Supplementary data for

Reciprocal interaction between carcinoma-associated fibroblasts and squamous carcinoma cells via interleukin 1α induces cancer progression

Jung Yoon Bae, Eun Kyoung Kim, Dong Hyun Yang, Xianglan Zhang, Young-Jin Park, Doo Young Lee, Chung Min Che, and Jin Kim*

Department of Oral Pathology, Oral Cancer Research Institute, Yonsei University College of Dentistry, Seoul 120-749, Korea

*Correspondence to: Jin Kim, Department of Oral Pathology, Oral Cancer Research Institute, Yonsei University College of Dentistry, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, Korea. Phone: 82-2-2228-3031; Fax: 82-2-392-2959; e-mail:

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Supplementary materials and methods

Cell culture

Three kinds of CAFs were obtained from the surgical specimens of OSCC patients. Three kinds of NFs were obtained from normal oral mucosa of the patients who third molar teeth were removed. All procedures were approved by the Institutional Review Board of Yonsei University College of Dentistry (IRB 2-2012-0027) in advance. After the CAFs were isolated according to the established procedure [1], their identities were verified withα-SMA and vimentin immunostaining (Supplementary Figure S1B). CAFs and NFs were cultured in a medium composed of Dulbecco’s Modified Eagles Medium (DMEM; Gibco BRL) and Ham’s Nutrient Mixture-F12 (Gibco BRL) mixed at a ratio of 3:1, supplemented with 10% FBS and 1% penicillin/streptomycin (F medium) at 37°C in a humidified chamber with 5% CO2 in air. Cancer cell lines were cultured in EF medium which is a mixture of F medium, supplemented with 0.01 μg/ml cholera toxin, 0.04 μg/ml hydrocortisone, 0.5 μg/ml insulin, 0.5 μg/ml apo-transferrin, and 0.2 μg/ml 3’-5-triodo-l-thyronine (Sigma) at 37°C in a humidified chamber with 5% CO2 in air. OSCC cell lines YD-10B, YD-32, and YD-38 were previously established in our laboratory [2,3]. Oral cancer cell line HSC-2 was provided by Prof. Takashi Muramatsu, Tokyo Dental College, Japan. The identities of all the cancer cell lines were validated by short tandem repeat (STR) DNA fingerprinting using the AmpFLSTR® Identifiler® PCR Amplification Kit according to the manufacturer's instructions (Applied Biosystems; catalogue no.: 4322288) at the department of Forensic Medicine, Yonsei University College of Medicine. The STR profiles were compared with the fingerprint database of Japanese Collection of Research Bioresources Cell Bank (JCRB, http://cellbank.nibio.go.jp) and previously published paper [2]. STR profiles of all the cell lines were identical with the known DNA fingerprints.

Immunofluorescent staining

The following antibodies were used for immunofluorescent staining: FITC-conjugated anti-α-SMA and Cy3-conjugated anti-vimentin antibodies (Sigma). The antibodies were diluted in PBS with 1% BSA (1: 200 for FITC-α-SMA, 1:200 for Cy3-anti-vimentin) and incubated at room temperature for 2 h. Nuclei were stained with 10 μg/ml of 4’, 6-diamidino-2-phenylindole (DAPI, Sigma). Cells were photomicrographed using confocal microscopy.

Immunohistochemistry

Samples obtained from mouse orthotopic xenograft model and human OSCC tissues were fixed with 10% neutral formalin, embedded in paraffin, cut into 4 µm-thick sections, and mounted on silane-coated slides (Muto Pure Chemicals Co. Ltd.). Sections were deparaffinized and re-hydrated in xylene and varying concentrations of ethanol. Antigen retrieval in mouse tissues was achieved by boiling sections in citrate buffer (pH 6.0) for 2 min. Endogenous peroxidase was blocked by incubating sections in a freshly prepared 3% hydrogen peroxide solution for 10 min. Sections were then allowed to cool and placed in PBS. Before applying primary antibodies, sections were blocked with 5% BSA (Sigma) at room temperature for 30 min. Sections from mouse orthotopic xenograft model were immunostained with antibodies specific for PCNA (1:100, mouse monoclonal, Dako) and vimentin (1:100, human monoclonal, Dako) at 4°C for 16 h. Sections from human OSCC tissues were immunostained with antibodies specific for IL1α (1:50, Rabbit polyclonal, Abcam) at 4°C for 16 h. Anti-IgG1 kappa antibody (1:100, mouse monoclonal, Dako) was used as a negative control. Peroxidase-labeled anti-mouse/rabbit IgG (Dako) was then applied at room temperature for 30 min. The sections were visualized with 3, 3-diaminobenzidine tetrachloride (DAB, Vector Labs), counter-stained with Mayer’s hematoxylin, mounted, and examined with a light microscope.

Reverse transcription-polymerase chain reaction (RT-PCR)

Total cellular RNA was extracted from YD-10B, YD-32, YD-38, HSC-2, and three kinds of CAFs (CAFs 1-3) cells using an RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA (1 µg) was used for first-strand cDNA synthesis. The cDNA product was amplified by PCR using a Gene Amp PCR System 9700 (Applied Biosystems) in a final reaction volume of 20 µl using Maxim PCR PreMix Kit (Intron). Primer sequences were as follows: 5’-TCTGTCACTGCCCAAGATGAA-3’ (sense) and 5’-CGTGAGTTTCCCAGAAGAAGA-3’ (antisense) for IL1α; 5’-GGCCGAGATCTCACTGACTA-3’ (sense) and 5’-AGTGGCCATCTCATTTTCAA-3’ (antisense) for α-SMA; 5’-GACAATGCGTCTCTGGCACGTCTT-3’ (sense) and 5’-TCCTCCGCCTCCTGCAGGTTCTT-3’ (antisense) for vimentin; 5’-CCAGGAGATCCACCTTTTCA-3’ (sense) and 5’-ACGCAGGGTAAGTGGTATCG-3’ (antisense) for FAP; 5’-GAAGGTGAAGGTCGGAGT-3’ (sense) and 5’-GAAGATGGTGATGGGATTTC -3’ (antisense) for GAPDH. Cycling conditions were as follows: 94°C for 1 min followed by 30 cycles of 94°C for 40 s, 53°C for 1 min, and 72°C for 40 s. Final extension step was performed at 72°C for 10 min. Amplified products were then separated on a 1.0% agarose gel stained with 0.1 µg/ml of ethidium bromide, and photographed under UV light.

Western blotting

CAFs co-cultured OSCC cells were lysed using RIPA buffer (Cell signaling). Subsequently, 25 μg of protein was subjected to SDS-PAGE with 10% gradient polyacrylamide gels (Bio-Rad). The separated proteins were then transferred to PVDF membranes (Bio-Rad). Membranes were probed with antibody specific for IL1R1 (1:1000, rabbit monoclonal, Abcam) at 4°C for 16 h. Anti-rabbit secondary antibody (1:2000, Cell signaling) were conjugated with horseradish peroxidase at room temperature for 1 h and visualized by western blotting luminal reagent (Santa Cruz).

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Supplementary references

[1] Liu Y, Hu T, Shen J, Li SF, Lin JW, Zheng XH, Gao QH, and Zhou HM (2006). Separation, cultivation and biological characteristics of oral carcinoma-associated fibroblasts. Oral Dis 12, 375-380.

[2] Kim EJ, Che ZM, Park YJ, Hwang YS, Kim KY, Jung da W, Jeon NK, Choi YW, Lee EJ, and Kim J (2009). Morphogenesis and biological significance of spindle cell transformation in a spindle cell carcinoma. Cancer Lett 275, 61-71.

[3] Lee EJ, Kim J, Lee SA, Kim EJ, Chun YC, Ryu MH, and Yook JI (2005). Characterization of newly established oral cancer cell lines derived from six squamous cell carcinoma and two mucoepidermoid carcinoma cells. Exp Mol Med 37, 379-390.


Supplementary figure legends

Figure S1. (A) mRNA expression of α-SMA, vimentin, and FAP in CAFs. Expression levels of mRNAs in CAFs cells were determined by RT-PCR. Micrographs shown in this figure are representative of three independent experiments that showed similar results. Results were normalized to GAPDH expression. (B) Human α-SMA and vimentin expressions in CAFs. The micrographs shown in this figure are representative of five different fields that showed similar results. All sections were viewed at 200X magnification (scale bar: 100 μm).

Figure S2. Correlation between the stromal proportion and α-SMA expression in 100 OSCC patients. Analysis of Pearson correlation was employed to assess correlation between the staining data of H&E and the staining data of α-SMA in 100 OSCC patients.

Figure S3. Tumor progression in mouse orthotopic xenograft model. (A) Different doses of CAFs were co-injected with OSCC cells into dorsal tongues of nude mice. The mice were sacrificed after 4 weeks and macroscopic morphology of the tumor mass was obtained. (B) Human vimentin expression in mouse OSCC (magnification: 200X, scale bar: 100 μm). A higher magnification view of the same region is presented in the inset micrograph (magnification: 500X, scale bar: 100 μm). The micrographs shown in this figure are representative of five different tissues that showed similar results. Human vimentin-positive cells were also counted in five randomly selected microscopic fields, and the average percentage of vimentin-positive cells was calculated. The results are shown as mean values ± SD (n = 5) and were analyzed by the Mann-Whitney U test.

Figure S4. Proliferation and invasiveness of OSCC cells co-cultured with NFs or CAFs. OSCC cells were co-cultured with either NFs or CAFs. OSCC cells co-cultured with OSCC cells or mono-cultured OSCC cells were used as controls. (A) Microscopic morphology was obtained (magnification: 100X, scale bar: 500 μm) and cell proliferation rate after 48 h was determined by the MTT assay. (B) Invasion assay was performed. After incubation at 37°C for 48 h, microscopic morphology was obtained (magnification: 40X, scale bar: 200 μm) and cells that penetrated the upper filter were counted manually under a light microscope. The micrographs shown in this figure are representative of three independent experiments that showed similar results. Quantitative results indicate average values of three independent experiments each of which was conducted in triplicate (n = 9). The results are shown as mean values ± SD (n = 9) and were analyzed by the Mann-Whitney U test (*p 0.05 and **p 0.01).

Figure S5. (A) Expression of IL1R1 proteins in CAFs co-cultured with or without OSCC cells was determined by Western blotting. Results were normalized to β-actin expression (*p 0.05). (B) Surgical specimens of two cell lines, YD-10B and YD-32, were stained with H&E (magnification: 40X, scale bar: 200 μm).

Figure S6. Cytokine secretion under various co-culture conditions. Cells under various co-culture conditions were incubated at 37°C, and conditioned media were harvested after 48 h. Secretion levels of (A) CCL7, (B) CXCL1, and (C) IL8 were evaluated by ELISA. Quantitative results indicate average values of three independent experiments each of which was conducted in triplicate (n = 9). The results are shown as mean values ± SD (n = 9) and were analyzed by the Mann-Whitney U test (*p 0.05 and ***p 0.001).

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