Quantum dots-based double-color imaging of HER2 positive breast cancer invasion

https://doi.org/10.1016/j.bbrc.2011.05.052Get rights and content

Abstract

It has been well recognized that human epidermal growth factor receptor 2 (HER2) level in breast cancer (BC) is closely related to the malignant biologic behaviors of the tumor, including invasion and metastasis. Yet, there has been a lack of directly observable evidence to support such notion. Here we report a quantum dots (QDs)-based double-color imaging technique to simultaneously show the HER2 level on BC cells and the type IV collagen in the tumor matrix. In benign breast tumor, the type IV collagen was intact. With the increasing of HER2 expression level, there has been a progressive decrease in type IV collagen around the cancer nest. At HER2 (3+) expression level, there has virtually been a total destruction of type IV collagen. Moreover, HER2 (3+) BC cells also show direct invasion into the blood vessels. This novel imaging method provides direct observable evidence to support the theory that the HER2 expression level is directly related to BC invasion.

Highlights

► HER2 level is closely related to the biologic behaviors of breast cancer cells. ► A new method to simultaneously image HER2 and type IV collagen was established. ► HER2 status and type IV collagen degradation predict breast cancer invasion. ► The complex interactions between tumor and its environment were revealed.

Introduction

Breast cancer (BC) is the most common female cancer both in developed and developing countries [1], [2]. Invasion and metastasis are the root causes of cancer death [3]. Cancer invasion is a complex process [4], with the initial step being the breakdown of extracellular matrix (ECM) by invading cancer cells through a concerted action of proteolytic enzymes such as matrix metalloproteinases (MMPs), particularly the gelatinases MMP2 and MMP9 [5], [6]. The secretion and activity of these enzymes are partially dependent on human epidermal growth factor receptor 2 (HER2) pathway [7], [8], which is amplified and overexpressed in 20–30% of BC [9]. HER2 overexpression drives BC cells growth and invasion by upregulating the expression of MMP2 and MMP9, resulting in accelerated degradation of type IV collagen [10], [11].

Such process has long been understood by biochemical studies or conventional Immunohistochemical (IHC) approaches [12]. Yet sufficient morphological evidence to directly show the dynamic process of invasion is not yet available, mainly because of the lack of appropriate technology platforms to simultaneously reveal the picture of cancer invasion from both perspectives of cancer cells and the ECM.

As an important class of emerging nanomaterial, quantum dots (QDs) are “among the most promising items in the nanotechnology toolbox” with unique optical and electronic properties producing different fluorescence signals depending on their size and components [13]. They have potential applications ranging from medicine to energy [14], [15]. Compared with traditional organic fluorophores and fluorescent proteins, QDs are superior in fluorescence brightness, photobleaching resistance, and fluorescence emission tuneability [16]. In addition, QDs have broad absorptions with narrow emission spectra, and different colors QDs can be excited simultaneously by a single-light source, with minimal spectral overlapping [17], [18]. These unique optical properties make QDs ideal candidates for multi-color imaging of molecules to investigate the dynamic cellular processes of cancer progression, such as continual cells migration, invasion and metastasis [3]. In our previous studies, we have applied QDs-based nanotechnology for BC and liver cancer molecular imaging, as well as multiplexed imaging of tumor microenvironment to reveal patterns of cancer invasion [2], [19], [20], [21], [22]. This study was to develop a QDs-based double-color imaging of HER2 on BC cells and the type IV collagen in the ECM, so as to investigate the dynamic changes of ECM degradation during BC invasion.

Section snippets

BC tissues and materials

Formalin-fixed paraffin-embedded human BC tissues were obtained from Hubei Cancer Hospital (Wuhan, China). The HER2 expression status had been validated by fluorescence in situ hybridization (FISH), as reported in our previous work [2]. The primary antibodies were rabbit anti-human polyclonal antibody against type IV collagen (ab-6586, Abcam, Elangnd, dilution 1/100), mouse anti-human monoclonal antibody against HER2 (Clone: CB11 MaiXin Bio Co., Ltd., Fuzhou, China, dilution 1/50). The QDs

Establishment of QDs-based double-color imaging

IHC plays important roles in pathology owing to high sensitivity, simplicity and low cost, providing not only geometrical but also functional information about biologic medical systems [24], [25]. We first studied the expression of HER2 and type IV collagen by conventional IHC in BC specimens. HER2 protein was stained brown in positive cells, mainly located on the BC cellular membrane (Fig. 1A). Type IV collagen, the most abundant constituent of the ECM [26], expressed mainly in the stroma

Acknowledgments

This research was supported by the grants from the New-Century Excellent Talents Supporting Program of the Ministry of Education of China (no. NCET-04-0669), the Natural Science Foundation of China (nos 20675058, 10874135 and 20833006), the Science Fund for Creative Research Groups of the National Natural Science Foundation of China (no. 20921062), the National Key Scientific Program (973)- Nanoscience and Nanotechnology (no. 2006CB933100), and the Ministry of Public Health (nos 2009ZX10004-107

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