We would like to thank Dr. Alana Welms, Huntsman Cancer Institute, University of Utah, for providing us with the patient-derived xenografts (HCI-010). This work was supported by NIH grants R01CA205632, R21CA226542, and in part, by a grant from Agilent Technologies.

The study was reviewed and considered as &#34;exempt&#34; by the Institutional Review Board of Georgetown University (IRB # 2002-022). Freshly harvested bone marrow tissues were collected from discarded healthy human bone marrow collection filters that had been de-identified.
1. New chamber design (Figure 2)
<ol>
	<li>Open a new dual-chamber cell analyzer plate. Set aside the top chamber with electrodes.</li>
	<li>Using a milling machine, shave off 2 mm of the U-shaped bottom wells of the cell analyzer plate.</li>
	<li>Attach a 2 cm x 7 cm polyethersulfone (PES) membrane with 0.2 &#181;m pore size to the bottom of the shaved wells using UV-curated adhesive. Allow 30 min curation time to ensure the glue is completely cured and inert.</li>
	<li>Using a milling machine, cut out two longitudinal slits (1.5 mm x 5.6 mm) along the sides to snap into the ridges of the newly fabricated third chamber.</li>
	<li>Using a milling machine, create a third polycarbonate chamber that replicates the overall dimensions of the cell analyzer plate; 72 mm x 18 mm (Table of Materials).</li>
	<li>Create wells 4.8 mm deep and 4.75 mm in diameter to replicate the 16-well design of the cell analyzer plate. This allows 90 &#181;L of volume per well.</li>
	<li>On the sides, create two triangular ridges so that the chamber locks into the original slits created in step 1.4. The horizontal part of the triangle is 1.5 mm, the vertical is 1.4 mm, and the hypotenuse is 2.052 mm.</li>
	<li>Create a knob on the short side that is 50.8 mm in diameter and 1.397 mm in height to fit into the original plate&#39;s notch (Figure 2, Middle).</li>
	<li>Use a 0.9 mm thick rubber washer for each well to provide a sealed fit.</li>
</ol>
2. Cell culture (MDA-MB-231, DCIS, DCIS-&#916;4, J2-fibroblasts)
<ol>
	<li>Wash adherent cell cultures (~70% confluence) with 1x phosphate-buffered saline (PBS).</li>
	<li>Add 0.05% trypsin-EDTA solution to lift the cells off.</li>
	<li>Neutralize the trypsin solution with cell culture media containing serum and count the cells using an aliquot of the cell suspension. 
	&#8203;NOTE: The specific cell culture media can be found in Table 1.</li>
</ol>
3. Patient-derived xenograft dissociation
<ol>
	<li>Chop a fresh tumor piece (1 cm2) into fine mush using a sterile scalpel.</li>
	<li>Place in a 50 mL conical tube with 20 mL of DMEM F12 media supplemented with 3 mg/mL trypsin and 2 mg/mL collagenase.</li>
	<li>Incubate in a thermal shaker (150 RPM) at 37 &#176;C for 20 min.</li>
	<li>Spin the tube at 500 x g for 5 min; remove the supernatant.</li>
	<li>Add 20 &#956;L of DMEM F12 + 2% FBS to wash the cells; spin at 300 x g for 5 min and remove the supernatant. Repeat wash two more times.</li>
	<li>Resuspend in 1 mL of PDX media (Table 1) to count the cells.</li>
</ol>
4. Bone marrow cell extraction
<ol>
	<li>Flush the bone marrow (BM) collection filter with 25 mL of 1x PBS. 
	NOTE: In this study, PBS was added to a used BM collection filter from the hospital to collect the remaining BM in the filter.</li>
	<li>Add the flushed BM slowly to a 50 mL conical tube with 25 mL of density gradient medium, taking care to keep the layers as separate as possible.</li>
	<li>Spin at 800 x g for 20 min at 18 &#176;C</li>
	<li>Siphon off the top layers after centrifugation (fat/plasma) and transfer 5 mL of the white layer above the density gradient medium that has the BM cells to a 15 mL conical tube. 
	&#8203;NOTE: Alternatively, dip a 5 mL pipette into the top layer until it touches the middle layer (BM) and pipette out the middle layer very slowly without moving the pipette.</li>
	<li>Fill the 15 mL conical tube with 1x PBS (~10 mL) and spin at 300 x g for 15 min.</li>
	<li>Remove the supernatant; the remaining white pellet is the BM.</li>
	<li>If red blood cells are observed in the pellet, add 5 mL of RBC lysis solution (Table of Materials) and let it sit for 5 min at room temperature (RT). Spin at 300 x g for 5 min and remove the supernatant.</li>
	<li>Add 10 mL of 1x PBS to wash the cells, spin at 300 x g for 5 min&#160;and remove the supernatant. Repeat the RBC lysis (step 4.7) until the pellet is white.</li>
</ol>
5. Cell seeding and assembly
<ol>
	<li>Place all three sterile chambers in the tissue culture hood.</li>
	<li>Locate the knob on the short side of the lower chamber. Orient the lower chamber so that the knob is facing the experimenter.</li>
	<li>Add 30,000-50,000 cells in 90 &#956;L of media to each well of the lower chamber. Avoid forming bubbles. These are the stromal cells that will provide secreted factors but will not be detected by the electrodes of the top chamber.</li>
	<li>Use 5% fetal bovine serum-supplemented media in two lower chamber wells as a positive control for cell motility. Use 0% serum-supplemented media as a negative control.</li>
	<li>Let the lower chamber with the cells sit for 10-15 min in the hood to settle. 
	NOTE: This step is recommended if cells are adherent or grow in suspension.</li>
	<li>Rotate the lower chamber at 90&#176; and place the middle chamber on top so that the knob on the lower chamber slides into the notch on the middle chamber. 
	NOTE: The knob on the lower chamber and the blue dot on the middle chamber are at opposite ends of the assembly.</li>
	<li>Push vertically down until a click sound is heard from each of the long sides of the assembly.</li>
	<li>Add 160 &#956;L&#160;of serum-free media to all the wells of the middle chamber.</li>
	<li>Make sure a dome-shaped meniscus is visible after wells are filled; otherwise, adjust the final volume based on the pipette calibration. Avoid forming bubbles.</li>
	<li>Place the top chamber with electrodes facing down onto the middle chamber making sure to align the blue dots on the middle and top chambers.</li>
	<li>Push vertically down until a click sound is heard from each of the long sides of the assembly.</li>
	<li>Add 25-50 &#956;L&#160;of serum-free media to the top chamber.</li>
	<li>Mount the assembly on the dual purpose cell analyzer in the tissue culture incubator and wait for 30 min before measuring the background. 
	NOTE: This time is necessary to equilibrate the array and can be used to prepare the cell lines to be added to the top chamber.</li>
	<li>Measure the background (see section 6) and place the assembly back into the tissue culture hood.</li>
	<li>Add 30,000-50,000 cells in 100 &#956;L&#160;of serum-free media to each well of the top chamber. These are the cells that the electrode will detect once they successfully migrate through the membrane. 
	NOTE: To achieve maximum response, it is recommended to grow cells in serum-free or low serum media for 6-18 h before performing the assay.</li>
	<li>Let the assembly stand in the hood for 30 min before mounting on the dual purpose cell analyzer for impedance measurement.</li>
</ol>
6. Background and impedance measurement
<ol>
	<li>Place the array into the cradle in the dual purpose cell analyzer instrument.</li>
	<li>Open the cell analyzer software and select the cradle to be used.</li>
	<li>Click on the Message tab and make sure it says Connections OK to ensure the array is well placed in the cradle and the electrodes are well aligned with the sensors.</li>
	<li>Click on the Experiment Notes tab and fill in as much information about the experiment as possible.</li>
	<li>Click on the Layout tab and fill in the description of the array layout.</li>
	<li>Click on the Schedule tab and add two steps from the Steps menu; a background step (one sweep) and a test step with 100 sweeps-a sweep every 15 min, totaling 25 h.</li>
	<li>After the array has been in the dual purpose cell analyzer incubator for 30 min, click on the Play button to start background measurement. A window asking to choose the folder to save the data will pop up.</li>
	<li>After the background measurement is done, remove the array from the cradle and place it back in the cell culture hood.</li>
	<li>Add cells to the top chamber as described in step 5.13, and keep the assembly in the tissue culture hood for 30 min for the cells to settle.</li>
	<li>Place the array back into the dual purpose cell analyzer and check the Message tab for the Connections OK message.</li>
	<li>Click on the Play button to start impedance measurement.</li>
	<li>Click on the Plot tab to monitor the progress of the signal.</li>
	<li>If the endpoint is reached before 25 h, click on the Abort step from the Execute drop-down menu.</li>
	<li>To export data, right-click on the graph, choose Copy in the list format, and then paste the data in a spreadsheet. 
	&#8203;NOTE: The data can be exported as cell index or delta cell index. Graph and/or layout information can also be chosen for export.</li>
</ol>
7. Detachment and cell collection
<ol>
	<li>Monitor the migration rate in real-time on the dual purpose cell analyzer to determine the stopping point of interest (6-18 h). 
	NOTE: The stopping point depends on the cell&#39;s invasion rate and when a distinct invasion signal from the negative control is achieved.</li>
	<li>Once achieved, unmount the assembly from the dual purpose cell analyzer and place it in the tissue culture hood.</li>
	<li>Prepare an appropriate number of 1.5 mL microcentrifuge tubes to collect the cells from the wells of interest.</li>
	<li>Place the assembly in a 10 cm dish to contain liquids when the chambers detach.</li>
	<li>Push the flexible snapping ends on the long side of the middle chamber inward until a click sound is heard.</li>
	<li>Dismantle the top chamber and invert it into a new 10 cm dish.</li>
	<li>Use a cell lifter with a 13 mm blade to collect the cells from all the wells harboring the same experimental condition (i.e., cell type, drug treatment, etc.). 
	NOTE: Design the setup to have at least two wells for each experimental condition to achieve statistically significant change from negative controls.</li>
	<li>Rinse or dip the blade in 1x phosphate buffered saline to collect the cells in 1.5 mL microcentrifuge tubes.</li>
	<li>Spin down the cells at 500 x g for 5 min.</li>
	<li>Propagate the collected cells (see section 8) or perform end point analysis such as single-cell RNA-seq. 
	&#8203;NOTE: For bulk RNA-seq, use a low cell number RNA extraction kit.</li>
</ol>
8. 3D cell propagation and retrieval
NOTE: Due to the small number of cells collected, seed the cells in 3D using an extracellular matrix (ECM) to enhance viability. That said, 2D culture is also an option at this point, especially if the cells used are from established cell lines.
<ol>
	<li>Thaw an aliquot of the basement membrane matrix at 4 &#176;C overnight. Keep the basement membrane matrix on ice until ready to use.</li>
	<li>Add 25 &#956;L&#160;of cold basement membrane matrix to the pellet of live cells collected and gently pipette up and down to mix. Avoid forming bubbles.</li>
	<li>Add the cell-basement membrane matrix mix to the bottom of a small tissue culture well (i.e., in a 96-well plate), forming a dome. Try not to touch the walls of the well. Incubate at 37 &#176;C for 20 min before gently adding 100 &#956;L&#160;of media dropwise.</li>
	<li>To retrieve cells, aspirate the media and add 100 &#956;L&#160;of dispase to each well.</li>
	<li>Incubate at RT for 10 min, pipetting up and down occasionally.</li>
	<li>Transfer the cells and the dissolved basement membrane matrix into a 1.5 mL microcentrifuge tube. Add 1 mL of 1x PBS, spin at 300 x g for 5 min and remove the supernatant. Repeat the wash once.</li>
	<li>Aspirate the supernatant. Split the cells in the pellet or perform endpoint analysis.</li>
</ol>

<ol>
	<li>Friedl, P., Gilmour, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cell+migration+in+morphogenesis,+regeneration+and+cancer.">Collective cell migration in morphogenesis, regeneration and cancer.</a> Nature Reviews Molecular Cell Biology. 10 (7), 445-457 (2009).</li><li>Boyden, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemotactic+effect+of+mixtures+of+antibody+and+antigen+on+polymorphonuclear+leucocytes.">The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes.</a> The Journal of Experimental Medicine. 115 (3), 453-466 (1962).</li><li>Xu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+impedance+measurements+of+whole+cells.">A review of impedance measurements of whole cells.</a> Biosensors &amp; Bioelectronics. 77, 824-836 (2016).</li><li>Stylianou, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+single-chain+antibody+targeting+of+the+ligand-binding+domain+in+the+anaplastic+lymphoma+kinase+receptor.">Effect of single-chain antibody targeting of the ligand-binding domain in the anaplastic lymphoma kinase receptor.</a> Oncogene. 28 (37), 3296-3306 (2009).</li><li>Abassi, Y. A., Wang, X., Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+time+electronic+cell+sensing+system+and+applications+for+cytotoxcity+profiling+and+compound+assays.">Real time electronic cell sensing system and applications for cytotoxcity profiling and compound assays.</a> United States Patent. , 1-88 (2013).</li><li>Abassi, Y. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free,+real-time+monitoring+of+IgE-mediated+mast+cell+activation+on+microelectronic+cell+sensor+arrays.">Label-free, real-time monitoring of IgE-mediated mast cell activation on microelectronic cell sensor arrays.</a> Journal of Immunological Methods. 292 (1-2), 195-205 (2004).</li><li>Morton, C. L., Houghton, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+human+tumor+xenografts+in+immunodeficient+mice.">Establishment of human tumor xenografts in immunodeficient mice.</a> Nature Protocols. 2 (2), 247-250 (2007).</li><li>DeRose, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+grafts+derived+from+women+with+breast+cancer+authentically+reflect+tumor+pathology,+growth,+metastasis+and+disease+outcomes.">Tumor grafts derived from women with breast cancer authentically reflect tumor pathology, growth, metastasis and disease outcomes.</a> Nature Medicine. 17 (11), 1514-1520 (2011).</li><li>Sharif, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+AIB1+isoform+alters+enhancer+access+and+enables+progression+of+early-stage+triple-negative+breast+cancer.">An AIB1 isoform alters enhancer access and enables progression of early-stage triple-negative breast cancer.</a> Cancer Research. 81 (16), 4230-4241 (2021).</li><li>Caileau, R., Olive, M., Cruciger, Q. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+human+breast+carcinoma+cell+lines+of+metastatic+origin:+preliminary+characterization.">Long-term human breast carcinoma cell lines of metastatic origin: preliminary characterization.</a> In Vitro. 14 (11), 911-915 (1978).</li><li>Sharif, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+growth+density+modulates+cancer+cell+vascular+invasion+via+Hippo+pathway+activity+and+CXCR2+signaling.">Cell growth density modulates cancer cell vascular invasion via Hippo pathway activity and CXCR2 signaling.</a> Oncogene. 34 (48), 5879-5889 (2015).</li><li>Miller, F. R., Santner, S. J., Tait, L., Dawson, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MCF10DCIS.com+xenograft+model+of+human+comedo+ductal+carcinoma+in+situ.">MCF10DCIS.com xenograft model of human comedo ductal carcinoma in situ.</a> Journal of the National Cancer Institute. 92 (14), 1185-1186 (2000).</li><li>Winkler, J., Abisoye-Ogunniyan, A., Metcalf, K. J., zenaa, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concepts+of+extracellular+matrix+remodelling+in+tumour+progression+and+metastasis.">Concepts of extracellular matrix remodelling in tumour progression and metastasis.</a> Nature Communications. 11 (1), 1-19 (2020).</li><li>Nkosi, D., Sun, L., Duke, L. C., Meckes, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epstein-Barr+virus+LMP1+manipulates+the+content+and+functions+of+extracellular+vesicles+to+enhance+metastatic+potential+of+recipient+cells.">Epstein-Barr virus LMP1 manipulates the content and functions of extracellular vesicles to enhance metastatic potential of recipient cells.</a> PLoS Pathogens. 16 (12), 1009023 (2020).</li><li>Eisenberg, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+modeling+of+the+effects+of+myoferlin+on+tumor+cell+invasion.">Mechanistic modeling of the effects of myoferlin on tumor cell invasion.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (50), 20078-20083 (2011).</li></ol>

Georgetown University filed a patent related to some of the approaches described in this manuscript. G.M.S, A.W, L.D, and M.P are named as inventors on this application and declare that as a potential conflict of interest.

We have modified the design of a dual-chambered array to include a third chamber for monitoring cell invasion in real-time in the presence of stromal cells. We have observed distinct effects of co-cultured fibroblasts on invasive and non-invasive cancer cells indicating that the array can be used to distinguish between cancer cell subpopulations that respond differently to factors produced by co-cultured stromal cells. The array was also used to monitor endothelial cell invasion into stromal tissues, a critical step duri...

Cell invasion is an important process that allows cells to cross basement membrane barriers in response to environmental cues provided by stromal cells. It is a crucial step during several stages of development for immune responses, wound healing, tissue repair, and malignancies that can progress from local lesions to invasive and metastatic cancers1. Assays developed early on to measure the invasive potential of cell populations typically generate a single endpoint measurement or require pre-labeling of invasive cells2. The integration of microelectronics and microfluidics techniques is now developed to detect different aspects of cell biology such as viability, movement, and attachment using the electric impedance of live cells on microelectrodes3,4. Impedance measurement allows for a label-free, non-invasive and quantitative assessment of cell status3. Here we describe a three-chambered array based on the design of the Real-Time Cellular Analysis (RTCA) system that was developed by Abassi et al.5. The three-chambered array allows for the assessment of co-cultured cells on cellular invasion and recovery of invasive cells for additional analyses or expansion.
In the cell analyzer system, cells invade through an extracellular matrix coated onto a porous membrane and reach an interdigitated electrode array positioned on the opposite side of the barrier. As the invasive cells continue to attach and occupy this electrode array over time, the electrical impedance changes in parallel. The current system comprises a cell invasion and migration (CIM) 16-well plate with two chambers. The RTCA-DP (dual purpose) (called dual purpose cell analyzer henceforth) instrument contains sensors for impedance measurement and integrated software to analyze and process the impedance data. Impedance values at baseline depend on the ionic strength of media in the wells and are changed as cells attach to the electrodes. The impedance changes depend on the number of cells, their morphology, and the extent to which cells attach to the electrodes. A measurement of the wells with media before the cells are added is considered as the background signal. The background is subtracted from impedance measurements after reaching equilibrium with cells attaching and spreading onto the electrodes. A unitless parameter of the status of the cells on an electrode termed Cell Index (CI) is calculated as follows: CI = (impedance after equilibrium - impedance in the absence of cells) / nominal impedance value6. When migration rates of different cell lines are compared, the Delta CI can be used to compare cell status regardless of the difference in attachment that is represented in the first few measurements.
The newly designed three-chambered array builds on the existing design and uses the top chamber from the dual purpose cell analyzer system that contains the electrodes. The modified middle and bottom chambers are adapted to fit the assembly into the dual purpose cell analyzer for impedance measurement and analysis using the integrated software. The two major advances that the new design provides over the existing dual-chamber CIM-plate (called cell analyzer plate henceforth) are: i) the ability to recover, and then analyze invasive cell subpopulations that are present in heterogeneous cell mixes and ii) the option to assess the impact of secreted factors from co-cultured stromal or immune cells on cell invasion (Figure 1).
This technology can be useful in studying the subpopulations of cells with different invasive capacities. That includes (a) invasive cancer cells that invade surrounding tissues or blood and lymphatic vessels or extravasate at metastatic seeding sites in distant organs, (b) cells from the immune system that invade tissues to tackle pathogens or diseased cells, (c) endothelial cells that invade tissues to form new blood vessels during tissue reorganization or wound healing, as well as (d) stromal cells from the tumor microenvironment that support and invade along with cancer cells. The approach allows the inclusion of stromal cross-talk that can modulate cell motility and invasion. The feasibility studies shown here use this modified array focused on cancer cell invasion and the interaction with the stroma as a model system, including endothelial invasion in response to differential signals from cancer cells. The approach can be extrapolated to isolate cancer cells and other cell types such as subpopulations of immune cells, fibroblasts, or endothelial cells. We tested invasive and non-invasive established breast cancer cell lines as a proof of principle. We also used cells from patient-derived xenograft (PDX) invasion in response to immune cells from human bone marrow to show feasibility for future use also in clinical diagnostic settings. PDX are patient tumor tissues that are implanted in immunocompromised or humanized mice model to allow for studying of growth, progression, and treatment options for the original patient7,8.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Thermofisher</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive</td>
			<td>Norland Optical Adhesive</td>
			<td>NOA63</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lifter</td>
			<td>Sarstedt</td>
			<td>83.1832</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera Toxin from Vibrio cholerae</td>
			<td>Thermofisher</td>
			<td>12585-014</td>
			<td></td>
		</tr>
		<tr>
			<td>CIM-plate</td>
			<td>Agilent</td>
			<td>5665817001</td>
			<td>Cell analyzer plate</td>
		</tr>
		<tr>
			<td>Collagenase from Clostridium histolyticum</td>
			<td>Sigma</td>
			<td>C0130</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>StemCell</td>
			<td>7913</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermofisher</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Thermofisher</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS), Heat Inactivated</td>
			<td>Omega Scientific</td>
			<td>FB-12</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermofisher</td>
			<td>15630106</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum (HS)</td>
			<td>Gibco</td>
			<td>16050-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Human EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Human umbilical Vein endothelail cells (HUVEC)</td>
			<td>LONZA (RRID:CVCL_2959)</td>
			<td>C-2517A</td>
			<td></td>
		</tr>
		<tr>
			<td>HUVEC media</td>
			<td>LONZA</td>
			<td>CC-3162</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma</td>
			<td>H4001</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Transferrin Selenium Ethanolamine (ITSX) (100x)</td>
			<td>Thermofisher</td>
			<td>51500056</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin, Human Recombinant, Zinc Solution</td>
			<td>Sigma</td>
			<td>C8052</td>
			<td></td>
		</tr>
		<tr>
			<td>J2 Fibroblasts</td>
			<td>Stemcell (RRID:CVCL_W667)</td>
			<td>100-0353</td>
			<td></td>
		</tr>
		<tr>
			<td>LymphoPrep</td>
			<td>Stemcell</td>
			<td>7851</td>
			<td>Density gradient medium for the isolation of mononuclear cells</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Basement membrane matrix</td>
		</tr>
		<tr>
			<td>MCFDCIS.com cells ( DCIS)</td>
			<td>RRID:CVCL_5552</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231 cells</td>
			<td>RRID:CVCL_0062</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Milling machine</td>
			<td></td>
			<td></td>
			<td>Bridgeport Series 1 Vertical</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (1x)</td>
			<td>Thermofisher</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethersulfone (PES) membrane</td>
			<td>Sterlitech</td>
			<td>PCTF029030</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC lysis solution</td>
			<td>Stemcell</td>
			<td>7800</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Micro Kit</td>
			<td>Qiagen</td>
			<td>74004</td>
			<td></td>
		</tr>
		<tr>
			<td>RTCA DP analyzer</td>
			<td>Agilent</td>
			<td>3X16</td>
			<td>Dual purpose cell analyzer</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>T4799</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time detection and capture of invasive cell subpopulations from co-cultures

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of cancer cell populations typically generate a single endpoint measurement that does not distinguish between cancer cell subpopulations with different invasive potential. Also, the tumor microenvironment consists of different resident stromal and immune cells that alter and participate in the invasive behavior of cancer cells. Invasion into tissues also plays a role in immune cell subpopulations fending off microorganisms or eliminating diseased cells from the parenchyma and endothelial cells during tissue remodeling and angiogenesis. Real-Time Cellular Analysis (RTCA) that utilizes impedance biosensors to monitor cell invasion was a major step forward beyond endpoint measurement of invasion: this provides continuous measurements over time and thus can reveal differences in invasion rates that are lost in the endpoint assay. Using current RTCA technology, we expanded dual-chamber arrays by adding a further chamber that can contain stromal and/or immune cells and allows measuring the rate of invasion under the influence of secreted factors from co-cultured stromal or immune cells over time. Beyond this, the unique design allows for detaching chambers at any time and isolating of the most invasive cancer cell, or other cell subpopulations that are present in heterogeneous mixes of tumor isolates tested. These most invasive cancer cells and other cell subpopulations drive malignant progression to metastatic disease, and their molecular characteristics are important for in-depth mechanistic studies, the development of diagnostic probes for their detection, and the assessment of vulnerabilities. Thus, the inclusion of small- or large-molecule drugs can be used to test the potential of therapies that target cancer and/or stromal cell subpopulations with the goal of inhibiting (e.g., cancer cells) or enhancing (e.g., immune cells) invasive behavior.

Using the newly designed three-chambered array, invasion of the cells was tested in the presence or absence of stromal cells such as fibroblasts. MDA-MB-231 cell invasion was enhanced when irradiated Swiss 3T3 fibroblasts (J2 strain) were seeded in the bottom chamber, allowing for the exchange of factors between the two cell lines. Interestingly, MDA-MB-231 invasion increased when 3T3-J2 cells were doubled in number (Figure 3A). On the other hand, the invasion rate of an invasive clone of MC...

Watch this Scientific Journal Video about Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures at JoVE.com

Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures

We describe an approach to detect and capture invasive cell subpopulations in real-time. The experimental design uses Real-Time Cellular Analysis by monitoring changes in the electric impedance of cells. Invasive cancer, immune, endothelial or stromal cells in complex tissues can be captured, and the impact of co-cultures can be assessed.

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of ...

real-time-detection-capture-invasive-cell-subpopulations-from-co

Research

JoVE Journal

Cancer Research

2.3K Views.  Georgetown University Medical Center. We describe an approach to detect and capture invasive cell subpopulations in real-time. The experimental design uses Real-Time Cellular Analysis by monitoring changes in the electric impedance of cells. Invasive cancer, immune, endothelial or stromal cells in complex tissues can be captured, and the impact of co-cultures can be assessed.

Video: Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures

Capture and Release of Viable Circulating Tumor Cells from Blood

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip for downstream analysis and/or culture. The strategy employs the use of a novel Parylene C membrane slot pore microfilter to capture CTC and a coating of poly (N-iso-propylacrylamide) (PIPAAm) for thermoresponsive viable release of the captured CTC. The capture of live cells is enabled by leveraging the design of a slot pore geometry with specific dimensions to reduce the shear stress typically associated with the filtration process. While the microfilter exhibits a high capture efficiency, the release of these cells is non-trivial. Typically, only a small percentage of cells are released when techniques such as reverse flow or cell scraping are used. The strong adhesion of these epithelial cancer cells to the Parylene C membrane is attributable to non-specific electrostatic interaction. To counteract this effect, we employed the use of PIPAAm coating and exploited its thermal responsive interfacial properties to release the cells from the filter. Blood is first filtered at room temperature. Below 32 &#176;C, PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture media or a buffer maintained at 37 &#176;C, which results in the PIPAAm turning hydrophobic, and subsequently releasing the electrostatically bound cells.

A protocol to utilize a poly(N-iso-propylacrylamide) (PIPAAm) coated microfilter for effective capture and thermoresponsive release of viable circulating tumor cells (CTC) is presented. This method allows capture of CTC from patients&#39; blood and subsequent release of viable CTC for downstream off-chip culture, analyses and characterization.

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip ...

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated cancer associated fibroblasts are a key component of the tumor stroma that interact with cancer cells and support their growth and survival. Models that recapitulate the interaction of cancer cells and activated fibroblasts are important tools for studying the stromal biology and for development of antitumor agents. Here, a method is described for the rapid generation of robust 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and activated pancreatic fibroblasts that can be used for subsequent biological studies. Additionally, described is the use of 3D spheroids in carrying out functional metabolic assays to probe cellular bioenergetics pathways using an extracellular flux analyzer paired with a spheroid microplate. Pancreatic cancer cells (Patu8902) and activated pancreatic fibroblast cells (PS1) were co-cultured and magnetized using a biocompatible nanoparticle assembly. Magnetized cells were rapidly bioprinted using magnetic drives in a 96 well format, in growth media to generate spheroids with a diameter ranging between 400-600 &#181;m within 5-7 days of culture. Functional metabolic assays using Patu8902-PS1 spheroids were then carried out using the extracellular flux technology to probe cellular energetic pathways. The method herein is simple, allows consistent generation of cancer cell-fibroblast spheroid co-cultures and can be potentially adapted to other cancer cell types upon optimization of the current described methodology.

Here, a method is described for the preparation of 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and fibroblasts, followed by measurement of metabolic functions using an extracellular flux analyzer.

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

Serum and Plasma Copy Number Detection Using Real-time PCR

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, and prediction of treatment resistance. Starting from the hypothesis that androgen receptor (AR) gene copy number (CN) gain is a frequent event in metastatic castration resistance prostate cancer (mCRPC), we propose to analyze this event in cfDNA as a potential predictive biomarker.
We evaluated AR CN in cfDNA using 2 different real-time PCR assays and 2 reference genes (RNaseP and AGO1). DNA amount of 60 ng was used for each assay combination. AR CN gain was confirmed using Digital PCR as a more accurate method. CN variation analysis has already been demonstrated to be informative for the prediction of treatment resistance in the setting of mCRPC, but it could be useful also for other purposes in different patient settings. CN analysis on cfDNA has several advantages: it is non-invasive, rapid and easy to perform, and it starts from a small volume of serum or plasma material.

This manuscript describes the copy number variation analysis performed in serum or plasma DNA using real-time PCR approach. This method is suitable for the prediction of drug resistance in castration resistant prostate cancer patients, but it could be informative also for other diseases.

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better understand the molecular mechanisms underlying glioma cell migration, an ability to study the interaction between hGCs and axons within the tumor microenvironment is essential. In order to model this cellular interaction, we developed a mixed culture system consisting of hGCs and dorsal root ganglia (DRG) axon-oligodendrocyte co-cultures. DRG cultures were selected because they can be isolated efficiently and can form the long, extensive projections which are ideal for migration studies of this nature. Purified rat oligodendrocytes were then added on purified rat DRG axons and induced to myelinate. After confirming the formation of compact myelin, hGCs were finally added to the co-culture and their interactions with DRG axons and oligodendrocytes was monitored in real-time using time-lapse microscopy. Under these conditions, hGCs form tumor-like aggregate structures that express GFAP and Ki67, migrate along both myelinated and non-myelinated axonal tracks and interact with these axons through the formation of pseudopodia. Our ex vivo co-culture system can be used to identify novel cellular and molecular mechanisms of hGC migration and could potentially be used for in vitro drug efficacy testing.

Here we present an ex-vivo mixed monolayer culture system for the study of human glioma cell (hGC) migration in real-time. This model provides the ability to observe interactions between hGCs and both myelinated and non-myelinated axons within a compartmentalized chamber.

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These acquired abnormalities in complex, multilayered molecular signaling networks induce aberrant cell proliferation and survival, extracellular matrix degradation, and metastasis to distant organs. Approximately 90% of cancer-related deaths are estimated to be caused by the direct or indirect effects of metastatic dissemination. Therefore, it is important to establish a highly reliable, comprehensive system to characterize cancer cell behaviors upon genetic and environmental manipulations. Such a system can give a clear understanding of the molecular regulation of cancer metastasis and the opportunity for successful development of stratified, precise therapeutic strategies. Hence, accurate determination of cancer cell behaviors such as migration and invasion with gain or loss of function of gene(s) allows assessment of the aggressive nature of cancer cells. The real-time measurement system based on cell impedance enables researchers to continually acquire data during a whole experiment and instantly compare and quantify the results under various experimental conditions. Unlike conventional methods, this method does not require fixation, staining, and sample processing to analyze cells that migrate or invade. This method paper emphasizes detailed procedures for real-time determination of migration and invasion of glioblastoma cancer cells.

Cancer is a lethal disease due to its ability to metastasize to different organs. Determining the ability of cancer cells to migrate and invade under various treatment conditions is crucial to assessing therapeutic strategies. This protocol presents a method to assess the real-time metastatic abilities of a glioblastoma cancer cell line.&#160;

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...