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This article was submitted to Cell Adhesion and Migration, a section of the journal Frontiers in Cell and Developmental Biology

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Up to 50% of head and neck squamous cell carcinoma (HNSCC) patients have lymph node (LN) metastasis, resulting in poor survival rate. Numerous studies have supported the notion that the alterations of gene expression and mechanical properties of cancer cells play an important role in cancer metastasis. However, which genes and how they regulate the biomechanical properties of HNSCC cells to promote LN metastasis remains elusive. In this study, we used an LN-metastatic mouse model

Lymph node (LN) metastasis is the major risk and cause of deaths in patients with head and neck squamous cell carcinoma (HNSCC) (

Tumor-induced lymphangiogenesis is crucial for tumor growth and LN metastasis (

In physiology and pathology, the alteration of the mechanical properties of cancer cells, including cytoplasm stiffness, cell traction force, and nuclear stiffness, plays a crucial role in cancer invasion and metastasis (

Characterization of the biomechanical properties and the expression of epithelial–mesenchymal transition marker proteins of lymph node (LN)-metastatic and non-metastatic head and neck squamous cell carcinoma (HNSCC) cells (SAS-LN and SAS cells).

The human head and neck squamous cell carcinoma (HNSCC) cell lines (OEC-M1 and SAS) were provided by Professor Muh-Hwa Yang, Institute of Clinical Medicine, National Yang Ming Chiao Tung University. OEC-M1 and SAS cells were maintained in the medium of RPMI 1640 (11875-093, Gibco) and DMEM (11995-065, Gibco), respectively, supplemented with 10% FBS (10270106, Gibco) and 1% penicillin–streptomycin solution (15140-122, Gibco) at 37°C and 5% CO_{2}. The SAS-Snail cell line was generated by transfecting PCDH-puro-Snail plasmid and PCDH-puro-ctrl; the latter served as a control (

Lymph node (LN)-metastatic HNSCC cells (SAS-LN) were generated and provided by Professor Muh-Hwa Yang (Institute of Clinical Medicine, National Yang Ming Chiao Tung University) (_{2}. Cell samples for all the experiments were within 10 passages. The expressions of Snail, N-cadherin, and E-cadherin in passages 1 and 10 did not have significant differences (

Cells were lysed in the protease inhibitor-containing lysis buffer for western blot analysis. The protein lysates were separated by SDS-PAGE, transferred onto a PVDF membrane (Millipore Corp., Bedford), and stained with proper antibodies. The antibodies used in this study include the antibodies against Snail (3895, Cell Signaling), E-cadherin (3195, Cell Signaling), N-cadherin (610920, BD), lamin A/C (8984, Abcam), lamin B1 (16048, Abcam), tubulin (T6074, Sigma-Aldrich), and GAPDH (2118s, Cell Signaling). Signals were developed using an enhanced chemiluminescence kit (Millipore Corp.) and photographed using Fujifilm LAS-4000.

The invasion capability of SAS-LN and SAS cells was assessed via microchannels (MC005, 4DCELL) of 5 μm height and varying widths (12, 16, and 20 μm). In total, 15 μL of cell suspension (10^{7} cell/mL) was added into the access ports of the microchannels and incubated at 37°C for 24 h. The invasion capability was determined by counting the number of cells invading into the microchannels from the access ports. In our microchannel assay, SAS-LN and SAS cells migrated into the microchannels from their opposite ends. We have demonstrated that the presence of each type of cell on the opposite sites of the microchannels did not affect cell invasion, which implied the absence of a significant interaction between SAS and SAS-LN cells on cell invasion in our microchannel assay (

The micropattern-based nuclear stiffness assay, consisting of traction force microscopy and rectangular micropattern to simultaneously measure the cell traction force and the longitudinal strain of cell nucleus and then calculate the nuclear stiffness, was described in a previous report (

Video particle tracking microrheology (VPTM) was used to analyze the cytoplasm stiffness as was described in our previous reports (^{12} particles/mL) into the cells via a biolistic particle delivery system (PDS-100, Bio-Rad; pressure 450 psi). The Brownian motion of the beads was tracked and recorded via an epi-fluorescence microscope system (Eclipse Ti, Nikon), equipped with a ×100 oil-immersion objective (MRD01991, Nikon, numerical aperture = 1.45) and a CMOS camera (Hamamatsu, OHCA-Flash 4.0; 100fps). The mean squared displacement (MSD) <Δr ^{2}(τ)> = <[x (t + τ) − x(t)]^{2} + [y (t + τ) − y(t)]^{2} > was calculated from the two-dimensional trajectory, x(t) and y(t), of each bead. The effective creep compliance J(τ) and the elastic modulus G’ (_{B} the Boltzmann constant, and T is the absolute temperature. The cytoplasmic stiffness was compared in terms of the value of the elastic modulus G′(

VPTM measures the local elastic modulus of cells based on the Brownian motion of individual intracellular particles without external contact forces. Hence, it has a great advantage in measuring the elastic modulus of cells cultured in 3D ECM environments. However, without applied external forces, it is a challenge to use VPTM to measure viscoelastic properties of materials in a non-equilibrium condition (

The primary antibodies used in this experiment are 1:200 dilution of anti-lamin A/C mouse monoclonal antibody (ab8984, Abcam), 1:1000 dilution of anti-lamin B1 rabbit polyclonal antibody (ab16048, Abcam), 1:200 dilution of anti-SNAI1 (GTX100754, GeneTex), and 1:200 dilution of anti-paxillin rabbit polyclonal antibody (GTX125891, GeneTex). The cell nuclei and F-actin were stained with 5 μg/mLof Hoechst 33342 (H3570, Thermo Fisher Scientific) and 165 nM of rhodamine–phalloidin (R415, Thermo Fisher Scientific), respectively. Immunofluorescent images were taken using a laser scanning microscope system (LSM 880, ZEISS) equipped with a 40× oil-immersion objective (plan-apochromat, ZEISS; numerical aperture = 1.3).

The individual focal adhesion (FA) area and number of FAs per cells were analyzed by MetaMorph software. The nuclear morphology was characterized in terms of nuclear area and nuclear aspect ratio (the ratio of the length of major and minor axes) via ImageJ software. The number of cells in the microchannel was analyzed by ImageJ software. The co-localization of paxillin and actin filaments was analyzed using ImageJ software.

Statistical analysis of data was performed using MATLAB and carried out by one-way ANOVA, followed by Tukey’s test. The statistical significance at *for

In this study, a lymph node metastatic mouse model system was used to generate LN-metastatic human HNSCC cells (SAS-LN), which were isolated from lymph nodes of nude mice in 4 weeks after tongue injection with non-metastatic HNSCC cells (SAS). In contrast to SAS cells, SAS-LN cells displayed a more mesenchymal phenotype with elongated morphology (

To comprehensively and systematically investigate the difference in the biomechanical properties of SAS-LN and SAS cells, we used microchannels, traction force microscopy (TFM), video particle tracking microrheology (VPTM), and nuclear stiffness assay to measure 1) cells’ invasion capability, 2) cells’ traction force, 3) cytoplasmic stiffness, and 4) nuclear stiffness (

Invasion capability of LN-metastatic HNSCC cells (SAS-LN) and non-metastatic HNSCC cells (SAS).

Previous studies have indicated that the cellular strain is determined by its unique stiffness, including nuclear stiffness and cytoplasmic stiffness (

The biomechanical properties of LN-metastatic HNSCC cells (SAS-LN) and non-metastatic HNSCC cells (SAS). _{x}) and transverse (S_{y}) strain of nuclei, along the major axis and the minor axis of the rectangular micropatterns, respectively, in SAS-LN cells and SAS cells before and after trypsin treatment. _{x}) repesent the reaction of traction forces projected along the major axis of the rectangular micropatterns. _{x}) according to Hooke’s law. Data represent mean ± SD (n = 20).

Actin cytoskeleton is one of the major cytoskeletal components that mediates cell shape and cell motility and participates in the regulation of cell traction forces and nuclear movement and deformation (

Previous studies have indicated that the biomechanical properties of cells are regulated by the corresponding biomolecular expression, including lamin, focal adhesions (FAs), and actin cytoskeleton (^{2}, has a positive correlation with the magnitude of local traction force (^{2}), a smaller number of paxillin-marked FAs (paxillin area < 1 μm^{2}) (

Characterization of the nuclear biomolecules and focal adhesion size of LN-metastatic HNSCC cells (SAS-LN) and non-metastatic HNSCC cells (SAS). ^{2} vs. those with FAs area <1 μm^{2}, in SAS-LN and SAS cells, in the regions (8 μm × 20 μm) bounded by the white dotted rectangles shown in

To investigate whether Snail could regulate the biomolecular and biomechanical properties of HNSCC cells for LN metastasis, we examined the effect of Snail overexpression on the biomolecular (^{2}) and a smaller number of small paxillin-marked FAs (paxillin area <1 μm^{2}) (

The effect of Snail on the biomolecular and biomechanical properties of SAS cells. _{x}), and reduced the cell nuclear stiffness, with much smaller effect in cytoplasmic stiffness. Data represent mean ± SD (n = 15). ^{2}) and reduced the number of nascent FAs (FAs area <1 μm^{2}); however, its effect on actin filaments was much less. Left: immunofluorescence micrographs of nuclear actin and paxillin filaments of control (PCDH) and Snail-overexpressing SAS cells (SAS-Snail). Scale bars = 20 μm. Right: the numbers of focal adhesions (FAs) in the regions (8 μm × 20 μm) bounded by the white dotted rectangles shown in the lower panel in

To further confirm that Snail can contribute to increase cell traction force and soften nuclear stiffness, we used shRNA sequences to inhibit Snail in LN-metastatic HNSCC cells, SAS-LN (^{2}), a larger number of small paxillin complexes (paxillin area <1 μm^{2}) (

The effect of Snail-knockdown on the biomolecular and biomechanical properties of LN-metastatic HNSCC cells (SAS-LN). _{x}), enhanced nuclear stiffness, but rendered smaller effect on cytoplasmic stiffness. Data represent mean ± SD (n = 15). ^{2}) and augmented the nascent FAs (FAs area <1 μm^{2}). Left: immunofluorescence micrographs of the cell nuclei, paxillin, and actin filaments of control (SAS-LN-pLKO) and Snail-knockdown SAS-LN cells (SAS-LN-shSnail). Scale bars = 20 μm. Right: the numbers of focal adhesions (FAs) in the regions (8 μm × 20 μm) bounded by the white dotted rectangles in the lower panel in

Consistently, Snail knockdown produced similar effects in another mesenchymal HNSCC cell line, OEC-M1 (

Lymph node (LN) metastasis is a complex process which requires coordinated changes in the biomolecular and biomechanical properties of cancer cells to invade from primary tumor to the surrounding ECM, tissues, blood vessels, and lymph nodes (

Lamin A/C plays a critical role in regulating the nuclear stiffness and maintaining nuclear morphology, which has a significant impact on the invasion capability of cancer cells (

Cancer invasion also requires dynamic alteration of cellular morphology and interaction with the surrounding ECM through dynamic assemblies and turnover FAs, which is stimulated by traction force or mechanical tension (

Summary of the changes in the biophysical and biochemical properties of HNSCC cells regulated by Snail for lymph node metastasis. A radar chart presents quantitatively the fold change in biophysical and biochemical properties in

The original contributions presented in the study are included in the article/

Y-QC: conceptualization, investigation, methodology, and writing—original draft preparation. C-YH and H-YC: investigation and validation. M-TW: conceptualization, visualization, and editing. J-CK and M-HY: supervision, conceptualization, resources, reviewing, and editing. AC: visualization and editing. All authors contributed to the article and approved the submitted version.

This work was financially supported by the “Cancer Progression Research Center, National Yang Ming Chiao Tung University,” from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan. This work was jointly supported by the Ministry of Science and Technology (107-2221-E-010-009-MY2 and 109-2221-E-010-018).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors, and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

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