A- CHD1 and beta-catenin expression in Vector-control and PAR-3 KD cells.

<p>A- CHD1 immunofluorescent staining of Vector-control and PAR-3 KD cells Vector-control and PAR-3 KD cultures were stained for e-cadherin immunofluorescence and photographed using 25× objective as described in Methods. Blue- DAPI nuclear counterstain. <b>B-</b> Beta-catenin immunocytochemical staining. Vector-control or PAR-3 KD cultures were processed and stained for beta-catenin as described in Methods. Photomicrographs were acquired using ×4 objective.</p

Increased adhesion of PAR-3 KD cells due to initial rate.

<p>Experiments were performed as described in Legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093879#pone-0093879-g001" target="_blank">Fig. 1B</a>. The bars represent ratios of 0 to 2 min or 2 to 20 min adhesion rate values of the indicated PANC-1 variants to those of vector-control cells. The data are presented as ratios of averages obtained from four independent experiments. * denotes p<0.02, all other differences not significant.</p

Cadherins' mRNAs levels of vector-controls and PAR-1 or -3 KD cells.

<p>Vector-control, PAR-1 or -3 KD cells were assayed for mRNA levels of the designated cadherins and normalized to GAPDH mRNA levels. The results are presented as fold difference of respective KDs mRNA levels to those of vector-controls. The results represent mean±SE, * denotes p<0.02</p

Integrins' mRNAs levels of vector-controls and PAR-1 or -3 KD cells.

<p>A- Vector-control, PAR-1 or -3 KD cells were assayed for mRNA levels of the designated integrins and normalized to GAPDH mRNA levels. The results are presented as fold difference of respective KDs mRNA levels to those of vector-controls. The results represent mean±SE of at least 3 independent experiments. B-Representative western blot analysis of ITGαV and GAPDH in Vector-Control, PAR-1 or -3 KD cells. Lowe panel-quantification of protein expression from three different western blots with 3 different cell lysates for each sample. The results represent mean±SE of 3 independent experiments.</p

The effect of E-cadherin inhibition on adhesion.

<p>Vector-control or PAR-3 KD cells were plated in 96-wells clusters at 25,000 cells/well and allowed to adhere for 30 min. EGTA (5 mM) or HAV inhibitor peptide (1 mM) were added at time 0.</p

The effects of PARs knockdown on wound closure on wound closure kinetics.

<p>Vector control and the desired PARs knockdown cells were seeded at approximately 80% density. The kinetics of wound closure were performed as described in Methods. The results represent mean±SE of 5 independent experiments performed in triplicates. P<0.05 for Vector vs. PAR-3 KD and >0.05 vs. PAR-1 KD.</p

Table_1_A clinical evaluation of an ex vivo organ culture system to predict patient response to cancer therapy.docx

IntroductionEx vivo organ cultures (EVOC) were recently optimized to sustain cancer tissue for 5 days with its complete microenvironment. We examined the ability of an EVOC platform to predict patient response to cancer therapy.MethodsA multicenter, prospective, single-arm observational trial. Samples were obtained from patients with newly diagnosed bladder cancer who underwent transurethral resection of bladder tumor and from core needle biopsies of patients with metastatic cancer. The tumors were cut into 250 μM slices and cultured within 24 h, then incubated for 96 h with vehicle or intended to treat drug. The cultures were then fixed and stained to analyze their morphology and cell viability. Each EVOC was given a score based on cell viability, level of damage, and Ki67 proliferation, and the scores were correlated with the patients’ clinical response assessed by pathology or Response Evaluation Criteria in Solid Tumors (RECIST).ResultsThe cancer tissue and microenvironment, including endothelial and immune cells, were preserved at high viability with continued cell division for 5 days, demonstrating active cell signaling dynamics. A total of 34 cancer samples were tested by the platform and were correlated with clinical results. A higher EVOC score was correlated with better clinical response. The EVOC system showed a predictive specificity of 77.7% (7/9, 95% CI 0.4–0.97) and a sensitivity of 96% (24/25, 95% CI 0.80–0.99).ConclusionEVOC cultured for 5 days showed high sensitivity and specificity for predicting clinical response to therapy among patients with muscle-invasive bladder cancer and other solid tumors.</p

Table_3_A clinical evaluation of an ex vivo organ culture system to predict patient response to cancer therapy.docx

IntroductionEx vivo organ cultures (EVOC) were recently optimized to sustain cancer tissue for 5 days with its complete microenvironment. We examined the ability of an EVOC platform to predict patient response to cancer therapy.MethodsA multicenter, prospective, single-arm observational trial. Samples were obtained from patients with newly diagnosed bladder cancer who underwent transurethral resection of bladder tumor and from core needle biopsies of patients with metastatic cancer. The tumors were cut into 250 μM slices and cultured within 24 h, then incubated for 96 h with vehicle or intended to treat drug. The cultures were then fixed and stained to analyze their morphology and cell viability. Each EVOC was given a score based on cell viability, level of damage, and Ki67 proliferation, and the scores were correlated with the patients’ clinical response assessed by pathology or Response Evaluation Criteria in Solid Tumors (RECIST).ResultsThe cancer tissue and microenvironment, including endothelial and immune cells, were preserved at high viability with continued cell division for 5 days, demonstrating active cell signaling dynamics. A total of 34 cancer samples were tested by the platform and were correlated with clinical results. A higher EVOC score was correlated with better clinical response. The EVOC system showed a predictive specificity of 77.7% (7/9, 95% CI 0.4–0.97) and a sensitivity of 96% (24/25, 95% CI 0.80–0.99).ConclusionEVOC cultured for 5 days showed high sensitivity and specificity for predicting clinical response to therapy among patients with muscle-invasive bladder cancer and other solid tumors.</p

Data_Sheet_3_A clinical evaluation of an ex vivo organ culture system to predict patient response to cancer therapy.docx

IntroductionEx vivo organ cultures (EVOC) were recently optimized to sustain cancer tissue for 5 days with its complete microenvironment. We examined the ability of an EVOC platform to predict patient response to cancer therapy.MethodsA multicenter, prospective, single-arm observational trial. Samples were obtained from patients with newly diagnosed bladder cancer who underwent transurethral resection of bladder tumor and from core needle biopsies of patients with metastatic cancer. The tumors were cut into 250 μM slices and cultured within 24 h, then incubated for 96 h with vehicle or intended to treat drug. The cultures were then fixed and stained to analyze their morphology and cell viability. Each EVOC was given a score based on cell viability, level of damage, and Ki67 proliferation, and the scores were correlated with the patients’ clinical response assessed by pathology or Response Evaluation Criteria in Solid Tumors (RECIST).ResultsThe cancer tissue and microenvironment, including endothelial and immune cells, were preserved at high viability with continued cell division for 5 days, demonstrating active cell signaling dynamics. A total of 34 cancer samples were tested by the platform and were correlated with clinical results. A higher EVOC score was correlated with better clinical response. The EVOC system showed a predictive specificity of 77.7% (7/9, 95% CI 0.4–0.97) and a sensitivity of 96% (24/25, 95% CI 0.80–0.99).ConclusionEVOC cultured for 5 days showed high sensitivity and specificity for predicting clinical response to therapy among patients with muscle-invasive bladder cancer and other solid tumors.</p

Table_2_A clinical evaluation of an ex vivo organ culture system to predict patient response to cancer therapy.docx

IntroductionEx vivo organ cultures (EVOC) were recently optimized to sustain cancer tissue for 5 days with its complete microenvironment. We examined the ability of an EVOC platform to predict patient response to cancer therapy.MethodsA multicenter, prospective, single-arm observational trial. Samples were obtained from patients with newly diagnosed bladder cancer who underwent transurethral resection of bladder tumor and from core needle biopsies of patients with metastatic cancer. The tumors were cut into 250 μM slices and cultured within 24 h, then incubated for 96 h with vehicle or intended to treat drug. The cultures were then fixed and stained to analyze their morphology and cell viability. Each EVOC was given a score based on cell viability, level of damage, and Ki67 proliferation, and the scores were correlated with the patients’ clinical response assessed by pathology or Response Evaluation Criteria in Solid Tumors (RECIST).ResultsThe cancer tissue and microenvironment, including endothelial and immune cells, were preserved at high viability with continued cell division for 5 days, demonstrating active cell signaling dynamics. A total of 34 cancer samples were tested by the platform and were correlated with clinical results. A higher EVOC score was correlated with better clinical response. The EVOC system showed a predictive specificity of 77.7% (7/9, 95% CI 0.4–0.97) and a sensitivity of 96% (24/25, 95% CI 0.80–0.99).ConclusionEVOC cultured for 5 days showed high sensitivity and specificity for predicting clinical response to therapy among patients with muscle-invasive bladder cancer and other solid tumors.</p