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 process<sup>1</sup>. 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.
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 <i>in vitro</i>. 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.
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.
The Nano Intravital Device, or NANIVID, is under development 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 hydrogel blend that contains growth factors or other chemicals to be
delivered to the tumor microenvironment. When the device is implanted in the tumor, the hydrogel will swell and release
these entrapped molecules, forming a gradient. Validation of the device has been performed in vitro using epidermal
growth factor (EGF) and Mena<sub>INV</sub>, a highly invasive, rat mammary adenocarcinoma cell line. In both 2-D and 3-D
environments, cells migrated toward the gradient of EGF released from the device. The chorioallantoic membrane
(CAM) of White Leghorn chicken eggs is being utilized to grow xenograft tumors that will be used for ex vivo cell
collection. Device optimization is being performed for in vivo use as a tool to collect the invasive cell population.
Preliminary cell collection experiments in vivo were performed using a mouse model of breast cancer. As a second
application, the device is being explored as a delivery vehicle for chemicals that induce controlled changes in the tumor
microenvironment. H<sub>2</sub>O<sub>2</sub> was loaded in the device and generated intracellular reactive oxygen species (ROS) in cells
near the device outlet. In the future, other induction targets will be explored, including hypoglycemia and the
manipulation of extracellular matrix stiffness.
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.
Microfluidic devices are currently being utilized in many types of BioMEMS and medical applications. In
these systems, the interaction between the surface and the biological specimen depends critically on surface properties.
The surface roughness and chemistry as well as the surface area to which the biomolecules or cells are exposed affect
this interaction. Modification of the surface of microfluidic channels can improve the operation of the device by
influencing the behavior of the biological specimens that are flowing through it. SU-8 is an epoxy-based, negative
photoresist that has been previously used to create covered channels. Once cured, it is both chemically and thermally
stable. It is also optically transparent above 360 nm, which allows optical measurements, including fluorescence
imaging, to be taken inside the channel. SU-8 microchannels have been fabricated with a porous layer on the sidewalls
by the photo-lithographic process, which is reproducible with precisely controlled channel dimensions. In order to attain
these porous sidewalls, no additional fabrication steps are required outside the standard photo-lithographic process. The
porosity of the sidewalls is a result of incomplete cross-linking of the polymer. The obtained porous surfaces can be
specially treated to provide conditions preferable for biological interactions. The porous layer increases the internal
surface area available on the sidewalls, which make these microfluidic channels preferable for biological applications.
This paper describes the details of the fabrication process and the experiments that verify the benefit of using SU-8
microchannels with porous sidewalls.