Fig 1: CSPG4 expression in pancreatic tumor cells.(A) Immunoblotting with H-300 antibody detected pCSPG4 protein in three of nine pancreatic cancer cell lines; shown in relation to melanoma SK-MEL-28 and a normal immortalized ductal epithelial cell line, HPDE. (B) QRT-PCR analysis of pancreatic cancer cell lines, normal epithelial HPDE cells and primary myofibroblasts (pancreatic stellate cell, PSC). (C) Whereas none of the ELISA kits was capable of detecting sCSPG4 in supernatants (not shown), FACS analyses with LHM2 and anti-mouse Cy2-conjugate (bold lines) revealed surficial exposure of CSPG4 by pCSPG4-postive Panc1 and HS766T but not pCSPG4-negative MiaPaca2. (D) The focal accumulation of CSPG4-forming dots and short fibrils was visualized by means of immunofluorescence in Panc1 cells. The specificity of staining was confirmed using mouse isotype IgG1 control (not shown).
Fig 2: Reduction of serum CSPG4 (sCSPG4) in pancreatic diseases and its selective preservation following the ‘drop and restoration’ pattern in advanced IPMN and PDAC.(A) Systemic levels of sCSPG4 were determined by ELISA in sera (USCN/Cloud-Clone Corp. kit; n = 83) of healthy volunteers and patients with chronic pancreatitis (CP), serous cystadenoma (SCA), premalignant (dysplastic IPMNdys and IPMNtis), and malignant (IPMN with an associated invasive carcinoma, IPMNinv) forms of intraductal papillary-mucinous neoplasm, as well as in ductal (PDAC), adenosquamous (AdSq) and anaplastic (Anapl) carcinomas. The data are summarized to show individual values, median level, and interquartile range (IQR). (B) The ROC analyses of the ELISA data showed the discriminatory power of sCSPG4 levels for patients with different pancreatic diseases. The dotted green line represents the median level of the particular cohort used to estimate the frequency of the sCSPG4 underexpressers in the panel (C), as detailed in Materials and Methods. (D–E) Validation of ELISA findings in an independent cohort (n = 221). Red-bordered areas indicate subgroups depicting IPMN and PDAC progression. The patients’ characteristics and results of statistical analyses are presented in the main text and Tables 1 and 2. (F) Validation of ELISA findings with a different ELISA kit (CUSABIO; n = 64).
Fig 3: Localization of CSPG4 in pancreatic tissues.Staining of tissues was performed using mouse LHM2 anti-CSPG4 antibody, followed by HRP-conjugated anti-mouse polymer and visualization with the Dako Envision system (immunohistochemistry, A–G), or using rabbit anti-CSPG4 antibody (H-300) and mouse anti-COL6 antibody followed by anti-rabbit-Cy2 and anti-mouse-Cy3 conjugates (immunofluorescence, I–J). The antigen-specific antibodies were replaced with isotype IgGs for negative control; the representative image is given as inset in (D). Shown (100x–200x) are tubular complexes/acinar-to-ductal metaplasia ADM (A), pre-malignant PanIN lesions of low (B) and high grade (C) in paratumoral areas of PDAC biopsies; perineural invasion of PDAC tumor cells (D); squamous compartment in adenosquamous carcinoma (E); and high-resolution images (630x) of an epithelium lining of cysts in serous cystadenoma, SCA (F) and tumor cells in PDAC lesion (G). (H) Co-expression of CSPG4 and COL6 RNA in pancreatic tissues according to microarray-based measurements. (I) Double immunofluorescent staining showed rare co-localization of pGSPG4 (green) and COL6 (red; white arrows) in PDAC lesions, and prevalence of COL6-free surfaces. The images were routinely recorded using Axiovision Software installed on a Carl Zeiss microscope, and (J) confirmed by confocal laser scanning microscopy (TCS-SP, Leica Microsystems, courtesy of Dr. N. Brady, Bioquant, Heidelberg University/DKFZ, Germany).
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