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: bioRxiv

Article Title: Cell Programmed Nutrient Partitioning in the Tumor Microenvironment

doi: 10.1101/2020.08.10.238428

Figure Lengend Snippet: a-b , Representative OCR ( a ) and ECAR ( b ) tracings of MC38 tumor cell fractions with indicated injections of oligomycin (O), FCCP (F), and rotenone and antimycin A (R/AA). c-d Basal mitochondrial OCR ( c ) and cellular ECAR ( d ) of MC38 tumor fractions (n=5 mice). e , Unsupervised cluster analysis of differentially expressed metabolic mRNA transcripts of whole tumor, CD45-cancer cell, CD45+ CD11b+ F4/80hi TAM, CD45+ CD11b+ F4/80lo myeloid cell, CD45+ CD3+ CD8a+ T cell, and CD45+ CD3+ CD8a-(CD4+) T cell flow-sorted MC38 tumor populations (n=2 mice). f-g , Glucose transporter ( f ) and hexokinase ( g ) mRNA transcript levels of indicated MC38 tumor populations (n=2 mice). Dotted line approximates limit of detection. hi , Representative flow cytometry plots ( h ) and quantification ( i ) of pS6 levels in indicated MC38 tumor and spleen populations. j-k , Representative flow cytometry histograms ( j ) and quantification ( k ) of pS6 levels in cancer cells (CD45-CA9+), myeloid cells (CD45+ CD11b+ CD14+), T cells (CD45+ CD3+), and other immune cells (CD45+ CD3-CD14-) from patient ccRCC tumor and PBMC (n=4 patients). Each data point represents a biological replicate and error bars are SEM. a-d and f-g are representative of at least two independent experiments. P values were calculated using Welch’s 2-tailed t-test for (c-d) and Brown-Forsythe ANOVA test for (h, j). * p <0.05, ** p <0.01, *** p <0.001. ECAR: extracellular acidification rate; FCCP: Trifluoromethoxy carbonylcyanide phenylhydrazone; FMO: fluorescence minus one; MFI: median fluorescence intensity; OCR: oxygen consumption rate; PBMC: peripheral blood mononuclear cells; pS6: phosphorylated ribosomal protein S6 (Ser235/236).

Article Snippet: The anti-mouse and cross-reactive antibodies used were: CD45 BV510 (40-F11, Biolegend 103138), B220 e450 (RA3-6B2, ThermoFisher 48-0452-82), CD11b e450 (M1/70, ThermoFisher 48-0112-82), CD11b FITC (M1/70, Biolegend 101206), CD8a AF488 (53-6.7, Biolegend 100723), Ly6C FITC (HK1.4, Biolegend 128006), CD11c PE (N418, BioLegend 117308), FOXP3 PE (FJK-16s, ThermoFisher 12-5773-82), pS6 Ser235/236 PE (D57.2.2E, Cell Signaling 5316S), CD4 PerCP-Cy5.5 (RM4-5, BioLegend 100540), Ly6G PerCP-Cy5.5 (1A8, BioLegend 127616), F4/80 PE-Cy7 (BM8, BioLegend 123114), NKp46 PE-Cy7 (29A1.4, BioLegend 137618), CD3 PE-Cy7 (17A2, BioLegend 100220), CD3 FITC (17A2, BioLegend 100204), CD3 APC (17A2, BioLegend 100236), CD206 APC (C068C2, BioLegend 141708), GLUT1 AF647 (EPR3915, Abcam ab195020), EPCAM PE (G8.8, BioLegend 118206), Thy1.1 PerCP-Cy5.5 (HIS51, ThermoFisher 45-090082), CD45 PE (30-F11, ThermoFisher 12-0451-83), Ly6C BV570 (HK1.4, BioLegend 128030), CD68 BV605 (FA-11, BioLegend 137021).

Techniques: Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: Cell Programmed Nutrient Partitioning in the Tumor Microenvironment

doi: 10.1101/2020.08.10.238428

Figure Lengend Snippet: a , Sorting gates of MC38 tumor cells used for mRNA transcript analyses. b , Expression of selected cell identity markers in flow sorted MC38 tumor cell population. Dotted line approximates limit of detection. c-d , GLUT1 levels determined by flow cytometry in MC38 ( c ) and CT26 ( d ) tumor populations. e, Quantification of pS6 levels determined by flow cytometry in indicated CT26 tumor and spleen populations. Each data point represents a biological replicate and error bars are SEM. c-e are representative of independent experiments performed at least twice. P values were calculated using the Brown-Forsythe ANOVA test. *** p <0.001.

Article Snippet: The anti-mouse and cross-reactive antibodies used were: CD45 BV510 (40-F11, Biolegend 103138), B220 e450 (RA3-6B2, ThermoFisher 48-0452-82), CD11b e450 (M1/70, ThermoFisher 48-0112-82), CD11b FITC (M1/70, Biolegend 101206), CD8a AF488 (53-6.7, Biolegend 100723), Ly6C FITC (HK1.4, Biolegend 128006), CD11c PE (N418, BioLegend 117308), FOXP3 PE (FJK-16s, ThermoFisher 12-5773-82), pS6 Ser235/236 PE (D57.2.2E, Cell Signaling 5316S), CD4 PerCP-Cy5.5 (RM4-5, BioLegend 100540), Ly6G PerCP-Cy5.5 (1A8, BioLegend 127616), F4/80 PE-Cy7 (BM8, BioLegend 123114), NKp46 PE-Cy7 (29A1.4, BioLegend 137618), CD3 PE-Cy7 (17A2, BioLegend 100220), CD3 FITC (17A2, BioLegend 100204), CD3 APC (17A2, BioLegend 100236), CD206 APC (C068C2, BioLegend 141708), GLUT1 AF647 (EPR3915, Abcam ab195020), EPCAM PE (G8.8, BioLegend 118206), Thy1.1 PerCP-Cy5.5 (HIS51, ThermoFisher 45-090082), CD45 PE (30-F11, ThermoFisher 12-0451-83), Ly6C BV570 (HK1.4, BioLegend 128030), CD68 BV605 (FA-11, BioLegend 137021).

Techniques: Expressing, Flow Cytometry