Journal: Cancers

Article Title: Integrity of the Antiviral STING-mediated DNA Sensing in Tumor Cells Is Required to Sustain the Immunotherapeutic Efficacy of Herpes Simplex Oncolytic Virus

doi: 10.3390/cancers12113407

Figure Lengend Snippet: Molecular characterization of Sting knockout cancer cell lines. ( A ) Analysis of human HER2 display on cell surface of LLC1-HER2 (left) and CT26_HER2 (right) by FACS analysis; an unrelated antibody was used as negative control. ( B ) The graphic shows Tmem173 (transcript ID ENSMUST00000115728.4) gene organization. Full and empty boxes represent, respectively, coding and untranslated exons. The positions of guide RNAs used for CRISPR/Cas9 genome editing to generate Sting knockout cancer cell lines are indicated by arrows. ( C ) Western blot analysis of Sting protein in CT26-HER2, LLC1_HER2 and their Sting knockout cell lines counterparts. Gamma tubulin was used as standard. ( D ) PCR screening of CT26-HER2_SKO and LLC1_HER2_SKO cell lines to assess the absence of eGFP and Cas9 residues in genomic DNA. Cas9/eGFP-encoding vector was used as positive control (C+). Genomic DNA from parental CT26-HER2 and LLC-HER2 cell lines was used as negative control (C−). ( E ) Cell doubling per day were assessed for Sting wild-type (grey lines) and Sting knockout (black lines) LLC1 (left) and CT26 (right) cell lines. The differences in cell doubling were calculated by Student’s t -test and were not statistically significant (Ns) to each passage.

Article Snippet: Human HER2 transduction of CT26 cells was performed by Origene, Rockville, MD, USA, RC212583L1V.

Techniques: Knock-Out, Negative Control, CRISPR, Western Blot, Plasmid Preparation, Positive Control

Journal: Cancers

Article Title: Integrity of the Antiviral STING-mediated DNA Sensing in Tumor Cells Is Required to Sustain the Immunotherapeutic Efficacy of Herpes Simplex Oncolytic Virus

doi: 10.3390/cancers12113407

Figure Lengend Snippet: Comparison of viral effectiveness in Sting knockout vs. parental wild-type cancer cell lines. ( A , B ) Spread of eGFP-encoding R-LM113 was evaluated by fluorescence microscopy in STING wild-type and knockout LLC1 (5×) ( A ) and CT26 (10×) ( B ) cell lines. ( C ) The lytic activity of R-LM113 was evaluated by extracellular LDH (lactate dehydrogenase) release in cell supernatants over the time course of infection (72, 96 and 120 h) in LLC1-HER2 (grey lines) and LLC1-HER2_SKO (black lines) at two different concentrations of viral particles (1 multiplicity of infection (MOI) continuous lines and 0.5 MOI dashed lines). ( D ) The same experiments performed in panel C were recapitulated in CT26-HER2 and CT26-HER2_SKO. All the infections were performed as biological replicates. The statistical significances for experiments described in panel c and d were calculated by Student’s t -test comparing MOI-matched Sting wild-type vs. knockout cell lines. The p -values were 0.00115 and 0.000219, respectively, for 1 and 0.5 MOI in panel C; 0.01583, 0.008543, respectively, for 1 and 0.5 MOI in panel D. ( E , F ) Evaluation of viral replication of R-LM113 in Sting wild-type and knockout LLC1 ( E ) and CT26 ( F ) infected with 0.3 PFU/cell. The qPCR-TaqMan analysis revealed the genome copies per mL (gc/mL) produced by the virus over time (24, 48, 72 h for LLC1 and 72, 96, 20 h for CT26). The statistical significances for experiments described in panel e and f were calculated by Student’s t -test comparing Sting wild-type vs. knockout cell lines. The p -values calculated on biological replicates were 0.0013 for LLC1 cell line and 0.0005 for CT26 cell line. ( G , H ) Analysis of the R-LM113 viral titers obtained in Sting wild-type and knockout LLC1 ( G ) and CT26 ( H ) cells infected with 0.3 PFU/cell. Plaque assay was performed as biological replicate. The statistical significance for experiments described in panel g and h was calculated by Student’s t -test comparing Sting wild-type vs. knockout cell lines. The p -values were 0.038 for LLC1 cell line and 0.02 for CT26 cell line. p < 0.05 *; p < 0.005 **; p < 0.0005 ***.

Article Snippet: Human HER2 transduction of CT26 cells was performed by Origene, Rockville, MD, USA, RC212583L1V.

Techniques: Knock-Out, Fluorescence, Microscopy, Activity Assay, Infection, Produced, Plaque Assay

Journal: Cancer Cell

Article Title: A living biobank of patient-derived ductal carcinoma in situ mouse-intraductal xenografts identifies risk factors for invasive progression

doi: 10.1016/j.ccell.2023.04.002

Figure Lengend Snippet: HER2 overexpression is a driver for invasive progression of DCIS (A) Representative IHC images of p-ERK and p-AKT. (B) Plotted H scores for p-ERK and p-AKT expression in HER2 + DCIS and HER2 – DCIS controls. (C) Tumor-free survival curves of responsive HER2 + DCIS models treated with vehicle (C) or herceptin (T) with log rank test. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. (D) Tumor-free survival curves of non-responsive HER2 + DCIS models treated with vehicle (C) or herceptin (T) with log rank test. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. (E) Representative whole-mount images and Ku80-stained sections of DCIS-injected mammary glands from DCIS063 mice treated with vehicle (left panels) or herceptin (right panels). (F) Schematic representation of the experimental setup for lentiviral overexpression of HER2 in HER2 – DCIS cells. (G) Western blot showing expression of HER2 and phospho-HER2 in parental 293T cells and 293T cells transduced with the HER2-GFP lentivirus. (H) Representative whole-mount images of intraductally injected mammary glands, showing non-invasive replacement growth of non-transduced HER2 – DCIS cells (left panel) and invasive expansive growth of the same DCIS cells transduced with the HER2-GFP lentivirus (right panel). Inset shows lesion without αSMA marker indicating GFP expression (green). Cells of human origin are marked with Ku80 (gray), myoepithelial cells are marked with αSMA (magenta). Inset shows lesion without αSMA marker indicating HER2-GFP expression (green). See also Figure S7 .

Article Snippet: Primary or PDX DCIS tissue was digested to a single-cell solution as described before and was transfected with a lentivirus (ERBB2-GFP lentivirus, Origene, RC222909L4, or Akaluciferase, courtesy of the lab of Shinae Kizaka-Kondo cloned into the pRRL vector backbone, Addgene plasmid #31485) or vehicle by putting the cells and the virus in a falcon tube and spin for 2 hours at 2500 rpm at RT.

Techniques: Over Expression, Expressing, Staining, Injection, Western Blot, Transduction, Marker

Journal: Cancer Cell

Article Title: A living biobank of patient-derived ductal carcinoma in situ mouse-intraductal xenografts identifies risk factors for invasive progression

doi: 10.1016/j.ccell.2023.04.002

Figure Lengend Snippet: A living biobank of MIND-PDX models of DCIS retaining histological and molecular features of primary lesions (A) Schematic overview of the generation and characterization of 130 MIND-PDX models of DCIS. (B) Pie chart of the grade distribution of the primary DCIS lesions. (C) Pie chart of the DCIS take rate in MIND-PDX models. (D) Examples of H&E-stained sections of the different growth patterns observed in primary DCIS lesions (top row) or DCIS-MIND outgrowths (middle row). Bottom row: human-specific Ku80 staining showing that DCIS-MIND outgrowths have a human origin. (E) Distribution of growth patterns of primary DCIS lesions and the corresponding DCIS-MIND outgrowths. A black line indicates concordance between primary and PDX, whereas an orange line indicates a discordance between primary and PDX. (F) Growth pattern analyses between an early (3–6 months) and late (12 months) time points. (G) Examples of immunohistochemistry for ER, PR, HER2, and Ki67 expression in DCIS-MIND lesions (top row) vs. matched primary DCIS lesions (bottom row). (H) Distribution of molecular subtypes (luminal A: ER + , PR +/− , HER2 − , Ki67 < 20%; luminal B: ER + , PR +/− , HER2 – , Ki67 ≥ 20%; HER2 + : ER +/− , PR +/− , and HER2 + ; basal: ER − , PR − , HER2 − ) of primary DCIS lesions and the corresponding DCIS-MIND lesions. A black line indicates concordance between primary and PDX, whereas an orange line indicates a discordance between primary and PDX. See also Figure S1 and Table S1 .

Article Snippet: Her2 (ERBB2) Human Tagged ORF Clone , Origene , Cat# RC222909.

Techniques: Staining, Immunohistochemistry, Expressing

Journal: Cancer Cell

Article Title: A living biobank of patient-derived ductal carcinoma in situ mouse-intraductal xenografts identifies risk factors for invasive progression

doi: 10.1016/j.ccell.2023.04.002

Figure Lengend Snippet: DCIS-MIND models retain mutational and transcriptional features of the primary DCIS lesions (A) Oncoprint showing the mutational landscape of the primary DCIS lesions, including amplifications, single-nucleotide variants, and insertion-deletions (indels) for the top mutated genes in our breast cancer gene panel. Annotations for each model includes ER, PR, and HER2 status. (B) Oncoprint showing amplifications, single-nucleotide variants, and insertion-deletions (indels) in cancer genes in primary DCIS lesions and corresponding DCIS-MIND lesions for the top mutated genes. (C) Unsupervised clustering of DCIS-MIND lesions based on PAM50 genes, showing clustering of luminal, HER2 + , and basal-like DCIS lesions. Annotations include origin (primary or PDX) and molecular subtype based on PAM50 or IHC. (D) Unsupervised clustering of DCIS-MIND lesions based on 90 informative genes resulting in three DCIS subtypes proposed by Strand et al. See also Figure S2 and Tables S2 and .

Article Snippet: Her2 (ERBB2) Human Tagged ORF Clone , Origene , Cat# RC222909.

Techniques:

Journal: Cancer Cell

Article Title: A living biobank of patient-derived ductal carcinoma in situ mouse-intraductal xenografts identifies risk factors for invasive progression

doi: 10.1016/j.ccell.2023.04.002

Figure Lengend Snippet: Comparison of clinical biomarkers related to invasive progression of DCIS (A) Whole-mount analysis (top row), H&E staining (middle row), and human-specific Ku80 staining (bottom row) of DCIS-MIND lesions showing non-invasive growth (left panel) or invasive growth (right panel). Cells of human origin are marked with Ku80 (green), myoepithelial cells are marked with alpha-smooth muscle actin (αSMA) (magenta). Blue arrows/circles indicate invasive cells. (B) Pie chart of the percentage of DCIS-MIND lesions with non-invasive growth, micro-invasion, or invasive progression. (C) Odds ratio table showing risk scores (univariate and multivariate linear regression models) for association between common clinical parameters, DCIS subtypes as proposed by Strand et al., and multiple invasive recurrence classifiers for invasive progression of DCIS-MIND lesions, identifying HER2 overexpression and solid growth patterns as independent risk factors. Molecular subtype and growth pattern parameters are based on DCIS-MIND characteristics while the other parameters are based on patient characteristics. See also Figure S3 .

Article Snippet: Her2 (ERBB2) Human Tagged ORF Clone , Origene , Cat# RC222909.

Techniques: Comparison, Clinical Proteomics, Staining, Over Expression

Journal: Cancer Cell

Article Title: A living biobank of patient-derived ductal carcinoma in situ mouse-intraductal xenografts identifies risk factors for invasive progression

doi: 10.1016/j.ccell.2023.04.002

Figure Lengend Snippet: HER2 overexpression is a driver for invasive progression of DCIS (A) Representative IHC images of p-ERK and p-AKT. (B) Plotted H scores for p-ERK and p-AKT expression in HER2 + DCIS and HER2 – DCIS controls. (C) Tumor-free survival curves of responsive HER2 + DCIS models treated with vehicle (C) or herceptin (T) with log rank test. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. (D) Tumor-free survival curves of non-responsive HER2 + DCIS models treated with vehicle (C) or herceptin (T) with log rank test. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. (E) Representative whole-mount images and Ku80-stained sections of DCIS-injected mammary glands from DCIS063 mice treated with vehicle (left panels) or herceptin (right panels). (F) Schematic representation of the experimental setup for lentiviral overexpression of HER2 in HER2 – DCIS cells. (G) Western blot showing expression of HER2 and phospho-HER2 in parental 293T cells and 293T cells transduced with the HER2-GFP lentivirus. (H) Representative whole-mount images of intraductally injected mammary glands, showing non-invasive replacement growth of non-transduced HER2 – DCIS cells (left panel) and invasive expansive growth of the same DCIS cells transduced with the HER2-GFP lentivirus (right panel). Inset shows lesion without αSMA marker indicating GFP expression (green). Cells of human origin are marked with Ku80 (gray), myoepithelial cells are marked with αSMA (magenta). Inset shows lesion without αSMA marker indicating HER2-GFP expression (green). See also Figure S7 .

Article Snippet: Her2 (ERBB2) Human Tagged ORF Clone , Origene , Cat# RC222909.

Techniques: Over Expression, Expressing, Staining, Injection, Western Blot, Transduction, Marker

Journal: Cancer Cell

Article Title: A living biobank of patient-derived ductal carcinoma in situ mouse-intraductal xenografts identifies risk factors for invasive progression

doi: 10.1016/j.ccell.2023.04.002

Figure Lengend Snippet:

Article Snippet: Her2 (ERBB2) Human Tagged ORF Clone , Origene , Cat# RC222909.

Techniques: Labeling, Polymer, Recombinant, Electron Microscopy, Plasmid Preparation, Blocking Assay, Software