Figure 1.
Characterization of stromal CD271 expression in pancreatic cancer.
(A) Immunohistochemical staining for CD271 in pancreatic tissues. (A-a) In normal pancreas (NP), stromal cells rarely stain positive for CD271. (A-b, -c) Stromal cells are strongly stained around pancreatic intraepithelial neoplasia (PanIN) (b) and intraductal papillary mucinous neoplasm (IPMN) (c). (A-d) In pancreatic ductal adenocarcinomas (PDAC), stromal cells are partly stained, and CD271+ stromal cells are not adjacent to pancreatic cancer cells. Black arrowheads and arrows indicate CD271+ cells and nerves as positive control in the serial sections. (A-e) α-smooth muscle actin (SMA) is expressed most stromal cells around cancer cells and neoplastic tubules. White arrowheads indicate α-SMA cells in the serial sections. Original magnification: 200×, insets: 600×. (B) Kaplan–Meier survival analysis for CD271 high expression in the stroma of PDAC. Stromal CD271 high expression is associated with a good prognosis (p = 0.0040).
Table 1.
Stromal CD271 high staining rates in surgical pancreatic tissues.
Table 2.
Relationships between stromal CD271 expression and clinicopathologic factors.
Table 3.
Multivariate survival analysis (Cox regression model) of conventional prognostic factors and stromal CD271 expression.
Figure 2.
Analyses of CD271 expression in human pancreatic stellate cells (PSCs) isolated from pancreatic tissues.
(A) Representative microphotograph of immunofluorescence staining for α-smooth muscle actin (SMA) (green) and vimentin (red) in PSCs. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). PSCs show a stellate-like or spindle-shaped morphology and express α-SMA. Original magnification: 200×. (B) The positive rates of CD271 expression in PSCs are 0.0–2.1% by flow cytometric analyses. Representative flow cytometric images of CD271 in activated PSCs are also shown (right).
Figure 3.
CD271+ pancreatic stellate cells (PSCs) are increased through cancer–stroma interactions.
(A) In flow cytometry analyses, the percentage of CD271+ cells is higher in coculture than in monoculture (0.7–0.8% vs. 0.1%). (B) The levels of CD271 mRNA expression in two cultures of PSCs are significantly higher in cocultures with pancreatic cancer cells than in monocultures (p<0.001). (C) The level of CD271 mRNA expression is higher in cultures with a cancer cell-derived supernatant than in monocultures (p = 0.0053). (D) Representative microphotograph of immunofluorescence staining for CD271 (green) and α-smooth muscle actin (SMA) (red) in PSCs. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). CD271 in PSCs was slightly expressed in cultures with cancer cell-derived supernatant, although it was difficult to detect CD271 expression in monocultures. Original magnification: 200×. *p<0.01; **p<0.001.
Figure 4.
CD271 expression in pancreatic stellate cells (PSCs) decreases after long-term interactions with pancreatic cancer cells.
(A) Real-time qRT-PCR analyses showed that the levels of CD271 mRNA expression in PSCs in coculture with pancreatic cancer cells start to increase on day 3 (p = 0.0249), are highest on day 4 (p<0.0001), and then decrease the following day after the peak expression (p<0.0001). (B) In cultures with addition of cancer cell-derived supernatant, the level of CD271 mRNA expression started to increase at day 2 (p<0.0001) and then decreased on day 4 (p<0.0001). (C) Evaluation of the differences in CD271 expression depending on the function of PSCs by real-time qRT-PCR. The level of CD271 mRNA expression in top side-derived PSCs, which did not move toward cancer cells through Matrigel and remained on upper chambers, is lower than that in bottom side-derived PSCs, which did move toward pancreatic cancer cells through Matrigel (p<0.01). *p<0.05; **p<0.01; ***p<0.001; NS, not significant.
Figure 5.
Expression of α-smooth muscle actin (SMA)+ and CD271+ stromal cells during in vivo tumor growth of pancreatic cancer.
(A) α-SMA+ stromal cells, as activated PSCs, exist in the xenograft tumor and normal pancreas (NP) areas (left side) around the tumor (right side). (B) Few CD271+ stromal cells were present in the NP area (left side) around the xenograft tumor, and were absent in the tumor core (right side). Original magnification: 200×.