Involvement of angiogenesis in cancer-associated acinar-to-ductal metaplasia lesion of pancreatic cancer invasive front

Abstract

Purpose: This study aimed to demonstrate the involvement of angiogenesis in cancer-associated acinar-to-ductal metaplasia (CA-ADM) lesion of invasive front pancreatic ductal adenocarcinoma (PDAC) and investigate the possible mechanism.

Methods: Tissue samples from 128 patients with PDAC and 36 LSL-Kras^G12D/+; LSL-Trp53^R172H/+; Pdx-1-Cre mice were analyzed. Immunohistochemical assay was performed using HE, anti-CK19 and anti-amylase to confirm the presence of CA-ADM lesions, using anti-CD34 and anti-CD31 to measure microvessel density (MVD), and using anti-CD68, anti-CD163, anti-iNOS, or anti-MMP9 to evaluate the immune microenvironment. We performed multiplex immunohistochemical assay to detect the co-expression of MMP9 and CD68 on macrophage. We examined clinical outcomes and other clinicopathological factors to determine the significance of high-level MVD of CA-ADM on survival and liver metastasis. We performed tube formation assay to evaluate the effect of macrophage on angiogenic capacity in vitro.

Results: Angiogenesis was significantly abundant in CA-ADM lesions compared with that in PDAC lesions in human and mouse tissues. High-level MVD in CA-ADM lesions was an independent predictor of poor prognosis (P = 0.0047) and the recurrence of liver metastasis (P = 0.0027). More CD68-positive and CD163-positive macrophages were detected in CA-ADM lesions than in PDAC. The percentage of CD68-positive macrophages was positively correlated with MVD in CA-ADM lesions. Multiplex-immunostaining revealed that MMP9 was expressed in CD68-positive macrophages of CA-ADM lesions. In CA-ADM lesions, the percentage of macrophages was positively correlated with MMP9 expression, which positively correlated with microvessel density.

Conclusion: CA-ADM related angiogenesis is a promising predictive marker for poor prognosis of PDAC and may provide an attractive therapeutic target for PDAC.

Keywords: Angiogenesis; Cancer-associated acinar-to-ductal metaplasia; Liver metastasis; Macrophages; Pancreatic cancer; Prognosis.

Fig. 1

Cancer-associated acinar-to-ductal metaplasia (CA-ADM) lesions are formed within the invasive front of pancreatic ductal adenocarcinoma (PDAC). a Human PDAC samples stained with HE. The boundaries between the tumor and the acini divide two histopathologically distinct regions, the non-invasive front and the invasive front. In the non-invasive front, the tumor is encapsulated by stromal cells. The invasive front is associated with CA-ADM lesions. b, c HE, CK19 and amylase staining in serial sections of human and mouse PDAC. Scale bars, 100 µm

Fig. 2

MVD level was higher in CA-ADM lesions than in PDAC lesions in KPC model mice. a Representative micrographs of CD34 staining in CA-ADM and PDAC lesions in KPC mice. The arrowheads indicate microvessels in CA-ADM and PDAC lesions. b Microvessel counts per field in CA-ADM and PDAC lesions. CA-ADM (n = 26), PDAC (n = 36). *P < 0.05. c, d The correlation plot shows that microvessel counts per field between CA-ADM and PDAC lesions are highly correlated (n = 26). Data shown represent the mean ± SD. Scale bars, 100 µm. ****P < 0.0001

Fig. 3

MVD level was higher in CA-ADM lesions than that in PDAC lesions in human PDAC samples. a Representative micrographs of CD31 staining in CA-ADM and PDAC lesions in human PDAC samples. The arrowheads indicate microvessels (stained with CD31) in the CA-ADM and PDAC lesion. b Microvessel counts per field in the CA-ADM lesions are higher than those in the PDAC lesions. CA-ADM (n = 120), PDAC (n = 128). ****P < 0.0001. c The comparison of microvessel counts per field between CA-ADM and PDAC lesions (n = 120). ****P < 0.0001. d The correlation plot shows that microvessel counts per field between CA-ADM and PDAC lesions are moderately correlated (n = 120). e Kaplan–Meier analysis of overall survival according to MVD in human ADM-like lesions (n = 120). High-level MVD in CA-ADM lesions is associated with shorter patient survival. f Kaplan–Meier analysis of overall survival based on MVD in human PDAC lesions (n = 128). High-level MVD of PDAC lesion is not associated with prognosis. Data shown represent the mean ± SD. Scale bars, 100 µm

Fig. 4

Macrophages were more frequently observed in CA-ADM lesions than in PDAC lesions. a Representative micrographs of CD68, iNOS and CD163 staining in CA-ADM and PDAC lesions of human PDAC samples. b Percentages of CD68-, CD163- and iNOS-positive cells in CA-ADM and PDAC lesions are shown in graphs. CD68, CD163 and iNOS staining was performed in CA-ADM lesions (n = 96, 96 and 79, respectively) and PDAC lesions (n = 97, 97 and 80, respectively). **P < 0.01, ****P < 0.0001. c, d The correlation plots show that the expression of CD68 and CD163 are positively correlated with the MVD in CA-ADM lesions (n = 96). e, f The correlation plots show that the expression of CD68 and CD163 are positively correlated with MVD in PDAC lesions (n = 97). g, h The correlation plots show that the expression of iNOS is not correlated with the MVD in CA-ADM or PDAC lesions (n = 79, 80 respectively). Data shown represent the mean ± SD. Scale bars, 100 µm

Fig. 5

MMP9 was co-localized with CD68-positive macrophages and correlated with MVD levels in CA-ADM lesions. a Representative micrographs of CD68 and MMP9 staining via mIHC analysis in CA-ADM lesions of human PDAC samples. The arrowheads indicate co-expression areas. b Representative micrographs of CD31, CD68 and MMP9 staining in CA-ADM and PDAC lesions in human PDAC samples. c MMP9 staining in CA-ADM and PDAC lesions of human PDAC samples. CA-ADM (n = 91), PDAC (n = 92). **P < 0.01. d, e The correlation plots show that the percentage of MMP9 positive cells is correlated with the percentage of CD68 positive cells and MVD in CA-ADM lesions (n = 91). Data shown represent the mean ± SD. Scale bars, 100 µm