in tumor progression. ADAM17-silencing inhibits in vivo growth of 
MC38CEA tumors We next decided to evaluate the effect of ADAM17-silencing on in vivo growth of MC38CEA tumors. After confirming that the mock-transfection does not influence in vivo MC38CEA tumor development we compared the growth of tumors induced by implanting the same number of cells of two different mock-transfected cell lines and three different ADAM17silenced cell lines. As shown in Fig. 2A all ADAM17-silenced tumors at the end of the experiment were over 70% smaller than the mock-transfected ones. Because individual mock-transfected and ADAM17-silenced cell lines differed in the level of CEA expression, we performed the majority of the following experiments using M2 and S3 cell lines which expressed almost identical levels of CEA as measured by flow cytometry to ensure correspondence between the two model cell lines. Immunohistochemical staining of blood- and lymphatic vessels Immunochemical staining of frozen tissue sections was performed according to the standard protocol. Briefly, 10 mmthick cryosections were air-dried and fixed with a zinc-based fixative for 24 h, blocked with 10% donkey- and 10% goat serum in PBS and stained with hamster anti-CD31/Alexa fluor 546conjugated anti-hamster IgG; rabbit anti-lymphatic endothelium specific antigen-1 /Alexa Fluor 488-conjugated anti-rabbit IgG; rat anti-CD11b/Alexa fluor 594-conjugated anti-rat IgG; APC-conjugated rat anti-CD45. In negative controls 16722652 primary antibodies were omitted. Cell nuclei were counterstained with DAPI. Sections were imaged on a Zeiss LSM 510 Meta confocal microscope at magnification 4006. Five randomly chosen fields of peritumoral area and five randomly chosen fields of tumor peripheral area were analyzed in each section. The area covered by CD31+LYVE-12 blood vessels in tumor periphery were calculated using MetaMorph imaging software and expressed as the percentage of the total area. Zymography Zymography was performed according to a standard protocol. Cells were cultured for 24 h in 6-well plates in DMEM containing 5% FCS and for the next 24 h in fresh, serumfree DMEM. The samples of cell media were subjected to SDS-PAGE in 10% gels containing 0.1% gelatin. The gels were stained with Coomassie Brilliant Blue, resulting in a blue background of nondegraded gelatin with cleared bands of proteolytic activity. ADAM17 silencing does not affect MC38CEA growth in vitro In some tumor models ADAM17 was shown to support autocrine stimulation of cell growth by shedding EGF family members. In those models, expression of ADAM17 positively correlated with in vivo tumor progression and in vitro proliferation rate of 24074843 tumor cells in serum-free medium. Does the same mechanism explain the requirement of ADAM17 for a robust MC38CEA development Surprisingly, the silencing of ADAM17 in MC38CEA cells did not affect in vitro cell growth and 5-Carboxy-X-rhodamine biological activity viability. All mock-transfected and ADAM17-silenced cell lines showed exactly the same 3Hthymidine incorporation during 6-h incubation with the radiolabeled nucleotide that followed a 24-h starvation period. In another type of experiment the cells were plated at a low density and cultured for 5 days in the absence of serum or at low serum concentration. The cells completely deprived of serum stopped growing no later than on the third day after plating, which is in contrast to many cancer cell Statistical analysis Statistical analysis was performed using the Student’s t-test, with P,
