Breast cancer induced nociceptor aberrant growth and collateral sensory axonal branching

Abstract

The tumour and neuron interaction has a significant impact upon disease progression and the patients quality of life. In breast cancer patients, it is known that there is an interaction between the tumour microenvironment and the sensory neurons to influence the progression of cancer as well as pain, though these mechanisms still need to be clearly defined. Here it is demonstrated that in a rodent orthotopic model of breast cancer (MDA MB 231) there was an increase in nerve fibre innervation into the tumour microenvironment (protein gene product 9.5), which were calcitonin gene related peptide positive C fibre nociceptors. In contrast, there was a reduction in myelinated nerve fibres (NF200). A sensory neuronal cell line was cultured in response to conditioned media from MDA MB231 and MCF7 as well as vascular endothelial growth factor-A (VEGF-A). All these experimental conditions induced sensory neuronal growth, with increased formation of collateral axonal branches. Furthermore, it was demonstrated that MDA MB231 and VEGF-A induced sensory neuronal sensitisation in response to capsaicin a TRPV1 agonist. MDA MB231 induced neuronal growth was suppressed by VEGFR2 inhibition (ZM323881 and neutralising antibody DC101), in addition both MDA MB231 and VEGF-A induced neurite growth was attenuated by the inhibition of ARP2/3 complex through co-treatment with CK666. This demonstrates that breast cancer can interact with the sensory nervous system to drive neuritogenesis through a VEGF-A/VEGFR2/ARP2/3 mediated pathway.

Keywords: MDA MB231; VEGF-A; breast; cancer; pain.

Figure 1. Breast cancer induced sensory nociceptor aberrant growth

(A) Pan-nerve fibre (PGP 9.5) and (B) nociceptor (CGRP) immunoreactivity was present in the mammary tissue of an adult female mouse indicative of nerve fibre innervation. In rodent orthotopic models (representative images of (C–D) Rag2; [E–F] CD1) of breast (MDA MB231) cancer there was an increase in (C, E, I, J) nerve fibre innervation (PGP9.5). (D, F, K, L) There was an increase in CGRP positive nociceptor nerve fibre innervation in the tumour. Representative images of no primary controls for (G) PGP9.5 and (H) CGRP. Myelinated nerve fibre innervation was present in the (M) normal mouse though this was decreased in the (N–P) tumour (CD1). Arrows = nerve fibres, arrowheads = blood vessel. Scale bar = 50 μm.

Figure 2. VEGF-A induced sensory neurogenesis mediated by VEGFR2-ARP2/3

(A) VEGF-A immunoreactivity in normal mouse mammary fat pad and in an (B) MDA MB231 orthotopic model of breast cancer (CD1 mice). (C) VEGF-A is upregulated in the breast tumour tissue. (*p < 0.05, Unpaired T test). (D) VEGFR2 is expressed in (E) peripheral nerve terminals (PGP9.5; (F) Overlay) in the plantar skin and in (G) L5 DRG sensory neurons (scale bar = 50 μm). (H) No primary control of L5 DRG sensory neurons. (I) Sensory neurons were treated with either (J) VEGF-A₁₆₅a + vehicle or (K) VEGF-A + ZM323881 and total neuronal growth was measured. (L) VEGF-A₁₆₅a led to a significant increase in neuronal growth versus untreated cells. ZM323881 treatment attenuated VEGF-A₁₆₅a induced sensory neuron growth. (M) Furthermore, VEGF-A₁₆₅a induced sensory neuron growth was inhibited following CK666 treatment to inhibit ARP2/3 function. Representative images of (N) sensory neurons in response to either (O) VEGF-A₁₆₅a + Vehicle and (P) VEGF-A+CK666. *P < 0.05, ***P < 0.001 Kruskal Wallis with Dunns multiple comparison. Scale bar = 20 μm.

Figure 3. MDA MB231 and MCF7 breast cancer cell lines induce sensory neuronal growth

A sensory neuronal cell line (A) 50B11 was exposed to conditioned media from (B) MDA MB231 and (C) MCF7 breast cancer cell lines as well as (D) VEGF-A and (E) unconditioned media. Scale bar = 20 μm. Conditioned media from MDA MB231 and MCF7 as well as VEGF-A₁₆₅a led to an increase in the (F) average neurite length, (G) increased neurite number and (H) total growth per cell. (I) MDA MB231 conditioned media and VEGF-A₁₆₅a led to increased maximum neurite length. (J) MDA MB231 conditioned media treated DRG sensory neurons had an increased capsaicin responses when compared to normal media treated DRG sensory neurons. Furthermore, (K) VEGF-A₁₆₅a increased capsaicin induced sensory DRG neuronal responses versus vehicle treated DRG neurons (n = 8 per experimental group). *P < 0.05, **P < 0.01, ***P < 0.001 Unpaired T test or Kruskal Wallis with Dunns multiple comparison. Scale bar = 20 μm.

Figure 4. MDA MB231 breast cancer cell line induces sensory neuronal growth via VEGFR2 activation

MDA MB231 conditioned media induced an increase in (A) average neurite length and (B) total neurite growth of sensory neurons when compared to normal media and conditioned media from MCF710A. (A and B) MDA MB231 conditioned media induced sensory neuroritogenesis was inhibited with VEGFR2 neutralising antibody DC101 when compared to rat IgG. Furthermore, inhibition of VEGFR2 using ZM323881 led to inhibition of the MDA MB231 conditioned media induced increase in sensory neuron (C) average neurite length and (D) total neuron growth. Representative images of sensory neurons in (E) normal media conditions, (F) MDA MB231 conditioned media + IgG, (G) MDA MB231 conditioned media + Vehicle, (H) MCF10A conditioned media, (I) MDA MB231 conditioned media + DC101 and (J) MDA MB231 conditioned media + ZM323881. ***P < 0.001 Unpaired T test or Kruskal Wallis with Dunns multiple comparison. Scale bar = 20 μm.

Figure 5. ARP 2/3 regulation controls MDA MB231 induced sensory neuronal growth

MDA MB231 induced increase in (A) maximum neurite length, (B) percentage of neurons with neurites and (C) total growth per neuron were attenuated by ARP2/3 inhibitor CK666. Representative images sensory neurons in response to experimental conditions; (D) normal media, (E) MDA MB231 unconditioned media, (F) MDA MB231 conditioned media + Vehicle, (G) MDA MB231 conditioned media + CK666. *P < 0.05, **P < 0.01, ***P < 0.001 Kruskal Wallis with Dunns multiple comparison. Scale bar = 20 μm.

Figure 6. MDA MB231 cancer cells drive sensory neuronal growth cone expansion and collateral branch formation

(A) Sensory DRG neurons treated with conditioned media from MDA MB231 and MCF7 as well as VEGF-A demonstrated increased growth cone area (Scale bar = 10 μm). (B) In addition, the number of collateral branches formed was increased in sensory neurons exposed to conditioned media from MDA MB231 cells when compared to normal media, unconditioned media, MCF7 and VEGF-A. (C) This was further demonstrated by MDA MB231 conditioned media inducing a greater percentage of sensory neurons to have greater numbers neurite branches (Scale bar = 20 μm). Representative images of sensory neuron (D) growth cone and (E) collateral branches. *P < 0.05, ***P < 0.001 Kruskal Wallis with Dunns multiple comparison.

Figure 7. CK666 inhibited sensory neuritogenesis induced by both MDA-MB-231 and MCF7

MDA MB231 and MCF7 induced sensory neuron growth; (A) average neurite length and (B) growth cone area versus normal media. This was inhibited by CK666 administration. Furthermore, (C) the tortuosity of the actin filaments was increased in CK666 sensory neurons indicative of a reduction in actin filament formation and straightness of such fibres Representative images of the sensory neuronal cell line 50B11 under varying experimental conditions; (D) normal media + vehicle, (E) MDA MB231 conditioned media + vehicle, (F) MCF7 conditioned media + vehicle, (G) normal media + CK666, (H) MDA MB231 conditioned media + CK666, (I) MCF7 conditioned media + CK666. *P < 0.05, ***P < 0.001 Kruskal Wallis with Dunns multiple comparison. Scale bar = 20 μm.

Figure 8. MDA MB231 and MCF7 induced neuritogenesis was inhibited by CK666 administration

MDA conditioned media induced (A) increased number of collateral branches with (B) percentage number of the neuron population had greater than 9 axonal collateral branches. Furthermore, (C) treatment of neurons with MDA MB 231 conditioned media increased mature (tubulin positive) axonal branches. ARP 2/3 inhibition with CK666 led to a (A) reduction in total number of collateral branches induced by MDA MB231 conditioned media treatment. In addition, CK66 administration prevented (B) the increase in the % of neurons with greater numbers of collateral branches induced by MDA MB231 conditioned media. (C) MDA MB231 induced mature sensory axonal fibre formation (phalloidin positive/tubulin positive) when compared to normal media treated neurons. CK666 treatment inhibited MDA MB231 induced mature collateral branch formation. *P < 0.05, ***P < 0.001 Kruskal Wallis with Dunns multiple comparison.

Figure 9. MDA MB231 induced mature sensory axonal neurite formation via ARP2/3

MDA MB231 induced sensory neuronal growth as well as increased neurite formation. Representative images demonstrate that when compared to (A–C) normal media + vehicle, (D–F) MDA MB231 conditioned media led to an increase in mature sensory neurite formation (phalloidin positive and tubulin positive). Furthermore, treatment with CK666 did not alter sensory neuronal growth in (G–I) normal + media but (J–L) inhibited MDA MB231 induced mature collateral branch formation. High magnification representative images demonstrate that (N) MDA MB231 conditioned media induces collateral branch formation when compared to (M, O) normal media ± CK666 and (P) MDA MB231+CK666. Low magnification images = 20 μm (A–L). High magnification images = 10 μm (M–P).