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.

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 w....

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