Periostin is expressed in pericryptal fibroblasts and cancer-associated fibroblasts in the colon - PubMed (original) (raw)

Periostin is expressed in pericryptal fibroblasts and cancer-associated fibroblasts in the colon

Yoshinao Kikuchi et al. J Histochem Cytochem. 2008 Aug.

Abstract

Periostin is a unique extracellular matrix protein, deposition of which is enhanced by mechanical stress and the tissue repair process. Its significance in normal and neoplastic colon has not been fully clarified yet. Using immunohistochemistry and immunoelectron microscopy with a highly specific monoclonal antibody, periostin deposition was observed in close proximity to pericryptal fibroblasts of colonic crypts. The pericryptal pattern of periostin deposition was decreased in adenoma and adenocarcinoma, preceding the decrease of the number of pericryptal fibroblasts. Periostin immunoreactivity appeared again at the invasive front of the carcinoma and increased along the appearance of cancer-associated fibroblasts. ISH showed periostin signals in cancer-associated fibroblasts but not in cancer cells. Ki-67-positive epithelial cells were significantly decreased in the colonic crypts of periostin-/- mice (approximately 0.6-fold) compared with periostin+/+ mice. In three-dimensional co-culture within type I collagen gel, both colony size and number of human colon cancer cell line HCT116 cells were significantly larger ( approximately 1.5-fold) when cultured with fibroblasts derived from periostin+/+ mice or periostin-transfected NIH3T3 cells than with those from periostin-/- mice or periostin-non-producing NIH3T3 cells, respectively. Periostin is secreted by pericryptal and cancer-associated fibroblasts in the colon, both of which support the growth of epithelial components.

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Figures

Figure 1

Periostin in non-neoplastic colorectal mucosa. (A) Pericryptal pattern of immunoreactive periostin in normal colonic mucosa. Note the linear staining around the crypts. No immunostaining in the epithelial cells. (B) Double immunostaining of α smooth muscle actin (αSMA) and periostin to evaluate the topographical relationship of both immunoreactivities. Immunofluorescent staining for αSMA shows the pericryptal fibroblasts (PCFs) rimming the crypts (left). Linear immunofluorescent staining for periostin (center). Merged image showing linear staining of periostin between basement membrane of the crypts and αSMA-positive PCFs (right). Note that condensed deposition of periostin faces αSMA-positive PCF (right; arrows). (C,D) Periostin in the inflamed colonic mucosa. (C) Mesh pattern of immunoreactive periostin in the colonic mucosa with inflammatory cell infiltration. Mesh-like deposition of immunoreactive periostin in the lamina propria in addition to the pericryptal pattern (inset, high-power magnification showing PCFs). (D) Merged image of the double immunofluorescent staining for αSMA and periostin in ulcerative colitis. Note the mesh pattern of periostin in the lamina propria without accompaniment of αSMA-positive cells. Bar = 50 μm.

Figure 2

Immunoelectron microscopy of periostin in normal colonic mucosa of the mouse. (A) Immunoelectron microscopy of the colonic mucosa of the mouse (arrowhead, PCF; star, colorectal crypt). Note the pericryptal pattern of immunoreactive periostin in the semithin section counterstained with toluidine blue. (B) Higher magnification of A. Electron dense materials, corresponding to the immunoreactive material, are admitted predominantly in the narrow space between the basement membrane (arrow) and PCF. Immunoreactivity is also observed at the cytoplasmic membrane of the PCF (arrowheads). Bar = 25 μm.

Figure 3

Periostin in adenoma and carcinoma of the colon. (A–F) Assessment of periostin expression in each component of cancer in adenoma. (A) Border area between non-neoplastic colorectal mucosa and adenoma [dotted line; hematoxylin and eosin (H&E)]. (B) Periostin immunohistochemistry of the adenoma component in A (star, same point at the star in A). Note marked decrease of periostin deposition in the pericryptal region of the adenomatous glands (arrow), in contrast to the preserving of pericryptal pattern in the normal crypts at the border (arrowhead). (C) Border area between adenoma and adenocarcinoma (dotted line; H&E). (D) Periostin immunohistochemistry of the carcinoma component in C, showing complete absence of periostin deposition (star, same point at the star in C). (E) Double immunostaining for αSMA and periostin in the adenoma. αSMA-positive PCFs are present just beneath the adenoma glands (arrowheads), but immunoreactive periostin is scant. (F) Semiquantitative evaluation of periostin and αSMA immunoreactivity relative to the glands of normal and neoplastic colon. The population of positive crypts or glands was scored in four groups according to prevalence: score 0, <10%; score 1, 10–49%; score 2, 50–80%; and score 3, >80%. Scores 2 and 3 are presented in the closed column for αSMA and in the open column for periostin. (G–N) Periostin in the invasive and metastatic colorectal carcinoma. (G) Periostin at the invasive front of the colorectal carcinoma at its early stage (arrowheads). (H,I) Periostin in invasive colorectal carcinoma. (H) Infiltrating pattern of periostin deposition in cancer stroma. (I) High-power magnification of H. No immunoreactivity was identified in the carcinoma cells. (J) H&E staining of the corresponding region to I. Mesenchymal cells showing spindle-shaped and plump nuclei, morphologically myofibroblasts, within the cancer stroma (arrowheads). (K) Immunofluorescent double staining for αSMA and periostin in advanced invasive colorectal cancer showing several portions of colocalization. (L) Periostin in the metastatic carcinoma in the liver. Periostin deposition at the capsule of metastatic carcinoma. Note no immunoreactivity in the carcinoma cells or in the normal liver. (Inset) Higher magnification showing fibroblasts within the periostin deposition and those without association of deposition. (M–O) Evaluation of periostin mRNA expression in neoplastic and non-neoplastic colon by ISH and real-time RT-PCR. (M) ISH of periostin mRNA in colorectal carcinoma. Positive signals were observed in the fibroblastic cells in cancer stroma, whereas no signal was observed in cancer cells. (N) No signal was observed in the negative control using sense probe. (O) Quantitative real-time RT-PCR analysis of periostin mRNA in neoplastic (T1–T4) and non-neoplastic (N) colon. Statistically significant overexpression was shown compared with the normal colonic tissue (**p<0.01, ***p<0.001). Bars: A,C,G,H,L = 500 μm; I = 25 μm; M,N = 100 μm.

Figure 4

Evaluation of effect of periostin on epithelial proliferation of normal and neoplastic colon. (A) Immunohistochemical study of Ki-67 for periostin+/+ and periostin−/− mice. The number of Ki-67–positive epithelial cells is larger in the periostin+/+ mice than in the periostin−/− mice. There is a statistically significant difference in the number of epithelial cells immunoreactive for Ki-67 per each crypt between periostin+/+ and periostin−/− mice (**p<0.01). Ki-67–positive epithelial cells are decreased in periostin−/− mice. (**B**) Proliferation assay of HCT116 supplemented with recombinant periostin (100 ng/ml) in culture medium. The increase of cell number is greater with 100 ng/ml periostin at 6, 12, 24, and 48 hr (**_p_<0.01). (**C**) Dose response of recombinant periostin. The increase of cell number at 24 hr is represented as fold change compared with the average of negative control. The difference is statistically significant at concentrations >50 ng/ml (**p<0.01) Bar = 50 μm.

Figure 5

Colony formation assay of HCT116 co-embedded with fibroblasts in type I collagen gel. (A) Colony formation assay of HCT116 co-embedded with periostin−/− or periostin+/+ primary-cultured fibroblast in type I collagen gel. Both colony size and number are larger in the gel co-embedded with periostin+/+ fibroblasts compared with those with periostin−/− fibroblasts (**p<0.01; *** p<0.001). (B) Colony formation assay of HCT116 co-embedded with NIH3T3 transfectants. Both colony size and number are larger in the gel co-embedded with periostin-transfected NIH3T3 compared with those with enhanced green fluorescent protein (EGFP)-transfected NIH3T3 (***p<0.001). Bar = 100 μm.

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Periostin is expressed in pericryptal fibroblasts and cancer-associated fibroblasts in the colon - PubMed (original) (raw)