Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Targeting COX-2 and EP4 to control tumor growth, angiogenesis, lymphangiogenesis and metastasis to the lungs and lymph nodes in a breast cancer model.

doi: 10.1038/labinvest.2012.90

Figure Lengend Snippet: Figure 1 COX-1/2 and COX-2 inhibitors and EP4 antagonist reduce VEGF-C and VEGF-D production in C3L5 cells. (a) Western blot analysis of proteins in C3l5 cell lysates revealed expression of COX-2 and all the EP receptors. (b, c) Both COX1/2 and COX-2 inhibitors as well as the EP4 (but not EP1, EP2 and EP3) antagonist suppressed VEGF-C and -D mRNA levels relative to GAPDH mRNA (presented as a fraction of control vehicle-treated cells) as well as secreted VEGF-C (d, e) measured with ELISA. C3L5 cells express VEGF-C (80 kDa) and VEGF-D (21 kDa) (f, control) measured with western blot, which were reduced by treatments with COX inhibitors and EP4 (but not EP1) antagonist, as compared with vehicle-treated control cells (f, g). siRNA-mediated knockdown of EP4 resulted in significant drop in VEGF-C and VEGF-D mRNA (h). Data presented in (a, b), (c, d) and (e, f) are from three different representative experiments. Data represent means (n ¼ 4)±s.e., *Po0.05, **Po0.01, ***Po0.001.

Article Snippet: EP4 siRNA products consisted of pools of three to five targetspecific 19–25 nucleotide siRNAs designed to knockdown mouse EP4 gene expression (SC-40174, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and silence select negative control siRNA (Cat. No: 4390843, Ambion) was used as EP4SC siRNA.

Techniques: Western Blot, Expressing, Control, Enzyme-linked Immunosorbent Assay, Knockdown

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Targeting COX-2 and EP4 to control tumor growth, angiogenesis, lymphangiogenesis and metastasis to the lungs and lymph nodes in a breast cancer model.

doi: 10.1038/labinvest.2012.90

Figure Lengend Snippet: Figure 2 Therapy with COX-1/2 and COX-2 inhibitors or EP4 antagonist, but not EP1 antagonist reduces primary tumor growth in C3H/HeJ mice. (a) Representative images of tumor-inclusive Matrigel implants retrieved on day 16 (a scale in mm shown in the background). (b) Image of only Matrigel implant retrieved on day 16. (c) Mean weights of tumors were reduced significantly in mice treated with indomethacin, celecoxib or EP4 antagonist ONO- AE3-208 (EP4A), but not EP1 antagonist ONO-8713 (EP1A), compared with respective vehicle-treated controls. This reduction was significant day 12 with celecoxib and EP4A, and highly significant on day 16 for all therapies except EP1A. Data represent mean (n ¼ 16 per group per day)±s.e. *Po0.05, **Po0.005.

Article Snippet: EP4 siRNA products consisted of pools of three to five targetspecific 19–25 nucleotide siRNAs designed to knockdown mouse EP4 gene expression (SC-40174, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and silence select negative control siRNA (Cat. No: 4390843, Ambion) was used as EP4SC siRNA.

Techniques:

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Targeting COX-2 and EP4 to control tumor growth, angiogenesis, lymphangiogenesis and metastasis to the lungs and lymph nodes in a breast cancer model.

doi: 10.1038/labinvest.2012.90

Figure Lengend Snippet: Figure 3 Therapy with non-selective or selective COX-2 inhibitors or EP4 antagonist reduced tumor-associated angiogenesis and lymphangiogenesis in C3H/HeJ mice. (a) Bright field images of Masson’s Tricrome-stained sections (first and third columns) showing peritumoral stroma inclusive of vessels (scale bars in mm given as insets), and fluorescence images of corresponding tumors representing ‘hot spots’ within the tumors (second and fourth columns). Tumor-associated angiogenesis (CD31 immuno-staining in red) and lymphangiogenesis (LYVE-1 immuno-staining in green), are shown as representative fluorescent merged images. There was practically no overlap between the vessels identified by two colors confirming the specificity of the markers. Therapy with COX-1/2 and COX-2 inhibitors or EP4 antagonist but not EP1 antagonist significantly reduced both angiogenesis and lymphangiogenesis. Representative images are shown in (a) and quantified as corresponding ‘hot spot’ scores for CD31 and LYVE-1 (b) (n ¼ 16, using the mean of three hot spots from each of the 16 tumors per group) ±s.e., *Po0.05, **Po0.005.

Article Snippet: EP4 siRNA products consisted of pools of three to five targetspecific 19–25 nucleotide siRNAs designed to knockdown mouse EP4 gene expression (SC-40174, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and silence select negative control siRNA (Cat. No: 4390843, Ambion) was used as EP4SC siRNA.

Techniques: Staining, Fluorescence, Immunostaining

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Targeting COX-2 and EP4 to control tumor growth, angiogenesis, lymphangiogenesis and metastasis to the lungs and lymph nodes in a breast cancer model.

doi: 10.1038/labinvest.2012.90

Figure Lengend Snippet: Figure 4 Therapy with COX-1/2 inhibitor or COX-2 inhibitor or EP4 antagonist abrogated regional and distant lymph node metastasis. Histological pictures (H&E stained) of representative lymph nodes: Day 8: inguinal nodes in control (vehicle treated) (a, inset magnified fourfold in (b) and EP4A treated (c, inset magnified fourfold in d) mice. Lymph node in (a) is completely replaced by tumor cells (marked as T) showing the trail of invasion from the primary tumor on the left. Lymph node in (c) is tumor free. Day 12: nodes in (e, f) show metastatic tumor cells (T), outlined by red markings; lymph nodes in (g, h) are tumor-free. Day 16: nodes in (i, j) show tumor cells (T), outlined by red markings. Nodes in (k, l) are tumor free. Scale bars for magnification in images are shown as insets (50 mm for all, except for a and c, 200 mm).

Article Snippet: EP4 siRNA products consisted of pools of three to five targetspecific 19–25 nucleotide siRNAs designed to knockdown mouse EP4 gene expression (SC-40174, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and silence select negative control siRNA (Cat. No: 4390843, Ambion) was used as EP4SC siRNA.

Techniques: Staining, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Targeting COX-2 and EP4 to control tumor growth, angiogenesis, lymphangiogenesis and metastasis to the lungs and lymph nodes in a breast cancer model.

doi: 10.1038/labinvest.2012.90

Figure Lengend Snippet: Figure 5 Therapy with COX-1/2 and COX-2 inhibitors or EP4 antagonist reduces metastatic lung colony formation. (a) Representative images of micro metastases in H&E-stained lung sections on day 16. They were noted as early as day 8 (data not presented). Scale bar represents 100 mm. (b) Median numbers of metastatic lung colonies were reduced with all treatments (except EP1A) at all-time points after tumor transplantation. (n ¼ 8 per group per day). Data represent median±quartile deviations. *Po0.05; **Po0.01.

Article Snippet: EP4 siRNA products consisted of pools of three to five targetspecific 19–25 nucleotide siRNAs designed to knockdown mouse EP4 gene expression (SC-40174, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and silence select negative control siRNA (Cat. No: 4390843, Ambion) was used as EP4SC siRNA.

Techniques: Staining, Transplantation Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Targeting COX-2 and EP4 to control tumor growth, angiogenesis, lymphangiogenesis and metastasis to the lungs and lymph nodes in a breast cancer model.

doi: 10.1038/labinvest.2012.90

Figure Lengend Snippet: Figure 6 Therapy with COX-1/2 inhibitor, COX-2 inhibitor and EP4 antagonist reduces the levels of VEGF-C, -D and phosphorylated AkT proteins in residual tumors. Total tumor lysate proteins (pooled from eight tumors per group, triplicate measurements) were subjected to western blots for pAkT and total AkT, VEGF-C and -D and GAPDH proteins. Densitometric measurements of pAkT relative to total AkT, and VEGF-C and -D relative to GAPDH reveal a significant reduction of all these parameters in the drug-treated groups at all intervals, as compared with vehicle-treated controls. Data represent mean±s.e., *Po0.05.

Article Snippet: EP4 siRNA products consisted of pools of three to five targetspecific 19–25 nucleotide siRNAs designed to knockdown mouse EP4 gene expression (SC-40174, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and silence select negative control siRNA (Cat. No: 4390843, Ambion) was used as EP4SC siRNA.

Techniques: Western Blot

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Targeting COX-2 and EP4 to control tumor growth, angiogenesis, lymphangiogenesis and metastasis to the lungs and lymph nodes in a breast cancer model.

doi: 10.1038/labinvest.2012.90

Figure Lengend Snippet: Figure 7 EP4 antagonist therapy increases the apoptotic/proliferative cell ratios in residual tumors. (a) Representative abundance of proliferative cells labeled for Ki67 marker (red), and apoptotic cells labeled for TUNEL (green) in situ are illustrated in serial sections for day 12 in vehicle-treated (control) and EP4A-treated mice. Images of corresponding H&E-stained tumors in adjacent sections are shown on the left (scale bars are 50 mm, shown as insets). (b) The ratios of apoptotic/proliferative cells at different days following vehicle or EP4A therapy. Data represent mean (n ¼ 12)±s.e., *Po0.05.; **Po0.005 comparing the two groups. The ratios showed a significant increase with time in EP4A-treated mice and decrease in control mice.

Article Snippet: EP4 siRNA products consisted of pools of three to five targetspecific 19–25 nucleotide siRNAs designed to knockdown mouse EP4 gene expression (SC-40174, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and silence select negative control siRNA (Cat. No: 4390843, Ambion) was used as EP4SC siRNA.

Techniques: Labeling, Marker, TUNEL Assay, In Situ, Control, Staining