Article Figures & Data
Figures
- Figure 1.
Detection of Brachyury protein in lung tumor cells and tissues. Western blot analysis using anti-Brachyury mAb for detection of (A) recombinant His6-Brachyury fusion protein derived from insect cells versus purified human serum albumin (HSA; Sigma-Aldrich) or (B) Brachyury protein in H460 lung carcinoma cells stably transfected with a control- or a Brachyury-specific shRNA-encoding vector. β-Actin was used as protein loading control; shown in the graph is the ratio Brachyury/β-actin. C, representative tissue sections stained with anti-Brachyury mAb corresponding to a grade 3 adenocarcinoma, patient 2 (i); a grade 1 squamous carcinoma, patient 12 (ii); and a grade 1 bronchioloalveolar carcinoma, mucinous type, patient 16 (iii). Tumor cells positive for Brachyury expression are indicated with (t); positive cells in the stroma adjacent to the tumor are indicated with (s). iv, staining of a grade 2 lung adenocarcinoma (patient 10) showing Brachyury-positive tumor cells invading a blood vessel. Arrows indicate endothelial cells. v, staining of a grade 2 adenocarcinoma (patient 9) showing Brachyury positivity in a fraction of tumor cells (t), and cells in the stroma directly adjacent to the tumor (s). Indicated with (n) are tumor cells negative for Brachyury expression (magnification 20×: panels i, ii; 40×: panels, iii–v). D, Western blot analysis of Brachyury expression in nuclear protein lysates prepared from primary lung tumor tissue (tumor) and corresponding normal lung adjacent to the tumor from patients 7 and 17.
- Figure 2.
Expression of EMT markers and mechanism of Brachyury overexpression. A, representative tissue sections stained for Brachyury, E-cadherin, and Vimentin. In one example (adenocarcinoma patient 2, top), the epithelial tumor compartment (indicated with white arrowheads) showed intense expression of Brachyury and simultaneous expression of epithelial E-cadherin and mesenchymal Vimentin. Another example of an adenocarcinoma (patient 6, bottom) showed expression of Brachyury in the epithelial tumor compartment (white arrowheads), with weak expression of E-cadherin and no expression of Vimentin in the tumor cells. In both cases, single disseminated cells within the adjacent stroma (indicated with black arrowheads) stained positive for Brachyury but negative for E-cadherin (magnification 20×). B, results of qPCR analysis for Brachyury copy number in indicated tumor specimens. Pos = positive control reaction with genomic DNA; neg = negative control reaction without DNA. SM = size marker.
- Figure 3.
Expression of Brachyury in human normal tissues. A, representative pictures of normal (i) positive testis, (ii) positive thyroid, (iii) negative lung alveoli, (iv) negative lung bronchioles, (v) negative spleen, and (vi) negative skeletal muscle (magnification 40×). B, real-time PCR analysis of Brachyury, Snail, and Twist mRNA expression in commercial panels of cDNA (*, normal tissue adjacent to tumor). C, Western blot analysis of Brachyury, Snail, and Twist protein expression in commercially available whole-tissue protein lysates obtained from normal testis and normal lung. The following antibodies were used: Snail clone H-130, Twist clone H-81, GAPDH clone 0411 (Santa Cruz Biotechnology). D, real-time PCR analysis of Brachyury, PAP, and CEA mRNA expression in cDNA from normal testis and thyroid tissues.
- Figure 4.
Brachyury and susceptibility to EGFR inhibition. A, real-time PCR analysis of Brachyury mRNA in human primary lung tumors and lung carcinoma lines. B, A549 and H460 lung carcinoma cell pairs were treated with various doses (μmol/L) of the EGFR kinase inhibitor AG1478 for 2 days and assayed for survival by the MTT assay. C, real-time PCR analysis of Brachyury expression in A549 cells grown for 2 weeks in the presence of control DMSO- or 1 μmol/L AG1478-containing medium. D, extracellular matrix invasion assay of A549 cells untreated (parental) or treated as indicated for 2 weeks in culture. *, P < 0.05; ***, P < 0.005.
- Figure 5.
Brachyury as a target for antitumor interventions. A, CTL-mediated lysis of H226 and H441 lung carcinoma cells with a normal donor–derived Brachyury-specific T-cell line I (25:1 effector-to-target ratio). B, lysis of H441 tumor cells with a Brachyury-specific T-cell line II derived from a prostate cancer patient in the presence of cold, competitor K562 A2.1 cells unpulsed or pulsed with the specific Brachyury peptide (Bra pep). C, lysis of H441 and H1703 lung carcinoma and control ASPC1 cells by tetramer-isolated, CD8+ Brachyury-specific T-cell line III derived from a different prostate cancer patient.
Tables
- Table 1.
Brachyury protein expression analyzed by IHC in human primary lung carcinoma tissues
Lung tumor tissues Brachyury positive (%) Adenocarcinoma 10/21 (48) Squamous carcinoma 3/12 (25) Undifferentiated carcinoma 2/4 (50) Bronchioloalveolar carcinoma 1/1 (100) Small cell lung carcinoma 0/1 (0) Total 16/39 (41) - Table 2.
Lung tumor tissues positive for Brachyury protein expression analyzed by IHC
Tumor cells Nuclear Cytoplasmic Adjacent tissue # Histology Grade Stage % positive intensity % positive intensity % positive intensity 1 Adenocarcinoma G2 pT2N0 85 +++ 85 + neg neg 2 Adenocarcinoma G3 pT2N2 80 +++ 80 + 30 +++ 3 Adenocarcinoma G2 pT1aN0 80 ++ 80 + 10 + 4 Adenocarcinoma G2 pT1aN0 60 ++ 60 + neg neg 5 Adenocarcinoma G3 pT2N1 60 + 60 + 50 + 6 Adenocarcinoma G2 pT2N0 40 ++ 20 + neg neg 7 Adenocarcinoma G3 pT2N0 30 + 30 + neg neg 8 Adenocarcinoma G2 pT1bN0 30 + neg neg neg neg 9 Adenocarcinoma G2 pT2N0 10 ++ 10 ++ 30 +++ 10 Adenocarcinoma G2 pT2N1 10 + 10 + neg neg 11 Squamous cell carcinoma G2 pT3N0 80 ++ neg neg neg neg 12 Squamous cell carcinoma G1 pT3N0 50 ++ neg neg 40 + 13 Squamous cell carcinoma — rec 10 + 10 + neg neg 14 Undifferentiated carcinoma G3 pT2N1 80 ++ 60 + 10 + 15 Undifferentiated carcinoma G3 pT2N1 25 + 15 + 50 + 16 Bronchioloalveolar carcinoma G1 pT1bN0 90 ++ 90 + neg neg NOTE: rec = recurrence; neg = negative.
Supplementary Data
Files in this Data Supplement:
- Supplementary Table 1 - PDF file, 62KB, Analysis of Brachyury gene copy number and promoter methylation status.
- Supplementary Table 2 - PDF file, 55KB, Number and percentage of tissues from adult non-cancer subjects that were positive for Brachyury expression by immunohistochemistry.
- Supplementary Table 3 - PDF file, 113KB, Patient characteristics for samples in human lung cancer cDNA arrays.
Volume 18, Issue 14
