Analysis of tumor template from multiple compartments in a blood sample provides complementary access to peripheral tumor biomarkers.

Targeted cancer therapeutics are promised to have a major impact on cancer treatment and survival. Successful application of these novel treatments requires a molecular definition of a patient\u27s disease typically achieved through the use of tissue biopsies. Alternatively, allowing longitudinal monitoring, biomarkers derived from blood, isolated either from circulating tumor cell derived DNA (ctcDNA) or circulating cell-free tumor DNA (ccfDNA) may be evaluated. In order to use blood derived templates for mutational profiling in clinical decisions, it is essential to understand the different template qualities and how they compare to biopsy derived template DNA as both blood-based templates are rare and distinct from the gold-standard. Using a next generation re-sequencing strategy, concordance of the mutational spectrum was evaluated in 32 patient-matched ctcDNA and ccfDNA templates with comparison to tissue biopsy derived DNA template. Different CTC antibody capture systems for DNA isolation from patient blood samples were also compared. Significant overlap was observed between ctcDNA, ccfDNA and tissue derived templates. Interestingly, if the results of ctcDNA and ccfDNA template sequencing were combined, productive samples showed similar detection frequency (56% vs 58%), were temporally flexible, and were complementary both to each other and the gold standard. These observations justify the use of a multiple template approach to the liquid biopsy, where germline, ctcDNA, and ccfDNA templates are employed for clinical diagnostic purposes and open a path to comprehensive blood derived biomarker access

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80°C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel

Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding. In some cases it may be desirable to reversibly bond devices to the Corning No. 1 cover glass. This could be due, perhaps, to a plasma cleaner not being available. In other instances, it may be desirable to remove the device from the glass after the culturing of neurons for certain types of microscopy or for immunostaining, though it is not necessary to remove the device for immunostaining since the neurons can be stained in the device. Some researchers, however, still prefer to remove the device. In this case, reversible bonding of the device to the cover glass makes that possible. There are some disadvantages to non-plasma bonding of the devices in that not as tight of a seal is formed. In some cases axons may grow under the grooves rather than through them. Also, because the glass and PDMS are hydrophobic, liquids do not readily enter the device making it necessary at times to force media and other reagents into the device. Liquids will enter the device via capillary action, but it takes significantly longer as compared to devices that have been plasma bonded. The plasma cleaner creates temporary hydrophilic charges on the glass and device that facilitate the flow of liquids through the device after bonding within seconds. For non-plasma bound devices, liquid flow through the devices takes several minutes. It is also important to note that the devices to be used with non-plasma bonding need to be sterilized first, whereas plasma treated devices do not need to be sterilized prior to use because the plasma cleaner will sterilize them

Examination of axonal injury and regeneration in micropatterned neuronal culture using pulsed laser microbeam dissection.

We describe the integrated use of pulsed laser microbeam irradiation and microfluidic cell culture methods to examine the dynamics of axonal injury and regeneration in vitro. Microfabrication methods are used to place high purity dissociated central nervous system neurons in specific regions that allow the axons to interact with permissive and inhibitory substrates. Acute injury to neuron bundles is produced via the delivery of single 180 ps duration, lambda = 532 nm laser pulses. Laser pulse energies of 400 nJ and 800 nJ produce partial and complete transection of the axons, respectively, resulting in elliptical lesions 25 mum and 50 mum in size. The dynamics of the resulting degeneration and regrowth of proximal and distal axonal segments are examined for up to 8 h using time-lapse microscopy. We find the proximal and distal dieback distances from the site of laser microbeam irradiation to be roughly equal for both partial and complete transection of the axons. In addition, distinct growth cones emerge from the proximal neurite segments within 1-2 h post-injury, followed by a uniform front of regenerating axons that originate from the proximal segment and traverse the injury site within 8 h. We also examine the use of EGTA to chelate the extracellular calcium and potentially reduce the severity of the axonal degeneration following injury. While we find the addition of EGTA to reduce the severity of the initial dieback, it also hampers neurite repair and interferes with the formation of neuronal growth cones to traverse the injury site. This integrated use of laser microbeam dissection within a micropatterned cell culture system to produce precise zones of neuronal injury shows potential for high-throughput screening of agents to promote neuronal regeneration

Results of intra and inter assay precision studies.

<p>Results of intra and inter assay precision studies.</p

Validation of Automated CTC flow cell operation on LiquidBiopsy platform.

<p>(A) Plot of CTC recovery as a function of spike-in density for MCF7 and H1650 cells. (B) Plot of sample purity as a function of spike in density for MCF7 and H1650 cells. The orange dots indicate the predicted purity of MCF7 cells if the non target recovery is held constant at 55 cells/mL. (C) Raw data and linear fit to MCF7 recovery curve from 9 to 90 cells/mL. (D) Raw data and linear fit to H1650 recovery curve from 9 to 300 cells/mL.</p

LiquidBiopsy automated platform.

<p>(A) Diagram of the LiquidBiopsy platform. (B) Closeup of the platform worksurface. A X-Y pipetting arm with 4 pipettor heads transfers sample, buffers and antibody stains into 4 flow cells in the manifold. Sheath buffers are controlled by pumps on the rear wall of the platform.</p

CTC Flow cell operation and performance.

<p>(A) The LiquidBiopsy CTC flow cell. (B) A cartoon illustrating how target cells are pulled from the sheath flow while non targets move through the flow cell unhindered. (C) Efficiency of recovery of target cells using EpCAM based recovery: Incremental numbers of MCF7 (N = 34), HCC1419 (N = 27) or A549 (N = 65) cells were spiked into NHD blood and purified on the CTC flow cell. Recovered cells were enumerated in the flow cell. Graphs show number of targets spiked per mL of blood against the yield +/−1 SD from an average of between 7 and 27 experiments.</p

Measurements of accuracy and precision of LiquidBiopsy capture of MCF7 and H1650 cells.

<p>Measurements of accuracy and precision of LiquidBiopsy capture of MCF7 and H1650 cells.</p