

Supplementary Figure 1

Supplementary Figure 1

Fascin-deficient mice have normal pancreas morphology and normal survival rate. (A) Dot plot of pancreas-to-body weight ratios in wild-type and fascin-deficient mice at 5 weeks. (B) Histology analysis of H&E-stained sections from pancreas of 5-week control or fascin-deficient mice. Representative images (C) and quantification (D) of BrdU+ or Ki67+ duct cells (left) and acinar cells (right) in pancreata from 5-week control and fascin-deficient mice measured by immunohistochemistry. Red arrows indicate BrdU or Ki67-positive nuclei (n = 4 mice per genotype). (E) Mendelian segregation of pups at weaning (3 weeks). Six litters from crosses between fascin heterozygous males and females in a mixed background were recorded. (F) Body mass of control and fascin-deficient mice in a mixed background at 8 weeks. At least 5 mice were measured for each genotype and sex. Scale bars in (B) and (C) = 50 μm. In (D), data are shown as mean ± SEM.

Supplementary Figure 2

Supplementary Figure 2

Fascin is not required for PanIN formation. (A) Gene targeting strategy for generating fascin−/−, KRasG12D Pdx1-Cre (FKC). (B) Quantitation of spontaneous (4 months) PanIN formation in KC and FKC mice. Left: Dot plot of measurements of pancreas-to-body weight ratios. Middle: Box plot of amylase-positive area (lower quartile, median, and upper quartile are shown). Right 2 bar graphs: Relative number and percentage of PanINs of grade 1−3 per histopathologic section of pancreas. Data are shown as mean ± SEM (n = 6 mice per genotype). (C) Panels show tissue sections with spontaneous PanIN in FKC compared with KC mice; H&E (top), Alcian blue (middle), and dual immunohistochemistry (IHC) for CK19 (brown) and amylase (blue) (bottom). (D) Images and quantification of BrdU incorporation assay measured by IHC in duct cells (top) and acinar cells (bottom) in 4-month KC and FKC pancreas. Red arrows indicate BrdU-positive nuclei (n = 4 mice per genotype). (E) Panels show spontaneous PanIN formation in 6-week FKPC or KPC mice, with H&E (top) and dual IHC for CK19 (brown) and amylase (blue) (bottom). (F) Quantitation of spontaneous (6 weeks) PanIN formation in KPC and FKPC mice. Left: Box plot of amylase-positive area (lower quartile, median, and upper quartile are shown). Right 2 bar graphs: Number and percentage of PanINs of grades 1−3 per histopathologic section of pancreas. Data are shown as mean ± SEM (n = 5 mice per genotype). Scale bars in (C) and (E) = 100 μm and in (D) = 50 μm.

Supplementary Figure 3

Supplementary Figure 3

Fascin is not required for acute pancreatitis−induced PanIN formation. (A) Protocol used for cerulein-induced PanIN formation in KC and FKC mice. (B) Western blot (top) and immunohistochemistry (IHC) analysis (bottom) of fascin expression in control and KC mice after phosphate-buffered saline or cerulein treatments (1 day, 7 days, and 21 days post cerulein). (C) Left: Panels show cerulein-induced (21 days post cerulein) PanIN formation in FKC compared with KC mice, shown by H&E (top), Alcian blue (middle) and dual IHC for CK19 (brown) and amylase (blue) (bottom). Right: Quantitation of cerulein-induced (21 days post cerulein) PanIN formation in KC and FKC mice. Graphs describe the following, as indicated: dot plot of pancreas-to-body mass ratios (n = 6 for KC, n = 15 for FKC mice); box plot of amylase-positive area (lower quartile, median, and upper quartile are shown); relative number of PanIN per field and percentage of PanINs of grade 1−3 per histopathologic section of pancreas. Data are shown as mean ± SEM (n = 6 mice per genotype). (D) Images and quantification of BrdU incorporation assay measured by IHC in duct cells (top) and acinar cells (bottom) in KC and FKC pancreas 21 days post cerulein injection. BrdU-positive nuclei are indicated by red arrows (n = 4 mice per genotype). (E) Quantification of neutrophil (anti-myeloperoxidase [MPO]) recruitment to normal and fascin-deficient pancreas 1 day after cerulein injection. Data are shown as mean ± SEM, n = 4 mice per genotype. (F) Dot plots of whole blood counts from 6-week control and fascin-deficient mice 21 days post cerulein injection. Left: monocytes, middle: lymphocytes, and right: neutrophils are shown. Data are shown as mean ± SEM. Scale bars in (B) and (C) = 100 μm and in (D) and (E) = 50 μm.

Supplementary Figure 4

Supplementary Figure 4

Tumor-intrinsic action of fascin. (A) Pancreata from Rosa26-LSL-tdRFP/+ Pdx1-Cre, fascin−/− Rosa26-LSL-tdRFP/+ Pdx1-Cre and Pdx1-Cre mice were analyzed using an OV100 in vivo imaging system. Red fluorescent protein signaling is detected in whole pancreata from Rosa26-LSL-tdRFP/+ Pdx1-Cre and fascin−/− Rosa26-LSL-tdRFP/+ Pdx1-Cre mice, but not in pancreata from mice only expressing Pdx1-Cre. (B) Representative images of immunohistochemical staining for Ki67, 2-hour BrdU pulse, phospho-histone H3 (PHH3), and cleaved capspase-3 (CC3). Quantification is shown in Figure 2_E_. Images (C) and quantifications (D) of infiltration of B cells (CD45R), T cells (CD3), macrophages (F4/80), and neutrophils (NIMP) into tumors. At least 16 medium-powered fields from 4 KPC and FKPC PDACs were analyzed. Data are shown as mean ± SEM. (E) Immunohistochemical visualization of platelet endothelial cell adhesion molecule (CD31) in tumors in KPC and FKPC mice. (F) Quantification of (D), no significant difference in vascularization. Data are shown as mean ± SEM (n = 4 mice from each genotype). Scale bars = 100 μm.

Supplementary Figure 5

Supplementary Figure 5

Slug signaling regulates fascin expression in PDAC cells. (A) Western blot analysis of fascin and EMT markers in primary ductal epithelial cells (PDEC) isolated from the pancreas of a wild-type mouse and a PDAC cell line 118739 harvested from a KPC mouse. Expression of CK-19 in the cell lines confirms their ductal origin and p53 in 118739 cell line confirms its PDAC origin. Flag-snail was used as positive control for snail antibody. Loading control: glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (B) Immunofluorescence microscopy analysis of PDEC and 118739 PDAC cells stained for the indicated proteins. (C) Representative images of example of E-cadherin−negative (127445) and E-cadherin−positive (022160) PDAC cells stained for fascin, E-cadherin, and slug. Co-expression of slug and E-cadherin in 022160 PDAC cells suggest partial EMT. Inserts show individual channels. (D) Expression of EMT markers in a representative panel of 5 independent FKPC PDAC cell lines. 083320 PKC PDAC cell line was used as positive control for fascin. (E) Spearman correlation analysis of (left) fascin and slug and (right) fascin and tubulin gene expression in human pancreatic cancer cell lines using Wagner cell line dataset. As a control, fascin does not correlate with tubulin expression. Scale bars in (B) and (C) = 10 μm.

Supplementary Figure 6

Supplementary Figure 6

Slug is an important regulator of fascin expression. (A) Immunofluorescence microscopy analysis of 070669 PDAC cells transduced with vector (control), Flag-slug, Flag-snail, and twist and stained for the proteins as indicated. Slug, snail, and twist overexpressing cells are indicated by yellow arrows. (B) Western blots (left) and quantitation (right) of proteins as indicated with 070669 PDAC cells treated with slug siRNA. Data are shown as mean ± SEM. ∗∗P < .01 by Student t test. Loading control: α-actinin. (C) Western blot analysis of proteins as indicated with 070669 PDAC cells treated with Zeb1 (top left), Zeb2 (top right), E-cadherin (bottom left), and fascin (bottom right) siRNA. Loading controls: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and α-actinin, as indicated. (D) Phase contrast images of 061843 PDAC cells transduced with vector (control), Flag-slug, Flag-snail, and twist as indicated. (E) Western blot analysis with control, Flag-slug, Flag-snail, and twist expressing 061843 PDAC cells for proteins as indicated. Loading control: α-actinin. (F) Western blot analysis with control, Flag-slug, Flag-snail expressing PANC-1 (left) and HT29 (right) human pancreatic and colon cancer cells for proteins as indicated. Loading control: α-actinin. Scale bars in (A) = 10 μm and in (D) = 50 μm.

Supplementary Figure 7

Supplementary Figure 7

Slug correlates with fascin in human pancreatic cancer. Spearman correlation analysis of fascin and slug (left) or tubulin (right) gene expression in human pancreatic cancer using the Badea dataset (A), Pei dataset (B), and Jimeno dataset (C). Fascin correlation with tubulin was used as a control.

Supplementary Figure 8

Supplementary Figure 8

Fascin regulates lamellipodia dynamics and migration but not cell proliferation. (A) Western blots of 105768 FKPC mouse PDAC cells expressing control vector (pLHCX), human GFP-fascin, and fascin siRNA treated rescue cells. Loading control: glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (B) Left: Control vector and GFP-fascin expressing FKPC 105768 cells expressing RFP or GFP-lifeact. Yellow stars indicate filopodia. Bar graphs show box plots of quantifications as indicated. ∗∗P < .01 by Student t test. (C) Top: Representative kymograph pictures of GFP-fascin rescued and fascin-deficient PDAC cells (derived from Video 2). Green lines indicate persistence time for each protrusion, yellow stars indicate each protrusion. Pixel intensities along a 1-pixel wide line were used to generate the kymograph from the corresponding Video 2; a magnified region (outlined in red) is displayed on the top. Red lines indicate the parameters for one protrusion. D, protrusion distance; P, protrusion persistence; and R, protrusion rate. Bottom bar graphs: Frequency of lamellipodial protrusion events, distance, protrusion rate, and persistence of individual lamellipodial protrusions. Values are the means of means from at least 30 cells. Data are shown as mean ± SEM. ∗∗P < .01 by Student t test. (D) Left: Migration speed of 105,768 cells expressing vectors/siRNA, as indicated. More than 300 cells from 3 experiments were randomly selected and mean migration speed during 6 hours was plotted according to frequency in the population. ∗∗P < .01 by t test. Right: 6-hour tracks of individual cell migration, black tracks migrated faster than 0.5 um/min, red indicates slower. (E) Top left: 2D proliferation assay, no difference between control vector and GFP-fascin expressing FKPC 105768 cells (n = 3). Top right: 3D collagen I cell proliferation assay, no difference between control vector and GFP-fascin expressing FKPC 105768 cells. Data are shown as mean ± SEM (n = 3). Bottom left: Western blot analysis of the effects on the viability of control vector and GFP-fascin−expressing FKPC 105768 cells grown in normal culture plates and ultra-low attachment plate for 24 hours to induce anoikis with cleaved-caspase 3 antibody. Bottom right: Fluorescence-activated cell sorting analysis using Annexin V and propidium iodide for cells undergoing anoikis with control vector and GFP-fascin−expressing FKPC 105768 cells cultured in ultra-low attachment plate for 6 and 24 hours, attached cells were used as negative control. Data are shown as mean ± SEM (n = 3).

Supplementary Figure 9

Supplementary Figure 9

Fascin regulates transmesothelial migration. (A) Representative image of PDAC cell transmesothelial migration. Met5A cells on a fibronectin-coated glass-bottom dish stained with β-catenin and 4′,6-diamidino-2-phenylindole (top left). Green CMFDA-labeled fascin-expressing PDAC 070669 cells were added to MCs for 2 hours (attachment_, top right_), 5 hours (junction open, bottom left), and 10 hours (intercalated, bottom right). Insert shows the junction opening of Met5A cells by PDAC cells. (B) Green CMFDA-labeled 070669 PDAC cells (green) were added to confluent MCs (labeled with β-catenin [_white_] and phalloidin [red_]) grown on a fibronectin-coated glass-bottom dish. Cells were fixed after 10 hours and their localization was analyzed by confocal microscopy. Top and bottom views of the MC monolayers are shown. (C) Western blots analysis of transient fascin knockdown in 070669 PDAC cells. Loading control: glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (D) Representative image of green CMFDA-labeled nontargeting (NT) or fascin siRNA expressing 070669 PDAC cells added to MCs for 5 hours. Top images show the MC monolayer. Middle and bottom images show the 3D top and bottom view of the MC monolayers. (E) Quantification of intercalation for NT and fascin siRNA-treated 070669 cells 5 hours after seeding on MC monolayers. Data are shown as mean ± SEM (n = 3). ∗∗_P < .01 by t test. Scale bars in (A) and (D) = 10 μm.

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References

    1. Warshaw A.L., Fernandez-del Castillo C. Pancreatic carcinoma. N Engl J Med. 1992;326:455–465. - PubMed
    1. Onkendi E.O., Boostrom S.Y., Sarr M.G. 15-year experience with surgical treatment of duodenal carcinoma: a comparison of periampullary and extra-ampullary duodenal carcinomas. J Gastrointest Surg. 2012;16:682–691. - PubMed
    1. Almoguera C., Shibata D., Forrester K. Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell. 1988;53:549–554. - PubMed
    1. Hingorani S.R., Wang L., Multani A.S. Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice. Cancer Cell. 2005;7:469–483. - PubMed
    1. Olive K.P., Tuveson D.A. The use of targeted mouse models for preclinical testing of novel cancer therapeutics. Clin Cancer Res. 2006;12:5277–5287. - PubMed

Supplementary References

    1. Rukstalis J.M., Habener J.F. Snail2, a mediator of epithelial-mesenchymal transitions, expressed in progenitor cells of the developing endocrine pancreas. Gene Expr Patterns. 2007;7:471–479. - PMC - PubMed
    1. Morton J.P., Jamieson N.B., Karim S.A. LKB1 haploinsufficiency cooperates with Kras to promote pancreatic cancer through suppression of p21-dependent growth arrest. Gastroenterology. 2010;139 586−597.e1. - PMC - PubMed
    1. Morton J.P., Karim S.A., Graham K. Dasatinib inhibits the development of metastases in a mouse model of pancreatic ductal adenocarcinoma. Gastroenterology. 2010;139:292–303. - PubMed
    1. Schreiber F.S., Deramaudt T.B., Brunner T.B. Successful growth and characterization of mouse pancreatic ductal cells: functional properties of the Ki-RAS(G12V) oncogene. Gastroenterology. 2004;127:250–260. - PubMed
    1. Li A., Dawson J.C., Forero-Vargas M. The actin-bundling protein fascin stabilizes actin in invadopodia and potentiates protrusive invasion. Curr Biol. 2010;20:339–345. - PMC - PubMed

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