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

Summary

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.

Abstract

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.

Introduction

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 microelectrodes^3,4. 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 patient^7,8.

Protocol

The study was reviewed and considered as "exempt" 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)

1. Open a new dual-chamber cell analyzer plate. Set aside the top chamber with electrodes.
2. Using a milling machine, shave off 2 mm of the U-shaped bottom wells of the cell analyzer plate.
3. Attach a 2 cm x 7 cm polyethersulfone (PES) membrane with 0.2 µm 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.
4. 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.
5. 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).
6. 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 µL of volume per well.
7. 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.
8. 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's notch (Figure 2, Middle).
9. Use a 0.9 mm thick rubber washer for each well to provide a sealed fit.

2. Cell culture (MDA-MB-231, DCIS, DCIS-Δ4, J2-fibroblasts)

1. Wash adherent cell cultures (~70% confluence) with 1x phosphate-buffered saline (PBS).
2. Add 0.05% trypsin-EDTA solution to lift the cells off.
3. Neutralize the trypsin solution with cell culture media containing serum and count the cells using an aliquot of the cell suspension.
   ​NOTE: The specific cell culture media can be found in Table 1.

3. Patient-derived xenograft dissociation

1. Chop a fresh tumor piece (1 cm²) into fine mush using a sterile scalpel.
2. 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.
3. Incubate in a thermal shaker (150 RPM) at 37 °C for 20 min.
4. Spin the tube at 500 x g for 5 min; remove the supernatant.
5. Add 20 μL 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.
6. Resuspend in 1 mL of PDX media (Table 1) to count the cells.

4. Bone marrow cell extraction

1. 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.
2. 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.
3. Spin at 800 x g for 20 min at 18 °C
4. 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.
   ​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.
5. Fill the 15 mL conical tube with 1x PBS (~10 mL) and spin at 300 x g for 15 min.
6. Remove the supernatant; the remaining white pellet is the BM.
7. 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.
8. Add 10 mL of 1x PBS to wash the cells, spin at 300 x g for 5 min and remove the supernatant. Repeat the RBC lysis (step 4.7) until the pellet is white.

5. Cell seeding and assembly

1. Place all three sterile chambers in the tissue culture hood.
2. Locate the knob on the short side of the lower chamber. Orient the lower chamber so that the knob is facing the experimenter.
3. Add 30,000-50,000 cells in 90 μL 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.
4. 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.
5. 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.
6. Rotate the lower chamber at 90° 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.
7. Push vertically down until a click sound is heard from each of the long sides of the assembly.
8. Add 160 μL of serum-free media to all the wells of the middle chamber.
9. 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.
10. 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.
11. Push vertically down until a click sound is heard from each of the long sides of the assembly.
12. Add 25-50 μL of serum-free media to the top chamber.
13. 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.
14. Measure the background (see section 6) and place the assembly back into the tissue culture hood.
15. Add 30,000-50,000 cells in 100 μL 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.
16. Let the assembly stand in the hood for 30 min before mounting on the dual purpose cell analyzer for impedance measurement.

6. Background and impedance measurement

1. Place the array into the cradle in the dual purpose cell analyzer instrument.
2. Open the cell analyzer software and select the cradle to be used.
3. 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.
4. Click on the Experiment Notes tab and fill in as much information about the experiment as possible.
5. Click on the Layout tab and fill in the description of the array layout.
6. 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.
7. 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.
8. After the background measurement is done, remove the array from the cradle and place it back in the cell culture hood.
9. 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.
10. Place the array back into the dual purpose cell analyzer and check the Message tab for the Connections OK message.
11. Click on the Play button to start impedance measurement.
12. Click on the Plot tab to monitor the progress of the signal.
13. If the endpoint is reached before 25 h, click on the Abort step from the Execute drop-down menu.
14. To export data, right-click on the graph, choose Copy in the list format, and then paste the data in a spreadsheet.
    ​NOTE: The data can be exported as cell index or delta cell index. Graph and/or layout information can also be chosen for export.

7. Detachment and cell collection

1. 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's invasion rate and when a distinct invasion signal from the negative control is achieved.
2. Once achieved, unmount the assembly from the dual purpose cell analyzer and place it in the tissue culture hood.
3. Prepare an appropriate number of 1.5 mL microcentrifuge tubes to collect the cells from the wells of interest.
4. Place the assembly in a 10 cm dish to contain liquids when the chambers detach.
5. Push the flexible snapping ends on the long side of the middle chamber inward until a click sound is heard.
6. Dismantle the top chamber and invert it into a new 10 cm dish.
7. 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.
8. Rinse or dip the blade in 1x phosphate buffered saline to collect the cells in 1.5 mL microcentrifuge tubes.
9. Spin down the cells at 500 x g for 5 min.
10. Propagate the collected cells (see section 8) or perform end point analysis such as single-cell RNA-seq.
    ​NOTE: For bulk RNA-seq, use a low cell number RNA extraction kit.

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.

1. Thaw an aliquot of the basement membrane matrix at 4 °C overnight. Keep the basement membrane matrix on ice until ready to use.
2. Add 25 μL of cold basement membrane matrix to the pellet of live cells collected and gently pipette up and down to mix. Avoid forming bubbles.
3. 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 °C for 20 min before gently adding 100 μL of media dropwise.
4. To retrieve cells, aspirate the media and add 100 μL of dispase to each well.
5. Incubate at RT for 10 min, pipetting up and down occasionally.
6. 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.
7. Aspirate the supernatant. Split the cells in the pellet or perform endpoint analysis.

Representative Results

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 MCFDCIS cells (DCIS-Δ4)⁹ appears to be inhibited by the cross-talk with 3T3-J2 cells (Figure 3B). This data shows the useful application of the three-chambered array to measure varying effects of the stroma, in this case, fibroblasts, on cell invasion.

Next, to monitor the change in endothelial cells motility and invasion in response to signals from either invasive (MDA-MB-231)^10,11 or non-invasive (DCIS)¹² cancer cells, human umbilical vein endothelial cells (HUVECs) that represent endothelial cells lining the walls of blood vessels were used. HUVECs were more invasive in response to factors secreted by MDA-MB-231 cells, unlike those secreted by DCIS cells (Figure 4). This is consistent with the ability of invasive tumors to recruit endothelial cells for blood vessel formation and later dissemination into the circulation.

The data above demonstrate the ability of the cell analyzer system to monitor different invasion rates of cell lines; when the invasion starts, progresses and plateaus. This allows the user to choose the time point of interest, for example, after the first 2-3 h of invasion, to capture the pioneer cells that initiate invasion and are distinct from the follower cells that invade thereafter by collective invasion.

While the above data demonstrates a solid proof of principle for the use of the three-chambered array to observe invasion in a co-culture setting, we wanted to test the potential usability of this array in clinical and diagnostic settings. For that, the invasion of cell suspensions from patient-derived xenografts (PDX)⁸ co-cultured with immune cells from human bone marrow samples were monitored. Total human bone marrow immune cells (BM) were seeded onto the bottom chamber with or without serum. PDX cells invasion from the top chamber increased in response to co-cultured BM immune cells (Figure 5). Interestingly, the presence of 2% serum in the bottom chamber with the BM cells was essential for PDX invasion.

Figure 1: Workflow of the cell invasion monitoring and cell collection system. (A) Stroma cells are added to the bottom chamber that is mounted on a middle chamber, which serves as a cell barrier that is permeable for soluble factors only. (B) The top chamber with impedance biosensors receives the cell line to be monitored. Real-time invasion is recorded until a user-defined timepoint for cell collection is reached. (C) The dismantled top chamber is inverted, and cells are harvested using a cell lifter. Please click here to view a larger version of this figure.

Figure 2: Images of the array chambers and modifications. (A) The three chambers used to build the array. No modification was made on the top chamber harboring the electrodes. (B) From the middle chamber wells, a height of 2 mm has been shaved off and a membrane attached to the open bottom; longitudinal slits (1.5 mm x 5.6 mm) were added to each side. (C) Lower chamber (72 mm x 18 mm) fabricated to replicate the 16-well design; wells are 4.8 mm deep, and 4.75 mm in diameter, triangle ridges (1.5 mm horizontal x 1.4 mm vertical) are added along the sides to click into the middle chamber slits. Please click here to view a larger version of this figure.

Figure 3: The effect of co-cultured fibroblasts on cancer cell invasion. (A) Real-Time Cellular Analysis of MDA-MB-231 cell invasion, alone or in co-culture with 3T3-J2 fibroblasts (bottom chamber). 3T3-J2 fibroblasts were seeded at either 30,000 or 60,000 cells per well. (B) Real-Time Cellular Analysis of DCIS-Δ4 cell invasion, alone or in co-culture with 3T3-J2 fibroblasts (bottom chamber). The solid circles represent the mean; the thin dotted lines represent the standard deviation. Please click here to view a larger version of this figure.

Figure 4: The effect of cancer cells on endothelial cell invasion. Real-Time Cellular Analysis of HUVEC invasion, alone, in co-culture with the invasive MDA-MB-231 cells (bottom chamber) or the non-invasive DCIS cells (bottom chamber). The solid circles represent the mean; the thin dotted lines represent the standard deviation. Delta cell index normalized to the impedance at time = 1 h. Please click here to view a larger version of this figure.

Figure 5: Cell invasion of patient-derived xenografts (PDX) co-cultured with bone marrow immune cells. Single cells disintegrated from PDX (top chamber) were co-cultured with human bone marrow cells (bottom chamber), and their invasion was monitored over time in the presence or absence of serum. The solid circles represent the mean; the thin dotted lines represent the standard deviation. Delta cell index normalized to the impedance at time = 1 h and 36 min. Please click here to view a larger version of this figure.

Media	Constituents	Concentration/proportion
MDA-MD-231 media	DMEM
	Fetal Bovine Serum (FBS)	10%
J2 Fibroblasts media	DMEM
	Fetal Bovine Serum (FBS)	10%
DCIS media	DMEM F12
	Horse serum (HS)	5%
	Epidermal growth factor (EGF)	20 ng/mL
	Insulin	10 μg/mL
	Hydrocortisone	0.5 μg/mL
	Cholera toxin	100 ng/mL
PDX media	DMEM F12
	Fetal Bovine Serum (FBS)	2%
	HEPES	1 M
	Insulin Transferrin Selenium Ethanolamine (ITS)	10 μg/mL
	Hydrocortisone	0.5 μg/mL
	Bovine serum albumin (BSA)	1 mg/mL

Table 1: Cell culture media composition. The table lists the compositions of MDA-MD-231 media, J2 Fibroblasts media, DCIS media, and PDX media.

Discussion

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 during blood vessels sprouting toward an angiogenic stimulus in the presence of cancer cells of varying invasive potential. These experiments show the versatile use of the array for cancer cell or other cell isolation.

It is recommended to optimize the number of cells to be added to each chamber. From our experience and the manufacturer's recommendation, 30,000-50,000 cells are optimal. Since two cell types can be co-cultured in this array, one of which may be a primary culture of stroma cells, it is suggested to monitor the effects of different media on cells used to maintain viability. We found that starvation of cells to be monitored reduces the variation between replicates. If serum-free growth conditions are harmful to the cells, low serum and shorter starvation times can be used. The addition of a low serum amount (1%-2%) may be critical for the survival of stromal cells (primary cells) in the bottom chamber, yet make sure to include the proper control conditions for data interpretation (i.e., 2% serum with and without stromal cells). If invasion rates are low, more wells per the experimental condition can increase the number of viable cells collected for downstream analyses. When analyzing patient biopsies, the disintegration of tissue into single cells is crucial before testing on the cell analyzer plate. Optimizing disintegration conditions to maintain cell viability is an important step before performing the invasion analysis. It is also possible to study additional aspects of invasion, such as sensitivity to drug treatment or the effect of extracellular basement matrix (ECM) components on invasion rate. ECM is an essential component of the microenvironment and has been reported to play a major role during cell invasion¹³. Several published studies have used ECM to coat the top chamber before invasive cells are added to monitor their interaction with various ECM components^14,15. The three-chambered array is a useful tool to study co-culture interactions between invasive and stroma cells. While the proposed design guarantees a cell collection specific to the invasive cells without any stromal cells, this setup may not be optimal if the cross-talk between cells requires the physical interaction of the different cell types. Additionally, non-adherent cells that grow in suspension may not be collected in the proposed methods here (scrapping), yet a different approach in which media in the disassembled chambers containing the non-adherent cells may be collected to harvest the non-adherent cells.

While this array can be utilized for multiple areas of research that monitor cell invasion in the presence of a stromal component, here we focused on cancer cell invasion and how this approach can uncover malignant cancer cell subpopulations present in heterogeneous biological samples. The new capacity of the three-chambered array used here provides a functional assay to isolate invasive subpopulations from heterogeneous cancer cell mixtures that contain more and less invasive cancer cells. Analysis of invasive and outcome-relevant cancer cell subpopulations is essential for appropriate mechanistic studies and molecular insights not obtainable or biased by the analysis of a mixed cell population. The co-culture chamber for stromal cells provides insights into cell-cell cross-talk in the tumor microenvironment during progression to invasive disease.

As a step toward application to tissue samples, e.g., tumor biopsies from patients, we used cancer cells that were isolated from patient-derived tumor xenografts (PDXs) and tested the impact of human bone marrow cells on the invasion of tumor cells. We were able to collect the invasive cells present in the PDXs for downstream analysis, i.e., RNA-seq. Assaying PDXs is the initial step toward analyzing cell subpopulations present in the heterogeneous mix of cancer cells in tumor biopsies obtained from patients. The ultimate goal will be to use such tumor biopsies and isolate subpopulations of cancer cells that invade and thus drive poor outcomes due to their potential for metastatic spread. Identification of the molecular features and testing the sensitivity of these invasive subpopulations to drug treatment are future applications.

Materials

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