Journal: JCI Insight

Article Title: Epigenetic drug screening defines a PRMT5 inhibitor–sensitive pancreatic cancer subtype

doi: 10.1172/jci.insight.151353

Figure Lengend Snippet: ( A ) Dose-response curves of 4 human PDOs after 6 days of treatment with JNJ-64619178. Viability was determined with CellTiter-Glo assay. Sensitive: red, resistant: blue. ( B ) Microscopy of a sensitive organoid treated with the indicated dose of JNJ-64619178 over 6 days. Scale bar: 500 μM. ( C ) GI 50 values of 24 human PDOs were calculated. Sensitive: red, resistant: blue. ( D ) MYC expression was analyzed in selected PDOs ( n = 7) by Western blot. β-Actin: loading control. Sensitive: red, resistant: blue. One Western blot was performed. ( E ) GI 50 values of 18 primary PDCLs were calculated. Sensitive: red, resistant: blue. ( F ) Relative MYC protein expression, determined by Western blot, in sensitive ( n = 3, red) and resistant ( n = 15, blue) PDCL was compared. P value of Mann-Whitney U test is depicted. MYC Western blot was performed once. ( G ) MYC mRNA expression based on RNA-Seq in JNJ-64619178–sensitive and –resistant PDCLs. * P < 0.05; Mann-Whitney U test. ( H ) JNJ-64619178 GI 50 values of PDOs were grouped into quartiles and differentially expressed genes of most sensitive (first quartile, n = 6) and most resistant (fourth quartile, n = 6) PDOs were calculated. The log-fold change was used as a rank to perform a preranked GSEA. Depicted are the top 10 HALLMARK signatures; q value is color coded. ( I ) JNJ-64619178 GI 50 values of PDCL were grouped into quartiles and analyzed corresponding to H . Sensitive: first quartile, n = 3, resistant: fourth quartile, n = 3. Depicted are the top 10 HALLMARK signatures; q value is color coded. ( J ) Proteomics-based protein expression of PRMT5i-sensitive (first quartile, n = 6) and -resistant (fourth quartile, n = 6) PDOs was used to determine differentially expressed proteins. All proteins upregulated in sensitive PDOs were analyzed using the Enrichr web tool. Combined scores of the HALLMARK signatures with an adjusted P value and q < 0.05 are shown.

Article Snippet: Normalized RNA-Seq data of PDCLs were obtained from the supplementary information of Brunton and colleagues ( ).

Techniques: Glo Assay, Microscopy, Expressing, Western Blot, Control, MANN-WHITNEY, RNA Sequencing

Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

Article Title: An Interleukin-15 Superagonist Enables Antitumor Efficacy of Natural Killer Cells Against All Molecular Variants of SCLC

doi: 10.1016/j.jtho.2022.11.008

Figure Lengend Snippet: Neuroendocrine SCLC express fewer factors that are inhibitory to NK cells. ( A ) Immunofluorescent staining of NCAM1 and MHC-class I proteins in SCLC tissues of a tumor microarray (NCAM1 = red, MHC-class I = white, DAPI = blue). Images representing tumor cases that are POS for NCAM1 and NEG for MHC-class I; double POS for NCAM1 and MHC-class I; NCAM1 NEG/MHC-class I POS; and double NEG for NCAM1 and MHC-class I. ( B ) Quantification of 80 SCLC tumors scored based on positivity for NCAM1 and MHC-class I expression. ( C ) Immunofluorescent staining of ASCL1, NEUROD1, POU2F3, YAP1, and MHC-class I in SCLC tissues of a tumor microarray (ASCL1 = turquoise, NEUROD1 = green, POU2F3 = pink, YAP1 = red, MHC-class I = white, DAPI = blue). Images representing a tumor classified as each of the four subtypes of SCLC. ( D ) Quantification of 79 evaluable tumors based on the predominant transcription factor expression and MHC-class I within each case. ( E ) SCLC tissues of a tumor microarray were analyzed by RNA in situ hybridization for the presence of NK cells. Representative image depicting RNA in situ hybridization staining of CD49b mRNA (pink) and NCR3 mRNA (NKp30, green). ( F ) Quantification displays the number of NK cells per 1000 DAPI POS cells within a standardized ROI; analysis completed on ASCL1 POS MHC-class I NEG , ASCL1 POS MHC-class I POS , NEUROD1 POS , POU2F3 POS (n = 5 tissues each), and YAP1 POS (n = 2 tissues). *p < 0.05 by one-way ANOVA with Tukey’s multiple comparison post-test. ( G ) Heatmap expression of ASCL1, NEUROD1, POU2F3, YAP1, and genes relevant to NK activity in 88 SCLC tumor biopsies obtained from 62 patients with metastatic SCLC previously analyzed by RNAseq analysis (NE = grey, non-NE = black, ASCL1 = purple, NEUROD1 = orange, POU2F3 = blue, YAP1 = green). ( H ) Heatmap of expression of selected NK activating and NK inhibitory ligand genes as determined by quantitative reverse transcription PCR across a panel of SCLC cell lines (NE = neuroendocrine; non-NE = non-neuroendocrine). Values illustrated correspond to log 2 -transformed gene expression relative to the control gene GAPDH in each cell line. ( I ) Neuroendocrine (DMS79, H69) and non-neuroendocrine (DMS114, H841) SCLC cells were assayed for susceptibility to NK cells at an E:Tratio of 10:1 in a 24-hour assay. **** p < 0.0001 comparing NE versus non-NE by unpaired t test. Data illustrated are representative of n = 8 healthy NK donors. #, number; ANOVA, analysis of variance; DAPI, ′,6-diamidino-2-phenylindole; E:T, effector-to-target; NE, neuroendocrine; NEG, negative; NK, natural killer; PCR, polymerase chain reaction; POS, positive; ROI, region of interest.

Article Snippet: Normalized RNAseq data from 88 biopsies from a range of metastatic sites obtained from 62 patients with SCLC available from a previously published report were analyzed on Partek for expression of selected genes relevant to NK activity, including ligands for NK activating and NK inhibitory receptors ( MICA , MICB , ULBP1–3 , RAET1E , RAET1G , RAET1L , NCR3LG1 , PVR , ICAM1 , HLA-A , HLA-B , HLA-C , HLA-E ) and a 20-gene signature of NK infiltration previously reported.

Techniques: Staining, Microarray, Expressing, RNA In Situ Hybridization, Comparison, Activity Assay, Reverse Transcription, Transformation Assay, Gene Expression, Control, Polymerase Chain Reaction