A Microfluidic Platform for Studying Roles of Mechanical Compression in Tumor-Immune Cell Interactions in a 3D Extracellular Matrix

Summary

We present a protocol for using a microfluidic platform to model tumor-immune interactions within a three-dimensional (3D) environment. Our results revealed that Natural Killer (NK) cells retained their anti-tumor activity under mechanical compression.

Abstract

Mechanical forces significantly influence the ability of immune cells to kill tumor cells in the context of cell-based immunotherapy. To kill tumor cells, immune cells must exert forces on the target cell and form an immune synapse, through which cytotoxic molecules —including granzyme B—are delivered. Despite their importance, how mechanical cues can be leveraged to enhance immune-mediated killing remains poorly understood. This knowledge gap is partly due to the lack of tools capable of providing well-controlled mechanical stress to cell cultures in a physiologically realistic environment. Here, we describe a microfluidic compression device that can apply static or dynamic compression to tumor spheroids embedded in extracellular matrix (ECM) while enabling real-time imaging of tumor-immune interactions via optical microscopy.

The microfluidic platform consists of 12 compartments (6 control and 6 functional). Each compartment contains a cell chamber positioned directly beneath the pressure control unit. Spheroids embedded in ECM are placed within the cell chamber. Using this platform, we investigated the killing efficiency of Natural Killer (NK) cells against breast tumor spheroids (MCF-7) under defined mechanical compression. The results showed that NK cells remained the primary drivers of tumor spheriods death regrardless of mechanical compression in 1.5 mg/mL collagen. Therefore, suggesting that NK cells can maintain their anti-tumor activity under compressive stress. These findings demonstrate the utility of this platform for investigating the role of mechanical forces in tumor-immune interactions. Ongoing studies are identifying the molecular mechanisms that allow immune cells to adapt to compressive stress. Insights gained from these studies may reveal a promising therapeutic avenue.

Introduction

Cell-based cancer immunotherapy is a therapeutic approach that utilizes immune cells to target cancer cells¹. This strategy has demonstrated remarkable success in the treatment of blood cancers^2,3,4. However, the level of success observed in blood cancers has not yet been fully translated to the treatment of solid tumors^5,6,7. One major reason for this limitation is the immunosuppressive tumor microenvironment (TME) that is specific to solid tumors.

The TME is characterized by distinct mechanical cues that can regulate cellular behavior. Effective killing of tumor cells by immune cells requires them to exert forces on target cells and establish an immune synapse through which cytotoxic molecules are delivered⁸. Therefore, understanding how mechanical forces influence immune cell function may uncover new therapeutic strategies to improve the efficacy of cell-based immunotherapies against solid tumors.

A defining mechanical feature of the solid TME is the compressive stress that the tumor microenvironment imposes on the growing tumor^9,10. To date, most compression studies have focused on the effects on tumor cell behavior^10,11. Only recently has attention shifted toward understanding how compression influences immune cells within the TME¹². As a result, the mechanisms by which compressive stress affects Natural Killer (NK) cell migration, immune synapse formation, and cytotoxicity activity remain poorly understood and insufficiently quantified. This gap represents a critical limitation in our understanding of how mechanical cues within TME can be leveraged to enhance immune targeting of solid tumors.

A major barrier to addressing this question is the lack of experimental platforms that can apply controlled and quantifiable compression within physiologically relevant 3D microenvironments while simultaneously enabling real-time imaging of tumor-immune interactions. Early experimental approaches to studying compressive stress involved embedding spheroids in agarose gels¹³. While this method effectively captured physical confinement, it eliminated native extracellular matrix (ECM)-cell interactions. Dolega et al. applied osmotic pressure to induce compressive stress; however, spheroids were suspended in culture medium and therefore remained devoid of ECM interactions⁹. Pandey et al. used a transwell insert and weights to compress spheroids embedded in collagen, which preserved ECM-cell interactions but limited the high-resolution imaging required to quantify immune cell motility and cytotoxic activity¹⁴. Together, these limitations underscore the need for experimental platforms that enable real-time imaging of the morphological responses of cells to compression within the TME.

Here, we present a protocol for a microfluidic compression device¹⁵ that enables controlled application of compressive stress while preserving physiologically relevant ECM-cell interactions and supporting high-resolution live-cell imaging of immune–tumor dynamics. The protocol consists of the preparation of the device components (Section 1), assembly of the device (Section 2), and the imaging of tumor–immune interactions (Section 3). We demonstrate the workflow using an NK killing assay against green fluorescent protein (GFP)-labeled tumor spheroids. Beyond this application, the platform can be readily adapted to investigate the effects of mechanical forces on a broad range of cell types and cellular interactions in 3D microenvironments.

Protocol

1. Preparation of the device components

1. Surface activation of the sample loading chamber
   1. Place a mask over the sample-loading chamber (Layer 1, L1) such that only the wells are exposed.
   2. Expose the masked Layer 1 to oxygen plasma for 15 s using the high-power setting (45 W).
   3. Place Layer 1 in a Petri dish, then continue the following steps in a biosafety cabinet.
   4. Treat each well with 10 µL of 1% polyethylenimine (PEI) for 10 min.
   5. Remove the PEI from the wells by vacuum aspiration from the four corners of each well to prevent scratching the glass surface.
   6. Wash the wells with 10 µL of phosphate-buffered saline (PBS), then remove the wash by vacuum aspiration from the side.
   7. Treat the wells with 10 µL of 0.1% glutaraldehyde for 30 min, then remove the glutaraldehyde by vacuum aspiration from the corners.
   8. Wash the wells with PBS three times, removing the wash from the corners after each wash.
   9. Wrap the Petri dish containing Layer 1 in foil and leave it in the biosafety cabinet overnight.
2. Sterilization of the metal frame
   1. Submerge the 38 mm × 88 mm metal frame and the twelve M2.5 pan-head screws in 70% ethanol overnight.
3. Priming of the pressure control unit
   1. Expose the pressure control unit to oxygen plasma for 30 s using the high-power setting (45 W).
   2. Submerge the pressure control unit, with the features facing downward, in de-ionized (DI) water overnight to fill the channels and the chambers of the pressure control unit.
   3. The next day, remove the pressure control unit from the DI water in which it was submerged.
   4. Fill a microfluidic reservoir with 50 mL of DI water.
      NOTE: The microfluidic reservoir consists of a 50 mL conical tube fitted with a custom lid containing two tubing connections: a pneumatic tubing line and a fluid delivery tubing line.
   5. Connect a syringe filled with air to the pneumatic tubing line.
   6. Slowly depress the syringe plunger to pressurize the reservoir and drive DI water through the fluid delivery tubing until the tubing is filled and water begins to drip from the tube.
   7. Connect the pre-filled, dripping fluid delivery tubing through the polycarbonate manifold and then to the 1.5 mm inlet located on the back of the pressure control unit.
   8. Allow the pressure control unit and manifold assembly to air dry for 2 h. Once completely dry, the pressure control unit and manifold assembly are ready for the next step.

2. Assembling the device

1. Preparation of cell-embedded collagen
   1. Remove and dry the metal frame and screws from the 70% ethanol.
   2. In the biosafety cabinet, place three 600 µL microtubes on ice. Label the tubes as follows: "N" (neutralization), "sp" (MCF-7 spheroids), and "sp+NK" (MCF-7 spheroids with NK cells).
   3. Prepare the neutralization mixture by adding 10× the required volume of Medium 199 (M199) and sodium hydroxide (NaOH) to the "N" tube.
   4. Transfer the appropriate volume of the M199:NaOH mixture to the "sp" and "sp+NK" tubes to achieve a final 1× concentration.
   5. Harvest tumor spheroids and centrifuge at 141 × g for 3 min to allow the spheroids to settle at the bottom of the tube.
      NOTE: Tumor spheroids approximately 200 µm in diameter were generated using a previously described agarose microwell platform¹⁶. Briefly, 3 × 10⁶ MCF-7 cells were seeded into a 36 × 36 agarose microwell array consisting of wells 200 µm in diameter and 200 µm in depth and then cultured for 7 days to produce the tumor spheroids used in the experiments.
   6. Carefully remove the supernatant and resuspend the spheroids in 200 µL of 50:50 MCF-7/NK complete medium (Table 1 and Table 2).
   7. Collect NK cells in a 50 mL conical tube, centrifuge at 141 × g for 4 min, and count the cells.
      NOTE: NK cell viability should be >85% the day of the experiment.
   8. Resuspend the resulting pellet in 200 µL of complete NK medium.
   9. Add the appropriate volume of collagen stock solution to each microcentrifuge tube.
   10. Add the appropriate volume of cell suspension to each tube to obtain a final volume of 200 µL containing 1.5 mg/mL collagen.
       NOTE: The "sp" suspension contains tumor spheroids only, whereas the "sp+NK" suspension contains tumor spheroids and 1.5 × 10⁶ NK cells.
   11. Mix gently by pipetting to ensure an even distribution of cells and collagen while minimizing bubble formation.
2. Device assembly and cell loading
   1. Place Layer 1 inside the dried metal frame.
   2. Load 9 µL of the collagen mixture into each well.
   3. Align the pressure control unit and manifold assembly with the cell chambers on Layer 1 using a microscope.
   4. Insert the dried screws through the manifold and into the metal frame, securing the pressure control unit and Layer 1 between the manifold and the metal frame.
   5. Place the assembled device in a 37 °C, 5% CO₂ incubator for 45 min to allow collagen polymerization.
   6. Add 50:50 MCF-7/NK complete cell culture media through the four media ports.

3. Imaging of tumor-immune interactions

1. Place the assembled device inside the stage incubator mounted on an inverted epifluorescence microscope for live-cell imaging.
   NOTE: The stage incubator maintained a temperature of 37 °C and a humidity of approximately 50% throughout the duration of imaging.
2. Place a secondary housing box over the assembled device to maintain the internal environment at 37 °C, 100% humidity, and 5% CO₂ throughout the imaging period.
3. Connect the device to the pressure controller using the pneumatic tubing.
4. Acquire brightfield and GFP images of the tumor spheroids before applying compression to enable assessment of spheroid deformation following compression.
5. Apply a constant pressure of 120 mbar throughout the imaging period.
   NOTE: The pressure controller software enables the user to define the pressure profile and magnitude applied during the experiment. Supplementary Figure 1 contains a schematic of the pressure control system.
6. Begin time-lapse imaging by acquiring brightfield and GFP images every 10 min for 18 h.
   NOTE: Images were acquired at the center Z-plane of each tumor spheroid using a 20× objective lens on an inverted epi-fluorescence microscope. Brightfield and GFP fluorescence images were captured with exposure times of 200.1 ms and 298.9 ms, respectively.

Results

For the following experiments, a microfluidic compression device¹⁵ was used to model tumor-immune interactions within a physiologically relevant 3D microenvironment, and at the same time, can apply static and dynamic compressions to tumor spheroids (Figure 1).  The device components (Figure 1A1) and their assembly order (Figure 1A2) are shown in Figure 1A...

Discussion

This method describes the use of a microfluidic compression device to investigate the effects of compressive stress on tumor–immune interactions. This system is able to apply controlled mechanical compression to tumor spheroids embedded within a 3D collagen matrix. The glass substrate that forms the base of the cell chamber (L1) enables high-resolution live-cell imaging, making the platform well-suited for studies aiming to quantify immune cell motility, infiltration, and cytotoxic activity. Importantly, the system...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 N NaoH	Sigma-Aldrich	79724	7 µL
10× M199 medium	Sigma-Aldrich	M0650	200 µL
2-mercaptoethanol	ThermoFisher	313500100
Air compressor	Elveflow	Jun-Air Model 3 Compressor
Automated X-Y microscope stage	Applied Scientific Instrumentation	MS-2000
CCD camera	Hamamtsu Photonics	ORCA-R2
Fetal bovine serum (FBS)	GeminiBio	S11150
Fluid delivery tubing	Eleveflow	PTFE tubing spare roll	1/16 in outer diameter
Fluorescence light source	Excelitas Technologies	X-Cite series 120 PC unit
Folic Acid	Sigma	F7876
Glutaraldehyde	EM Sciences	16019	0.10%
Harrick Plasma Cleaner	Harrick Plasma	PDC-001-HP	High power setting (45W)
Horse Serum (HS)	ThermoFisher	16050-122
Inverted epi-fluorescent microscope	Olympus	IX81	20x objective lens (NA = 0.25), operated using the Olympus CellSens software
Mask	Artus	10 Sheet Aluminum Shim Stock	Thinkness is 0.0125" (0.75 mm)
MCF-7/GFP (Puromycin) stable cell line	GenTarget Inc	SC050-G
Metal Frame	Custom made	Custom made
Microfluidic reservoir	Eleveflow	50 mL microfluidic reservoir
Microscope	Olympus	CKX41	4x objective lens              (NA = 0.1)
Myo-inositol	Sigma	I7508
NK-92 MI	ATCC	CRL-2407
Penicillin/streptomycin	Gibco	15140122
Phosphate-buffered saline (PBS)	ThermoFisher	10010072
Pneumatic tubing	Eleveflow	PUR polyether clear pipe input	4" mm outer diameter
Polyethyleneimine (PEI)	Sigma-Aldrich	P3143	1%
Pressure controller	Elveflow	OB1 NK4	Operated using the Elveflow Smart Interface software
Rat tail type I collagen	Corning	354249	31.3 µL of 9.6 mg/mL stock collagen
Screws	Phillips	M2.5 Pan-head	12 screws
Stage incubator	Precision Plastic	Custom made
Syringe	Becton Dickinson	303134	Paired with a Male luer Integral lock to 3/32" OD Barb from Eleveflow
α-MEM media	ThermoFisher	12561056