Journal: EMBO Molecular Medicine

Article Title: A clinically compatible drug‐screening platform based on organotypic cultures identifies vulnerabilities to prevent and treat brain metastasis

doi: 10.15252/emmm.202114552

Figure Lengend Snippet: Schema of experimental design. Organotypic cultures with established brain metastases from H2030‐BrM cells were treated with DEBIO‐0932 at 1 µM for 6 h and subjected to laser capture microdissection (LCM) and proteomic profiling. Representative image of a fully established brain metastasis from H2030‐BrM before and after laser capture microdissection (LCM). The dotted line delimits the metastasis. Scale bar: 100 µm. Volcano plot with deregulated proteins (red: upregulated; green: downregulated) found in brain metastases treated with DEBIO‐0932 compared to DMSO ( n = 3 biological replicates (mice) per condition, n ≥ 12 brain metastases per mouse were pooled together). Proteins with a P < 0.05 and a log 2 ratio > 1 or < −1 were defined as deregulated. Gray dotted lines indicate P value and log 2 ratio cut offs. The names of the top deregulated proteins are shown. GSEA of top 25 upregulated (red) and downregulated (green; only four fulfill the filter) pathways upon DEBIO‐0932 treatment. Those biological processes represented with more than one signature are labeled with colored lines. Representative images showing AHR and DDA1 levels in brain metastases (generated by intracardiac inoculation of H2030‐BrM) found at endpoint of vehicle and DEBIO‐0932‐treated animals. This result was reproduced in 2 independent staining with different brains. BB: bisbenzamide. Scale bars: low magnification (HSP90 and GFP), 50 µm; high magnification (DDA1), 6 µm (dotted lines). Representative images of squash preparations showing nuclear AHR and UBE4B in established brain metastases from H2030‐BrM generated by intracardiac inoculation. Scale bar: 5 µm. The dashed line surrounds the nucleus. Kaplan–Meier curves showing significant correlation between worse survival post‐brain metastasis and high expression levels of the HSP90 signature ( AHR , DDA1 , UBE4B , GPATCH8 ) in a cohort of 45 breast cancer brain metastasis patients. Representative images (selected cases obtained from Fig EV6M) and histological score of AHR, DDA1 and UBE4B in human brain metastases ( n = 16) according to the signal intensity of the corresponding protein in cancer cells. Schema of the experimental design. H2030‐BrM cells carrying the corresponding shRNA against AHR or the non‐targeting control were inoculated intracardially into nude mice. Ex vivo BLI of brains and thoracic regions were analyzed 5 weeks after injection of cancer cells. Brains were processed for histological analysis. Representative images of brains and thorax from shControl and sh AHR #1 mice at the endpoint of the experiment. Quantification of ex vivo BLI of brains and thoracic regions at the endpoint of the experiment. Values are shown in box‐and‐whisker plots where every dot represents a different animal and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles, and the whiskers go from the minimum to the maximum value ( n = 9 shControl mice and n = 10 sh AHR #1 mice). P value was calculated using two‐tailed t ‐test. Quantification of the bioluminescence signal emitted by H2030‐BrM established metastases in organotypic cultures at Day 7 normalized by the initial value obtained at Day 0 and normalized to the organotypic cultures treated with DMSO. Day 0 is considered right before addition of the treatment or DMSO. Values are shown in box‐and‐whisker plots where each dot is an organotypic culture and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value ( n = 17 organotypic cultures treated with DMSO; n = 18 organotypic cultures treated with BAY‐218, 2 independent experiments). P value was calculated using two‐tailed t ‐test. Schema depicting the evaluation of a clinical cohort composed of 251 ER + breast cancer primary tumors with follow‐up to determine the correlation of UBE4B, DDA1 or AHR with relapse. Representative images of primary tumors with high (red dot) or low (gray dot) UBE4B levels. A few cases of matched primary metastases allowed to evaluate the HSP90‐dependent protein. Scale bar: 100 µm. H‐score analysis of UBE4B in primary tumors with (red) or without (gray) associated relapse. Values are shown in a scattered plot where each dot is a primary tumor and the line corresponds to the median ( n = 100 primary tumors with relapse; n = 147 primary tumors without relapse). P value was calculated using two‐tailed t ‐test. Kaplan–Meier curve comparing relapse‐free survival of primary tumors with high and low values of UBE4B. P value was calculated using log‐rank (Mantel‐Cox) test.

Article Snippet: Slides were incubated with the corresponding primary antibodies as follows: HSP90α/β (clone F‐8, 1:3,000; sc‐13119, Santa Cruz Biotechnology, 8 min), DDA1 (1:2,400; 14995‐1‐AP, ProteinTech), UBE4B (1:500; ab97697, Abcam) and AHR (1:400; 031714, USBiologicals Life Sciences).

Techniques: Laser Capture Microdissection, Labeling, Generated, Staining, Expressing, shRNA, Control, Ex Vivo, Injection, Whisker Assay, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: A clinically compatible drug‐screening platform based on organotypic cultures identifies vulnerabilities to prevent and treat brain metastasis

doi: 10.15252/emmm.202114552

Figure Lengend Snippet: A Representative images showing RPLP1 levels in brain metastases (generated by intracardiac inoculation of H2030‐BrM) found at endpoint of vehicle and DEBIO‐0932‐treated animals. This result was reproduced in two independent staining with different brains. Scale bars: 50 µm. B Quantification of RPLP1 and AHR levels shown in (Figs and ) in arbitrary fluorescent units (A.F.U.). Values are shown in box‐and‐whisker plots where each dot is a metastatic lesion and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles, and the whiskers go from the minimum to the maximum value ( n = 8–16 metastatic lesions from 2 to 4 brains per condition, two independent staining with different brains were performed). P value was calculated using two‐tailed t ‐test. C Quantification of percentage of nuclear DDA1 + BB + cells shown in (Fig ). Values are shown in box‐and‐whisker plots where each dot is a metastatic lesion, and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles, and the whiskers go from the minimum to the maximum value ( n = 16 metastatic lesions from 4 brains per condition, 2 independent staining with different brains were performed). P value was calculated using two‐tailed t ‐test. D Representative images showing p‐ERK levels in organotypic cultures from (Fig ). This result was reproduced in three independent staining with organotypic cultures from different mice. Scale bar: 20 µm. E–H Kaplan‐Meier curves showing significant correlation between worse survival post‐brain metastasis and high gene expression levels of AHR (E), DDA1 (F), UBE4B (G), and GPATCH8 (H) in a cohort of 45 breast cancer brain metastasis patients. I, J Distribution of poor prognosis breast cancer subtypes HER2 + and TNBC within the low and high gene expression level cohorts considering the signature (I) or individual genes (J). K, L Quantification of ex vivo BLI of brains (K) and thoracic regions (L) of mice inoculated with H2030‐BrM cells carrying shControl or sh AHR #2 at the endpoint of the experiment (5 weeks after injection of cancer cells). Values are shown in box‐and‐whisker plots where every dot represents a different animal and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value ( n = 10 shControl mice and n = 10 sh AHR #2 mice). P value was calculated using two‐tailed t ‐test. M Representative sections of brains from shControl and sh AHR #1 mice 5 weeks (experimental endpoint) after intracardiac inoculation of cancer cells. The dotted lines surround the metastases (GFP + ). Scale bar: 1 mm. N Quantification of metastases found in brains inoculated with H2030‐BrM cells with sh AHR . Relative metastatic load was normalized to the respective control. Values are shown in dot plots where every dot represents a different brain and the dotted line corresponds to the mean ± s.e.m. ( n = 8 shControl; n = 5 sh AHR #1; n = 3 sh AHR #2 mice). P value was calculated using two‐tailed t ‐test. O, Q H‐score analysis of AHR (O) and DDA1 (R) in primary tumors with (red) or without (gray) associated relapse. Values are shown in a scattered plot where each dot is a primary tumor and the line corresponds to the median ( n = 100/103 primary tumors with relapse; n = 101/103 primary tumors without relapse, respectively). P value was calculated using two‐tailed t ‐test. P, R Kaplan‐Meier curve comparing relapse‐free survival of primary tumors with high and low values of AHR (P) and DDA1 (R). P value was calculated using log‐rank (Mantel‐Cox) test.

Article Snippet: Slides were incubated with the corresponding primary antibodies as follows: HSP90α/β (clone F‐8, 1:3,000; sc‐13119, Santa Cruz Biotechnology, 8 min), DDA1 (1:2,400; 14995‐1‐AP, ProteinTech), UBE4B (1:500; ab97697, Abcam) and AHR (1:400; 031714, USBiologicals Life Sciences).

Techniques: Generated, Staining, Whisker Assay, Two Tailed Test, Gene Expression, Ex Vivo, Injection, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Protective effects of dexmedetomidine combined with flurbiprofen axetil on remifentanil-induced hyperalgesia: A randomized controlled trial

doi: 10.3892/etm.2016.3687

Figure Lengend Snippet: First analgesic requirement time, hyperalgesic area and sufentanil consumption during the postoperative 24 h.

Article Snippet: The PCIA pump contained sufentanil (150 μg; Humanwell Pharmaceutical Co., Ltd.), ondansetron (8 mg) and normal saline in a total volume of 100 ml, and was set to deliver a 2-ml bolus dose with a 10-min lockout interval with a background infusion of 1 ml/h.

Techniques: