Hyaluronan deposition in pancreatic ductal adenocarcinoma as determined by hyaluronic acid binding protein (HABP) ligand histochemistry.

<p>Some tumours showed no extracellular HA (A) deposition while other tumours showed moderate (B) or high amounts of HA (C).</p

PaCa 5061 primary human pancreatic adenocarcinoma xenograft immunohistochemically stained with an anti-hyaluronan polyclonal antibody.

<p>Note the staining of the cancer cells themselves, which were more intensively stained than the surrounding extracellular matrix (A). If hyaluronan was detected by hyaluronic acid binding protein, it was present in the extracellular matrix and not in the cancer cells themselves (C). If the section was pre-treated with hyaluronidase, the hyaluronan immunoreactivity did not vanish after antibody IHC (B); in contrast, hyaluronidase pre-treatment abolished its reactivity completely according to HABP staining (D). Collectively these results indicate that the anti-hyaluronan antibody binding is not caused by hyaluronan and that this approach is therefore unsuitable for its detection.</p

Clinico-pathological data of patients with pancreatic adenocarcinoma correlated with hyaluronan (HA) expression levels (<i>n</i>.<i>a</i>. not available).

<p>Clinico-pathological data of patients with pancreatic adenocarcinoma correlated with hyaluronan (HA) expression levels (<i>n</i>.<i>a</i>. not available).</p

Is hyaluronan deposition in the stroma of pancreatic ductal adenocarcinoma of prognostic significance? - Fig 2

<p>Hyaluronan (HA) deposition in human nasal conchae as determined by anti-HA antibody staining (A) and after hyaluronidase pretreatment (B). Changes in staining patterns and intensity were not seen, unlike when detecting HA by hyaluronic acid binding protein (HABP) ligand histochemistry (C), which showed a complete absence of HA after pretreatment (D).</p

Clinicopathological correlation of BLCAP expression in 2,197 sample TMA.

*<p>Kruskal-Wallis one way analysis of variance on ranks.</p>**<p>Mann-Whitney rank-sum test.</p

Clinical characteristics of DCTB tissue specimens.

<p>Clinical characteristics of DCTB tissue specimens.</p

2D PAGE and 2D Western blot analysis of BLCAP protein spot patterns.

<p>(A) COS-1 cells transfected with pZeoSV2 empty vector and labeled with ³⁵S-methionine. (B) COS-1 cells transfected with pZeoSV2– BLCAP overexpressing construct and labeled with ³⁵S-methionine. Radioactive metabolic labeling (³⁵S-methionine) of COS-1 cells was used to ensure the highest detection sensitivity. (C) 2D Western blot of COS-1 cells transfected with pZeoSV2– BLCAP construct detected with anti-BLCAP antibody (10 sec film exposure). (D) 2D gel of proteins from breast tumor 63 stained with silver. (E) 2D Western blot of protein lysate from breast tumor 63 (see D) reacted with anti-BLCAP antibody (1 min film exposure). The positions of the BLCAP protein in the 2D-PAGE gels and corresponding 2D Western blots, are indicated by black arrows. The positions of several reference proteins are indicated by red arrows: ACTB – beta actin; ENO1 -alpha enolase 1; CANX – calnexin; PDI - Protein disulfide-isomerase; TUBA1A - tubulin alpha-1A chain; YWHAZ - 14-3-3 protein zeta/delta. The identity of all reference spots were confirmed by MS analysis.</p

Differential expression of BLCAP in tumor samples.

<p>The DCTB 123 patient set, including normal, tumor and lymph node metastasis samples (A), or a subset of matched 62 normal and tumor samples (B) were analyzed by quantitative IHC. Mean intensity scores for each group are indicated by red lines.</p

Immunohistochemical expression analysis of BLCAP in FFPE breast tissue samples.

<p>(A) No immunostaining was observed in a normal breast tissue section reacted with BLCAP antibody preincubated with immunizing peptide. (B) Immunohistochemical staining of BLCAP protein in a normal breast tissue sample demonstrated the presence of the BLCAP antigen in luminal epithelial cells with weak cytoplasmic expression (black arrow). Marked nuclear expression was also observed occasionally (red arrow). Yellow arrow points to a vessel with moderate immunoreactivity for BLCAP. (C) In a few cases IHC analysis of tumor samples showed that BLCAP was expressed in tumor cells with weak cytoplasmic expression (red arrow). (D) Most cases showed moderate to strong cytoplasmic expression with no detectable nuclear presence (red arrow) but in some cases (E) we could observe strong nuclear expression of BLCAP (red arrow). (F) In a few cases, samples were heterogenous with some cells showing distinct perinuclear immunoreactivity for BLCAP (red arrow). (G) Malignant cells showed stronger immunoreactivity (red arrows) than adjacent normal-looking ducts (black arrow), demonstrating up-regulation of this protein in tumor cells. Yellow arrows point to vessels with strong immunoreactivity for BLCAP. (H) We also observed up-regulation of BLCAP in early lesions where lobular carcinoma in situ cells showed overexpression of this protein (grey arrows) in relation to normal adjacent areas (black arrow), and at levels comparable to invasive carcinoma cells (red arrow).</p

Expression analysis of BLCAP by quantitative IHC of normal specimens (blue bars) and corresponding tumor samples (red bars) of 62 matched cases from the DCTB dataset.

<p>Illustrative IHC images are shown for normal and tumor samples with weak immunoreactivity (N11 and T76, respectively), and for normal and tumor samples showing substantial immunoreactivity for BLCAP (N14 and T63, respectively). Magnification 20X.</p