Engineering Tumor Stroma Morphogenesis Using Dynamic Cell-Matrix Spheroid Assembly.

The tumor microenvironment consists of resident tumor cells organized within a compositionally diverse, three-dimensional (3D) extracellular matrix (ECM) network that cannot be replicated in vitro using bottom-up synthesis. We report a new self-assembly system to engineer ECM-rich 3D MatriSpheres wherein tumor cells actively organize and concentrate microgram quantities of decellularized ECM dispersions which modulate cell phenotype. 3D colorectal cancer (CRC) MatriSpheres were created using decellularized small intestine submucosa (SIS) as an orthotopic ECM source that had greater proteomic homology to CRC tumor ECM than traditional ECM formulations such as Matrigel. SIS ECM was rapidly concentrated from its environment and assembled into ECM-rich 3D stroma-like regions by mouse and human CRC cell lines within 4-5 days via a mechanism that was rheologically distinct from bulk hydrogel formation. Both ECM organization and transcriptional regulation by 3D ECM cues affected programs of malignancy, lipid metabolism, and immunoregulation that corresponded with an in vivo MC38 tumor cell subpopulation identified via single cell RNA sequencing. This 3D modeling approach stimulates tumor specific tissue morphogenesis that incorporates the complexities of both cancer cell and ECM compartments in a scalable, spontaneous assembly process that may further facilitate precision medicine.

"Tumor Treating Fields" delivered via electromagnetic induction have varied effects across glioma cell lines and electric field amplitudes.

Previous studies reported that alternating electric fields (EFs) in the intermediate frequency (100-300 kHz) and low intensity (1-3 V/cm) regime - termed "Tumor Treating Fields" (TTFields) - have a specific, anti-proliferative effect on glioblastoma multiforme (GBM) cells. However, the mechanism(s) of action remain(s) incompletely understood, hindering the clinical adoption of treatments based on TTFields. To advance the study of such treatment in vitro, we developed an inductive device to deliver EFs to cell cultures which improves thermal and osmolar regulation compared to prior devices. Using this inductive device, we applied continuous, 200 kHz electromagnetic fields (EMFs) with a radial EF amplitude profile spanning 0-6.5 V/cm to cultures of primary rat astrocytes and several human GBM cell lines - U87, U118, GSC827, and GSC923 - for a duration of 72 hours. Cell density was assessed via segmented pixel densities from GFP expression (U87, U118) or from staining (astrocytes, GSC827, GSC923). Further RNA-Seq analyses were performed on GSC827 and GSC923 cells. Treated cultures of all cell lines exhibited little to no change in proliferation at lower EF amplitudes (0-3 V/cm). At higher amplitudes (> 4 V/cm), different effects were observed. Apparent cell densities increased (U87), decreased (GSC827, GSC923), or showed little change (U118, astrocytes). RNA-Seq analyses on treated and untreated GSC827 and GSC923 cells revealed differentially expressed gene sets of interest, such as those related to cell cycle control. Up- and down-regulation, however, was not consistent across cell lines nor EF amplitudes. Our results indicate no consistent, anti-proliferative effect of 200 kHz EMFs across GBM cell lines and thus contradict previous in vitro findings. Rather, effects varied across different cell lines and EF amplitude regimes, highlighting the need to assess the effect(s) of TTFields and similar treatments on a per cell line basis.

Optogenetic Signaling Activation in Zebrafish Embryos.

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally manipulate signaling are required to understand how signaling is interpreted in these wide-ranging contexts. Molecular optogenetic tools can provide reversible, tunable manipulations of signaling pathway activity with a high degree of spatiotemporal control and have been applied in vitro, ex vivo, and in vivo. These tools couple light-responsive protein domains, such as the blue light homodimerizing light-oxygen-voltage sensing (LOV) domain, with signaling effectors to confer light-dependent experimental control over signaling. This protocol provides practical guidelines for using the LOV-based bone morphogenetic protein (BMP) and Nodal signaling activators bOpto-BMP and bOpto-Nodal in the optically accessible early zebrafish embryo. It describes two control experiments: A quick phenotype assay to determine appropriate experimental conditions, and an immunofluorescence assay to directly assess signaling. Together, these control experiments can help establish a pipeline for using optogenetic tools in early zebrafish embryos. These strategies provide a powerful platform to investigate the roles of signaling in development, health, and physiology.

"Tumor Treating Fields" delivered via electromagnetic induction have varied effects across glioma cell lines and electric field amplitudes.

Previous studies reported that alternating electric fields (EFs) in the intermediate frequency (100 - 300 kHz) and low intensity (1 - 3 V/cm) regime - termed "Tumor Treating Fields" (TTFields) - have a specific, anti-proliferative effect on glioblastoma multiforme (GBM) cells. However, the mechanism(s) of action remain(s) incompletely understood, hindering the clinical adoption of treatments based on TTFields. To advance the study of such treatment in vitro , we developed an inductive device to deliver EFs to cell cultures which improves thermal and osmolar regulation compared to prior devices. Using this inductive device, we applied continuous, 200 kHz electromagnetic fields (EMFs) with a radial EF amplitude profile spanning 0 - 6.5 V/cm to cultures of primary rat astrocytes and several human GBM cell lines - U87, U118, GSC827, and GSC923 - for a duration of 72 hours. Cell density was assessed via segmented pixel densities from GFP expression (U87, U118) or from staining (astrocytes, GSC827, GSC923). Further RNA-Seq analyses were performed on GSC827 and GSC923 cells. Treated cultures of all cell lines exhibited little to no change in proliferation at lower EF amplitudes (0 - 3 V/cm). At higher amplitudes (> 4 V/cm), different effects were observed. Apparent cell densities increased (U87), decreased (GSC827, GSC923), or showed little change (U118, astrocytes). RNA-Seq analyses on treated and untreated GSC827 and GSC923 cells revealed differentially expressed gene sets of interest, such as those related to cell cycle control. Up- and down-regulation, however, was not consistent across cell lines nor EF amplitudes. Our results indicate no consistent, anti-proliferative effect of 200 kHz EMFs across GBM cell lines and thus contradict previous in vitro findings. Rather, effects varied across different cell lines and EF amplitude regimes, highlighting the need to assess the effect(s) of TTFields and similar treatments on a per cell line basis.

Whole Animal Feeding FLat (WAFFL): a complete and comprehensive validation of a novel, high-throughput fly experimentation system.

Non-mammalian model organisms have been essential for our understanding of the mechanisms that control development, disease, and physiology, but they are underutilized in pharmacological and toxicological phenotypic screening assays due to their low throughput in comparison with cell-based screens. To increase the utility of using Drosophila melanogaster in screening, we designed the Whole Animal Feeding FLat (WAFFL), a novel, flexible, and complete system for feeding, monitoring, and assaying flies in a high-throughput format. Our 3D printed system is compatible with inexpensive and readily available, commercial 96-well plate consumables and equipment. Experimenters can change the diet at will during the experiment and video record for behavior analysis, enabling precise dosing, measurement of feeding, and analysis of behavior in a 96-well plate format.

Quantitative evaluation of the dynamic activity of HeLa cells in different viability states using dynamic full-field optical coherence microscopy.

Dynamic full-field optical coherence microscopy (DFFOCM) was used to characterize the intracellular dynamic activities and cytoskeleton of HeLa cells in different viability states. HeLa cell samples were continuously monitored for 24 hours and compared with histological examination to confirm the cell viability states. The averaged mean frequency and magnitude observed in healthy cells were 4.79±0.5 Hz and 2.44±1.06, respectively. In dead cells, the averaged mean frequency was shifted to 8.57±0.71 Hz, whereas the magnitude was significantly decreased to 0.53±0.25. This cell dynamic activity analysis using DFFOCM is expected to replace conventional time-consuming and biopsies-required histological or biochemical methods.

A Novel In Vitro Device to Deliver Induced Electromagnetic Fields to Cell and Tissue Cultures.

We have developed a novel, to our knowledge, in vitro instrument that can deliver intermediate-frequency (100-400 kHz), moderate-intensity (up to and exceeding 6.5 V/cm pk-pk) electric fields (EFs) to cell and tissue cultures generated using induced electromagnetic fields (EMFs) in an air-core solenoid coil. A major application of these EFs is as an emerging cancer treatment modality. In vitro studies by Novocure reported that intermediate-frequency (100-300 kHz), low-amplitude (1-3 V/cm) EFs, which they called "tumor-treating fields (TTFields)," had an antimitotic effect on glioblastoma multiforme (GBM) cells. The effect was found to increase with increasing EF amplitude. Despite continued theoretical, preclinical, and clinical study, the mechanism of action remains incompletely understood. All previous in vitro studies of "TTFields" have used attached, capacitively coupled electrodes to deliver alternating EFs to cell and tissue cultures. This contacting delivery method suffers from a poorly characterized EF profile and conductive heating that limits the duration and amplitude of the applied EFs. In contrast, our device delivers EFs with a well-characterized radial profile in a noncontacting manner, eliminating conductive heating and enabling thermally regulated EF delivery. To test and demonstrate our system, we generated continuous, 200-kHz EMF with an EF amplitude profile spanning 0-6.5 V/cm pk-pk and applied them to exemplar human thyroid cell cultures for 72 h. We observed moderate reduction in cell density (<10%) at low EF amplitudes (<4 V/cm) and a greater reduction in cell density of up to 25% at higher amplitudes (4-6.5 V/cm). Our device can be readily extended to other EF frequency and amplitude regimes. Future studies with this device should contribute to the ongoing debate about the efficacy and mechanism(s) of action of "TTFields" by better isolating the effects of EFs and providing access to previously inaccessible EF regimes.

Rodent Arena Tracker (RAT): A Machine Vision Rodent Tracking Camera and Closed Loop Control System.

Video tracking is an essential tool in rodent research. Here, we demonstrate a machine vision rodent tracking camera based on a low-cost, open-source, machine vision camera, the OpenMV Cam M7. We call our device the rodent arena tracker (RAT), and it is a pocket-sized machine vision-based position tracker. The RAT does not require a tethered computer to operate and costs about $120 per device to build. These features make the RAT scalable to large installations and accessible to research institutions and educational settings where budgets may be limited. The RAT processes incoming video in real-time at 15 Hz and saves x and y positional information to an onboard microSD card. The RAT also provides a programmable multi-function input/output pin that can be used for controlling other equipment, transmitting tracking information in real time, or receiving data from other devices. Finally, the RAT includes a real-time clock (RTC) for accurate time stamping of data files. Real-time image processing averts the need to save video, greatly reducing storage, data handling, and communication requirements. To demonstrate the capabilities of the RAT, we performed three validation studies: (1) a 4-d experiment measuring circadian activity patterns; (2) logging of mouse positional information alongside status information from a pellet dispensing device; and (3) control of an optogenetic stimulation system for a real-time place preference (RTPP) brain stimulation reinforcement study. Our design files, build instructions, and code for the RAT implementation are open source and freely available online to facilitate dissemination and further development of the RAT.

Rodent Activity Detector (RAD), an Open Source Device for Measuring Activity in Rodent Home Cages.

Physical activity is a critical behavioral variable in many research studies and is, therefore, important to quantify. However, existing methods for measuring physical activity have limitations which include high expense, specialized caging or equipment, and high computational overhead. To address these limitations, we present an open-source, cost-effective, device for measuring rodent activity. Our device is battery powered and designed to be placed in vivarium home cages to enable high-throughput, long-term operation with minimal investigator intervention. The primary aim of this study was to assess the feasibility of using passive infrared (PIR) sensors and microcontroller-based dataloggers in a rodent home cages to collect physical activity records. To this end, we developed an open-source PIR based data-logging device called the rodent activity detector (RAD). We publish the design files and code so others can readily build the RAD in their own labs. To demonstrate its utility, we used the RAD to collect physical activity data from 40 individually housed mice for up to 10 weeks. This dataset demonstrates the ability of the RAD to (1) operate in a high-throughput installation, (2) detect high-fat diet (HFD)-induced changes in physical activity, and (3) quantify circadian rhythms in individual animals. We further validated the data output of the RAD with simultaneous video tracking of mice in multiple caging configurations, to determine the features of physical activity that it detects. The RAD is easy to build, economical, and fits in vivarium caging. The scalability of such devices will enable high-throughput studies of physical activity in research studies.

Spatial control of oxygen delivery to three-dimensional cultures alters cancer cell growth and gene expression.

Commonly used monolayer cancer cell cultures fail to provide a physiologically relevant environment in terms of oxygen delivery. Here, we describe a three-dimensional (3D) bioreactor system where cancer cells are grown in Matrigel in modified six-well plates. Oxygen is delivered to the cultures through a polydimethylsiloxane (PDMS) membrane at the bottom of the wells, with microfabricated PDMS pillars to control oxygen delivery. The plates receive 3% oxygen from below and 0% oxygen at the top surface of the media, providing a gradient of 3-0% oxygen. We compared growth and transcriptional profiles for cancer cells grown in Matrigel in the bioreactor, 3D cultures grown in 21% oxygen, and cells grown in a standard hypoxia chamber at 3% oxygen. Additionally, we compared gene expression of conventional two-dimensional monolayer culture and 3D Matrigel culture in 21% oxygen. We conclude that controlled oxygen delivery may provide a more physiologically relevant 3D system.

SCORHE: a novel and practical approach to video monitoring of laboratory mice housed in vivarium cage racks.

The System for Continuous Observation of Rodents in Home-cage Environment (SCORHE) was developed to demonstrate the viability of compact and scalable designs for quantifying activity levels and behavior patterns for mice housed within a commercial ventilated cage rack. The SCORHE in-rack design provides day- and night-time monitoring with the consistency and convenience of the home-cage environment. The dual-video camera custom hardware design makes efficient use of space, does not require home-cage modification, and is animal-facility user-friendly. Given the system's low cost and suitability for use in existing vivariums without modification to the animal husbandry procedures or housing setup, SCORHE opens up the potential for the wider use of automated video monitoring in animal facilities. SCORHE's potential uses include day-to-day health monitoring, as well as advanced behavioral screening and ethology experiments, ranging from the assessment of the short- and long-term effects of experimental cancer treatments to the evaluation of mouse models. When used for phenotyping and animal model studies, SCORHE aims to eliminate the concerns often associated with many mouse-monitoring methods, such as circadian rhythm disruption, acclimation periods, lack of night-time measurements, and short monitoring periods. Custom software integrates two video streams to extract several mouse activity and behavior measures. Studies comparing the activity levels of ABCB5 knockout and HMGN1 overexpresser mice with their respective C57BL parental strains demonstrate SCORHE's efficacy in characterizing the activity profiles for singly- and doubly-housed mice. Another study was conducted to demonstrate the ability of SCORHE to detect a change in activity resulting from administering a sedative.