Breast cancer-induced bone remodeling, skeletal pain, and sprouting of sensory nerve fibers

Abstract

Breast cancer metastasis to bone is frequently accompanied by pain. What remains unclear is why this pain tends to become more severe and difficult to control with disease progression. Here we test the hypothesis that with disease progression, sensory nerve fibers that innervate the breast cancer bearing bone undergo a pathological sprouting and reorganization, which in other nonmalignant pathologies has been shown to generate and maintain chronic pain. Injection of human breast cancer cells (MDA-MB-231-BO) into the femoral intramedullary space of female athymic nude mice induces sprouting of calcitonin gene-related peptide (CGRP(+)) sensory nerve fibers. Nearly all CGRP(+) nerve fibers that undergo sprouting also coexpress tropomyosin receptor kinase A (TrkA(+)) and growth-associated protein-43 (GAP43(+)). This ectopic sprouting occurs in periosteal sensory nerve fibers that are in close proximity to breast cancer cells, tumor-associated stromal cells, and remodeled cortical bone. Therapeutic treatment with an antibody that sequesters nerve growth factor (NGF), administered when the pain and bone remodeling were first observed, blocks this ectopic sprouting and attenuates cancer pain. The present data suggest that the breast cancer cells and tumor-associated stromal cells express and release NGF, which drives bone pain and the pathological reorganization of nearby CGRP(+)/TrkA(+)/GAP43(+) sensory nerve fibers.

Perspective: Therapies that block breast cancer pain by reducing the tumor-induced pathological sprouting and reorganization of sensory nerve fibers may provide insight into the evolving mechanisms that drive breast cancer pain and lead to more effective therapies for attenuating this chronic pain state.

Copyright © 2011 American Pain Society. All rights reserved.

Figures

Figure 1. Human breast cancer cells produce osteolytic lesions when injected into the femur of an immunocompromised mouse

Radiograph and μCT images from naïve (A, D), sham + vehicle (B, E), or MDA-MB-231-BO + vehicle mice (C, F) at 42 days post-injection showing the difference in bone remodeling in the distal femur following the different experimental procedures. Note that the sham-injected femur does not display a significant amount of bone destruction when compared to the naïve bone. However, when MDA-MB-231-BO cells are injected into the femur, they produce osteolytic lesions in the cortical bone. These indices of bone destruction provide the tumor with an avenue to escape from the intramedullary space and invade the periosteum. Serial sections (7μm-thick) stained with H&E demonstrate this phenomenon as well as the difference in morphology observed between normal marrow (G) versus cancerous tissue (H) and the apparent loss of trabecular bone in the tumor-bearing femur.

Figure 2. Breast cancer-induced pain behaviors increase with disease progression, and are reduced with sustained anti-NGF therapy

Injection of MDA-MB-231-BO breast cancer cells into the intramedullary space of the femur results in significant osteolytic bone destruction (A) and bone pain-related behaviors (B–D). Anti-NGF therapy (10 mg/kg, i.p.) appeared to slow the bone destruction induced by the tumor; however, this effect was not statistically significant. Tumor-bearing mice treated with vehicle exhibited significantly greater pain behaviors compared to sham mice from day 28 until day 42 post cell injection (B, C, D). Whereas anti-NGF administration did not have a statistically significant effect on tumor-induced bone destruction, the therapy did significantly reduce cancer pain behaviors in the mid to late stages of disease. Each bar represents the mean ± SEM. The number of animals used for this analysis was n≥5 for each experimental group. Dotted arrows (B) indicate the dates of anti-NGF therapy administration.

Figure 3. Sprouting of sensory nerve fibers in the periosteum of the tumor-bearing bone

Representative confocal images of non-decalcified whole-mount preparations of periosteum immunostained with calcitonin gene-related peptide (CGRP) (A, B), and their accompanying higher-power camera-lucida renderings (C, D), show that there is a striking change in the density and morphology of CGRP+ nerve fibers innervating the periosteum of naïve (A, C) vs. tumor-bearing femurs (B, D) 42 days post-tumor injection. In the presence of cancer, very thin CGRP+ nerve fibers are apparent which have a more disorganized and non-linear pattern. Confocal images were acquired with a 40× objective, projected from 80 optical sections at 0.5μm intervals. Camera-lucida renderings were created using the ‘trace’ function in Image Pro Plus Version 3.0.

Figure 4. Human breast cancer cells induce nerve sprouting in the periosteum, and this sprouting is blocked by the administration of anti-NGF

Representative confocal images of femoral sections from sham + vehicle (A, D), MDA-MB-231-BO + vehicle (B, E), and MDA-MB-231-BO + anti-NGF (C, F) treated mice. Decalcified bone sections were immunostained with an antibody against CGRP (A, B, C) or antibody against GAP43 (D, E, F). Note that at day 42 post-injection there is a greater density of CGRP+ (B) and GAP43+ (E) nerve fibers in MDA-MB-231-BO + vehicle mice and the nerve fibers have a disorganized morphology as compared to nerve fibers innervating sham bones (A, D). Anti-NGF therapy (10 mg/kg; i.p., given at days 14, 19, 24, 29, 34, and 39 post cell injection) significantly reduces this pathological tumor-induced reorganization of CGRP+ and GAP43+ nerve fibers (C, F). Confocal images were acquired in the distal metaphyseal periosteum (~2 mm distal from the growth plate, 20μm in thickness) using sequential acquisition mode to reduce bleed-through and were projected from 40 optical sections at 0.5μm intervals with a 40× objective.

Figure 5. Schematic depicting how breast cancer cells induce sprouting of CGRP+ and GAP43+ nerve fibers in the bone

The periosteum of the bone is a thin cellular and fibrous sheath that envelops the entire outer surface of bone but is absent from areas of articular cartilage. The periosteum is innervated by and sensory and sympathetic nerve fibers and is sensitive to distortion of the underlying cortical bone (A). Sensory nerve fibers that innervate the normal periosteum have a net-like morphology (B) and appear linear when viewed in cross-section (C). As breast cancer colonizes and remodels the bone, extensive osteoclast-driven bone resorption occurs (D), forming lesions in the cortical bone that allows cancer cells to escape into the periosteum (E, F) where these cancer and their associated stromal cells induce sprouting of CGRP+ and GAP43+ nerve fibers (E, F).

Figure 6. The majority of CGRP+ and GAP43+ sensory nerve fibers in the tumor bearing periosteum co-express the neurotrophin receptor TrkA (yellow, C, F)

Confocal images of tumor-bearing (day 42 post-cell injection) bone sections that were immunostained with CGRP (red, A), a widely used marker of nociceptors, or GAP43 (red, E) which labels regenerating and sprouting nerve fibers. These sections were co-labeled with an antibody to TrkA (green, B, D), which is the cognate receptor for NGF. Note that the great majority of CGRP+ and GAP43+ nerve fibers co-express TrkA. Each confocal image was acquired with a 40× objective, projected from 40 optical sections at 0.5μm intervals.

Figure 7. Quantitative analysis of the effects of anti-NGF therapy on tumor-induced nerve sprouting

At day 42 post cell injection, the density of CGRP+ (A), GAP43+ (B) and TrkA+ (C) nerve fibers is significantly greater in MDA-MB-231-BO + vehicle-treated mice compared to sham + vehicle-treated mice. This tumor-induced increase in the density of CGRP+, GAP43+ and TrkA+ nerve fibers is attenuated by administration of anti-NGF (10 mg/kg; i.p., given at days 14, 19, 24, 29, 34, and 39 post cell or vehicle injection). The volume of periosteum that was analyzed was an average 400 μm (length), 70 μm (width), 20 μm (depth). The Z-stacked images were analyzed with Image-Pro Plus v. 6.0 (Media Cybernetics) and nerve fibers were manually traced to determine the length of nerve fibers. Nerve sprouting was reported as total length of nerve fibers per volume of periosteum. Both imaging acquisition and analysis were performed in a blinded fashion. Brackets indicate the groups being compared. *p<0.05. Bars represent the mean ± SEM. The number of animals used for this analysis was n≥5 for every experimental group and marker.

Figure 8. Schematic depicting how Nerve Growth Factor (NGF) released by breast cancer and associated stromal cells appears to drive sprouting of TrkA+, CGRP+ and GAP43+ primary afferent nerve fibers in the tumor-bearing bone

Primary afferent neurons have their cell body in the dorsal root ganglia (DRG) and transmit sensory information from the periphery to the spinal cord and brain (A). Cancer and tumor-associated stromal cells produce a variety of pro-nociceptive factors, such as NGF, that may directly activate or sensitize nociceptors. NGF binds to it’s cognate receptor TrkA, and the NGF/TrkA complex is retrogradely transported to the nucleus of the sensory neuron resulting in increased synthesis and anterograde transport of neurotransmitters, receptors, ion channels, and scaffolding molecules from the cell body to the peripheral nerve terminals located in the peripheral tissue and spinal cord. Thus, the NGF/TrkA complex may serve as an upstream regulator of nociceptor function by modulating the sensitivity or increasing the expression of several other receptors and ion channels, contributing to increased excitability of nociceptors in the vicinity of the tumor. The binding of NGF to TrkA may also induce a pathological sprouting and neuroma formation by sensory nerve fibers that may contribute to ongoing and breakthrough cancer pain (B). Anti-NGF therapy resulted in a significant reduction of the tumor-induced pain and nerve sprouting without significantly modifying disease progression (C).