High interstitial fluid pressure promotes tumor cell proliferation and invasion in oral squamous cell carcinoma

  • Authors:
    • Tao Yu
    • Kun Liu
    • Yingying Wu
    • Jinchuan Fan
    • Jianchao Chen
    • Chao Li
    • Guiquan Zhu
    • Zhaohui Wang
    • Longjiang Li
  • View Affiliations

  • Published online on: September 13, 2013     https://doi.org/10.3892/ijmm.2013.1496
  • Pages: 1093-1100
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Abstract

It has been shown that interstitial fluid pressure (IFP) is elevated in many solid tumors. The elevated IFP in tumors is responsible, at least in part, for the poor blood supply, inadequate delivery of therapeutic agents to solid tumors and poor treatment response in patients. The present study was carried out to examine alterations in malignant phenotypes in oral squamous cell carcinoma cells subjected to conditions mimicking IFP and to identify the relevant molecular mechanisms. We investigated tumor cell proliferation and invasion using SCC-4 and SCC-9 cells subjected to an increased extracellular pressure of 0, 15 and 30 mmHg in vitro. The results revealed that the increased IFP resulted in a marked increase in cancer cell proliferation, survival and invasion in vitro and altered the expression of >1,800 genes involved in invasion and metastasis, the heat shock pathway, the p38 and JNK signaling pathway, apoptosis and the cell growth and differentiation signaling pathway. These results suggest the important potential clinical application of measuring IFP, which can be used as a generic marker of prognosis and response to therapy.

Introduction

Tumors are characterized by high geometric and viscous resistance to blood flow, high microvascular hydrostatic pressure, low resistance to transcapillary fluid flow, high resistance to interstitial fluid flow, impaired lymphatic drainage and an abnormal extracellular matrix (1,2). Excess fluid leaks from the vasculature into the interstitium, where it accumulates and distends the elastic extracellular matrix (ECM), elevating interstitial fluid pressure (IFP). Solid tumors present with an IFP that is elevated above that of normal tissues. A number of studies have shown that solid malignant tumors have IFP values of 5 to 40 mmHg, whereas the IFP of most normal tissues ranges from −3 to +3 mmHg (36). Gutmann et al (3) measured IFP in squamous cell carcinomas of the head and neck region in humans using the wick-in-needle technique and identified that the IFP ranged from 4 to 33 mmHg. The increase in IFP leads to a positive pressure gradient, which is a driving force for a convective transport back into the capillaries or to adjacent regions with low IFP. Such convective forces inhibit the transfer of drugs to the tumor interstitium and facilitate tumor cell intravasation into the vascular or lymphatic circulation, and hence promote metastasis (7). Furthermore, there are clinical data showing that tumor IFP correlates with response to treatment, with strong evidence for high IFP as a poor prognostic indicator in patients with cervical cancer treated with radiotherapy (6,8). Patients are significantly more likely to develop distant recurrence if they present with a tumor IFP value above the group median (19 mmHg), which suggests a role for IFP in metastatic spread (9).

Although local oral squamous cell carcinoma (OSCC) can be effectively controlled by surgical excision and radiotherapy, metastasis to the lymph nodes and distant organs significantly decreases the survival rate (10). Metastasis is the final step in solid tumor progression and is the most common cause of mortality in cancer patients (11). How tumor cells become metastatic is largely unknown. However, several mechanisms have been suggested, including the possibility that tumors with a high IFP may have a high rate of cell proliferation and metastasis (2). It was widely believed that metastatic cells are rare and evolve during thelate stages of tumor progression from a series of changes in gene expression that enable the cells to progress through the sequential steps that finally result in growth in distant organ microenvironments.

Experimental studies attempting to relate the outcome of cancer treatment to IFP are rare. Thus, the mechanisms linking an elevated IFP to poor survival rates in patients with OSCC are not yet well understood. In this study, we subjected SCC-4 and SCC-9 OSCC cells to conditions mimicking IFP in vitro. The cells were incubated for 24 h under 0, 15 and 30 mmHg increased extracellular pressure and we then investigated the alterations in malignant phenotypes and the relevant molecular mechanisms. Furthermore, we identified that an elevated extracellular pressure significantly promotes cancer cell proliferation and invasion by altering the expression of >1,800 genes, which are involved in invasion and metastasis, the heat shock pathway, the p38 and JNK signaling pathway, apoptosis and the cell growth and differentiation signaling pathway using microarray analysis.

Materials and methods

Cell culture and reagents

SCC-4 and SCC-9 human tongue squamous cell carcinoma cell lines were obtained from ATCC. The cells were cultured at 37ºC in 5% CO2 in RPMI-1640 containing 10% fetal bovine serum (FBS; Gibco Life Technologies, Carlsbad, CA, USA), 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/ml penicillin G and 100 mg/ml streptomycin (Sigma, USA). The cell proliferation and cytotoxicity assay kit was from Dojindo Laboratories (Kumamoto, Japan). The real-time PCR primers were synthesized commercially (Takara, Kyoto, Japan). The Invasion Assay kit was from Millipore (Billerica, MA, USA). All tissue culture media and sera were from Gibco Life Technologies. Peroxidase-labeled anti-rabbit antibody and enhanced chemiluminescence (ECL) system were from Millipore.

Pressure regulation

The pressure system used in the present study was custom designed (Sichuan University, Chengdu, China) to expose cultured tumor cells to well-defined stable pressure regimes appropriate for modeling IFP levels in tumors. The cells were placed in an airtight stainless steel box with an inlet valve through which the air flowed into the box and an air pump was connected to this box. There is an absolute pressure sensor (measuring range 30–120 kPa; VTI Technologies, Finland) in the box, which can promptly sense and respond to variations ins pressure. Control and data acquisition programs utilized a pre-calibrated differential pressure transducer to acquire pressure data (needed to control the opening and closing of the appropriate solenoid valves). The pressure box was warmed and maintained at a temperature of 37ºC for at least 1 h prior to each experiment to prevent pressure fluctuations due to temperature shifts of the pressurizing gas. Temperature was maintained within ±2ºC and pressure within ±1.5 mmHg. The whole experimental facility apart from the electronic control panel was placed in a 5% CO2 incubator. The desired pressure was achieved within 1 min for the experiments with the cells. Cells subjected to pressure were placed inside this pressure box for 24 h with the pressure set at 15 and 30 mmHg above atmospheric pressure. The blank group cells were incubated at 37ºC in 5% CO2 under ambient pressure for the same amount of time (Fig. 1).

Cell proliferation assay

Cell proliferation assays were performed using the Cell Counting kit-8 (CCK-8; Dojindo Laboratories). Cells were plated in 96-well plates at 1×104 cells/well and incubated for five days. Each day, at a certain time, 10 μl of CCK-8 solution were added to each well and the cells were incubated for a further 2 h. The optical density (OD) value was obtained by the differences in absorbance at a wavelength of 450 nm using a microplate reader (Varioskan Flash 3001; Thermo Scientific, Waltham, MA, USA). The amount of formazan dye, generated by the activities of dehydrogenases in the cells, is directly proportional to the number of living cells. In addition, we used plate colony formation assay to evaluate the colony forming ability of the tumor cells. The tumor cells were cultured at 500 cells/5 ml with RPMI-1640 medium and 10% FBS in a 6-well plate. After ten days in culture, the cells were fixed with methanol for 10 min and stained with 1% crystal violet solution for 20 min to visualize the colonies for cell counting.

Cell invasion assay

The cell invasion assay was performed using the QCM™ 12-well Invasion Assay kit (Chemicon International, Inc., Temecula, CA, USA). This cell invasion assay is performed in an invasion chamber, based on the Boyden chamber principle. This kit contains 12 inserts; each insert contains an 8-μm pore size polycarbonate membrane coated with a thin layer of ECMatrix™. Briefly, the cells were resuspended in serum-free RPMI-1640, and 2×105 cells were added to the interior of the inserts, which had been previously rehydrated at room temperature for 30 min. A total of 500 μl of RPMI-1640 containing 10% FBS was added to the lower chamber as a chemoattractant. The cells were incubated for 48 h at 37ºC in a CO2 incubator (5% CO2). Subsequently, the upper surface of the chamber was scraped to remove non-migratory cells. The migrated cells were fixed and stained with 0.1% crystal violet and photographed under a light microscope (x100). The stained insert was transferred to a clean well containing 500 μl of 10% acetic acid for 15 min at room temperature. The OD of the stained cells was measured at 560 nm.

Microarray analysis

The 44K Human Genome Oligo Microarray including 44,000 60-mer oligonucleotide probes representing 41,000 unique genes and transcripts was purchased from Agilent Technologies (Palo Alto, CA, USA). In brief, total RNA from the SCC-4 and SCC-9 cells treated with normal ambient pressure, 15 and 30 mmHg, were harvested using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and the RNeasy kit (Qiagen, Hilden, Germany). The amplification and labeling of 500 ng of total RNA was performed according to the protocol provided with the Agilent Quick Amp labeling kit using Cy3. Hybridization was performed for 16 h at 50ºC. Following hybridization and washing, the processed slides were scanned using the Agilent DNA microarray scanner (part no. G2505B; Agilent Technologies). The resulting text files extracted using Agilent Feature Extraction Software (version 10.5.1.1) were imported into the Agilent GeneSpring GX software (version 10.0) for further analysis. To identify the genes that were differentially expressed, we performed a fold-change screening between the two groups obtained from the experiment. The threshold we used to screen the upregulated or downregulated genes was a fold-change of >2. Hierarchical clustering and a tree diagram were generated using Cluster 3.0 software.

Quantitative reverse transcription PCR (qRT-PCR)

Total RNA was isolated from 1×106 cells using TRIzol reagent (Invitrogen). Total RNA was subsequently reverse transcribed into cDNA using the SuperScript First-Strand cDNA Synthesis kit (Invitrogen). qRT-PCR was performed using the SYBR premix Ex Taq™ II kit (Takara). The specific primers used in this study are listed in Table I. The comparative threshold cycle (CT) method was used to calculate the amplification fold. The GAPDH gene was used as a reference control gene to normalize the expression value of the target genes. Triple replicates were performed for each gene and the average expression value was computed for subsequent analysis. The relative expression level of the genes was calculated using the 2−ΔΔCt method as previously described (12).

Table I

Genes and primer sequences used for quantitative RT-PCR.

Table I

Genes and primer sequences used for quantitative RT-PCR.

GenesForward primer (5′-3′)Reverse primer (5′-3′)
HSPA4 GGAGGAACCACATGTTGAAG TGGATCCAGCTTGAGAGGTC
VEGFC AACCTCCATGTGTGTCCGTC TGGCAAAACTGATTGTTACTGG
VEGFR3 CTACAAAGACCCCGACTACG CGTAGTCGGGGTCTTTGTAG
KISS1 CTTGGCAGCTACTGCTTTTC GTAGCAGCTGGCTTCCTCTC
TWIST1 GGCTCAGCTACGCCTTCTC CTAGTGGGACGCGGACAT
VEGFD GTATGGACTCTCGCTCAGCAT AGGCTCTCTTCATTGCAACAG
MAPK8 ACGCCTTATGTAGTGACTCGCTACT TTGTAGCCCATGCCAAGGA
PAI2 GCATCCACTGGCTTGGAA GGGAATGTAGACCACAACATCAT
FOS TCGGGCTTCAACGCAGACTACG TGACCGTGGGAATGAAGTTGGC
HIF1A CATAAAGTCTGCAACATGGAAGGT ATTTGATGGGTGAGGAATGGGTT
CDKN1A CTGCCCAAGCTCTACCTTCC CAGGTCCACATGGTCTTCCT
BAX GCCCTTTTGCTTCAGGGTTT TCCAATGTCCAGCCCATGAT
FOSL1 ATCCCCGACCTCTGACCTAT CAAGGCGTTCCTTCTGCTT
CD44 CCTCCAGTGAAAGGAGCAGCAC GTGTCTTGGTCTCTGGTAGCAGGGAT
CCND1 ACAAGTGTGTCTTACGTGCCACCAC ACGACAGACAAAGCGTCCCTCAAG
STAB1 GTTTGTCACTCACACACCCTGT ATAGCGGCAGTCCAGAAGTATC
BAK1 GAACAGGAGGCTGAAGGGGT TCAGGCCATGCTGGTAGACG
CASP1 AATGATTGAGAAACTCTTCACTGTGT CGGGGTACCAAGCCTAGGAAACACAAGGAGA
ERBB2 AGCAGACCCAGTACCTGTCC AGGGTTGGTCCTTCTATGAGAAT
NM23 ATGGCCAACTGTGAGCGTACC TTCATAGATCCAGTTCTGAGCACAAGC
BCL2 GACAGAAGATCATGCCGTCC GGTACCAATGGCACTTCAAG
ANKK1 CGGCTGGCCAAGTTGTCTAA AGCACCTTCCTGAGTGTCATCA
TNFRSF6 TATGCTTCTTCGTGCAGCAGTT GCTGCCACACGCTCCTCTAG
SMAD2 CCAGGTCTCTTGATGGTCGT TATATCCAGGAGGTGGCGTT
GAPDH GAAGGTGAAGGTCGGAGTC GAAGATGGTGATGGGATTTC
Statistical analysis

Data are expressed as the means ± standard deviation (SD), when normally distributed. The statistical significance of differences was determined using the Student's two-tailed t-test for two groups and one-way ANOVA for multiple groups. A P-value <0.05 was considered to indicate a statistically significant difference. All data were analyzed using SPSS 15.0 software.

Results

Elevated extracellular pressure promotes OSCC cell proliferation and survival in vitro

To detect the effects of extracellular pressure on OSCC cell proliferation and survival, we used the CCK-8 and plate colony formation assays to assess the effect of extracellular pressure on the proliferation and survival of SCC-4 and SCC-9 cells (0, 15 and 30 mmHg). The results revealed that elevated extracellular pressure significantly increased cancer cell proliferation and colony formation, particularly in the group exposed to 30 mmHg pressure (P<0.05) (Fig. 2A–E).

Increased extracellular pressure promotes OSCC cell invasion in vitro

The proteolytic degradation of the ECM components is critical for tumor cell invasion. Matrigel-coated Millicell Chambers were used to determine the influence of extracellular pressure on the invasion capacity of the cancer cells. Quantitative analysis demonstrated that the invasion ability of the cells from the group exposed to 30 mmHg pressure was significantly high, compared with the blank group and the group exposed to 15 mmHg pressure (P<0.01) (Fig. 3). In addition, 15 mmHg extracellular pressure enhanced the invasion ability of the SCC-4 cells, compared with the blank group (P<0.05).

Gene expression profile analysis for OSCC cells subjected to elevated extracellular pressure

To further investigate the effects of increased extracellular pressure on downstream targets in OSCC cells, we performed gene expression profiling analysis on the SCC-4 and SCC-9 cells exposed to 0, 15 and 30 mmHg pressure. With the increase in extracellular pressure, unsupervised clustering analysis of 1,827 genes identified two groups of genes (trees 1 and 2) with significantly altered expression levels (by >2-fold) in the SCC-4 and SCC-9 cells. Tree 1 included 542 upregulated genes, and tree 2 contained 319 downregulated genes. Functional profiling of these genes revealed that the majority of these genes are involved in invasion and metastasis, as well as the heat shock pathway, the p38 and JNK signaling pathway, apoptosis and the cell growth and differentiation signaling pathway (Fig. 4A).

qRT-PCR for tumor-related genes

We then used qRT-PCR to confirm the pressure-dependent expression of over 20 genes identified in the microarray analysis. Apart from the expression of the KISS1 gene in SCC-4 cells, the expression levels of the other genes were consistent with those from microarray analysis (Fig. 4B–C).

Discussion

Since Young et al 13) firstly identified that IFP is significantly higher in tumors compared with normal tissues, an enhanced IFP has been identified in many solid tumors, and it has been used as an important prognostic factor. It has been shown to be the single best indicator of survival for patients with cancer of the cervix (5). Several studies have demonstrated the correlation between IFP and the malignant characteristics of tumors (1416). van der Voort van Zyp et al (16) identified that an increased extracellular pressure of 15 mmHg can stimulate colon cancer cell adhesion to surgical wounds, promoting tumor recurrence. Roh et al (5) found that the IFP increased with the grade (degree of differentiation) of squamous cell carcinoma of the uterine cervix. Although there is no doubt that the IFP is related to tumor size, invasion and metastasis, we wished to determine whether the increased IFP would in turn promote tumor cell proliferation and metastasis. Therefore, in this study, to further investigate the effect of IFP on OSCC cells, we investigated the effects of increased IFP on SCC-4 and SCC-9 cells exposed to conditions mimicking IFP (0, 15 and 30 mmHg above atmospheric pressure) in vitro. Of note, the proliferation and invasion of SCC-4 cells significantly increased in the group exposed to 30 mmHg pressure in vitro. Furthermore, to our knowledge, this is the first study to demonstrate the effect of a high IFP on OSCC cells. The exposure of OSCC cells to extracellular pressure induces an aggressive cancer phenotype that promotes cancer cell growth and metastasis.

To identify the potential molecular mechanisms responsible for the effects of elevated extracellular pressure on tumor growth and invasion, we used microarray analysis to detect the gene expression profiles in SCC-4 and SCC-9 cells treated with different pressure values. A total of 1,827 genes with altered expression levels (by >2-fold) was identified. A total of 542 genes were upregulated with the increase in pressure in the SCC-4 and SCC-9 cells; these genes were mainly involved in invasion and metastasis, the heat shock pathway, the p38 and JNK signaling pathway and the hypoxia signaling pathway. In addition, 319 genes were found to be downregulated with the elevation in pressure values in the SCC-4 and SCC-9 cells, which were mainly involved in apoptosis, cell growth and differentiation and metastasis. Subsequently, we used qRT-PCR to confirm the pressure-dependent expression of over 20 genes identified in the microarray analysis. Heat shock protein (HSP)A4, also known as HSP70, has been shown to be significantly associated with low differentiation and poor prognosis in a number of malignant tumors (1719). CCND1, also known as cyclin D1, is overexpressed in several human tumors; its expression is induced by growth factors and occurs at multiple levels, including increased transcription, translation and protein stability (2022). Cyclin D1 plays an important role in G1 phase transition, which is a kinase-independent function, by sequestering cyclin-dependent kinase (CDK) inhibitors, such as CDKN1A (p21) and CDKN1B (p27) for the efficient activation of CDK2-containing complexes (23,24). Since hypoxia is clearly related to radioresistance, it has been suggested that the abnormal vascularization of tumors leads to increased hypoxia, whereas reperfusion improves the radiosensitivity of tumors (25,26). It has been reported that the hypoxic environment consists of rapidly growing cancer cells that proliferate faster than the vasculature within the environment, creating low oxygen tension in the central and intermediate regions of the tumor mass. This activates the transcriptional factor, hypoxia-inducible factor (HIF-1a), that is known to promote angiogenesis for tumor cell survival and the formation of a central hypoxic region and a normoxic peripheral region; this is possibly regulated by IFP (2729).

Taken together, to our knowledge, this study demonstrates for the first time the important role of an elevated IFP and the corresponding molecular mechanisms, particularly, the effect of lymphatic metastasis-related gene activation on this process, in promoting OSCC proliferation, as well as invasion and metastasis. Our data suggest the important potential clinical application of measuring IFP; measuring tumor IFP may be a generic marker of prognosis and response to therapy.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (grant no. 30973345), the Doctoral Fund of Ministry of Education of China (grant no. 20090181110082) and the Fund of the Department of Health of Sichuan Province (grant no. 130231).

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November 2013
Volume 32 Issue 5

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Spandidos Publications style
Yu T, Liu K, Wu Y, Fan J, Chen J, Li C, Zhu G, Wang Z and Li L: High interstitial fluid pressure promotes tumor cell proliferation and invasion in oral squamous cell carcinoma. Int J Mol Med 32: 1093-1100, 2013
APA
Yu, T., Liu, K., Wu, Y., Fan, J., Chen, J., Li, C. ... Li, L. (2013). High interstitial fluid pressure promotes tumor cell proliferation and invasion in oral squamous cell carcinoma. International Journal of Molecular Medicine, 32, 1093-1100. https://doi.org/10.3892/ijmm.2013.1496
MLA
Yu, T., Liu, K., Wu, Y., Fan, J., Chen, J., Li, C., Zhu, G., Wang, Z., Li, L."High interstitial fluid pressure promotes tumor cell proliferation and invasion in oral squamous cell carcinoma". International Journal of Molecular Medicine 32.5 (2013): 1093-1100.
Chicago
Yu, T., Liu, K., Wu, Y., Fan, J., Chen, J., Li, C., Zhu, G., Wang, Z., Li, L."High interstitial fluid pressure promotes tumor cell proliferation and invasion in oral squamous cell carcinoma". International Journal of Molecular Medicine 32, no. 5 (2013): 1093-1100. https://doi.org/10.3892/ijmm.2013.1496