Journal: Nature communications

Article Title: Disruption of the HER3-PI3K-mTOR oncogenic signaling axis and PD-1 blockade as a multimodal precision immunotherapy in head and neck cancer.

doi: 10.1038/s41467-021-22619-w

Figure Lengend Snippet: Fig. 1 HER3 is a candidate driver of the PI3K/mTOR oncogenic signaling circuitry in HNSCC. a Experimental scheme of the kinome siRNA library screen. A “smart pool” of four individual siRNAs targeting each protein kinase gene of the human kinome is distributed in each well of the experimental plates. Cal27 cells were incubated for 72 h and assayed for cell viability, and the Z-score for viability was calculated (Z = (x −µ)/σ) (x stands for each value of cell viability; µ stands for average value; σ stands for standard deviation). b siRNA library targeting human kinases using the kinome siRNA library with Cal27 cells was conducted to search for genes that affect proliferation of HNSCC. Shown are the genes whose knockdown decrease cell viability (Z-score). The blue are the top 20 genes and HER3 is shown in red. (See Supplementary Data 1 for complete list). c Cal27 cells were transfected with the corresponding siRNAs (top 20 hits) for 72 h and cell lysates were analyzed for pS6 by western blotting. Densitometry analysis of western blots was performed using ImageJ. Shown are the top 20 hits of the kinome siRNA screen and their Z-scores, together with changes in pS6 levels after each gene was knocked down, as compared to the non-targeting siRNA group. Similar levels of S6 and GAPDH as loading control were confirmed (Supplementary Fig. 1). d The TCGA (The Cancer Genome Atlas) database was used to determine the relationship between HER3 phosphorylated on tyrosine 1289 (PY1289) and overall survival (OS) (n = 122 HNSCC patients, two sided log-rank test; p = 0.033). e Histogram demonstrating HER3 expression in EpCAM+ E-CAD+ tumor cells (red) and tumor-infiltrating CD4+ and CD8+ T cells (blue) in a fresh surgical specimen from a Stage II T2N0M0 primary tongue squamous cell carcinoma, compared to Cal27 cells (orange) as control. FMO (fluorescence minus one) samples were used to create HER3 + staining gates. f Immunofluorescent staining of CK5, CD8, and HER3 in the same specimen in panel (e), showing HER3 co-expressed with cancer cells (CK5 positive), but not with CD8+

Article Snippet: The following antibodies were used: pS6 (Cell Signaling Technology #2211, 1:200), Brdu (Bio-Rad #OBT0030S, 1:100), and Cleaved Caspase-3 (Cell Signaling Technology # 9661, 1:400).

Techniques: Incubation, Standard Deviation, Knockdown, Transfection, Western Blot, Control, Expressing, Staining

Journal: Nature communications

Article Title: Disruption of the HER3-PI3K-mTOR oncogenic signaling axis and PD-1 blockade as a multimodal precision immunotherapy in head and neck cancer.

doi: 10.1038/s41467-021-22619-w

Figure Lengend Snippet: Fig. 4 Anti-tumor effect of HER3 kinase inhibition with CDX-3379 antibody in vivo. a WT Cal27 cells, Cal27 cells expressing PIK3CA H1047R or Detroit 562 were transplanted into the flanks of athymic nude mice, and when they reached 150–200 mm3, mice were treated with vehicle diluent or CDX-3379 (10 mg/kg, three times/week) for the indicated days (n = 10 for Cal27; n = 10 for Cal27 PIK3CA H1047R; n = 6 for Detroit 562). Data were reported as mean ± SEM; two-sided Student’s t-test. b Representative H&E stains of mouse tumors from the experiment from panel a. c Representative immunohistochemical analysis of pS6 and BrdU in the short-term treatment (every other day for three times) groups from panel (a) (n = 4 mice per group). Brown chromogen deposition reflects the immunoreactivity; hematoxylin was used as a nuclear counterstain (blue). Scale bars represent 25 μm. Quantification from images using Qupath software and the percentage of positive staining are shown on each image. Data were reported as mean ± SEM, two-sided Student’s t-test, p > 0.05, non-significant or ns; ***p < .001 when compared with the control-treated group. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used: pS6 (Cell Signaling Technology #2211, 1:200), Brdu (Bio-Rad #OBT0030S, 1:100), and Cleaved Caspase-3 (Cell Signaling Technology # 9661, 1:400).

Techniques: Inhibition, In Vivo, Expressing, Immunohistochemical staining, Software, Staining, Control

Journal: Nature communications

Article Title: Disruption of the HER3-PI3K-mTOR oncogenic signaling axis and PD-1 blockade as a multimodal precision immunotherapy in head and neck cancer.

doi: 10.1038/s41467-021-22619-w

Figure Lengend Snippet: Fig. 6 Anti-tumor effect of HER3 inhibition in syngeneic HNSCC models and increased durable responses to PD-1 blockade. a C57Bl/6 mice were implanted with 1 × 106 of 4MOSC1 cells into the tongue. After tumors reached ~30 mm3, mice were treated IP with of isotype control, CDX-3379 (20 mg/ kg), anti-PD-1 (10 mg/kg), or a combination of CDX-3379 and PD-1 three times per week for 3 weeks. Individual growth curves of 4MOSC1 tumor-bearing mice are shown (n = 10 per group). b C57Bl/6 mice were implanted with 2 × 106 MOC1 cells into the flanks. After tumors reached approximate 50 mm3, mice were treated same as panel (a). Individual growth curves of MOC1 tumor-bearing mice are shown (n = 8 per group). c A Kaplan–Meier curve showing the survival of mice from panels (a) and (b). The death of animals occurred either naturally, when tumor compromised the animal welfare, when tongue tumor volume (panel a) reached 100 mm3 (n = 10 mice per group), or when flank tumor volume (panel b) reached 500 mm3 (n = 8 mice per group). Two sided log-rank/Mantel–Cox test. d Representative immunohistochemical analysis of pS6 and BrdU in the short-term treatment groups (every other day for three treatments) from panel (a). Brown chromogen deposition reflects the immunoreactivity; hematoxylin was used as a nuclear counterstain (blue). Scale bars represent 25 μm. Quantification from images on the left using Qupath software and the percentage of positive staining are shown on each image. e Immunofluorescent staining of CD8 and CK5 in the short-term treatment from panel (a). Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used: pS6 (Cell Signaling Technology #2211, 1:200), Brdu (Bio-Rad #OBT0030S, 1:100), and Cleaved Caspase-3 (Cell Signaling Technology # 9661, 1:400).

Techniques: Inhibition, Control, Immunohistochemical staining, Software, Staining

Journal: Cancer cell international

Article Title: MicroRNA-550a-3-5p controls the brain metastasis of lung cancer by directly targeting YAP1.

doi: 10.1186/s12935-021-02197-z

Figure Lengend Snippet: Fig. 7 The effects of miR-550a-3-5p on cleaved-PARP, pRB, CDK6, YAP1, CTGF, and CYR61. A Western blotting images. B Cleaved-PARP protein expression. C pRB protein expression. D CDK6 protein expression. E YAP1 protein expression. F CTGF protein expression. (G) CYR61 protein expression. *P < 0.05, compared with the control group; #P < 0.05, compared with the miR-550a-3-5p mimics group

Article Snippet: After blocking with 5% skim milk, the membranes were incubated with anti-cleaved-Poly(ADP-ribose) polymerase (PARP) antibody (1:1000, Abcam), anti-RB transcriptional corepressor 1 (pRB) antibody (1:1000, Abcam), anti-cyclin dependent kinase 6 (CDK6) antibody (1:1000, Abcam), anti-Yes1 associated transcriptional regulator (YAP1) antibody (1:1000, Abcam), anti- connective tissue growth factor (CTGF) antibody (1:1000, Proteintech Group, Inc.), anti-cysteine rich angiogenic inducer 61 (CYR61) antibody (1:1000, Abcam), and anti-GAPDH antibody (1:10,000, Proteintech Group, Inc.) overnight at 4 °C.

Techniques: Western Blot, Expressing, Control