Overexpression of TGF-beta by infiltrated granulocytes correlates with the expression of collagen mRNA in pancreatic cancer

Abstract

Pancreatic cancer is often associated with an intense production of interstitial collagens, known as the desmoplastic reaction. To understand more about desmoplasia in pancreatic cancer, the expression of mRNA for type I and III collagens and potent desmoplastic inducing growth factors transforming growth factor-beta (TGF-beta), connective tissue growth factor (CTGF), acidic and basic fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) A and C and epidermal growth factor (EGF) was analysed by quantitative RT-PCR. Expression of both collagens in 23 frozen primary pancreatic cancer nodules was significantly higher than that in 15 non-neoplastic pancreatic tissues. The expressions of mRNAs for TGF-beta, acidic FGF, basic FGF and PDGF C were likewise higher in surgical cancer nodules, while that of CTGF, PDGF A and EGF were not. Among these growth factors, the expression of TGF-beta mRNA showed the most significant correlation with that of collagens (P<0.0001). By immunohistochemistry, TGF-beta showed faint cytoplasmic staining in cancer cells. In contrast, isolated cells, mainly located on the invasive front surrounding cancerous nests, were prominently and strongly stained. These TGF-beta-positive cells contained a segmented nucleus, were negative for anti-macrophage (CD68) and positive for anti-granulocyte antibodies, indicating their granulocytic nature. In conclusion, TGF-beta seemed to play a major role among the various growth factors in characteristic overproduction of collagens in pancreatic cancer. Moreover, the predominant cells that express TGF-beta were likely to be infiltrated granulocytes (mostly are neutrophils) and not pancreatic cancer cells.

Figure 1

Expressed copy number per 100 ng of total RNA in pancreatic cancerous (C) and noncancerous (N) lesions from surgical specimens as measured by real-time RT–PCR. (A) Expressions of type I collagen and type III collagen in C were significantly higher than that of N. (B) Expressions of TGF-β, aFGF, bFGF and PDGF C in C were significantly higher than that of N, while those of CTGF, PDGF A and EGF were not significant. Bars indicate mean values. NS: not significant.

Figure 2

Correlation between TGF-β and type I collagen (A), and TGF-β and type III collagen (B) mRNA expression in surgical specimens. The expressed copy number of TGF-β and collagens in pancreatic cancer tissues from surgical specimens showed a correlation.

Figure 3

Expression of collagens and TGF-β mRNA in various cancer cell lines. (A) Expressions of type I and type III collagens were negative in pancreatic cancer cells, except for a small amount of type III in PSN1 compared with fibroblasts. (B) Since the expression of TGF-β in pancreatic cancer cell lines was the same or less than that in fibroblasts, gastric cancer and colon cancer cell lines, it was presumed that TGF-β overexpression is not a specific feature for pancreatic cancer cells.

Figure 4

TGF-β immunohistochemistry in pancreatic adenocarcinoma. Transforming growth factor-β immunostaining was visualised by short (≈1 min) and long (≈10 min) reactions with DAB. Note that staining for cancer cells is barely visible at short DAB staining times (closed arrows) in both the tumour periphery (A) and core (B), and only slightly apparent after a 10-min reaction (closed arrows) in both the tumour periphery (C) and core (D). Intense TGF-β immunoreactivity was found in granular cells adjacent to the pancreatic cancer nests, even at short DAB incubation periods (open arrow heads) (A, B). These TGF-β-positive cells were predominantly observed at the tumour periphery (A), and are rare in the tumour core (B). Immunohistochemistry using anti-GFP rabbit polyclonal antibodies as a negative control against anti-TGF-β rabbit polyclonal antibody resulted in negative staining in both the tumour periphery (E) and core (F). NC: negative control.

Figure 5

Distribution of TGF-β, CD68 and antigranulocyte-positive cells in pancreatic adenocarcinoma. The distribution of isolated TGF-β-positive cells in pancreatic cancer (A) was similar to that of macrophage (i.e. CD68+ cells) (C) and granulocytes (E) in low power field observation. However, in high power field observation, the morphology of TGF-β1-positive isolated cells (B) coincided with antigranulocyte-positive segmented nucleus cells (F) but not CD68+ mononuclear cells (D).

Figure 6

TGF-β immunoreactivity in pancreatic, gastric, and colon cancer tissues. TGF-β immunoreactivity was found in isolated cells around cancer nests in pancreatic cancer tissue (A) and many of these cells harboured segmented nuclei (B). Transforming growth factor-β-positive segmented nuclei cells were likewise observed in gastric (C, D) and colon (E, F) cancers, especially in the tumour periphery. Low power field (A, C, E). High power field (B, D, F).

Figure 7

Confocal immunofluorescence images showing TGF-β (red) (A, D), CD68 as a marker of macrophages (green) (B) and granulocytes (green) (E). Transforming growth factor-β staining was topographically different from the staining of CD68+ cells (C). However, double staining with anti-TGF-β and antigranulocyte antibodies resulted in a consistent overlap (F). AG: antigranulocyte.

Figure 8

Morphological demonstration of infiltrated granulocytes in pancreatic cancer in Haematoxylin–eosin section. (A, B) Majorities of isolated cells in stromal, that was the precipitated area of TGF-β-staining positive cells, harboured segmented or polymorpho nuclei with neutrally stained cytoplasmic granules, indicating their neutrophilic nature. (C, D) Bilobed or trinuclei cells with acidic stained granules (=red), characteristic presentation of eosinophils, are also observed, but the proportion of these cells was at most 3 – 5% of the entire granulocyte. Low power field (A, C). High power field (B, D).