Nerve fibers infiltrate the tumor microenvironment and are associated with nerve growth factor production and lymph node invasion in breast cancer

Abstract

Infiltration of the tumor microenvironment by nerve fibers is an understudied aspect of breast carcinogenesis. In this study, the presence of nerve fibers was investigated in a cohort of 369 primary breast cancers (ductal carcinomas in situ, invasive ductal and lobular carcinomas) by immunohistochemistry for the neuronal marker PGP9.5. Isolated nerve fibers (axons) were detected in 28% of invasive ductal carcinomas as compared to only 12% of invasive lobular carcinomas and 8% of ductal carcinomas in situ (p = 0.0003). In invasive breast cancers, the presence of nerve fibers was observed in 15% of lymph node negative tumors and 28% of lymph node positive tumors (p = 0.0031), indicating a relationship with the metastatic potential. In addition, there was an association between the presence of nerve fibers and the expression of nerve growth factor (NGF) in cancer cells (p = 0.0001). In vitro, breast cancer cells were able to induce neurite outgrowth in PC12 cells, and this neurotrophic activity was partially inhibited by anti-NGF blocking antibodies. In conclusion, infiltration by nerve fibers is a feature of the tumor microenvironment that is associated with aggressiveness and involves NGF production by cancer cells. The potential participation of nerve fibers in breast cancer progression needs to be further considered.

Keywords: Breast cancer; Nerve fibers (axons); Nerve growth factor; Tumor microenvironment.

Figure 1

Detection of nerve fibers in breast cancers. IHC for the neuronal marker PGP9.5 was performed on a series of 319 breast cancer samples. A) Nerve trunks (composed of many nerve fibers), occasionally present in breast tumors, were positive for PGP9.5. Perineural invasion (PNI) could be observed, as shown here. B–H) In some breast cancers, isolated nerve fibers (axons) positive for PGP9.5 were observed and are indicated by arrows. B) Nerve fibers around cancer cells and adipocytes (Ad). C) Nerve fibers in the tumor stroma (St) adjacent to cancer cells (CC). D) Enlargement of C. E, F) Nerve fibers among scattered breast cancer cells and in tumor stroma. G) Nerve fibers around an arteriole (Ar). H) Nerve fibers close to a thin walled blood vessel in the tumor stroma. Scale bar = 50 μm.

Figure 2

Frequency distribution of NGF level in breast cancers according to the presence of nerve fibers. NGF levels were obtained after digital quantification. A) Distribution of NGF intensity staining (h‐score) in ductal carcinomas in situ (DCIS), invasive lobular carcinomas (ILC) and invasive ductal carcinomas (IDC). Box and Whisker plots comparing median NGF levels using h‐scores as a measure of IHC staining (n = 50, 160 and 160, respectively). The box limits indicate the 25th and 75th percentiles with the whiskers extending 1.5 times the interquartile range from the 25th and 75th percentiles (outliers are represented by dots; prepared using BoxPlotR). B) Distribution of NGF staining intensity in DCIS, ILC and IDC. Categorization is presented as 0 = h‐score <50, 1 = h‐score 50–100, 2 = h‐score 101–150, 2 = h‐score>150. C) Distribution of NGF staining intensity in invasive tumors (nerve fibers positive versus nerve fibers negative tumors). Categories of NGF staining (0, 1, 2, 3) were the same as in B. Tumors presenting with nerve fibers were more likely to have higher NGF expression than tumors without nerve fibers. Number of cases (n) is indicated. ***One‐way ANOVA was used for A and Chi square for B and D.

Figure 3

Co‐localization between nerve fibers and NGF in breast cancer. A) IHC for PGP9.5 indicating the presence of many nerve fibers in the stroma and along cancer cells of an invasive ductal carcinoma. Arrows point to few nerve fibers. B) IHC against NGF in a section serial to that presented in panel A. NGF immunoreactivity (indicated by stars) was observed in cancer cells adjacent to nerve fibers. C) Enlargement of the area boxed in panel A. D) Enlargement of the area boxed in panel B. Scale bar = 50 μm.

Figure 4

NGF‐mediated neurotrophic effect of breast cancer cells. A) Co‐culture experiments were performed in Transwell Boyden chambers with breast epithelial cells in the upper part and PC12 cells in the lower part. B) Some breast cancer cell lines were able to induce a neurotrophic effect on PC12 cells. Neurite outgrowth was induced in presence of MDA‐MB‐231, 231‐BR, MCF‐7, SKBR‐3, JIMT‐1, but not in presence of BT‐474 and the non‐tumorigenic HME. A negative control (with no breast cancer cells) and a positive control (addition of 50 ng/ml NGF) have been added. The results represent the mean of 3 independent experiments ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 for comparison with control. C) Representative pictures showing the effect of breast cancer cell lines and HME on PC12 cells. Quantifications are presented in panel B. D) Dot‐blot analysis for the detection of NGF in breast cancer cell conditioned media and quantification of NGF signal intensity in different breast epithelial cells. E) Impact of blocking anti‐NGF antibodies on breast cancer cell‐induced neurite outgrowth. MCF‐7 cells were co‐cultured for 72 h with PC12 cells in presence or absence of anti‐NGF blocking antibodies (1 μg/ml). Control was without MCF‐7 cells, and isotype antibodies were also tested. The results represent the mean of 3 independent experiments ± SD. ***p < 0.001. F) Representative pictures corresponding to the experiment described in panel E.