A microfluidic 3D in vitro model for specificity of breast cancer metastasis to bone

Abstract

Cancer metastases arise following extravasation of circulating tumor cells with certain tumors exhibiting high organ specificity. Here, we developed a 3D microfluidic model to analyze the specificity of human breast cancer metastases to bone, recreating a vascularized osteo-cell conditioned microenvironment with human osteo-differentiated bone marrow-derived mesenchymal stem cells and endothelial cells. The tri-culture system allowed us to study the transendothelial migration of highly metastatic breast cancer cells and to monitor their behavior within the bone-like matrix. Extravasation, quantified 24 h after cancer cell injection, was significantly higher in the osteo-cell conditioned microenvironment compared to collagen gel-only matrices (77.5 ± 3.7% vs. 37.6 ± 7.3%), and the migration distance was also significantly greater (50.8 ± 6.2 μm vs. 31.8 ± 5.0 μm). Extravasated cells proliferated to form micrometastases of various sizes containing 4 to more than 60 cells by day 5. We demonstrated that the breast cancer cell receptor CXCR2 and the bone-secreted chemokine CXCL5 play a major role in the extravasation process, influencing extravasation rate and traveled distance. Our study provides novel 3D in vitro quantitative data on extravasation and micrometastasis generation of breast cancer cells within a bone-like microenvironment and demonstrates the potential value of microfluidic systems to better understand cancer biology and screen for new therapeutics.

Introduction

The systemic nature of cancer metastases coupled with the resistance to most current therapeutic agents explains why metastases are responsible for as much as 90% of cancer-related mortality [1], [2]. The dissemination of circulating tumor cells (CTCs) represents a “hidden” process leading to micrometastases where quiescent cells can survive for prolonged periods before their activation [3], [4].

In order to generate secondary tumors, CTCs must survive in the circulation and undergo a process known as extravasation [5], [6], [7]. Extravasation into the parenchyma of distant tissues represents a multistep sequence within the metastatic cascade, in which cancer cells establish transient, metastable contacts with the endothelium [8], [9], [10], firmly adhere to the vascular walls [11] and finally transmigrate across the endothelial and pericyte layers [12] as microcolonies or isolated cells [13], [14].

Although it is well known that circulatory patterns play a pivotal role in the spread of metastatic cells to secondary sites, the cross-talk between specific cancer cell types and receptive environments also preferentially guides the dissemination process [15]. In this context, it has been shown that breast cancer metastasizes to bone, liver, lung and brain while prostate cancer frequently disseminates to bone [3]. Particularly, autoptic studies have demonstrated that 70% of advanced breast cancer patients have skeletal metastases, leading to pain, due to spinal cord compression and fractures, and often mortality [16], [17].

Despite the clinical importance of metastases, research has largely focused on the oncogenic transformations leading to the development of primary tumors and much remains to be learned about the metastatic process [5]. Moreover, a deeper understanding of the metastatic cascade and particularly of extravasation to a specific organ could promote the development of new therapeutic strategies, thus improving cancer survival rates [12].

In vivo and ex vivo models have been developed to study the extravasation process in mice and zebrafish embryos through intravital microscopy [13], [18], [19] and advanced models of bone metastasis employ intravenous, intracardiac or direct skeletal injection of breast cancer cells [20], [21]. Although these experiments replicate physiological conditions, they cannot model all aspects of the interaction and cross-talk between human cancer cells, human endothelial cells and human tissue parenchyma. Moreover, strictly regulated, reproducible parametric studies are difficult to perform.

In vitro models, although unable to fully replicate the in vivo situation, can overcome some of these limitations by using human cells throughout and providing highly controllable environments where single culture parameters can be modified [22], [23]. Traditional assays (e.g. Boyden chamber, wound assay, and others) have been widely used to study cell migration in response to chemotactic gradients, particularly cancer cell invasion and migration. However, they do not provide tight control over the local environment, complex interactions cannot be accurately analyzed, and imaging is limited [24], [25], [26].

Microfluidics can provide useful model systems to investigate complex phenomena under combination of multiple controllable biochemical and biophysical microenvironments, coupled with high resolution real time imaging [27], [28], [29], [30]. The synthesis of these features is technically impossible with traditional assays as the Boyden chamber [31], [32]. Toward this goal, several microfluidic devices have been developed to investigate cancer cell transition to invasion and migration from a primary site [33], [34], [35], cell transition effects across mechanical barriers [36], intravasation [37], adhesion [38] and extravasation [39], [40], [41], [42], [43], [44] processes.

However, despite supporting experimental evidence, none of the previously reported in vitro systems has reproduced the specific cross-talk among several cell types in a complex cancer microenvironment during extravasation and none has gone beyond the study of transendothelial migration towards a non-organ-specific extracellular matrix (ECM). Indeed, the importance of organ-specific cancer models lies in the possibility to better clarify the mutual interactions between different cell populations in a well-defined microenvironment, in order to develop highly focused and more effective therapies.

We develop here a new tri-culture microfluidic 3D in vitro model demonstrating the key role played by an osteo-cell conditioned microenvironment, a collagen gel with embedded osteo-differentiated human bone marrow-derived mesenchymal stem cells (hBM-MSCs) [45] and lined with endothelium, in the extravasation process of highly-metastatic MDA-MB-231 human breast cancer cells [16], [46].