Patterns of cancer invasion revealed by QDs-based quantitative multiplexed imaging of tumor microenvironment

Abstract

Tumor growth and progression depends on their microenvironment, which undergoes constant co-evolution because of the dynamic tumor–stormal interactions. Such co-evolution has long been under appreciated due to the lack of appropriate technology platforms to simultaneously reveal these complex interactions. Here we report on a quantum dots based multiplexed imaging and spectrum analysis technology to simultaneously study major components of tumor stroma, including type IV collagen, tumor angiogenesis, macrophages infiltration and tissue destructive proteolytic enzyme matrix metalloproteinase 9. The new technology revealed a panoramic picture of the tempo-spatial co-evolution of tumor cells and their stroma at the architecture level. Four patterns of tumor invasion with distinctive co-evolution features were identified as Washing pattern, Ameba-like pattern, Polarity pattern and Linear pattern. This quantum dots based multiplexed technology could help gain new insight into the complex process of tumor invasion, and formulate new anti-cancer strategies.

Introduction

Invading tumor cells in vivo are confronted with three-dimensional (3D) extracellular matrix (ECM) networks that form physical barriers against the advancing cells [1]. Traditionally, invading depth is the center of attention, and few studies have concurrently focused on the dynamic co-evolution of the cancer cells and stroma. It has been recognized that invasion is regulated not only by intrinsic genetic changes in cancer cells as ‘initiators’ of carcinogenesis, but also by stromal cells as ‘promoters’ [2], [3]. A seminal event in cancer progression is the ability of invading cells to migrate through tissue barriers particularly the basement-membrane, a specialized form of ECM that separates the epithelium from the stroma [4]. This process requires the co-evolution of cancer microenvironment that includes several simultaneous events, such as up-regulation and activation of proteolytic enzymes such as matrix metalloproteinases (MMPs), remodeling of ECM barrier (ECMB) mainly by cleaving and re-patterning type IV collagen, tumor angiogenesis, and recruitment and conversion of immune cells [5]. In recent years, the ECMB has been recognized as an important regulator of cell behavior, not only just a tissue structure scaffold. The ECMB mediates tissue compartmentalization and sends signals to epithelial cells about the external microenvironment [6]. Human cancer is especially complex because it evolves over a long time course and shows a multitude of molecular, cellular, and architectural heterogeneity [7], [8]. Neither the studies at purely molecular and cellular levels, nor the studies at the purely clinical level can decipher the co-evolution of cancer microenvironment. At the architectural level, however, major critical molecular and cellular events and stroma changes can be visualized, revealing a real-time panoramic picture of the co-evolution of cancer cells and their microenvironment [9]. Unfortunately, such co-evolution of cancer microenvironment has long been under appreciated due to the lack of appropriate technology platforms to reveal the dynamic spatiotemporal processes.

Quantum dots (QDs) are engineered nanoparticles with unique optical and electronic properties which have potential applications ranging from medicine to energy [10], [11]. Compared with organic dyes and fluorescent proteins, QDs have unique features such as size- and composition-tunable light emission, enhanced signal brightness, resistance to photobleaching [12], [13], [14]. In addition, different QDs colors can be simultaneously excited by a single light source, with minimal spectral overlapping, which provides significant advantages for multiplexed detection of targets. This property is very suitable for investigating the co-evolution of cancer cells and tumor microenvironment at the architectural level, a key issue in studying the mechanisms of cancer progression and also in developing more specific targeting therapeutic approaches [3], [15], [16], [17].

In this work, we report on a new strategy to directly reveal the co-evolution of cancer cells and their microenvironment on human cancer tissues in order to gain better insights into the complex and dynamic biology of cancer invasion. Four common invasion patterns in 15 cases of gastric cancer specimens and 10 breast cancer specimens were revealed by QDs-based fluorescent imaging and spectrum analysis of type IV collagen. In addition, major components involved in the critical processes of cancer invasion were revealed simultaneously by multiplexed QDs imaging. This new multiplexed QDs mapping provides new spatiotemporal information on the co-evolution of cancer cells and microenvironment.