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 millin...

<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 ...

This technique enables the identification of the cells with different invasive capacities under the influence of secreted factors from co-cultured stromal cells. The technique assesses the impact of secreted factors from co-cultured stromal or immune cells on cell invasion. It also allows the recovery and analysis of invasive cell subpopulations present in heterogeneous cell mixes.The technique can determine which tumors harbor invasive cell subpopulations that lead to metastasis. The capture and analysis of invasive cancer cells may inform therapy decisions. To begin the cell culture, wash the adherence cell cultures having approximately 70%confluency with 1X phosphate buffered saline.Then add 0.05%trypsin and EDTA solution to lift off the cells. Neutralize the trypsin solution with cell culture media containing serum and count the cells using an aliquot of the cell suspension. Place all three sterile chambers in the tissue culture hood.Locate the knob on the short side of the lower chamber and orient the lower chamber so that the knob is facing the experimenter. Add 30, 000 to 50, 000 cells in 90 microliters of media to each well from the lower chamber without forming any bubbles. Use 5%of fetal bovine serum supplemented media in two lower chambers as a positive control for cell motility, and use 0%serum supplemented media as a negative control.Rotate the lower chamber at 90 degrees after letting it settle for 10 to 15 minutes in the hood. Then place the middle chamber on top so that the knob on the lower chamber slides into the notch on the middle chamber. Push the chamber vertically downward until a click is heard from each of the long sides of the assembly.Add 160 microliters of serum-free media to all the wells of the middle chamber. Make sure a dome-shaped meniscus is visible after wells are filled and place the top chamber with electrodes facing down onto the middle chamber to align the blue dots on the middle and top chambers. Push the chamber vertically down until a click sound is heard from each of the long sides of the assembly.Add 20 to 50 microliters of serum-free media to the top chamber. Mount the assembly on the dual-purpose cell analyzer in the tissue culture incubator and wait 30 minutes before measuring the background. Open the cell analyzer software, then select the cradle to be used, followed by a click on the message tab, and make sure it says Connections okay"to ensure the array is well placed in the cradle and the electrodes are well aligned with the sensors.Click on the experiment notes tab and fill in all the possible information about the experiment. Then click on the layout tab and fill in the description of the array layout. Then click on the schedule tab and add two steps from the steps menu, a background step with one sweep, and a test step with 100 sweeps.Once the array has been in the dual-purpose cell analyzer incubator for 30 minutes, click on the play button to start background measurement. After the background measurement is done, remove the array from the cradle and place it back in the cell culture hood. Then add 30, 000 to 50, 000 cells in 100 microliters 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. Let the assembly stand in the hood for 30 minutes before mounting on the dual-purpose cell analyzer for impedance measurement. Place the array back into the dual-purpose cell analyzer and check the message tab for the Connections okay"message.Click on the play button to start impedance measurement and then click on the plot tab to monitor the progress of the signal. If the endpoint is reached before 25 hours, click on the abort step from the execute dropdown menu. To export data, right-click on the graph, choose copy in the list format, and then paste the data into a spreadsheet.Monitor the migration rate in real time on the dual-purpose cell analyzer to determine the stopping point of interest. Once achieved, unmount the assembly from the dual-purpose cell analyzer and place it in the tissue culture hood. Prepare an appropriate number of 1.5 milliliters of microcentrifuge tubes to collect the cells from the wells of interest.Place the assembly in a 10 centimeter dish to contain liquids when the chambers detach. Push the flexible snapping ends on the long side of the middle chamber inward until a click sound is heard, followed by dismantling the top chamber and inverting it into a new 10 centimeter dish. Use a cell lifter with a 13 millimeter blade to collect the cells from all the wells harboring the same experimental condition.Rinse or dip the blade in 1X phosphate buffered saline to collect the cells in 1.5 milliliter microcentrifuge tubes. Then spin down the cells at 500 times G for five minutes and propagate these collected cells or perform endpoint analysis such as single-cell RNA-sequencing. Assessment of invasion of the cells in the presence or absence of stromal cells showed that MDA-MB-231 cell invasion was enhanced when irradiated Swiss-3T3 fibroblasts J2 strain were seated in the bottom chamber for the exchange of factors between the two cell lines.Interestingly, MDA-MB-231 invasion increased when 3T3 J2 cells were doubled in number. On the other hand, the invasion rate of an invasive clone of MCFDCIS cells, DCIS-Delta Four, appears to be inhibited by the crosstalk with 3T3 J2 cells, suggesting the valuable application of the three chamber array to measure varying effects of the stroma. In this case, fibroblasts on cell invasion.Human umbilical vein endothelial cells were more invasive in response to factors secreted by MDA-MB-231 cells, unlike those secreted by DCIS cells, which is consistent with the ability of invasive tumors to recruit endothelial cells for blood vessel formation and later dissemination into the circulation. Patient-derived xenograft cells invasion from the top chamber increased in response to co-cultured human bone marrow immune cells. Interestingly, the presence of 2%serum in the bottom chamber with the human bone marrow immune cells was essential for patient derived xenografts invasion.Wells can be coated with an extracellular matrix to mimic the basement membrane barrier. Therapeutic agents can be added to the media in the wells to monitor their effect on invasion.

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Cancer Research

2.3K Views.  Georgetown University Medical Center. This technique enables the identification of the cells with different invasive capacities under the influence of secreted factors from co-cultured stromal cells. The technique assesses the impact of secreted factors from co-cultured stromal or immune cells on cell invasion. It also allows the recovery and analysis of invasive cell subpopulations present in heterogeneous cell mixes.The technique can determine which tumors harbor invasive cell subpopulations that lead to metastasis. The ...