Name: A Microfluidic Platform for Studying Roles of Mechanical Compression in Tumor-Immune Cell Interactions in a 3D Extracellular Matrix (Video) | JoVE Journal
Uploaded: 2026-08-28T13:45:00.000+00:00
Duration: 9 min 15 s
Description: Learn a microfluidic platform for studying tumor-immune cell interactions under mechanical compression in 3D ECM. This method enables real-time imaging of NK cell-mediated killing of breast tumor spheroids.

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This work is supported by a grant from the National Cancer Institute (Grant R01CA221346), the Lance R. Collins Fellowship from the College of Engineering at Cornell University to FM, and the Cornell Provost&#8217;s Fund for Research Resilience to FM. This work was performed in part at the Cornell NanoScale Facility, a member of the National Nanotechnology Coordinated Infrastructure (NNCI), which is supported by the National Science Foundation (Grant NNCI-2025233).

1. Preparation of the device components
<ol>
	<li>Surface activation of the sample loading chamber
	<ol>
		<li>Place a mask over the sample-loading chamber (Layer 1, L1) such that only the wells are exposed.</li>
		<li>Expose the masked Layer 1 to oxygen plasma for 15 s using the high-power setting (45 W).</li>
		<li>Place Layer 1 in a Petri dish, then continue the following steps in a biosafety cabinet.</li>
		<li>Treat each well with 10 &#181;L of 1% polyethylenimine (PEI) for 10 min.</li>
		<li>Remove the PEI from the wells by vacuum aspiration from the four corners of each well to prevent scratching the glass surface.</li>
		<li>Wash the wells with 10 &#181;L of phosphate-buffered saline (PBS), then remove the wash by vacuum aspiration from the side.</li>
		<li>Treat the wells with 10 &#181;L of 0.1% glutaraldehyde for 30 min, then remove the glutaraldehyde by vacuum aspiration from the corners.</li>
		<li>Wash the wells with PBS three times, removing the wash from the corners after each wash.</li>
		<li>Wrap the Petri dish containing Layer 1 in foil and leave it in the biosafety cabinet overnight.</li>
	</ol>
	</li>
	<li>Sterilization of the metal frame
	<ol>
		<li>Submerge the 38 mm &#215; 88 mm metal frame and the twelve M2.5 pan-head screws in 70% ethanol overnight.</li>
	</ol>
	</li>
	<li>Priming of the pressure control unit
	<ol>
		<li>Expose the pressure control unit to oxygen plasma for 30 s using the high-power setting (45 W).</li>
		<li>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.</li>
		<li>The next day, remove the pressure control unit from the DI water in which it was submerged.</li>
		<li>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.</li>
		<li>Connect a syringe filled with air to the pneumatic tubing line.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
2. Assembling the device
<ol>
	<li>Preparation of cell-embedded collagen
	<ol>
		<li>Remove and dry the metal frame and screws from the 70% ethanol.</li>
		<li>In the biosafety cabinet, place three 600 &#181;L microtubes on ice. Label the tubes as follows: &#34;N&#34; (neutralization), &#34;sp&#34; (MCF-7 spheroids), and &#34;sp+NK&#34; (MCF-7 spheroids with NK cells).</li>
		<li>Prepare the neutralization mixture by adding 10&#215; the required volume of Medium 199 (M199) and sodium hydroxide (NaOH) to the &#34;N&#34; tube.</li>
		<li>Transfer the appropriate volume of the M199:NaOH mixture to the &#34;sp&#34; and &#34;sp+NK&#34; tubes to achieve a final 1&#215; concentration.</li>
		<li>Harvest tumor spheroids and centrifuge at 141 &#215; g for 3 min to allow the spheroids to settle at the bottom of the tube. 
		NOTE: Tumor spheroids approximately 200 &#181;m in diameter were generated using a previously described agarose microwell platform16. Briefly, 3 &#215; 106 MCF-7 cells were seeded into a 36 &#215; 36 agarose microwell array consisting of wells 200 &#181;m in diameter and 200 &#181;m in depth and then cultured for 7 days to produce the tumor spheroids used in the experiments.</li>
		<li>Carefully remove the supernatant and resuspend the spheroids in 200 &#181;L of 50:50 MCF-7/NK complete medium (Table 1 and Table 2).</li>
		<li>Collect NK cells in a 50 mL conical tube, centrifuge at 141 &#215; g for 4 min, and count the cells. 
		NOTE: NK cell viability should be &#62;85% the day of the experiment.</li>
		<li>Resuspend the resulting pellet in 200 &#181;L of complete NK medium.</li>
		<li>Add the appropriate volume of collagen stock solution to each microcentrifuge tube.</li>
		<li>Add the appropriate volume of cell suspension to each tube to obtain a final volume of 200 &#181;L containing 1.5 mg/mL collagen. 
		NOTE: The &#34;sp&#34; suspension contains tumor spheroids only, whereas the &#34;sp+NK&#34; suspension contains tumor spheroids and 1.5 &#215; 106 NK cells.</li>
		<li>Mix gently by pipetting to ensure an even distribution of cells and collagen while minimizing bubble formation.</li>
	</ol>
	</li>
	<li>Device assembly and cell loading
	<ol>
		<li>Place Layer 1 inside the dried metal frame.</li>
		<li>Load 9&#160;&#181;L of the collagen mixture into each well.</li>
		<li>Align the pressure control unit and manifold assembly with the cell chambers on Layer 1 using a microscope.</li>
		<li>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.</li>
		<li>Place the assembled device in a 37 &#176;C, 5% CO&#8322; incubator for 45 min to allow collagen polymerization.</li>
		<li>Add 50:50 MCF-7/NK complete cell culture media through the four media ports.</li>
	</ol>
	</li>
</ol>
3. Imaging of tumor-immune interactions
<ol>
	<li>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 &#176;C and a humidity of approximately 50% throughout the duration of imaging.</li>
	<li>Place a secondary housing box over the assembled device to maintain the internal environment at 37 &#176;C, 100% humidity, and 5% CO&#8322; throughout the imaging period.</li>
	<li>Connect the device to the pressure controller using the pneumatic tubing.</li>
	<li>Acquire brightfield and GFP images of the tumor spheroids before applying compression to enable assessment of spheroid deformation following compression.</li>
	<li>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.</li>
	<li>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&#215; 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.</li>
</ol>

<ol>
	<li>Cao J-Z, Zhao W, Xia X-J. Cell-based cancer immunotherapy: milestones, mechanistic insights, and emerging therapeutic directions. Acta Pharmacol Sin. 2026:https://doi.org/10.1038/s41401-026-01811-y.</li>
	<li>Yang R, Yang Y, Liu R, et al. Advances in CAR-NK cell therapy for hematological malignancies. Front Immunol. 2024;15:1414264.</li>
	<li>Neelapu SS, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med. 2017;377(26):2531-44.</li>
	<li>Sheykhhasan M, Manoochehri H, Dama P. Use of CAR T-cell for acute lymphoblastic leukemia (ALL) treatment: a review study. Cancer Gene Ther. 2022;29(8-9):1080-96.</li>
	<li>Tan S, Li D, Zhu X. Cancer immunotherapy: Pros, cons and beyond. Biomed Pharmacother. 2020;124:109821.</li>
	<li>Chen T, Wang M, Chen Y, Liu Y. Current challenges and therapeutic advances of CAR-T cell therapy for solid tumors. Cancer Cell Int. 2024;24(1):133.</li>
	<li>Peng L, et al. CAR-T and CAR-NK as cellular cancer immunotherapy for solid tumors. Cell Mol Immunol. 2024;21(10):1089-108.</li>
	<li>Basu R, et al. Cytotoxic T cells use mechanical force to potentiate target cell killing. Cell. 2016;165(1):100-10.</li>
	<li>Dolega ME, et al. Cell-like pressure sensors reveal increase of mechanical stress towards the core of multicellular spheroids under compression. Nat Commun. 2017;8(1):14056.</li>
	<li>Linke JA, Munn LL, Jain RK. Compressive stresses in cancer: characterization and implications for tumour progression and treatment. Nat Rev Cancer. 2024;24(11):768-91.</li>
	<li>Luo M, Cai G, Ho KKY, et al. Compression enhances invasive phenotype and matrix degradation of breast cancer cells via Piezo1 activation. BMC Mol Cell Biol. 2022;23(1):1.</li>
	<li>Ren T, Sun L, Zheng Y, et al. Mechanical forces and immune cells in the tumor microenvironment: from regulation mechanisms to therapeutic strategies. Int J Surg. 2025;111(8):5420-34.</li>
	<li>Helmlinger G, Netti PA, Lichtenbeld HC, Melder RJ, Jain RK. Solid stress inhibits the growth of multicellular tumor spheroids. Nat Biotechnol. 1997;15(8):778-83.</li>
	<li>Pandey M, et al. Mechanical compression regulates tumor spheroid invasion into a 3D collagen matrix. Phys Biol. 2024;21(3):036003.</li>
	<li>Suh YJ, et al. A microfluidic rheometer for tumor mechanics and invasion studies. Lab Chip. 2025;25(22):6018-32.</li>
	<li>Song W, et al. Dynamic self-organization of microwell-aggregated cellular mixtures. Soft Matter. 2016;12(26):5739-46.</li>
	<li>Pandey M, et al. Viscoelastic properties of tumor spheroids revealed by a microfluidic compression device and a modified power law model. Soft Matter. 2026;22(7):1618-29.</li>
	<li>Steff A-M, Fortin M, Arguin C, Hugo P. Detection of a decrease in green fluorescent protein fluorescence for the monitoring of cell death: An assay amenable to high-throughput screening technologies. Cytometry. 2001;45(4):237-43.</li>
</ol>

The authors have nothing to disclose.

This method describes the use of a microfluidic compression device to investigate the effects of compressive stress on tumor&#8211;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...

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 cancers2,3,4. However, the level of success observed in blood cancers has not yet been fully translated to the treatment of solid tumors5,6,7. 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 tumor9,10. To date, most compression studies have focused on the effects on tumor cell behavior10,11. 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&#8211;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&#8211;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.

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

a microfluidic platform for studying roles of mechanical compression in tumor-immune cell interactions in a 3d extracellular matrix

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

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 &#8212;including granzyme B&#8212;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.&#160;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.

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...

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.

Mechanical forces significantly influence the ability of immune cells to kill tumor cells in the context of cell-based immunotherapy. To kill tumor cells, ...

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Learn a microfluidic platform for studying tumor-immune cell interactions under mechanical compression in 3D ECM. This method enables real-time imaging of NK cell-mediated killing of breast tumor spheroids.

A Microfluidic Platform for Studying Roles of Mechanical Compression in Tumor-Immune Cell Interactions in a 3D Extracellular Matrix (Video) | JoVE Journal

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

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