Cells
293T human embryonic kidney cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nissui, Tokyo, Japan) containing 10% fetal bovine serum (FBS, Gibco®-Life Technologies, Grand Island, NY, USA). Two human colon cancer cell lines, HCT116 and WiDr cells, were cultured in McCoy’s 5A (Gibco®-Life Technologies) and RPMI 1640 (Nissui) medium containing 10% FBS, respectively.
Transfection of Rac1 V12 and Rac1 N17 in 293T cells
For the GST pull-down assay, immunofluorescence, and construction of the cell block, 293T cells were transiently transfected with pCXN2-Flag-Rac1 V12 or pCXN2-Flag-Rac1 N17 using Fugene HD transfection reagent (Promega, Madison, WI, USA).
GST pull-down assay and immunoprecipitation for detecting activated Rac1
293T cells with or without enforced Rac1 V12 or Rac1 N17 were lysed with lysis buffer (25 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% NP40, 10 mM MgCl2, 10% glycerol, 1 mM PMSF). For the GST pull-down assay, 1 mg of protein lysate was incubated with 10 µg GST-PAK2 RBD (recombinant protein) with rotation for 30 min at 4 °C, followed by incubation with 20 µl glutathione Sepharose 4B beads (GE Healthcare, Bio-Sciences Uppsala, Sweden) with additional rotation for 30 min at 4 °C. After brief centrifugation, the supernatant was discarded, and the precipitates were washed with lysis buffer three times. Final precipitates were suspended in 20 µl of 2 × SDS sample buffer (100 mM Tris–HCl (pH 6.8), 4% SDS, 10% β-mercaptoethanol, 20% glycerol, and 0.01% BPB), boiled for 5 min, and subjected to electrophoresis using a 12% SDS–PAGE gel. PVDF membrane-transferred separated proteins were immunoblotted with mouse monoclonal anti-Rac1 antibody (Ab) (BD Transduction Laboratories, CA, USA). For immunoprecipitation, 1 mg of protein lysate was incubated with anti-active Rac1 Ab (New East, Malvern, PA, USA) at 4 °C overnight. After brief centrifugation, the subsequent steps were performed as described above.
To reveal activation of Rac1 in human colon cancer cell lines, HCT116 and WiDr cells were serum-starved overnight and stimulated with FBS for 30 min. The cell lysate was subjected to GST pull-down assay as described above.
Immunofluorescence for activated Rac1 in 293T cells
293T cells expressing Rac1 V12 were cultured on glass-based dishes (IWAKI, Tokyo, Japan) and fixed in 3% paraformaldehyde (Nacalai Tesque, Kyoto, Japan) in PBS for 15 min. The cells were permeabilized with 0.1% Triton X-100 for 4 min and blocked with 1% BSA for 20 min. To detect activated Rac1, the cells were treated with 2 µg/ml GST-PAK-RBD for 2 h at room temperature (RT), followed by anti-GST Ab overnight at 4 °C and Alexa Fluor 488-conjugated anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) for 1 h at RT. In comparison, the fixed and permeabilized cells were stained with anti-active Rac1 Ab (New East) at 4 °C overnight. Images were acquired using a fluorescence microscope (Olympus, Tokyo, Japan).
Immunostaining of activated Rac1 in 293T cell block
To establish a method for detecting activated Rac1 in formalin-fixed paraffin embedded (FFPE) tissues, optimal conditions of immunostaining were investigated using cell blocks of 293T cells with or without enforced Rac1 V12. 293T cells were transfected as described above and fixed in formalin, and cell blocks were constructed. The concentration of GST-PAK (2, 5, 10 µg/ml), dilution ratio of α-GST Ab (× 50, × 100 dilution), formalin-fixed duration (30 min, 2 h, 1 day, 2 days, 3 days), and requirement of antigen retrieval (pressure cooker: PC, in pH 6.0 citric acid buffer) were investigated. The established protocol was as follows: incubation at 37 °C for 10 min, deparaffinization for 10 min, endogenous peroxidase (3% H2O2) for 5 min, GST-PAK-RBD for 5 min using an R-IHC machine, 1st Ab (α-GST Ab) at 37 °C for 30 min and for an additional 10 min at RT, polymer 2nd Ab for 30 min (Dako). During each step, specimens were washed with PBS containing 0.5% Tween 20. After coloring with diaminobenzidine (DAB, Dako) within 50 s, the tissues were nuclear stained (Mayer) for 30 s, dehydrated, penetrated, and mounted (Supplementary Fig. 1). In principle, this method also recognizes activated Cdc42.
Detection of activated Rac and Cdc42 in mouse xenografts of human colon cancer cell lines
HCT116 and WiDr cells (1 × 107) were subcutaneously injected into six- to eight-week-old female 10 nude mice, Balb/cA Jcl nu/nu (Clea Japan, Inc., Tokyo, Japan) per cell type. After approximately one month, the mice were sacrificed by cervical fracture under isoflurane inhalation anesthesia, and the growing tumor tissues were resected. The tumors that grew to almost equal volume were randomly divided into two groups within the weight range group (3 mice/group) and fixed in formalin or the PAXgene Tissue system (Qiagen), which is a formalin-free system designed to improve the quality of molecular analysis without disturbing those of histopathological analysis. Rac and Cdc42 activation in the excised tumors was evaluated as described above. All animal experiments were conducted in accordance with the guidelines of the Hokkaido University Manual for Implementing Animal Experimentation, which was consistent with the ARRIVE Guidelines for the Care and Use of Laboratory Animals and for study design and approved by the Institutional Animal Care and Use Committee at Hokkaido University Graduate School of Medicine (Number 12-0092). All researchers who performed procedures using live animals were preapproved by the Animal Welfare Committee of Hokkaido University. For each animal experiment, four different investigators were involved as follows: two investigators (RH, LW) performed the surgical procedure and all steps for making tumor specimens and were aware of treatment group allocation (formalin or PAX gene fixation). Two investigators (MaTs, MiTa) who were unaware of treatment evaluated the staining intensity and pattern of active Rac1/Cdc42.
Analysis of activated Rac and Cdc42 in human colon cancer tissue microarray (TMA) specimens
In tissue microarray (TMA) specimens from 33 patients with colon cancer (Stage 0–IV: 33 cases) diagnosed at the Department of Cancer Pathology, Hokkaido University Graduate School of Medicine between 2003 and 2015 with informed consent from participants, activated Rac and Cdc42 were immunostained, as shown in Supplementary Fig. 1. DAB coloring indicating Rac/Cdc42 activity was analyzed in the tumor area and normal mucosa using Histoquest software (Novel Science Co., Ltd, Tokyo, Japan). Correlations between Rac/Cdc42 activity and lymphatic vessel invasion, venous invasion, and lymph node metastasis were analyzed. Human studies followed the ethical guidelines for clinical application, in accordance with the Declaration of Helsinki. All ethical issues related to human pathological specimens were discussed and approved by the Ethics Committee of Hokkaido University Graduate School of Medicine (Number 15-022).
Analysis of activated Rac and Cdc42 in the human colon cancer FFPE tissues
The colon cancer FFPE tissues of 50 patients diagnosed with colon cancer at the Department of Cancer Pathology, Hokkaido University Graduate School of Medicine between 2003 and 2015 with informed consent from participants are summarized in Supplementary Table 1 (Stage 0: 3 cases; Stage I, II, IIIA, and IV: each 10 cases; and Stage IIIB: 7 cases). Activated Rac and Cdc42 were immunostained and evaluated with the R-IHC system as described above. In identical patients, Rac/Cdc42 activity in normal mucosa and tumor areas was measured, and the activation patterns of Rac/Cdc42 were investigated in the tumor areas. Statistical analyses were performed between the Tis, T1, and T2 groups (low invasion) and the T3 and T4 groups (high invasion) using Student’s t test. A value of P < 0.05 was considered statistically significant. All ethical issues related to human pathological specimens were discussed and approved by the Ethics Committee of Hokkaido University Graduate School of Medicine (Number 15-022).
Statistical analysis
Graphical data are presented as the mean and standard deviation (S.D.), and Student’s t test was used for comparisons, with P < 0.05 considered significant. The F test was used to investigate the equal or unequal variance of samples.
