Study patients
We retrospectively surveyed ESCC patients who underwent radical esophagectomy in Keio University hospital between 1997 and 2007. Of these patients, we selected 105 consecutive patients from 1997 to 2002 who were diagnosed with Stage IA-IIIC and 61 patients from 1997 to 2007 who were diagnosed with pathological T1 cancer (tumor invasion into the lamina propia or submucosa), according to the UICC staging system (7th edition).
Immunohistochemistry
CCR7 expression in tissue samples was assessed by immunohistochemistry (IHC) using the ENVISION + system (Dako, Glostrup, Denmark). A rabbit anti-human polyclonal IgG CCR7 antibody (1:100; Medical and Biological Laboratories, Aichi, Japan) was used as a primary antibody. Paraffin-embedded human normal spleen and tonsil tissues were used as positive controls for CCR7. Negative control sections were treated with a non-immunized rabbit immunoglobulin fraction (Dako) under equivalent conditions.
To assess the immunoreactivity, the sections were scored in terms of their proportion (score 0: -10%, 1; 10%–40%, 2: 40%–70%, 3: >70%) and intensity (score 0: none, 1: weak, 2: strong) by two investigators (T.I. and H.T.) who had no knowledge of clinicopathological factors. The total score was calculated by multiplying the two scores and they were defined as positive at ≥1.
Esophageal cell line
We used 10 established ESCC cell lines from the TE series (TE-1, 4, 5, 6, 8, 9, 10, 11, 14, and 15) kindly provided by Dr. Nishihira (Tohoku University, Miyagi, Japan). Identity of each cell line was confirmed by short tandem repeat (STR) analysis (data not shown).
RNA extraction and quantitative real-time RT-PCR
Total RNA from each of the 10 TE cell lines was extracted and analyzed by quantitative real-time RT-PCR using the 7300 Real Time PCR system (Applied BioSystems, Carlsbad, CA), TaqMan Gene Expression Master Mix (Applied BioSystems), and ready-to-use CCR7 primers (Assay ID: Hs99999080_m1; Applied BioSystems). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. We used human lymphocyte from a healthy donor as a positive control and distilled water without the template as a negative control. The relative quantity of CCR7 mRNA in 10 TE cell lines was calculated using ΔΔCt method in which TE1 expression level is defined as 1.
Establishment of a stable CCR7-overexpressing cell line
CCR7 mRNA was extracted from TE8 and its full-length open reading frame (ORF) was amplified and inserted into the plasmid vector pFLAG-CMV-4 (Sigma Aldrich, St. Louis, MO). The plasmids were electroporated into E.coli 5DHα (Takara Bio Inc., Shiga, Japan) and transfected to TE4 cells using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA). We defined this stable transfectant as TE4CCR7+ in which CCR7 overexpression was confirmed by western blotting, cell enzyme-linked immunosorbent assay (ELISA), and real-time RT-PCR.
Adhesion assay
TE4 and TE4CCR7+ cells were labeled with green 5-chloromethylfluorescein diacetate (CMFDA; Invitrogen). To investigate the effect of epithelial cells on cell adhesion, normal human lung lymphatic microvascular endothelial cells (HMVEC-LLy; Lonza Walkersville Inc., Walkersville, MD) were used in this assay, and HMVEC-LLy was labeled with a red fluorescent dye (CMPTX; Invitrogen). Chambers were confluent with a monolayer of HMVEC-LLy. We seeded 5 × 104 cells/well TE4 or TE4CCR7+ and incubated for 10 min at 37°C with or without human recombinant CCL21/SLC (R&D Systems). A blocking assay was performed using mouse monoclonal anti-CCR7 antibody (10 μg/ml; R&D Systems), mouse monoclonal anti-intercellular adhesion molecule (ICAM)-1 (10 μg/ml; Abcam, Cambridge, England), or anti-ICAM-2 antibody (10 μg/ml; Abcam). The number of attached cells in five randomly-selected fields was semiautomatically counted using BIOREVO BZ-9000 microscope (Keyence, Osaka, Japan). These procedures were repeated at least three times.
Development of lymph node metastasis model and quantification of DNA from tumor cells in lymph nodes
Our heterotopic transplantation mouse model of lymph node metastasis was developed as follows: 1) a subcutaneously growing tumor was established by injecting 1 × 107 TE4 or TE4CCR7+ cells into five-week-old nude mice; 2) after the tumors had developed (4–6 weeks after injection), they were excised and a small piece (approximately 1 mm3) was transplanted into the right and left elbows of additional five-week-old nude mice (defined as the TE4 and TE4CCR7+ groups, respectively); 3) the accessory axillary lymph nodes were excised and examined at 3, 4, and 5 weeks after transplantation. Ten lymph nodes from five nude mice were examined each week, and DNA of each node was extracted.
We used a human Alu sequence to quantitatively assess lymph node metastasis. We quantified 90 ng of DNA from each lymph node using primers for the Alu sequence [10]. The primers were 5’-CGCCTGTAATCCCAGCACTTT-3’ (forward), 3’-CCCAGGCTGGAGTGCAGT-5’ (reverse), and 5’-FAMCGAGGCGGGCGGATCACCTBHQ1-3’ (TaqMan Probe). We used DNA extracted from TE4 cells as a positive control while that from the lymph nodes of a non-treated nude mouse was used as a negative control. The estimated number of cells was calculated based on a standard curve prepared prior to this experiment (data not shown, r
2 = 0.998).
Statistical analyses
For statistical analyses, Student’s t-test, Pearson’s χ2 test or Fisher’s exact probability test was used for assessing the correlation between CCR7 expression and clinicopathological characteristics. The Kaplan–Meier and log-rank test were used for survival analysis. Student’s t-test or Welch’s t-test with Bonferroni correction was used for analysis in adhesion assay. In the mouse model, the Mann–Whitney U test was used to calculate statistical significances of the estimated number of cells. All statistical procedures were performed using IBM SPSS Statistics (Version 19; IBM, Armonk, NY). A p value of less than 0.05 was considered statistically significant. This study was approved by the Institutional Review Board at Keio University School of Medicine (ref. 20–125).