Cancer cells create a unique microenvironment in vivo that enables migration to distant organs. To better understand the tumor microenvironment, special tools and devices are required to monitor the interactions between different cell types and the effects of particular chemical gradients. Our study presents the design and optimization of a versatile chemotaxis device, the nano-intravital device (NANIVID), which consists of etched and bonded glass substrates that create a soluble factor reservoir. The device contains a customized hydrogel blend that is loaded with epidermal growth factor (EGF), which diffuses from the outlet to create a chemotactic gradient that can be sustained for many hours in order to attract specific cells to the device. A microelectrode array is under development for quantification of cell collection and will be incorporated into future device generations. Additionally, the NANIVID can be modified to generate gradients of other soluble factors in order to initiate controlled changes to the microenvironment including the induction of hypoxia, manipulation of extracellular matrix stiffness, etc. The focus of the article is to present the design and optimization of the device towards wide ranging applications of cancer cell dynamics in vitro and, ultimately, implantation for in vivo investigations.
Cancer cells create a unique microenvironment in vivo which enables migration to distant organs. To better understand
the tumor microenvironment, special tools and devices are required to monitor the interactions between different cell
types and the effects of particular chemical gradients. This study presents the design and optimization of a new, versatile
chemotaxis device called the NANIVID (NANo IntraVital Device). The device is fabricated using BioMEMS techniques
and consists of etched and bonded Pyrex substrates, a soluble factor reservoir, fluorescent tracking beads and a
microelectrode array for cell quantification. The reservoir contains a customized hydrogel blend loaded with EGF which
diffuses out of the hydrogel to create a chemotactic gradient. This reservoir sustains a steady release of growth factor
into the surrounding environment for many hours and establishes a concentration gradient that attracts specific cells to
the device. In addition to a cell collection tool, the NANIVID can be modified to act as a delivery vehicle for the local
generation of alternate soluble factor gradients to initiate controlled changes to the microenvironment such as hypoxia,
ECM stiffness and etc. The focus of this study is to design and optimize the new device for wide ranging studies of
breast cancer cell dynamics in vitro and ultimately, implantation for in vivo work.
Metastatic cancer cells respond to chemical and mechanical stimuli in their microenvironment that guide invasion into
the surrounding tissue and eventually the circulatory/lymph systems. The NANIVID is designed to be an in vivo device
used to collect metastatic cancer cells by providing a gradient of epidermal growth factor through the controlled release
from a customized hydrogel. The model cells, MTLn3 and Mena<sup>Inv</sup>, both derived from a rat mammary adenocarcinoma,
will migrate toward the device and be collected in the chamber. A set of electrodes inside the chamber will provide real-time
data on the density of cells collected in the device. The characterization and optimization of the electrodes in vitro
will be reported, as will the development of an equivalent circuit model used to describe electrode behavior. The ultimate
goal of this work is for the NANIVID to be used for in vivo investigations of a rat model of mammary cancer.
Furthermore, since the morphology, mechanical properties, and movement of cells are influenced by the
microenvironment, a combined scanning confocal laser microscope and atomic force microscope will be used to study
these relationships. This work will further the understanding of the dynamics and mechanics of metastatic cancer cells as
they leave the primary tumor and metastasize.
In-vivo cancer cells create a unique microenvironment which enables their spread to other organs. To understand the
tumor microenvironment, special tools and devices are required to monitor the interaction among different cell types as
well as the effects of particular chemical gradients. We are reporting on the status of a new device (the NANIVID:
NANoIntraVItal Device) that will collect chemotactic cells from the tumor environment. Due to the transparency of this
implantable device, direct in-vivo cell imaging both inside and outside the device is possible. The cell collection chamber
of the device consists of a micro-electrode system based on patterning of transparent, conducting films that deliver real
time data including cell density and dynamics. The current development and testing status of the device will be
presented. This will include the modeling of ligand gradient profile results produced from the device and the cell
migration in the EGF (epidermal growth factor) gradient created by the device. Further, prototype electrode arrays were
designed, fabricated and cells were cultured on the arrays at selected degrees of confluence to measure the device
sensitivity. The development path of the NANIVID will be integrated with an existing animal model protocol for in-vivo
testing. This will result in a clearer understanding of the dynamics of a tumor's metastatic progression.
The Tumor MicroEnvironment for Metastasis (TMEM) is a critical determinant which will presage the evolution of
primary tumors and the resulting metastatic dynamics. Primary tumor cells up and down regulate certain genes which
increase motility and cause a disregard for positional information. We report on the development of a new tool for the
documentation of cancer cell migration (initial targets: the rat mammary adenocarcinoma cell lines MTLn3 with an over
expression of Mena+++). This tool, the NANo IntraVital Device (NANIVID), is a multi-functional nanosystem
composed of a chemoattractant source (hydrogel-EGF), capsule (cell trap), counter (transparent, interdigitated electrode
arrays for sensing cell arrival), and remote reporter (readout electronics). The device will be retrieved from the tumor
site and the cells will be expelled for subsequent assay. The NANIVID will be used in conjunction with the current
catheter-based approach in which a needle is loaded with a chemoattractant source and injected into the tumor. A major
drawback in the catheter approach is the short cell collection time and lack of real time registering and reporting of cell
arrival. This paper will present the current status of the NANIVID prototypes developed in which a transparent
implantable device is loaded with chemoattractant source and placed near candidate mammary gland tumors in an
established rat model for multiple days or weeks. This series of experiments will allow the comparison of methods and
to benchmark the NANIVID for use in research. Initial results of these experiments and NANIVID design modifications
will be presented.
Cancerous tumors are dynamic microenvironments that require unique analytical tools for their study. Better
understanding of tumor microenvironments may reveal mechanisms behind tumor progression and generate new strategies for diagnostic marker development, which can be used routinely in histopathological analysis. Previous studies have shown that cell invasion and intravasation are related to metastatic potential and have linked these activities to gene expression patterns seen in migratory and invasive tumor cells <i>in vivo</i>. Existing analytical methods for tumor microenvironments include collection of tumor cells through a catheter needle loaded with a chemical or protein attractant (chemoattractant). This method has some limitations and restrictions, including time constraints of cell collection, long term anesthetization, and <i>in vivo</i> imaging inside the catheter. In this study, a novel implantable device was designed to replace the catheter-based method. The 1.5mm x 0.5mm x 0.24mm device is designed to controllably release chemoattractants for stimulation of tumor cell migration and subsequent cell capture. Devices were fabricated using standard microfabrication techniques and have been shown to mediate controlled release of bovine serum albumin (BSA) and epidermal growth factor (EGF). Optically transparent indium tin oxide (ITO) electrodes have been incorporated into the device for impedance-based measurement of cell density and have been shown to be compatible with <i>in vivo</i> multi-photon imaging of cell migration.