Chemokine-mediated directed tumor cell migration within a three dimensional (3D) matrix, or chemoinvasion, is an important early step in cancer metastasis. Despite its clinical importance, it is largely unknown how cytokine and growth factor gradients within the tumor microenvironment regulate chemoinvasion. We studied tumor cell chemoinvasion in well-defined and stable chemical gradients using a robust 3D microfluidic model. We used CXCL12 (also known as SDF-1α) and epidermal growth factor (EGF), two well-known extracellular signaling molecules that co-exist in the tumor microenvironment (e.g. lymph nodes or intravasation sites), and a malignant breast tumor cell line, MDA-MB-231, embedded in type I collagen. When subjected to SDF-1α gradients alone, MDA-MB-231 cells migrated up the gradient, and the measured chemosensitivity (defined as the average cell velocity along the direction of the gradient) followed the ligand – receptor (SDF-1α – CXCR4) binding kinetics. On the other hand, when subjected to EGF gradients alone, tumor cells increased their overall motility, but without statistically significant chemotactic (directed) migration, in contrast to previous reports using 2D chemotaxis assays. Interestingly, we found that the chemoinvasive behavior to SDF-1α gradients was abrogated or even reversed in the presence of uniform concentrations of EGF; however, the presence of SDF-1α and EGF together modulated tumor cell motility cooperatively. These findings demonstrate the capabilities of our microfluidic model in re-creating complex microenvironments for cells, and the importance of cooperative roles of multiple cytokine and growth factor gradients in regulating cell migration in 3D environments.
SUMMARY
It is known that the calcium-dependent transcription factor NFAT initiates transcription in response to pulsatile loads of calcium signal. However, the relative contributions of calcium oscillation frequency, amplitude, and duty cycle to transcriptional activity remain unclear. Here, we engineer HeLa cells to permit optogenetic control of intracellular calcium concentration using programmable LED arrays. This approach allows us to generate calcium oscillations of constant peak amplitude, in which frequency is varied while holding duty cycle constant, or vice versa. Using this setup and mathematical modeling, we show that NFAT transcriptional activity depends more on duty cycle, defined as the proportion of the integrated calcium concentration over the oscillation period, than on frequency alone. This demonstrates that NFAT acts primarily as a signal integrator of cumulative load rather than a frequency-selective decoder. This approach resolves a fundamental question in calcium encoding and demonstrates the value of optogenetics for isolating individual dynamical components of larger signaling behaviors.
Rapid, accurate,
and low-cost detection of SARS-CoV-2 is crucial
to contain the transmission of COVID-19. Here, we present a cost-effective
smartphone-based device coupled with machine learning-driven software
that evaluates the fluorescence signals of the CRISPR diagnostic of
SARS-CoV-2. The device consists of a three-dimensional (3D)-printed
housing and low-cost optic components that allow excitation of fluorescent
reporters and selective transmission of the fluorescence emission
to a smartphone. Custom software equipped with a binary classification
model has been developed to quantify the acquired fluorescence images
and determine the presence of the virus. Our detection system has
a limit of detection (LoD) of 6.25 RNA copies/μL on laboratory
samples and produces a test accuracy of 95% and sensitivity of 97%
on 96 nasopharyngeal swab samples with transmissible viral loads.
Our quantitative fluorescence score shows a strong correlation with
the quantitative reverse transcription polymerase chain reaction (RT-qPCR)
Ct values, offering valuable information of the viral load and, therefore,
presenting an important advantage over nonquantitative readouts.
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