Supplementary information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Reagents. The monoclonal antibodies against TATI and Ki-67 were purchased from MO Bi Tec (PSKAN2, Göttingen, Germany) and DakoCytomation (Glostrup, Denmark), respectively. The polyclonal antibody against murine collagen IV was purchased from Abcam (Cambridge, UK). Clostridium botulinum exoenzyme C3 transferase (abbreviated C3T) was a generous gift from Dr Flatau (INSERM U627, Nice, France), Y27632 was kindly provided by Yoshitomi Pharmaceutical Industries Ltd. (Osaka, Japan). Wortmannin, Gö6976, GF109203X, PD98059, and SB203580 were from Calbiochem (Darmstad, Germany).

Cell culture. HT-29 STD and HT-29 5M21 cells were a gift from Dr T. Lesuffleur. The human cell lines HCT-8/E11, PC/AA/C1, PCmsrc, and MCF7 were cultured by conventional methods, as described (Empereur et al., 1997). The PC/AA/C1 cell line was established from a colonic adenoma in a patient with familial adenomatous polyposis. This cell line is nontumorigenic and exhibits a mucinous phenotype. After transfer of the c-src oncogene, PCmsrc cells became tumorigenic in the athymic nude mice and were invasive upon addition of HGF. HT-29 STD and 5M21 cells were cultured for 24h in serum-free DMEM containing 0.01% BSA and the corresponding CM was collected for the identification of proinvasive factors. CM was concentrated by ultrafiltration using Centricon-3 and -10 (Amicon, Bedford, MA).

Quantitative Polymerase Chain Reaction (qPCR). RNA (1.5 µg) was reverse-transcribed with the 1st-STRAND cDNA Synthesis Kit (Clontech) using hexamer primers. Primers for SPINK1 were designed using the Primers Express software (Applied Biosystems). 18S rRNA was used as an internal standard and was amplified with the target in the same PCR. The 18S rRNA primers and probe were from the Taq Man 18S control reagents kit (Applied Biosystems). For the PCR, 2µl of 1/10 diluted reverse-transcriptase (RT) reaction was mixed with 12.5 µl of Taq Man Universal Master Mix and 200 nM of each specific primer, 200nM probe, 15nM of 18S rRNA primers, and 200 nM of 18S rRNA probe. PCR was performed for 10 min at 95°C, followed by 45 two-step cycles (15 s at 95°C and 1 min at 60°C). All samples were analyzed in triplicate PCR. The cycle threshold values of all samples were measured by the ABI Prism 7700 sequence detector system (Applied Biosystems).

Cellular invasion, tumor growth and metastasis. Collagen type I invasion assays were done as described (Truant et al., 2003). Invasive and superficial cells adherent to collagen type I gels were counted in 12 fields of 0.157 mm2 using an inverted microscope, representing a total of 250- 300 cells examined and screened for each experimental condition. The invasion index is expressed as the percentage of invading cells versus the total cell number of cells. Data are means +/- SEM and the significance of differences between experimental values was assessed by the unpaired Student’s t-test. HT-29 5M21 cancer cells and their corresponding counterparts (2x106 cells) stably transfected by the KY-TATI expression vector were injected subcutaneously into the flanks of female SCID CB17 mice (6 to 8-weeks old, Institut Pasteur de Lille, 3-4 mice per group). Tumor dimensions were measured twice a week, and the volume (V, mm3) calculated as V= (L2xW)/6, L and W being the length and width of the tumor xenografts. Lungs were examined for the presence of metastases by hematein eosin saffron astra blue staining (HESAB). Primary tumor xenografts were examined by histochemistry (HESAB) and immunohistochemistry in order to identify the collagen IV and Ki-67 antigens as relevant angiogenesis and proliferation markers. The Ki-67 proliferation index was assessed in 1000 cells at the tumor periphery, i.e. in areas associated with the highest Ki-67 nuclear signal. Angiogenesis was evaluated by immunostaining of murine collagen IV (Larrivée et al., 2007). Specificity of labelling was checked by control staining performed in the absence of the primary antibody. All the experiments were conducted in agreement with the guidelines of the Animal Care Committee.

Reference List

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