Breast cancer is the most common cancer among women worldwide and ranks second in terms of overall cancer deaths. One of the difficulties associated with treating breast cancer is that it is a heterogeneous disease with variations in benign and pathologic tissue composition, which contributes to disease development, progression, and treatment response. Many of these phenotypes are uncharacterized and their presence is difficult to detect, in part due to the sparsity of methods to correlate information between the cellular microscale and the whole-breast macroscale. Quantitative multiscale imaging of the breast is an emerging field concerned with the development of imaging technology that can characterize anatomic, functional, and molecular information across different resolutions and fields of view. It involves a diverse collection of imaging modalities, which touch large sections of the breast imaging research community. Prospective studies have shown promising results, but there are several challenges, ranging from basic physics and engineering to data processing and quantification, that must be met to bring the field to maturity. This paper presents some of the challenges that investigators face, reviews currently used multiscale imaging methods for preclinical imaging, and discusses the potential of these methods for clinical breast imaging.
KEYWORDS: Tumors, In vitro testing, In vivo imaging, Microfluidics, Etching, Glasses, Content addressable memory, Cancer, Quantitative analysis, Scanning electron microscopy
The NANIVID – or Nano Intravital Device – is an implantable delivery tool designed to locally affect the tumor microenvironment in vivo. This technology is being redesigned and validated as a cell collection tool for the study of metastatic cancer cells. A methodology has been developed to facilitate this transition, consisting of microfluidic analysis of the device microchannels and a series of cell-related collection experiments building up to in vivo collection. Single-chamber designs were first used to qualitatively demonstrate the feasibility of cell collection ex vivo. This was followed by the development and implementation of devices containing a second, negative-control chamber for quantitative analysis. This work sets the foundation for in vivo cancer cell migration studies utilizing the NANIVID.
The tumor microenvironment is a complex system which is not fully understood. New technologies are needed to provide a better understanding of the role of the tumor microenvironment in promoting metastasis. The Nano Intravital Device, or NANIVID, has been developed as an optically transparent, implantable tool to study the tumor microenvironment. Two etched glass substrates are sealed using a thin polymer membrane to create a reservoir with a single outlet. This reservoir is loaded with a custom hydrogel blend that contains selected factors for delivery to the tumor microenvironment. When the device is implanted in the tumor, the hydrogel swells and releases these entrapped molecules, forming a sustained concentration gradient. The NANIVID has previously been successful in manipulating the tumor microenvironment both in vitro as well as in vivo. As metastatic cells intravasate, it has been shown that some are able to do so unscathed and reach their new location, while others are cleaved during the process1. There appears to be a correlation between cell migration and the mechanical properties of these cells. It is believed that these properties can be detected in real time by atomic force microscopy. In this study, metastatic MTLn3 rat mammary cells are seeded onto 1-dimensional microfibers and directed up a stable gradient of growth factor. The NANIVID device is placed behind our AFM tip, where it generates a stable chemotactic gradient of epidermal growth factor. Scanning confocal laser microscopy is also used to monitor movement of the cells over time. This experiment will shed light on the mechanical changes in metastatic cells as they undergo directed migration.
Second-harmonic generation (SHG) imaging can help reveal interactions between collagen fibers and cancer cells. Quantitative analysis of SHG images of collagen fibers is challenged by the heterogeneity of collagen structures and low signal-to-noise ratio often found while imaging collagen in tissue. The role of collagen in breast cancer progression can be assessed post acquisition via enhanced computation. To facilitate this, we have implemented and evaluated four algorithms for extracting fiber information, such as number, length, and curvature, from a variety of SHG images of collagen in breast tissue. The image-processing algorithms included a Gaussian filter, SPIRAL-TV filter, Tubeness filter, and curvelet-denoising filter. Fibers are then extracted using an automated tracking algorithm called fiber extraction (FIRE). We evaluated the algorithm performance by comparing length, angle and position of the automatically extracted fibers with those of manually extracted fibers in twenty-five SHG images of breast cancer. We found that the curvelet-denoising filter followed by FIRE, a process we call CT-FIRE, outperforms the other algorithms under investigation. CT-FIRE was then successfully applied to track collagen fiber shape changes over time in an in vivo mouse model for breast cancer.
Multiple changes within the tumor microenvironment have been correlated with an increase in metastasis, yet the mechanisms are not fully understood. Tumor cells can be stimulated by the release of chemoattractant factors such as epidermal growth factor (EGF) from nearby stromal cells, resulting in increased intravasation and metastasis. Additionally, altered extracellular matrix density can result in changes in gene expression patterns governing increased cellular proliferation and motility. The Nano Intravital Device (NANIVID) has been used to produce gradients of select soluble factors in the tumor microenvironment and to study the role of these changes on cell migration. In previous studies, the NANIVID utilized a synthetic hydrogel to produce an EGF gradient to attract metastatic breast cancer cells. In this work, a matrigel insert will be introduced into the outlet to provide a substrate for cells to migrate on when entering the device. The concentration of the chemoattractant and matrigel comprising the insert will be optimized to produce a suitable gradient for inducing chemotaxis in metastatic breast cancer cells in vitro. Additionally, silk and alginate matrices will be explored as improved soluble factor release mediums. Delivery of larger molecules such as collagen cross-linkers requires an alternative hydrogel material. Future NANIVID experiments will utilize these materials to gauge the cellular motility response when a stiffer matrix is encountered.
Tumor-Associated Collagen Signatures (TACS) have been identified that manifest in specific ways during breast tumor
progression and that correspond to patient outcome. There are also compelling metabolic changes associated with
carcinoma invasion and progression. We have characterized the difference in the autofluorescent properties of metabolic
co-factors, NADH and FAD, between normal and carcinoma breast cell lines. Also, we have shown in vitro that
increased collagen density alters metabolic genes which are associated with glycolysis and leads to a more invasive
phenotype. Establishing the relationship between collagen density, cellular metabolism, and metastasis in
physiologically relevant cancer models is crucial for developing cancer therapies. To study cellular metabolism with
respect to collagen density in vivo, we use multiphoton fluorescence excitation microscopy (MPM) in conjunction with a
rodent mammary imaging window implanted in defined mouse cancer models. These models are ideal for the study of
collagen changes in vivo, allowing determination of corresponding metabolic changes in breast cancer invasion and
progression. To measure cellular metabolism, we collect fluorescence lifetime (FLIM) signatures of NADH and FAD,
which are known to change based on the microenvironment of the cells. Additionally, MPM systems are capable of
collecting second harmonic generation (SHG) signals which are a nonlinear optical property of collagen. Therefore,
MPM, SHG, and FLIM are powerful tools with great potential for characterizing key features of breast carcinoma in
vivo. Below we present the current efforts of our collaborative group to develop intravital approaches based on these
imaging techniques to look at defined mouse mammary models.
Multiphoton laser scanning microscopy (MPLSM) utilizing techniques such as multiphoton excitation (MPE), second harmonic generation (SHG), and multiphoton fluorescence lifetime imaging and spectral lifetime imaging (FLIM and SLIM, respectively) are greatly expanding the degree of information obtainable with optical imaging in biomedical research. The application of these nonlinear optical approaches to the study of breast cancer holds particular promise. These noninvasive, multidimensional techniques are well suited to image exogenous fluorophores that allow relevant questions regarding protein localization and signaling to be addressed both in vivo and in vitro. Furthermore, MPLSM imaging of endogenous signals from collagen and fluorophores such as nicotinamide adenine dinucleotide (NADH) or flavin adenine dinucleotide (FAD), address important questions regarding the tumor-stromal interaction and the physiologic state of the cell. We demonstrate the utility of multimodal MPE/SHG/FLIM for imaging both exogenous and/or endogenous fluorophores in mammary tumors or relevant 3-D systems. Using SLIM, we present a method for imaging and differentiating signals from multiple fluorophores that can have overlapping spectra via SLIM Plotter—a computational tool for visualizing and analyzing large spectral-lifetime data sets.
Multiphoton fluorescence lifetime imaging microscopy (FLIM) is a noninvasive, cellular resolution, 3-D functional imaging technique. We investigate the potential for in vivo precancer diagnosis with metabolic imaging via multiphoton FLIM of the endogenous metabolic cofactor nicotinamide adenine dinucleotide (NADH). The dimethylbenz[]anthracene (DMBA)-treated hamster cheek pouch model of oral carcinogenesis and MCF10A cell monolayers are imaged using multiphoton FLIM at 780-nm excitation. The cytoplasm of normal hamster cheek pouch epithelial cells has short (0.29±0.03 ns) and long lifetime components (2.03±0.06 ns), attributed to free and protein-bound NADH, respectively. Low-grade precancers (mild to moderate dysplasia) and high-grade precancers (severe dysplasia and carcinoma in situ) are discriminated from normal tissues by their decreased protein-bound NADH lifetime (p<0.05). Inhibition of cellular glycolysis and oxidative phosphorylation in cell monolayers produces an increase and decrease, respectively, in the protein-bound NADH lifetime (p<0.05). Results indicate that the decrease in protein-bound NADH lifetime with dysplasia is due to a shift from oxidative phosphorylation to glycolysis, consistent with the predictions of neoplastic metabolism. We demonstrate that multiphoton FLIM is a powerful tool for the noninvasive characterization and detection of epithelial precancers in vivo.
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