Intravital imaging reveals systemic ezrin inhibition impedes cancer cell migration and lymph node metastasis in breast cancer

Abdi Ghaffari,Yolanda Madarnas,Graeme Mullins,Bruce E Elliott,Victoria Hoskin,Andrew G Day,Gulisa Turashvili,Friedemann Kiefer,Sonal Varma,Jeff Mewburn,Sandip Sengupta,Peter A Greer

doi:10.1186/s13058-018-1079-7

Abstract

BackgroundLimited understanding of the cancer biology of metastatic sites is a major factor contributing to poor outcomes in cancer patients. The regional lymph nodes are the most common site of metastasis in most solid cancers and their involvement is a strong predictor of relapse in breast cancer (BC). We have previously shown that ezrin, a cytoskeletal–membrane linker protein, is associated with lymphovascular invasion and promotes metastatic progression in BC. However, the efficacy of pharmacological inhibition of ezrin in blocking cancer cell migration and metastasis remains unexplored in BC.MethodsWe quantified ezrin expression in a BC tissue microarray (n = 347) to assess its correlation with risk of relapse. Next, we developed a quantitative intravital microscopy (qIVM) approach, using a syngeneic lymphatic reporter mouse tumor model, to investigate the effect of systemic ezrin inhibition on cancer cell migration and metastasis.ResultsWe show that ezrin is expressed at significantly higher levels in lymph node metastases compared to matched primary tumors, and that a high tumor ezrin level is associated with increased risk of relapse in BC patients with regional disease. Using qIVM, we observe a subset of cancer cells that retain their invasive and migratory phenotype at the tumor-draining lymph node. We further show that systemic inhibition of ezrin, using a small molecule compound (NSC668394), impedes the migration of cancer cells in vivo. Furthermore, systemic ezrin inhibition leads to reductions in metastatic burden at the distal axillary lymph node and lungs.ConclusionsOur findings demonstrate that the tumor ezrin level act as an independent biomarker in predicting relapse and provide a rationale for therapeutic targeting of ezrin to reduce the metastatic capacity of cancer cells in high-risk BC patients with elevated ezrin expression.

Highlights

Limited understanding of the cancer biology of metastatic sites is a major factor contributing to poor outcomes in cancer patients
High tumor ezrin levels correlate with increased risk of relapse in invasive breast cancer (BC) To assess the association between ezrin and risk of metastasis in BC, we quantified ezrin protein expression in primary tumors (n = 347, Additional file 1: Figure S1A) and a subset of matched benign ductal tissues (n = 90) in tissue microarray (TMA) cores, using immunohistochemistry (IHC) and HALO automated quantitative image analysis (Fig. 1a)
As ezrin plays a critical role in cancer cell invasion, we explored its prognostic potential in patients with higher risk of metastatic disease

Summary

Introduction

Limited understanding of the cancer biology of metastatic sites is a major factor contributing to poor outcomes in cancer patients. The addition of regional nodal irradiation to conventional treatment, after mastectomy or breast-conserving surgery in node-positive patients, reduces the rate of locoregional and systemic recurrence [6, 7] Despite this clinical importance, and partly due to a lack of appropriate single-cell resolution imaging models of LN metastasis, we have a very limited understanding of the fate of cancer cells within LNs and of the efficacy of therapeutic targeting of prometastatic molecules to prevent further metastatic spread of cancer cells beyond regional LNs. We have previously shown that tumor ezrin levels correlate with lymphovascular invasion in a locally accrued BC cohort and that ezrin acts cooperatively with Src in regulating tumor lymphangiogenesis [8]. These findings prompted us to examine whether pharmacological inhibition of ezrin could have therapeutic benefits by suppressing the spread of highly metastatic cancer cells from lymph node micrometastases

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Discussion

Conclusion