A micropattern-based assay to study contact inhibition of locomotion and entosis of adherent human and canine cells in vitro

Summary

We present a protocol for using micropatterns to study post-collision locomotion and entosis of human and canine cells in vitro. We describe steps for lentiviral transduction and the preparation of micropatterned slides consisting of narrow matrix-coated stripes separated by cytophobic spacers. We then detail cell seeding, chamber assembly, and live cell analysis. We provide steps for analysis by live cell imaging using fluorescence microscopy as well as fixing for subsequent analysis by confocal microscopy or correlative light and electron microscopy.

For complete details on the use and execution of this protocol, please refer to Kummer et al. (2022)¹ and Schwietzer et al. (2022).²

Graphical abstract

Highlights

• •Cells are grown on matrix-functionalized stripes separated by cytophobic spacers
• •Stripes provoke cell collisions facilitating post-collision cell behavior analysis
• •Cell behavior can be studied by live microscopy and in fixed samples
• •Assay useful to study contact inhibition of locomotion and cell-in-cell formation

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

We present a protocol for using micropatterns to study post-collision locomotion and entosis of human and canine cells in vitro. We describe steps for lentiviral transduction and the preparation of micropatterned slides consisting of narrow matrix-coated stripes separated by cytophobic spacers. We then detail cell seeding, chamber assembly, and live cell analysis. We provide steps for analysis by live cell imaging using fluorescence microscopy as well as fixing for subsequent analysis by confocal microscopy or correlative light and electron microscopy.

Before you begin

The use of micropatterns has dramatically facilitated the study of cell behavior in vitro. The main advantage of growing cells on micropatterns is that specific patterns imprinted on the surface impose physical constraints on the cells, allowing, for example, the measurement of the influence of physical forces on cellular components such as specific organelles or the analysis of cellular responses at the level of single cells or in multicellular systems.^3,4 Micropatterns can also be used to study developmental processes.^3,4,5 We have used micropatterns to study contact inhibition of locomotion (CIL), a process that regulates cell motility in response to a collision between two cells.⁶ The use of micropatterned stripes (1D kinematic assay) is extremely useful for the study of CIL since the culture of cells on narrow ECM-functionalized stripes prevents cells from avoiding each other upon initial contact formation⁷ and thus favors cell collisions. However, micropatterns can be used as two-dimensional substrates as well (2D kinematic assays)⁶ The following protocol uses micropatterned stripes of 5 μm width to study CIL in MCF-7 cells,⁸ in which the cell adhesion molecule JAM-A has been depleted. We also performed micropattern experiments with MDCK II cells, a polarized epithelial cell line derived from kidney distal tubules.⁹ This protocol should be applicable to any type of adherent cell.

The following general considerations should be made before experiments are started.

• 1.When studying cell collisions, it is important to distinguish individual cells in a pair of collided cells from each other. For example, differential labeling of colliding cells strongly facilitates the identification of inner vs. outer cells within cell-in-cell structures or the identification of top-vs-bottom cells when cells migrate across each other.
• 2.
  In collision assays, two cells collide with each other. Keep in mind that either the cell-of-interest (CoI) or the control cell collide with a second cell type, e.g., WT cells.

  • a.
    Make sure that control cells (e.g., scrambled siRNA-transfected cells) and gene-of-interest (GoI) knockdown (KD) cells (GoI siRNA-transfected cells) are transfected with the same fluorescence marker, whereas the collision partner (WT cell) is transfected with a different fluorescence marker.
  • b.
    Analyze Ctrl – WT collisions and GoI KD – WT collisions on separate slides.
• 3.The micropatterned chips need to be coated with an extracellular matrix (ECM) protein to enable and/or facilitate cell attachment and migration. Keep in mind that the ECM protein can have a strong influence on the attachment or migratory behavior, in particular when the role of a specific integrin is analyzed.
• 4.Cells analyzed in collision assays should have approximately the same passage number.
• 5.Cells are cultured in serum-containing (10% FCS) medium on the micropatterns. However, depending on the cell type the addition of growth factors may be necessary to stimulate motility.

Lentiviral transduction of MCF-7 cells

Timing: 3 days

This step describes the generation of stably transfected cell lines using lentiviral transduction.

Plasmid vectors: psPAX (lentiviral packing vector, Addgene #12260); pMD2G (lentiviral envelope vector, Addgene #12259); LifeAct-mCherry in pLV-PGK-Puro (lentiviral expression vector); LifeAct-eGFP in pFUGW (lentiviral expression vector); hJAM-A shRNA 5′-GAAGTGAAGGAGAATTCAA-3′ in pLVTHM (Addgene #12247).

Note: The lentiviral expression vectors pLV-PGK-Puro_LifeAct-mCherry and pFUGW_LifeAct-eGFP are examples for fluorescent labeling of the cells used in the experiments. Other fluorescent markers that are not based on LifeAct can be used as well.

Cell lines: HEK293T cells (prepare a 100 mm cell culture dish of HEK293T cells (confluency ca. 90%); MCF-7 wildtype cells (prepare for the day of transfection a subconfluent cell dish with approximately 70% of confluency).

• 6.
  Day -2.

  • a.
    Preparation and co-transfection of HEK293T producer cells. Split HEK293T cells at a ratio of 1:3 (seed approximately 1.2 × 10⁶ cells per dish) for each lentiviral expression vector.

Note: Recommended cell density at the day of transfection ca. 50%–70%.

• 7.
  Day -1 (day before lentiviral transduction): Preparation of transfection mix.

  • a.
    Prepare 2 reaction tubes of 1.5 mL (Tube #1, Tube #2).
  • b.
    In Tube #1: pipette 500 μL Xfect buffer, 15 μg lentiviral expression vector, 10 μg psPAX and 5 μg pMD2, vortex on medium power for 10 s.
  • c.
    In Tube #2: pipette 500 μL Xfect buffer and 0.3 μL Xfect polymer per μg plasmid DNA (here 8 μL are used), vortex for 10 s at half maximal power.
  • d.
    Add solution from Tube # 1 to Tube # 2 and vortex again for 10 s at half power.
  • e.
    Incubate for 10 min at 20°C–25°C.

• 8.
  Polymer-based co-transfection of HEK293T cells using X-fect transfection reagent (Takara, Kyoto).

  • a.
    Transfect HEK293T cells with plasmids psPAX, pMD2G and the respective lentiviral expression vector.

    Note: For 100 mm cell culture dish: mix 4 mL medium for HEK293T cells (DMEM) with 4 mL chloroquine (25 μM in PBS w/o Ca²⁺ and Mg²⁺, PBS^−/−) and add to HEK293T cells.

    Incubate for 1 h at 37°C.
  • b.
    Carefully (dropwise) add the transfection mix (approx. 1 mL) to the medium of the HEK239T cells.

    Note: it is important to add the transfection mix dropwise since HEK293T cells tend to detach from the tissue culture dish.

  • c.
    Incubate cells for 4–6 h at 37°C.
  • d.
    Change medium to MCF-7 standard medium (10 mL per 100 mm dish), wash cells once with PBS (w Mg²⁺/Ca²⁺, PBS^+/+).

    Note: All subsequent steps must be carried out in Biosafety Level S2 facilities.

  • e.
    Incubate cells for 12–16 h at 37°C.
• 9.
  Day 0: Lentiviral transduction of MCF-7 cells.

  • a.
    Prepare transduction solution.

    • i.
      Collect supernatant (SN) from transfected HEK293T cells.
    • ii.
      Pass SN through a 0.45 μm sterile filter.
    • iii.
      Add 5 μL of polybrene per 6.5 mL of medium.
    • iv.
      Vortex at half maximal power for approximately 10 s.
  • b.
    Add transduction solution to the MCF-7 cells (target cells).
  • c.
    Incubate MCF-7 cells for 12–16 h at 37°C.
• 10.
  Day +1:

  • a.
    Wash MCF-7 cells 5 times with PBS^+/+.
  • b.
    Split MCF-7 cells 1:3 in MCF-7 standard medium.
• 11.
  Day +4:

  • a.
    Add antibiotics (puromycin, 3 μg/mL) to select for stable integrations of the lentiviral vector.
  • b.
    Grow cells under regular culture conditions with puromycin (3 μg/mL) added to medium.

Note: This concentration has been proven to be effective for MCF-7 cells. For other cell lines, the effective concentration of puromycin should be determined in advance in dose response experiments (“kill curve”).

• 12.
  Beyond Day +4:

  • a.
    Replace culture medium every 3 days with culture medium supplemented with puromycin (3 μg/mL); wash cells with PBS^+/+ to remove dead cells.
  • b.
    After 5–7 days, most untransduced cells should be removed and single colonies should be apparent.
  • c.
    Isolate and expand single colonies using standard methods, e.g., by using Raschig glass cylinders to isolate individual colonies or by limiting dilution of the bulk culture in 96-well plates; analyze cells for transgene expression or knockdown efficiency using appropriate methods.
  • d.
    If desired, establish clones.

    • i.
      For this purpose, prepare and seed a highly diluted cell solution; for a 96-well plate, this means a concentration of 1 cell/200 μL culture medium so that only one cell remains per well (filled with 200 μL medium).
    • ii.
      Test the different subclones according to their transduction efficiency.

Note: Verify cell concentration by simple light microscopy before expanding the individual clones.

Note: For a series of experiments, the same subclone should always be used for best comparability of results.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti LAMP1 (1:600)	Developmental Studies Hybridoma Bank	#H4A3 RRID: AB_2296838
Anti β-Catenin (1:500)	BD Biosciences	#610154 RRID: AB_397555
Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647 (1:800)	Invitrogen	#A-21236
Bacterial and virus strains
NEB5α	NEB	#C2987H
Stbl3	Dr. Hans Schnittler, University Hospital Münster, Germany	NA
Chemicals, peptides, and recombinant proteins
Recombinant human vitronectin	PeproTech	#140-09
RPMI	Thermo Fisher	#32404014
DMEM	Pan Biotech	#P04-03500
FBS	Capricorn Scientific	FBS-11A
PBS (w/o Mg2+/Ca2+, PBS−/−)	PAN Biotech	P04-36500
PBS (w Mg2+/Ca2+, PBS+/+)	PAN	P04-35500
L-Glutamine	Lonza AG	BE17-605E
Penicillin/Streptomycin	Lonza AG	DE17-602E
Na-pyruvate	Sigma-Aldrich	S8636-100ML
Non-essential amino acids (NEAA)	Capricorn Scientific	NEAA-B
2,4,Diamidino-2-phenylindole (DAPI) (1:1000)	Sigma-Aldrich	#D9542
Paraformaldehyde	Sigma-Aldrich	#P6148
Triton X-100	AppliChem	A1388,1000
Tween-20	AppliChem	A1389,1000
BSA	Roth	8076.3
Polybrene	Sigma-Aldrich	TR-1003-G
Puromycin-dihydrochloride	AppliChem	A2856,0100
Glycine	Applichem	A1067,5000
Mowiol 4-88	Sigma-Aldrich	#81381
EDTA-trypsin	PAN	P10-24100
Glutaraldehyde, 25% (EM grade)	Polysciences	#01909-10
Osmium tetroxide, crystalline 99.95%	Polysciences	#0223A-5
Uranyl acetate 98%	Polysciences	# 21447-25
Epon epoxy resin	Agar Scientific	#AGR1031
Propylene oxide	Serva	#33715
Gelatin capsule (Size 3)	Plano	#G29203
Pb(II) citrate	Sigma-Aldrich	#15326
D-PBS	Sigma-Aldrich	#D5652
Chloroquine	Sigma-Aldrich	C6628
Xfect™ Transfection Reagent (Xfect Polymer and Xfect Reaction Buffer)	TaKaRa	631318
Mowiol 4-88	Merck Millipore	#475904
Critical commercial assays
CYTOOchips™ Motility Ax18 (Set of 18 chips = approx. 950 €; pricing is subject to change and available upon quote requests)	CYTOO INC, Grenoble, France	#10-031-00-18
CYTOOchamber™ – 1 well (Figure 1) (1 chamber = approx. 350 €)	CYTOO INC, Grenoble, France	#30-010
Experimental models: Cell lines
MCF-7: human adenocarcinoma cells derived from a female patient	ATCC	#HTB-22 RRID: CVCL_0031
HEK293T: human epithelial-like cells derived from a female embryonic kidney	ATCC	#CRL-3216 RRID: CVCL_0063
MDCKII: canine epithelial cells derived from the kidney (distal tubule) of a female cocker spaniel	Sigma-Aldrich	#00062107 RRID: CVCL_0424
Oligonucleotides
5′-GAAGTGAAGGAGAATTCAA-3′, hJAM-A shRNA	in pLVTHM	NA
5′-CCAGTAAGAAGGTGATTTA-3′, cJAM-A shRNA	in pEmU6-proT	NA
5′-GAAGTGAAGGAGAATTCAA-3′, hJAM-A shRNA	in pEmU6-proT	NA
5′-TAATGAGGCGCGTACAGAG-3′, hCsk shRNA	in pTRIPZ (Horizon-Dharmacon)	#RHS4696-200701764
5′-CCTAAGGTTAAGTCGCCCTCG-3′, scrambled shRNA	in pLKO.1 (Addgene)	#1864
Recombinant DNA
pLVTHM	Addgene	#12247
psPAX2	Addgene	#12260
pMD2.G	Addgene	#12259
pEmU6-proT	Dr. Karl Matter, University College London, UK	NA
pLKO.1	Addgene	#10878
pFUGW_LifeAct-eGFP	Dr. Hans Schnittler, University Hospital Münster, Germany	The pFUGW backbone plasmid can be otained from Addgene (#14883)
pLV-PGK-Puro_ LifeAct-mCherry	Dr. Henner Farin, Institute for Tumor Biology and Experimental Therapy, Gerog-Speyer-Haus, Frankfurt	The pLV-PGK-Puro backbone plasmid can be otained from Addgene (#108543)
Software and algorithms
Adobe Photoshop CS4	Adobe	https://www.adobe.com/de/
GraphPad Prism 5.0	GraphPad Software	http://www.ddooo.com/softdown/44298.htm
Image J Version 1.53k	ImageJ Software	https://imagej/nih/gov/ij/
ZEISS ZEN lite	ZEN 2 (blue edition) software	https://www.zeiss.com/
Other
LSM780 microscope	Carl Zeiss	NA
LSM800 Airyscan microscope	Carl Zeiss	NA
FEI-Tecnai 12 electron microscope	FEI, Eindhoven, The Netherlands	NA
Objective Plan-Apochromat × 20/0.5 oil differential interference contrast	Carl Zeiss	NA
Objective Plan-Apochromat × 40/1.3 oil differential interference contrast	Carl Zeiss	NA
Objective Plan-Apochromat × 63/1.4 oil differential interference contrast	Carl Zeiss	NA

Materials and equipment

Here is a list of reagents needed.

4% Paraformaldehyde (PFA)

Reagent	Final concentration	Amount
PFA	4%	0.4 g
NaOH 10 M	2 mM	2 μL
10 × PBS+/+	1 × PBS+/+	1 mL
Total	N/A	10 mL

Note: Pre-warm 9 mL of water in a glass bottle with a lid in a water bath at 65°C–70°C. Add 2 μL of 10 M NaOH and 0.4 g of PFA powder. Heat and stir the bottle in the water bath at 65°C–70°C until the PFA powder is almost dissolved. Mix occasionally to aid in dissolving PFA. Chill the solution on ice, add 1 mL of 10× PBS. Adjust volume with H₂O to 10 mL. Filter (0.45 μm) the PFA solution to remove any precipitates.

Note: Keep the PFA solution at 2°C–8°C for up to a week.

Glycine wash buffer

Reagent	Final concentration	Amount
Glycine (75.07 g/mol)	100 mM	3.75 g
1 × PBS−/−	1 × PBS	500 mL
Total	N/A	500 mL

Note: Keep the Glycine wash buffer solution at 20°C–25°C for up to three months.

Blocking buffer for IF experiments

Reagent	Final concentration	Amount
Bovine Serum Albumin (BSA)	0.002%	10 mg
Triton-X 100	0.2%	100 μL
100% Tween 20	0.05%	25 μL
Fetal Calf Serum (FCS)	10%	5 mL
4% NaN3 (Sodium Azide) solution in water	0.5%	250 μL
1 × PBS−/−	1 × PBS	adjust vol. to 50 mL
Total	N/A	50 mL

Note: Keep the Blocking buffer solution at 2°C–8°C for up to a week, at −20°C it can be stored for several months.

Cell permeabilization buffer for IF experiments

Reagent	Final concentration	Amount
Triton-X 100	0.5%	0.5 mL
1 × PBS−/−	1 × PBS	99.5 mL
Total	N/A	100 mL

Note: Keep the cell permeabilization buffer at 2°C–8°C for up to three months.

Cell washing solution

EDTA stock solution 0.5 M

Reagent	Final concentration	Amount
EDTA disodium salt (372.24 g/mol)	0.5 M	186.12 g
MilliQ water	N/A	800 mL
NaOH to adjust the pH to 8.0	N/A	ca. 18–20 g
MilliQ water	N/A	adjust vol. to 1 L

Note: Keep the EDTA stock solution at 2°C–8°C for up to six months.

Final EDTA washing solution

Reagent	Final concentration	Amount
EDTA 0.5 M	5 mM	150 μL
1 × PBS (wo Mg2+/Ca2+)	1 × PBS	Adjust vol. 15 mL
Total	N/A	15 mL

Note: Keep the cell permeabilization buffer at 20°C–25°C for one day.

MCF-7 standard medium

Reagent	Final concentration	Amount
RPMI 1640 medium without phenol red	1 ×	435 mL
FCS	10%	50 mL
Penicillin/ Streptomycin 100 U/mL	1%	5 mL
Na-pyruvate 1 mM	1%	5 mL
Non-essential amino acids 100 ×	1%	5 mL
Total	N/A	500 mL

Note: Keep medium at 2°C–8°C for up to two month.

HEK293T cell medium

Reagent	Final concentration	Amount
DMEM	1 ×	460 mL
FCS	10%	50 mL
Glutamine 2 mM	1%	5 mL
Penicillin/ Streptomycin 100 U/mL	1%	5 mL
Total	N/A	500 mL

Note: Keep medium at 2°C–8°C for up to two months.

Step-by-step method details

Part one: Preparation of micropatterned slides

Timing: 2.5 h

Cells are seeded on CYTOOchip™ Motility glass slides (Figure 1A). These glass slides (chips) are patterned with stripes of different widths that are separated by cytophobic spacer regions (Figure 1B). Incubating the slides with a solution containing an extracellular matrix (ECM) protein results in coating, i.e., functionalization, of the stripes to support cell adhesion and migration. The cytophobic spacer regions between the functionalized stripes prevent cell migration in lateral directions, thereby promoting cell-cell collisions.

Figure 1.

Chamber with micropatterned chip

(A) Components of the chip-containing chamber. After coating with an extracellular matrix, the micropatterned chip (4) is placed into the bottom plate (5). The silicone gasket (3) seals the interior of the chamber. The main body (2) of the chamber adheres magnetically to the bottom part. The chamber can be closed with a glass lid (1). The assembled chamber is placed without glass lid into the CO₂ incubation chamber of the microscope for live cell imaging. At the end of the experiment, the chip can be fixed for antibody stainings and IF analysis. The individual parts are cleaned and incubated in 70% ethanol for 12–16 h. The chamber can be re-used for further experiments. The outer diameter of the chamber (bottom plate) is 35 mm; it thus fits to microscope stage inserts with holes of a diameter smaller than 35 mm.

(B) Cartoon of a micropatterned chip. The chip contains functionalized stripes of different widths (2.5 μm–20 μm) that are coated with extracellular matrix (ECM) proteins. The ECM-coated stripes are separated by cytophobic spacer regions which prevent adhesion and migration outside of the functionalized stripes.

Note that cells must be handled under sterile culture conditions. The following steps are performed under a laminar air flow bench to maintain sterile culture conditions. Keep in mind that all regarents are autoclaved or sterile-filtered before use. The chips can be used directly from the original packaging without further measures. The chamber is incubated for 12–16 h in 70% ethanol. additional measures are not required.

• 1.
  Prepare coating solution with a 2× concentration of the ECM protein in PBS^−/− (for vitronectin we used a final concentration for coating 10 μg/mL; we generated a stock solution of 1 mg/mL in PBS^−/−).

  • a.
    For each micropatterned slide, prepare 2 mL of the 2× concentrated (20 μg/mL) coating solution.
• 2.For easier handling when coating and washing the chip, place the micropatterned slide into a well of a 6 well tissue culture dish (required area: approx. 9 cm²). The chip can be used directly and does not need to be pretreated or sterilized beforehand.

Note: The micropatterned slide must be placed with the pattern (top side) facing up.

Note: Avoid touching the slide to prevent any damage to the micropattern areas by using forceps. Alternatively, place the 6-well plate face down over the opened blister tray with the chip, flip them over and let the chip fall into the well plate.

Note: The coating of the chip may also be performed directly in the chamber which reduces handling steps. We preferred to perform the coating in 6-well plates to ensure sterility of the samples.

• 3.Add 2 mL of PBS^−/− to the 6-well containing the micropatterned slide, make sure that the slide is completely covered.

Add 2 mL of the 2×-concentrated coating solution.

Note: Adding the 2× coating solution to the chip submerged in PBS^−/− ensures that the chip is completely covered by liquid when the coating is added. This prevents heterogeneous coating which may result in non-specific attachment of cells.

• 4.Incubate the chip for 2 h at 20°C–25°C.
• 5.
  Washing the chip: The functionalized stripes must be prevented from drying out. Therefore, the coating solution is gradually diluted by several washing steps.

  • a.
    Add 4 mL of PBS^−/−, swirl the plate gently, then remove a volume of 4 mL.
  • b.
    Repeat this procedure 5 times.

Note: for the last two washing steps use cell culture medium instead of PBS^−/−.

• 6.Transfer the chip into the bottom plate of the 1 well - CYTOOchamber™ with the patterns again facing up using tweezers (Figure 1A). Assemble the chamber.
• 7.Immediately add 1 mL PBS^−/− to prevent the micropatterned slide from drying out.
• 8.Transfer the chamber into a cell culture dish with lid.

CRITICAL: The surface of the chip must be prevented from drying out as drying of the chips’ surface may lead to non-specific attachment of cells to the cytophobic spacer regions. Therefore, make sure that the chip is always covered with liquid.

Note: After coating, the chip should be stored for no more than one day in the refrigerator at 2°C–8°C before starting the experiment. Uncoated chips can be stored at 2°C–8°C for about 2 months.

Part two: Seeding of MCF-7 cells

Timing: 3.5 h

When seeding the cells, it is important to have a homogeneous single cell suspension without cell clusters. Likewise, the cells should be spread evenly over the chip in order to make optimal use of the micropatterned surface. It is also important to ensure that the cells are completely adherent to the surface of the chip before starting live cell recording.

Note: Make sure that you prepare cell suspensions of both collision partners, which may be the same or different cell types, depending on the experimental setting.

Note: The following instructions (regarding cell handling, trypsinization, centrifugation and washing) are based on MCF-7 cells. For other cell lines, different volumes and incubation times may apply. In particular, the incubation of cells with Trypsin/EDTA may vary depending on the cell line.

• 9.
  Harvest the cells.

  • a.
    Wash cells with PBS^−/−/5 mM EDTA.
  • b.
    Add Trypsin/EDTA solution for 5 min at 37°C (for a 10 cm cell culture dish 1.5 mL Trypsin/EDTA).

    • i.
      Stop the reaction by adding 5 volumes of cell culture medium.
• 10.Centrifuge cells (200 g, 4 min, 20°C–25°C).
• 11.
  Resuspend cell pellet in 5 mL cell culture medium.

  • a.
    Pass the cells through a sieve with 40 μm pore size to remove large cell clusters and cellular debris.
• 12.Count the cells.
• 13.Resuspend cells in medium to obtain a cell suspension of 50.000 cells/mL for each cell type.
• 14.Mix equal volumes of the two cell types (1 + 1) to generate a cell suspension with a concentration of 50.000 cells/mL.
• 15.Aspirate the PBS^−/− solution from the chip, preferably with a 1,000-μL pipette from the edge of the chip without touching the surface; carefully add 450 μL of the mixed cell suspension (22.500 cells in total) to the chip.

Note: Cells should be added slowly and dropwise to cover the entire chip.

• 16.Close the lid of the cell culture dish and leave the chamber under the hood for 10 min, allowing cells to settle down; then transfer the cells to the incubator.

Troubleshooting 1 Avoid swirling of the chamber to prevent accumulation of the cells in the center of the chip.

• 17.Incubate the cells for an additional 30 min to allow the cells to settle and spread at the chip surface.
• 18.Fill up the CYTOOchamber™ to 2 mL in total with medium.
• 19.Incubate again for additional 3 h to ensure the complete spreading of the cells.

Troubleshooting 2 If cells are not completely attached to the surface of the chip, extend the incubation time for 1–2 h before starting image acquisition. MCF-7 cells should have settled almost completely after this time. For other cell types the optimal time period may have to be tested. Optional: An immunostaining against the coated ECM protein may be performed to confirm the correct functionalization of the stripes.

Part three: Microscopic observation and live cell imaging

Timing: 16 h

Live-cell microscopy images of the cells were taken with a LSM780 (Carl Zeiss) confocal microscope. The Plan-Neofluar ×20/0.5 objective allows the identification of single cells. However, this magnification does not provide a complete overview of the entire chip.

The time of image recording may be adjusted for each cell type. For MCF-7 cells, image acquisition over a time period of approximately 15 h in 10-min intervals has turned out to be suitable. The first cell collisions events can be observed after approx. 10 h.

• 20.
  Place the CYTOOchamber™ under the microscope. Make sure that the incubation chamber is heated to 37°C and that there is a constant inflow of 5% CO₂.

  Note: The main body of the chamber has an outer diameter of 35 mm. The microscope inlay must have a suitable recess for this. We have used an inlay with an approximately 30 mm round cutout.

  • a.
    Set the focus on the cells using the Differential Interference Contrast (DIC) mode of the microscope.

    • i.
      Move around the entire chip by zooming in and out, specifying several Z-positions that will be later successively approached when capturing the microscopy images.

      Note: Selecting several Z-positions helps to optimize focus stability over the time period of 15 h. In these experiments, a number of 7 Z- positions has proven to be appropriate.

  • b.
    Search for suitable coordinates (X and Y position) that could lead to a collision provided that the cells continue to migrate.
  • c.
    Identify two collision partners (e.g., JAM-A KD cells and JAM-A WT cells) in close proximity. Cells can be distinguished by their different fluorescent markers.

    Note: Cells can migrate in both dimensions. MCF-7 migrate approx. 60 μm in 15 h, which corresponds to approximately 5% of a stripe at a speed of about 0.3–0.4 μm/min. Therefore, cells must not be too far away from each other at the beginning of the acquisition. With a 20× objective, a track length of approximately 600–1,200 μm can be set - depending on the focus chosen. Thus, there would be more track length available for cell migration, even if the collision partners ultimately move at different speeds. If the remaining track length is too long and the cells do not meet in the predicted time, a denser seeding of the cells can be attempted. Should the cells migrate significantly faster, a smaller magnification must be selected.

    Note: Approx. 10 different positions can be selected for a total acquisition time of 15 h, and several cell collision events can be observed in one position. However, this will vary depending on various factors including the used microscope, the area of the set position, or the desired image quality (with lower resolution, the exposure time can be reduced and, consequently, more positions can be addressed). In this study, we have routinely scanned an area of 716 × 716 × 7 pixels (607 × 607 × 17 μm) with a zoom of 0.7, requiring 6 s of scanning time per image. Using these settings, approx. 40–60 collision events can be observed in a single experiment. The optimal settings should be determined in a pilot experiment.

  • d.
    After starting the recording of cell behavior, follow the first image acquisition cycle to make sure that all positions are approached and that the focus is correctly set.
  • e.
    End of image acquisition: after 15 h (corresponds to 90 cycles with images taken at 10-min intervals).

    CRITICAL: The CYTOOchip™ Motility chips contain functionalized stripes of different widths (2.5 μm, 5 μm, 10 μm, 20 μm). For MCF-7 cells (cell diameter approx. 15 μm), stripes of 5 μm width turned out to be optimal. On 2.5 μm stripes, cell migration was less efficient, most likely because of the elongated cell shapes forced by the narrow stripes. On stripes wider than 5 μm, the frequencies of cell collisions were reduced. The optimal widths may vary and should be determined for each cell type. It is important, however, to analyze collision events that occurred on stripes with identical widths only.

Part four: Cell fixation after live cell image acquisition

Fixation for subsequent immunofluorescence analysis

Timing: 2 days

The cells adhering to the micropatterned stripes can be fixed and processed for further microscopic analysis. This allows a more detailed characterization of collided cells. For example, entotic cell-in-cell structures can be distinguished from other types of cell-in-cell structures on the basis of a Lamp1- and β-Catenin positive vacuole. Also, true cell-in-cell structure formation can be verified by confocal analysis of the fixed samples. The following steps describe the processing of the micropatterned slides for subsequent antibody stainings and immunofluorescence analysis.

Note: when using a microscope with 4-color imaging, only one other molecule can be stained at a given time besides the two fluorescent colors of the collision partners and DAPI staining.

• 21.Transfer the chip back into a cell culture dish, for example in a well of a 6-well plate.
• 22.Wash the cells with PBS^−/−.
• 23.Fix in 4% PFA for 10 min at 20°C–25°C.
• 24.Permeabilize the cells for 10  min in PBS^−/−/0.5% Triton X-100.
• 25.Wash samples 3 times for 5 min with 100 mM glycine in PBS^−/−.
• 26.Incubate samples with antibody blocking buffer (PBS^−/−, 10% FCS, 0.2% Triton X-100, 0.05% Tween 20, 0.02% BSA) for 1 h at 20°C–25°C.
• 27.Incubate samples with primary antibodies (here: anti-LAMP1 (dilution 1:600 in blocking buffer) or anti-β-Catenin (dilution 1:500)) in antibody blocking buffer for 12–16 h at 4°C.
• 28.Wash samples 3 times for 5 min with PBS^−/−.
• 29.Incubate samples with fluorochrome-conjugated secondary antibodies for 2 h at 20°C–25°C (dilution 1:800), stain DNA with 4,6-diamidino-2-phenylindole (DAPI, dilution 1:1000).
• 30.Wash again 3 times for 5 min with PBS^−/−.
• 31.The cells can then be covered with a drop of Mowiol and embedded with a coverslip. Allow the sample to harden for 12–16 h at 20°C–25°C.
• 32.Analysis of cell engulfments by confocal microscopy: take Z-stacks (0.36 μm-intervals) using the 63× Plan-Apochromat oil-immersion objective (LSM800 Airyscan).

CRITICAL: When studying cell-in-cell structures using LifeAct-EGFP- or LifeAct-mCherry-expressing cells, the samples must be fixed with PFA as organic solvent-based fixation methods result in a loss of EGFP or mCherry fluorescent signals.

Fixation for subsequent analysis of cell engulfment by correlative light and electron microscopy (CLEM)

Timing: 10 days

The cells adhering to the micropatterned stripes can also be processed for correlative light and electron microscopy (CLEM). For this, cells or cell pairs of interest are identified on the basis of their fluorescence using the confocal microscope. The position of the cells of interest must be noted using the coordinates imprinted on the chips. Confocal sections of cells of interest are taken for subsequent overlay with electron microscopy (EM) pictures. The cells can subsequently be fixed and processed for EM.

• 33.Identify cell pairs of interest on live cell samples using a 20× objective lens.
• 34.Notice the position(s) of the cell pairs of interest using the coordinates imprinted on the micropatterned chip (Figure 2).
• 35.Take confocal sections Z-stacks (smallest interval possible) using a 63× objective.
• 36.Fix samples in 5% PFA, 5% glutaraldehyde (GLUT) in D-PBS (pH7.2), mixed at a 1:1 ratio with normal growth medium for 10 min at 20°C–25°C (final concentration of PFA and GLUT is 2.5% each).
• 37.Replace fixation solution by 2.5% PFA, 2.5% GLUT in D-PBS (pH7.2); incubate for 4 h at 20°C–25°C. After this step, samples can be stored at 4°C for processing at later time points.
• 38.Wash samples with D-PBS for a total of 3 times (20 min per wash) at 20°C–25°C.
• 39.Incubate with 1% osmium tetroxide in double-distilled water (ddH₂O) for 1 h at 20°C–25°C.
• 40.Wash samples with ddH₂O; perform 2 washes for 20 min each at 20°C–25°C and 1 wash for 20 min at 4°C.
• 41.Perform block staining for 12–16 h with 0.5% uranyl acetate in ddH₂O at 4°C.
• 42.Wash samples 2 times for 20 min with ddH₂O at 20°C–25°C.
• 43.Perform alcohol dehydration by incubation the samples for 20 min each in 50% (in ddH₂0), 70%, 90%, 96%, 99.8%, 99.8%, 99.8% EtOH (pure) at 20°C–25°C.
• 44.Incubate samples in propylene oxide (100%) for 5 min at 20°C–25°C; repeat this step 1 time.
• 45.Incubate samples with epon-propylene oxide mixtures at 20°C–25°C: epon-propylene oxide 1 + 3 (1 part epon + 3 parts propylene oxide) for 1 h; epon-propylene oxide 1 + 1 for 1 h; epon-propylene oxide 3 + 1 for 1 h; epon pure for 1 h.
• 46.Add fresh epon pure to sample, place a gelatin capsule above the position of interest on the micropatterned chip, incubate for 12–16 h at 20°C–25°C.
• 47.Fill the gelatin capsule with epon. Allow the epon to polymerize by incubation for 3d at 60°C.
• 48.Separate the gelatin capsule from the chip by incubating the chip-capsule device at two different temperatures: 60°C water bath and 4°C ice bath; the temperature shift will result in the blowing up of the glass chip; repeat step if necessary. The coordinates imprinted on the chip will be visible at the surface of the epoxy resin.
• 49.Place the epoxy block under a stereomicroscope and identify the region of interest using the coordinates.

CRITICAL: Keep in mind that the coordinates imprinted on the micropatterns appear mirror-inverted at the bottom of the epoxy block.

• 50.Using a razer blade remove areas of the epoxy block which are outside the region of interest.
• 51.Using an ultramicrotome suitable for EM studies, generate serial sections of the cell starting from the basal side of the cell. The thickness of the sections should be 50 nm for optimal resolution.
• 52.Use the section with optimal information content and identify the correlating confocal image (Figure 2); overlay the two images using an imaging software.

Figure 2.

Micropatterned chip and identification of cell-in-cell structures by fluorescence

The chips consist of zones (indicated by different colors) that are imprinted with stripes of different width (2.5 μm, 5 μm, 10 μm, 20 μm, left panel). Zones are subdivided in blocks (indicated by broken lines), which are specified by coordinates (letters and numbers, middle panel). The coordinates allow the specification of blocks that contain events of interest, for example entotic cell-in-cell structures, for the subsequent analysis by electron microscopy (right panel).

Part five: Analysis of contact inhibition of locomotion (CIL), entosis and cell motility

Timing: 4 h

To characterize CIL in response to cell collisions it is useful to categorize cell behavior. We have distinguished four types of cell behavior in response to a collision with another cell (Post-collision events) (Figure 3): Type -2: Cells migrate in the opposite direction. Type -1: Cells do not form stable contacts but also do not actively migrate away (anergy). Type 0: Cells form stable contacts, i.e., cells remain closely associated for at least 7–8 h after collision. Type +1: Cells migrate across the collided cells. In addition to these types of CIL behavior, we have also observed cell-in-cell structures (entosis) after collisions. These events were analyzed separately.

• 53.
  Analysis of CIL.

  Note: Live cell recordings of examples of CIL behaviours (Opposite migration (Type -2), Anergy (Type -1), Contact Formation (Type 0), Continuous Migration (Type +1), examples of entosis and different types of inner cell fates (engulfment, inner cell death, inner cell escape, inner cell division), and immunofluoresence stainings of entotic vacuoles using β-catenin or Lamp1 as markers are published in references.^1,2

  • a.
    Select appropriate collision events: For the evaluation of cell collisions it is important that all analyzed cells have comparable chances of a collision with a neighboring cell (Figure 4, flowchart).

    • i.
      First, exclude cells that are in close contact at the beginning of the observation period (e.g., as a result of aggregation or cell division) from the analysis.

      Note: A head-to-head migration for 1–2 h prior to collision was set as prerequisite for inclusion in the analysis of collision events.

    • ii.
      Second, check that the type of migratory behavior is the dominant type during the observation period.

      Note: We arbitrarily defined an observation period of 7–8 h (half of the recording time) during which the cell behavior must have prevailed in order to be included into one of the four categories of post-collision behavior (Figure 3).

    • iii.
      Include only cells on tracks with the same width into an evaluation.

      Note: For MCF-7 cells we exclusively analyzed cell behavior on tracks with 5 μm.

      CRITICAL: For the analysis of CIL behavior it should be kept in mind that the post-collision behavior is dynamic and can change. For example, cells that behaved anergic after collison may regain migratory behaviour. Therefore, post-collision cell behavior should have prevailed for at least half of the observing time (for 15 h recording: 7.5 h–45 frames) before behavior is attributed to one of the four CIL categories.

  • b.
    Quantification of cell collisions:
    All appropriate cell collisions are assigned to the dominant type of migratory behavior. Calculate the proportion in percentage of this type in the total of analyzed cell collisions.
  • c.
    Test the values of at least three independent experiments of the same experimental setup for normality of data sample distributions using the D’Agostino-Pearson normality test.
  • d.
    Compare the individual migration types between control experiments and experiments with GoI cell collision.
• 54.
  Analysis of entosis.

  • a.
    Select the cells according to the same criteria as for the analysis of CIL behavior.

    Note: In parallel, the cells must not be engulfed already at the beginning of the acquisition, but the engulfment must take place during the time of observation, so that the fate of the involved cells can be followed completely.

  • b.
    Divide entotic events into two groups depending on which of the collision partners is the host cell and which is the engulfed cell.
  • c.
    Calculate the percentage of engulfed cell types in the total entotic events.

    Note: For MCF-7 cells, between 5% (Ctrl – WT) and 50% (JAM-A KD – WT) of cell collisions result in entotic engulfments.

  • d.
    Quantify among the entotic events whether the inner cell is fragmentated in its host cell, undergoes cell division, or is able to escape.

• 55.
  Analysis of cell motility.

  • a.
    Cell motility and track displacement of the cells were analyzed using ImageJ software. Cells were tracked semi-automatically using the TrackMate plugin.
  • b.
    Further settings were made as follows:

    • i.
      Algorithm: Downsample LoG detector.
      Diameter: 60.000 px.
      Threshold: 0,01.
      Filter: median filter.
      Particle-linking algorithm: simple LAP tracker.
      View: Hyperstack Displayer.
      Linking max Distance: 250 px.
      Gap-closing max distance: 100 px.
      Gap-closing max frame gap: 5.
    • ii.
      The track displacement covered by single cells is defined as Euclidean distance and converted from pixel to μm. The mean velocity of the cells is calculated as track displacement/total acquisition time and is given in μm/min.

      Note: When analyzing cell motility of cells grown on stripes of defined width (e.g., 5 μm), keep in mind that the physical constraints imposed by the narrow stripes could influence the motility of cells, for example by adopting an elongated shape. This should be considered when comparing data obtained from narrow stripes (1D) with data obtained from regions in the absence of constraints (2D).

Figure 3.

Classification of CIL phenotypes in response to cell collisions

Type -2: Cells migrate in the opposite direction. Type -1: Cells do not form stable contacts but also do not actively migrate away (anergy). Type 0: Cells form stable contacts. Type +1: Cells migrate across the collided cells. Arrows indicate direction of migration after collision. Double arrows indicate anergic behavior.

Figure 4.

Flowchart of live cell imaging and analysis of post-collision CIL behavior

Cells with different fluorescent labeling suitable for live cell imaging (e.g., LifeAct-GFP, Life-Act-mCherry) are seeded on micropatterned slides, allowed to adhere, and observed for 15 h by live cell imaging. For the subsequent analysis of cell behavior in response to cell-cell collisions, it is important to include only events observed on tracks with identical width (step 1). For MCF-7 cells, a track width of 5 μm was useful. Criteria for events to be included in the analysis and classification are i) head-to-head migration for 1–2 h before collision, and ii) type-specific behavior for at least 7–8 h during the remaining observation period of 13–14 h (post-collision, steps 2 and 3).

Expected outcomes

The analysis of CIL and entosis events is based on a qualitative evaluation of the migratory behavior after the collision of two migrating cells. The experimental setup as a 1D-assay is designed to provoke single cell collisions with high frequency. Approximately 40–60 cell-cell collisions per experiment can be observed under the given experimental conditions and analysis settings. For CIL analysis, the different post-collision behaviors were categorized into 4 types (-2, -1, 0, +1) using arbitrarily defined criteria outlined in this work and in previous publications^1,2,6 (Figure 4). The frequency of entotic cell engulfments among cell-cell collisions is relatively low (approximately 5%), which is consistent with a low frequency of entotic events in matrix-adherent cells grown under standard culture conditions, i.e., in 2D conditions in the presence of regular culture medium.¹⁰ This frequency, however, may significantly increase but also decrease under conditions which impair or support cell motility and migration, respectively. Note that the use of collision partners with different fluorescent labels allows for additional analyses after fixation, such as the characterization of inner cell fates, the type of ingestion (“who eats whom”), or the ultrastructures of cell pairs by CLEM.

Quantification and statistical analysis

The normality of data sample distributions was tested using the D’Agostino-Pearson normality test. Data were statistically compared using unpaired, two-tailed Student’s t test. If not-normally distributed, non-parametric, Mann–Whitney U test was used. All statistical analyses were performed using GraphPad Prism version 6. P-values are defined as: ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001.

Limitations

An intrinsic limitation of the approach with micropatterns is that it cannot reflect in vivo conditions. In tissues, migrating cells can move in three dimensions. This has to kept in mind when studying cell behavior on linear micropatterns in vitro.

Troubleshooting

Problem 1

For optimal experimental conditions, it is crucial that the cells are evenly distributed across the chip. Local cell accumulations may result in cells hindering each other in their migration. At the same time, local accumulations would result in distinct areas with too low cell densities. In such regions the distance between two cells would be too big, and the cells would not be able to collide within the observation period (15 h).

Potential solution

“Part 2: Seeding of MCF-7 cells”, Instruction #7:

After adding the cell suspension to the chamber try to swirl the chamber as little as possible. Make sure to distribute the cell suspension evenly over the chip using the pipette tip (do not just add it to the center of the chip.

Problem 2

Cells are not sufficiently attached and spread on the surface of the micropatterned chip.

Potential solution

“Part 2: Seeding of MCF-7 cells”, Instruction #9:

After adding the medium in the chamber, incubate the cells for additional 1–2 h to ensure that the cells have settled and spread completely.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Klaus Ebnet (ebnetk@uni-muenster.de).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate or analyze data sets.