2026 年 51 巻 1 号 p. 227-235
Cancer cells form clusters and invade the surrounding stroma. This phenomenon is associated with metastasis and poor prognosis in patients with cancer. Therefore, prevention of invasion may improve cancer therapy. A previous study suggested that the C-C motif chemokine ligand 5 (CCL5) is more highly expressed in the highly invasive subclone than in the less invasive subclone derived from A431 cell line, a model of human skin squamous cell carcinoma. However, the role of CCL5 and the mechanisms through which it regulates invasion in A431 cell clusters remain unclear. Herein, we investigated the molecular mechanisms underlying CCL5-induced invasion in A431 cell clusters. Knockdown of CCL5 suppressed invasion on A431 cell clusters, whereas treatment with recombinant CCL5 promoted invasion of them. Mechanistically, CCL5 increased the phosphorylation levels of Src and ERK, leading to the invasion of A431 cell clusters. Furthermore, the knockdown of syndecan-1 (SDC1), a transmembrane protein known to interact to CCL5, suppressed CCL5-induced invasion in A431 cell clusters. Consistently, knockdown of SDC1 reduced the level of phosphorylated ERK. These findings demonstrate that the CCL5 promotes the invasion of A431 cells clusters through SDC1-dependent ERK signaling and Src phosphorylation.
Key words: invasion, CCL5, syndecan-1, ERK signaling, Src
Graphical Abstract
Metastasis, the process by which cancer cells spread from the primary tumor to distant organs, is the leading cause of cancer-related deaths. In the early stages of metastasis, cancer cells invade the surrounding normal tissues in a process known as invasion (Chaffer and Weinberg, 2011). Cancer cells use various invasive strategies. Recent studies have shown that cancer cells invade the surrounding stroma in clusters while maintaining cell–cell contact, a process termed collective invasion (Friedl et al., 2012). Collective invasion has been observed in various cancers, including squamous cell carcinoma (SCC) (Wang et al., 2016). Previous studies have reported that circulating tumor cell (CTC) clusters have a higher metastatic potential than single CTCs and that the abundance of such clusters is associated with poor prognosis in patients with breast cancer (Aceto et al., 2014). Therefore, inhibition of collective invasion is a potential therapeutic strategy for improving cancer therapy.
In a previous study, we established a highly invasive subclone (A431-6 cells) and a minimally invasive subclone (A431-7 cells) derived from human skin squamous cell carcinoma (A431 cells) (Kumagai et al., 2022). DNA microarray analysis of each subclone revealed that C-C motif chemokine ligand 5 (CCL5) was highly expressed in A431-6 cells compared to that in A431-7 cells (Kumagai et al., 2022). CCL5 is a chemokine secreted by T cells that induces immune cell migration (Aldinucci et al., 2020). Previous studies have demonstrated that the expression of chemokines, including CCL5, increases with the disease stage in patients with skin SCC (Tuong et al., 2019). However, the role of CCL5 in the invasion and skin SCC remains unclear.
Recent studies have shown that CCL5 interacts with glycosaminoglycans (GAGs), such as heparan sulfate (Liang et al., 2016; Yu et al., 2024). Increasing evidence indicates that CCL5 promotes cancer cell invasion and migration in hepatocellular carcinoma and pancreatic ductal adenocarcinoma cells through syndecan-1 (SDC1), a heparan sulfate proteoglycan (Charni et al., 2009; Chen et al., 2022). However, the detailed molecular mechanisms underlying the interaction between CCL5 and SDC1 remain unclear.
Herein, we demonstrated that CCL5 promoted the invasion of A431 cell clusters, a model of collective invasion (Kumagai et al., 2022). Mechanistically, CCL5 increased the phosphorylation levels of Src and Extracellular signal-regulated kinases (ERK), leading to invasion of A431 cell clusters. Furthermore, SDC1 is critical for the CCL5-induced invasion of A431 cell clusters. These results suggest that the CCL5 promotes the invasion of A431 cell clusters via SDC1-dependent ERK signaling and Src phosphorylation.
A431-6 and A431-7 cells, subcloned from A431 cells (American Type Culture Collection, Manassas, VA, USA), were cultured in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich Co. LLC, St Louis, MO, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich Co. LLC) and 1% antibiotic/antimycotic solution (Sigma-Aldrich Co. LLC). Cells were cultured at 37°C in a humidified incubator with 5% CO2. All the cells were tested for mycoplasma contamination using the MycoAlert Assay Control Set (LT07-518; Lonza Bioscience, Basel, Switzerland) and were not authenticated.
Three-dimensional collagen gel cultureThree-dimensional collagen gel cultures were prepared as previously described (Kumagai et al., 2022). Briefly, cell suspensions were embedded in a 1.6 mg/mL type I collagen solution (Cell matrix I-P; Nitta Gelatin, Osaka, Japan) and incubated for 30 min at 37°C to complete gelation. In this experimental system, single cells formed spheroids and invaded the surrounding extracellular matrix with invasive protrusions. This invasive morphology was used to assess the invasive potential of the spheroids.
Quantitative PCRRNA extraction, reverse transcription, and quantitative PCR of cells on collagen gels were performed as previously reported (Kumagai et al., 2022). The primer sequences were as follows; SDC1: 5'-CACCATGAGACCTCAACCCC-3' (forward), 5'-AAAGGTGAAGTCCTGCTCCC-3' (reverse), ACTB: 5'-TGGGACGACATGGAGAAAATCTG-3' (forward), 5'-AGGTCTCAAACATGATCTGGGTC-3' (reverse), CCL5: 5'-CTTTGTCACCCGAAAGAACCGC-3' (forward), 5'-GACAAGAGCAAGCAGAAACAGGC-3' (reverse), SRC: 5'-AGTGAGACCACGAAAGGTGC-3' (forward), 5'-GGGAGGTGATGTAGAAGCCG-3' (reverse).
Western blottingThe samples prepared from the cells in collagen gels were fixed with 10% trichloroacetic acid (TCA) (Sigma-Aldrich Co., LLC) in phosphate-buffered saline (PBS) for 20 min at 4°C and washed thrice with PBS. 0.1% collagenase (Wako, Osaka, Japan) in PBS was added and incubated for 10 min at 37°C. After collagen digestion, collagenase was removed by centrifugation at 10,000 × g for 5 min at 4°C. Proteins from the cells were extracted using sodium dodecyl sulfate (SDS) sample buffer (60 mM Tris HCl, 2.2% SDS, 10% glycerol, 100 mM dithiothreitol, pH 6.8) and boiled at 95°C for 5 min. A431-7 cells in 6-well plates under the condition of 0.5 to 2 h of treatment with recombinant human CCL5 were extracted without TCA fixation and collagenase treatment. Western blotting was performed as previously described (Kumagai et al., 2022). Primary and secondary antibodies were diluted in Tris-buffered saline with Tween 20 (TBS-T). Anti-CCL5 (1:1,000, 25HCLC, Invitrogen, Waltham, MA, USA), anti-Phospho-p44/42 MAPK (Thr202/Tyr204) (p-ERK1/2; 1:2,000, 4377, Cell Signaling Technology, Danvers, MA, USA), anti-p44/42 MAPK (ERK1/2; 1:2,000, 4695, Cell Signaling Technology), and anti-Src (phospho Y419) (p-Src; 1:5,000, ab185617, abcam, Cambridge, UK) were used as primary antibodies. Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Cell Signaling Technology) was used as a secondary antibody at 1:10,000 (for CCL5), 1:20,000 (for p-ERK1/2 and ERK1/2), or 1:1,000 (for p-Src).
Immunofluorescent stainingSamples were prepared from the cells in collagen gels. The cells were fixed with 4% paraformaldehyde in PBS for 1 h at 4°C and washed thrice with PBS. The cells were permeabilized with 0.05% saponin in PBS for 10 min at room temperature and washed thrice with PBS. The cells were blocked with 2% bovine serum albumin (BSA) in PBS for 1 h at room temperature and washed thrice with PBS. The cells were then incubated with a primary antibody solution (1:50 anti-CCL5, 25HCLC, Invitrogen) in PBS for 3 h at room temperature. After three washes with PBS, the cells were incubated with a secondary antibody solution (1:200 Goat anti-Rabbit IgG (H + L), Superclonal Recombinant Secondary Antibody, Alexa Fluor 555, A27039, Invitrogen; 1:5,000 Hoechst 33342, H1300, Invitrogen; 1:500 Alexa Fluor 488 phalloidin, A12379, Invitrogen) in PBS for 45 min at room temperature. After three washes with PBS, fluorescent images were captured using an A1 confocal imaging system (Nikon, Tokyo, Japan) with a 60× objective.
Fluorescent staining of cell clustersFluorescent staining of cell clusters in collagen gels was performed as previously reported (Kumagai et al., 2022), with some modifications. Cell clusters in collagen gels were fixed with 4% paraformaldehyde in PBS for 1 h at 4°C and washed thrice with PBS. The cells were permeabilized, blocked with 1% BSA and 0.3% Triton X-100 (Sigma-Aldrich Co. LLC, Saint Louis, MO, USA) in PBS for 1 h at room temperature, and washed thrice with PBS. The cells were then incubated with Alexa Fluor 488 phalloidin (1:500, A12379, Invitrogen) for 1 h at room temperature. After three washes with PBS, fluorescent images were captured using an A1 confocal imaging system with a 60× objective lens. Cell clusters were imaged from top to bottom at 1.5-μm intervals.
Morphological analysis using Imaris softwareMorphological analysis using the Imaris software was performed as previously reported (Kumagai et al., 2022). Briefly, we constructed 3D images of spheroids using Imaris software (Bitplane AG, Zurich, Switzerland). The sphericity (36πV2/S3) of the spheroid was calculated from the ratio of its circumference (π), surface area (S), and volume (V).
Small interfering RNA (siRNA) transfectionCells were seeded in 6-well plates at a density of 2 × 105 cells/well and transfected with the appropriate siRNA using the Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Transfection was performed using siRNAs at a concentration of 4 or 5 nM. Nucleotide blasts demonstrated that there were no homologous sequences in the control siRNA. The following target sequences were used: CCL5:5'-GAGCTTACTGGCAAACATGAAAA-3' (sense sequence), SDC1:5'-AAATTAGTAGCCACAATTTGCGG-3' (sense sequence), SRC: 5'-TAAATATGGACGCACATGGAG-3' (sense sequence), and negative control: 5'-TTCAACTGTGATAGATCCAAT-3' (sense sequence).
Reagent treatmentRecombinant human CCL5 (rhCCL5) was expressed as a CaM-fusion protein as previously reported (Ishida et al., 2016; Yan et al., 2025). A construct consisting of an N-terminal His-tag, CaM, an EK cleavage site, and CCL5 was cloned into the pET15 vector. This vector was transformed into Escherichia coli BL21(DE3) cells, grown in Luria–Bertani (LB) medium, and induced with IPTG. After harvesting and sonication, soluble fusion proteins were purified using IMAC. Following EK cleavage, CCL5 was purified by reverse-phase HPLC. The purity and folding of the final product were confirmed using SDS-PAGE, MS, and NMR spectroscopy.
rhCCL5 in PBS (500 ng/mL) and U0126 (1 μM) (Cell Signaling Technology) were added to A431-6 or A431-7 cells in a three-dimensional collagen gel culture system.
Statistical analysisEach experiment was independently repeated at least twice. The threshold for statistical significance was set at 0.05. Statistical analyses were performed using confidence intervals (western blotting and qPCR in the two groups). The sphericity of the cell clusters in the two groups was tested using the Student’s t-test or Welch’s t-test. The similarity of variances between groups was examined using the F-test. When variances were equal, the Student’s t-test was used; when they were unequal, Welch’s t-test was used. For more than three groups, a Bonferroni correction was performed. All the tests were two-sided.
We examined CCL5 expression in A431-6 and A431-7 cells. Quantitative PCR (qPCR) and western blotting were performed to compare the mRNA and protein levels of CCL5 in each subclone. Both analyses revealed that CCL5 was highly expressed at both the mRNA and protein levels in A431-6 cells compared to A431-7 cells (Fig. 1A, B, C). In addition, immunofluorescence staining for CCL5 showed higher fluorescence intensity in A431-6 than A431-7 cells on cell surface (Fig. 1D). These results demonstrate that CCL5 was upregulated in A431-6 cells compared to A431-7 cells.
CCL5 promotes invasion of A431 cell clusters
(A) qPCR of CCL5 and ACTB in A431-6 and A431-7 cells. Bars represent the mean with SE of three independent experiments. (B) Western blot of CCL5 and β-actin in A431-6 and A431-7 cells. (C) Quantification of (B). Bars represent the mean with SE of three independent experiments. (D) Immunofluorescence of CCL5 (red) with F-actin (green) and nucleus (blue) in A431-6 and A431-7 cell clusters. (E) Representative three-dimensional constructed images of siControl or siCCL5-transfected A431-6 cell clusters. (F) Quantification of sphericity in (E). n = 29 (siControl) and 30 (siCCL5) clusters by two independent experiments. Box plots with mean, maximum, and minimum values. (G) Representative three-dimensional constructed images of Control- or rhCCL5-treated A431-7 cell clusters. Cells were stimulated by PBS (control) or rhCCL5 for 96 h. (H) Quantification of sphericity in (G). n = 29 (Control) and 32 (+rhCCL5) clusters from two independent experiments. Box plots with mean, maximum, and minimum values. * Statistically significant. Scale bars represent 30 μm.
Next, we examined the role of CCL5 in the invasion of A431 cell clusters. A431 cells were embedded in type I collagen gel to induce spheroid formation and invasion. Invasion was evaluated by calculating the sphericity of the cell clusters, as previously reported (Kumagai et al., 2022). When a cell cluster invades the surrounding matrix and forms protrusions, its sphericity approaches zero. In contrast, when a cell cluster does not invade and maintains a round morphology, its sphericity approaches one. Knockdown of CCL5 by siRNA (Fig. S1A) prevented the formation of protrusions and significantly increased the sphericity of A431-6 cell clusters (Fig. 1E, F). Conversely, treatment with recombinant human CCL5 (rhCCL5) significantly promoted the formation of protrusions and decreased the sphericity of A431-7 cell clusters (Fig. 1G, H). These results suggest that CCL5 triggers invasion of A431 cell clusters.
Phosphorylation of ERK is induced by CCL5 and promotes invasion of A431 cell clustersWe investigated the downstream signaling pathways activated by CCL5 in A431 cells. ERK signaling is involved in tumor progression (Guo et al., 2020) and is activated by CCL5 (Aldinucci et al., 2020). Therefore, we examined the association between ERK signaling and CCL5-induced invasion of A431 cell clusters. Western blotting was performed to compare total and phosphorylated ERK1/2 expression levels in each subclone. Both total ERK1/2 and phosphorylated ERK1/2 were more induced in A431-6 than in A431-7 cells (Fig. 2A, B, S1B). Subsequently, we examined whether ERK1/2 was upregulated by CCL5. Treatment with rhCCL5 increased the level of phosphorylated ERK1/2 in A431-7 cells (Fig. 2C, D, S1C). These results indicated that the amount of phosphorylated ERK1/2 was increased by CCL5 in A431 cells. Next, we examined the association between ERK signaling and invasion of A431 cell clusters. MEK is an upstream kinase that phosphorylates ERK1/2 (Guo et al., 2020). Treatment with U0126, a MEK inhibitor, significantly reduced phosphorylated ERK1/2 in A431-6 cells (Fig. S1D, E) and increased the sphericity of A431-6 cell clusters (Fig. 2E, F), indicating that phosphorylation of ERK is critical for invasion of A431 cell clusters. These findings suggest that ERK signaling promotes the invasion of A431 cell clusters upon activation by CCL5.
ERK pathway promotes CCL5-induced invasion of A431 cell clusters
(A) Western blot of p-ERK1/2, ERK1/2 and β-actin in A431-6 and A431-7 cells. (B) Quantification of (A). Bars represent the mean with SE of three independent experiments. (C) Western blot of p-ERK1/2, ERK1/2 and β-actin of Control or rhCCL5-treated A431-7 cells. A431-7 cells were cultured in a 6-well plate after treatment with rhCCL5 for 0.5–2 h. (D) Quantification of (C). Bars represent the mean with SE of three independent experiments. (E) Representative three-dimensional constructed images of DMSO- or U0126-treated A431-6 cell clusters. (F) Quantification of sphericity in (E). Box plot with the mean, with maximum and minimum values for n = 23 (DMSO) and 22 (U0126) clusters by two independent experiments. * Statistically significant. n.s. represents non-statistical significance. Scale bars represent 30 μm.
Next, we investigated the transmembrane proteins that interact with CCL5 and regulate invasion in A431 cell clusters. Syndecan-1 (SDC1) is a cell-surface proteoglycan with a binding affinity for CCL5 (Slimani et al., 2003; Charnaux et al., 2005). Therefore, we examined the association between SDC1 and invasion of A431 cell clusters. Knockdown of SDC1 by siRNA (Fig. S1F) significantly increased the sphericity of A431-6 cell clusters (Fig. 3A, B). Additionally, knockdown of SDC1 by siRNA (Fig. S1G) significantly inhibited the decrease in sphericity induced by rhCCL5 treatment in A431-7 cell clusters (Fig. 3C, D). These results suggest that SDC1 is critical for the CCL5-induced invasion of A431 cell clusters. Next, we examined the association between SDC1 and ERK signaling. SDC1-knockdown by siRNA (Fig. S1F) suppressed the phosphorylation level of ERK1/2 (Fig. 3E, F, S1H), suggesting that SDC1 contributes to the CCL5-induced invasion of A431 cell clusters through ERK signaling.
CCL5 promotes invasion of A431 cell clusters in a SDC1-dependent manner
(A) Representative three-dimensional constructed images of siControl or siSDC1-transfected A431-6 cell clusters. (B) Quantification of sphericity in (A). n = 35 (siControl) and 36 (siSDC1) clusters by three independent experiments. Box plots with mean, maximum, and minimum values. (C) Representative three-dimensional constructed images of siControl, siControl + rhCCL5, or siSDC1 + rhCCL5-transfected/treated A431-7 cell clusters. Cells were stimulated by PBS (control) or rhCCL5 for 96 h. (D) Quantification of sphericity in (C). n = 20 (siControl), 22 (siControl + rhCCL5), and 23 (siSDC1 + rhCCL5) clusters by two independent experiments. Box plots with mean, maximum, and minimum values. (E) Western blot of p-ERK1/2, ERK1/2 and β-actin of siControl or siSDC1 treated A431-6 cells. (F) Quantification of (E). Bars represent the mean with SE of three independent experiments. * Statistically significant. n.s. indicates non-statistical significance. Scale bars represent 30 μm.
Finally, we investigated the mediator molecule between the CCL5/SDC1 axis and ERK signaling pathway. Previous studies have reported that Src interacts with the cytoplasmic domain of SDC1 (Roper et al., 2012; Stepp et al., 2015), and acts as an upstream molecule of ERK signaling (Pelaz and Tabernero, 2022). In addition, a previous study showed that CCL5 activates ERK1/2 via Src (Tyner et al., 2005). Therefore, we examined the role of Src in A431 cells. qPCR for SRC and western blotting for phosphorylated Src were performed to compare SRC mRNA expression and phosphorylation levels in each subclone. At mRNA levels, SRC was more highly expressed in A431-6 cells than in A431-7 cells (Fig. 4A). A431-6 cells exhibited increased Src phosphorylation compared to A431-7 cells. (Fig. 4B, C). In addition, treatment with rhCCL5 enhanced the phosphorylation level of Src in A431-7 cells (Fig. 4D, E). Knockdown of SRC by siRNA (Fig. S1I) significantly increased the sphericity of A431-6 cell clusters (Fig. 4F, G). These results indicate that Src transduces signals from the CCL5 to promote invasion in A431 cell clusters.
Src is activated by CCL5 and promotes invasion of A431 cell clusters
(A) qPCR of SRC and ACTB in A431-6 and A431-7 cells. Bars represent the mean with SE of three independent experiments. (B) Western blot of p-Src and β-actin in A431-6 and A431-7 cells. (C) Quantification of (B). Bars represent the mean with SE of three independent experiments. (D) Western blot of p-Src and β-actin of Control or rhCCL5-treated A431-7 cells. A431-7 cells were cultured in 6-well plates after treatment with rhCCL5 for 0.5–2 h. (E) Quantification of (D). Bars represent the mean with SE of three independent experiments. (F) Representative three-dimensional constructed images of siControl or siSRC-transfected A431-6 cell clusters. (G) Quantification of sphericity in (F). Box plot with the mean, with maximum and minimum values for n = 23 (siControl) and 28 (siSRC) clusters by two independent experiments. * Statistically significant. n.s. indicates non-statistical significance. Scale bars represent 30 μm.
In the present study, we demonstrated that CCL5 plays a critical role in the invasion of A431 cell clusters. CCL5 was highly expressed in the highly invasive subclone, which drove invasion via Src and ERK signaling. Consistently, exogenous CCL5 enhanced invasion by increasing in phosphorylated levels of Src and ERK in the low-invasiveness subclone. In addition, SDC1 is essential for CCL5-induced invasion of A431 cell clusters. Previous studies showed that Src interacts with SDC1 (Roper et al., 2012; Stepp et al., 2015) and acts as an upstream molecule of ERK signaling (Pelaz and Tabernero, 2022), therefore CCL5 may transduce signaling pathways including SDC1-Src-ERK. CCL5 is highly expressed in various tumor tissues and is associated with malignancy (Huang et al., 2023). CCL5 expression is higher in invasive skin SCC than in intraepidermal carcinoma, actinic keratosis, and normal tissue (Tuong et al., 2019). Therefore, highly invasive SCC cells secrete CCL5 to promote invasion in vivo.
ERK plays crucial roles in tumor progression, including cell invasion, and metastasis (Guo et al., 2020). ERK phosphorylates cytoskeletal proteins such as myosin light chain kinase and focal adhesion kinase, which are important for cell invasion (Kohno and Pouyssegur, 2006). Recent studies demonstrated that ERK is activated at the wound edge to promote cell migration (Aoki et al., 2017). Our previous study showed that highly invasive A431-6 cells localize to the invasive front and promote collective invasion when co-cultured with less invasive A431-7 cells (Kumagai et al., 2022). Therefore, CCL5 may activate ERK signaling in highly invasive cells, thereby driving collective polyclonal invasion in A431 cells. Although exogenous CCL5 enhances the invasion of low-invasive A431-7 cell clusters, it remains unclear whether CCL5 secreted by A431-6 cells promotes this process. Further studies are required to clarify the role of CCL5 in the collective polyclonal invasion of heterogeneous cell clusters composed of A431-6 and A431-7 cells.
While previous studies have focused on the CCL5/C-C chemokine receptor type 5 (CCR5) axis (Aldinucci et al., 2020), recent studies have indicated that CCL5 promotes cancer cell invasion and migration through SDC1 (Charni et al., 2009; Chen et al., 2022). However, its underlying mechanisms remain poorly understood. In this study, we showed that CCL5-promoted invasion of A431 cell clusters was dependent on the SDC1 expression, suggesting that SDC1 is critical for CCL5-induced invasion in A431 cell clusters. Nevertheless, the direct interaction between CCL5 and SDC1 was not examined in this study; therefore, further investigation is required.
Our findings showed that CCL5 enhanced the invasion of A431 cell clusters through SDC1, Src, and ERK. Targeting these molecules may be a potential therapeutic strategy for suppressing the invasion of SCC.
This work was supported by Japan Agency for Medical Research and Development (AMED) Grant Number JP16gm0810007 to HH, JP17gm0810011 to HH, JP22ym0126814 to HH; JSPS KAKENHI Grant Numbers JP17K07150 to HH, JP21K07142 to HH, JP18K15232 to SI, JP21K07141 to SI, JP23KK0143 to SI, JP24H01917 to SI, JP24K10302 to SI; the cancer research grant of SGH Foundation to SI; Grants-in-Aid for Regional R&D Proposal-Based Program from Northern Advancement Center for Science & Technology of Hokkaido Japan to SI; the research grant of Astellas Foundation for Research on Metabolic Disorders to SI; the research grant of The Uehara Memorial Foundation to SI; the research grant of The Yasuda Medical Foundation to SI; the research grant of The Akiyama Life Science Foundation to SI; recurring donations from MD. Mariko Takamura to HH; Co-Creation Core for Soft Materials Aspiring Research & Translation (C3-SMART) and Japan Society for the Promotion of Science (JSPS) Program for Forming Japan’s Peak Research Universities (J-PEAKS) Grant Number JPJS00420230001 “Soft Materials Platform Aspiring the Unique Properties from Natural Polymers” and “Analysis of cancer specific mechanoresponse for drug discovery.”
Conflict of Interest StatementThe authors declare no competing interests.
Data Availability StatementMicroarray data in this study were published (https://www.nature.com/articles/s41389-022-00403-9).
Author Contribution StatementToko Yamazaki: Writing – original draft, Methodology, Investigation
Yuji Kumagai: Writing – review & editing, Methodology
Kosuke Maeda: Writing – review & editing, Resources
Tomoyasu Aizawa: Writing – review & editing, Resources
Kosuke Maekawa: Writing – review & editing, Methodology
Atsushi Enomoto: Writing – review & editing, Resources
Seiichiro Ishihara: Writing – review & editing, Project administration, Methodology, Supervision, Funding acquisition
Hisashi Haga: Writing – review & editing, Project administration, Supervision, Funding acquisition
Ethics Approval and Consent to ParticipateEthics approval is not required in this study.
Patient Consent for PublicationNot applicable.
The authors thank all the present and former members of the Haga laboratory at Hokkaido University for helpful support.
