Journal: Cancer Science

Article Title: Regulation of programmed cell death ligand 1 expression by atypical protein kinase C lambda/iota in cutaneous angiosarcoma

doi: 10.1111/cas.13981

Figure Lengend Snippet: Expression of programmed cell death 1 ( PD ‐1) and its ligand ( PD ‐L1) in cutaneous angiosarcoma tissue. A,B, Representative confocal images of PD ‐L1 + and PD ‐L1 − (red or white) (A) and PD ‐1 + / PD ‐1 − (red or white) (B) tumors. Transformed endothelial cells were stained with an antivascular endothelial ( VE )‐cadherin Ab (green). Nuclei were stained with Hoechst (blue). Scale bar = 50 μm. Middle and right panels are higher magnification images of areas indicated on left panels

Article Snippet: Formaldehyde‐fixed paraffin‐embedded angiosarcoma tissue samples were deparaffinized, and antigen retrieval was carried out by boiling the slides in EDTA buffer (pH 8.0) for 15 minutes, blocked with 5% BSA/5% FBS/0.1% Tween‐20 for 30 minutes, and treated with rabbit anti‐human PD‐L1 Ab (1:100 dilution; Cell Signaling Technology, Danvers, MA, USA), mouse anti‐human PD‐1 Ab (1:100 dilution; Cell Signaling Technology), and goat anti‐human vascular endothelial (VE) ‐cadherin Ab (1:100 dilution; R&D Systems, Minneapolis, MN, USA) at 4°C overnight.

Techniques: Expressing, Transformation Assay, Staining

Journal: Cancer Science

Article Title: Regulation of programmed cell death ligand 1 expression by atypical protein kinase C lambda/iota in cutaneous angiosarcoma

doi: 10.1111/cas.13981

Figure Lengend Snippet: Correlation of programmed cell death ligand 1 ( PD ‐L1) expression with poor prognosis of patients, the presence of pS er218 Forkhead box protein O1 (FoxO1), and atypical protein kinase C lambda/iota ( aPKC λ) expression. A, Kaplan‐Meier curve of patients with PD ‐L1 + / PD ‐L1 − tumors revealed the poor prognosis of patients with PD ‐L1 + tumors (log‐rank, * P < .05). B, Kaplan‐Meier curve of the patients with PD ‐1 + / PD ‐1 − tumors failed to show any significant difference between these 2 groups (log‐rank, P = 1.00). C, PD ‐L1 expression strongly correlated with pS er218 FoxO1 expression on cutaneous angiosarcoma tissue, analyzed by Fisher's exact test using 2 × 2 contingency table ( P = .018*). PD ‐L1 expression related to strong aPKC λ expression with marginal statistical difference analyzed by Fisher's exact test ( P = 0.068). D, Representative confocal images of PD ‐L1 (red)/ pF oxO (blue) or aPKC (red)/ pF oxO (blue) with vascular endothelial‐cadherin ( VE cad; green)

Article Snippet: Formaldehyde‐fixed paraffin‐embedded angiosarcoma tissue samples were deparaffinized, and antigen retrieval was carried out by boiling the slides in EDTA buffer (pH 8.0) for 15 minutes, blocked with 5% BSA/5% FBS/0.1% Tween‐20 for 30 minutes, and treated with rabbit anti‐human PD‐L1 Ab (1:100 dilution; Cell Signaling Technology, Danvers, MA, USA), mouse anti‐human PD‐1 Ab (1:100 dilution; Cell Signaling Technology), and goat anti‐human vascular endothelial (VE) ‐cadherin Ab (1:100 dilution; R&D Systems, Minneapolis, MN, USA) at 4°C overnight.

Techniques: Expressing

Journal: Cancer Science

Article Title: Regulation of programmed cell death ligand 1 expression by atypical protein kinase C lambda/iota in cutaneous angiosarcoma

doi: 10.1111/cas.13981

Figure Lengend Snippet: Suppression of programmed cell death ligand 1 (PD‐L1) expression by atypical protein kinase C lambda/iota (aPKCλ) knockdown (KD) in physiologic and pathologic endothelial cells (ECs) in vitro with RT‐PCR and western blotting. A,B, PRKCI KD HUVECs showed markedly reduced PD‐L1 expression compared to control ECs. Reduced transcription of CD274 was analyzed by real‐time RT‐PCR (n = 3) (A), and decreased PD‐L1 protein expression was shown by western blot analysis (B) (n = 3). C,D, PRKCI KD AS‐M cells showed markedly reduced PD‐L1 expression compared to control ECs. Reduced transcription of CD274 was analyzed by real‐time RT‐PCR (n = 3) (C), and decreased PD‐L1 protein expression was shown by western blot analysis (n = 3) (D). Relative amount of PD‐L1 in PRKCI KD ECs compared to control EC is shown. Data represent mean ± SEM by 2‐tailed unpaired t test. * P < .05. FoxO1, Forkhead box protein O1

Article Snippet: Formaldehyde‐fixed paraffin‐embedded angiosarcoma tissue samples were deparaffinized, and antigen retrieval was carried out by boiling the slides in EDTA buffer (pH 8.0) for 15 minutes, blocked with 5% BSA/5% FBS/0.1% Tween‐20 for 30 minutes, and treated with rabbit anti‐human PD‐L1 Ab (1:100 dilution; Cell Signaling Technology, Danvers, MA, USA), mouse anti‐human PD‐1 Ab (1:100 dilution; Cell Signaling Technology), and goat anti‐human vascular endothelial (VE) ‐cadherin Ab (1:100 dilution; R&D Systems, Minneapolis, MN, USA) at 4°C overnight.

Techniques: Expressing, In Vitro, Reverse Transcription Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR

Journal: Cancer Science

Article Title: Regulation of programmed cell death ligand 1 expression by atypical protein kinase C lambda/iota in cutaneous angiosarcoma

doi: 10.1111/cas.13981

Figure Lengend Snippet: Suppression of programmed cell death ligand 1 ( PD ‐L1) expression by atypical protein kinase C lambda/iota ( aPKC λ) knockdown ( KD ) in physiologic and pathologic endothelial cells in vitro confirmed with immunostaining. A,B, PRKCI KD and control HUVEC s (A) and AS ‐M cells (B) were immunostained with Ab against PD ‐L1 (green), aPKC λ (red), vascular endothelial‐cadherin ( VE ‐cad; gray), and Hoechst (blue). Fluorescence intensity of the green and red channels in each cell was measured and analyzed (n ≥ 20 each group). The scatter diagram of the mean fluorescence intensity of these 2 colors revealed the significantly proportional relationship between PD ‐L1 and aPKC λ in HUVEC s and AS ‐M cells, and the decreased expression of PD ‐L1 in PRKCI KD HUVEC s was confirmed by comparing the mean fluorescence intensity of PD ‐L1 in PRKCI KD and control HUVEC s or AS ‐M cells. Scale bar = 20 μm. Data represent mean ± SEM by 2‐tailed unpaired t test. * P < .05

Article Snippet: Formaldehyde‐fixed paraffin‐embedded angiosarcoma tissue samples were deparaffinized, and antigen retrieval was carried out by boiling the slides in EDTA buffer (pH 8.0) for 15 minutes, blocked with 5% BSA/5% FBS/0.1% Tween‐20 for 30 minutes, and treated with rabbit anti‐human PD‐L1 Ab (1:100 dilution; Cell Signaling Technology, Danvers, MA, USA), mouse anti‐human PD‐1 Ab (1:100 dilution; Cell Signaling Technology), and goat anti‐human vascular endothelial (VE) ‐cadherin Ab (1:100 dilution; R&D Systems, Minneapolis, MN, USA) at 4°C overnight.

Techniques: Expressing, In Vitro, Immunostaining, Fluorescence

Journal: Cancer Science

Article Title: Regulation of programmed cell death ligand 1 expression by atypical protein kinase C lambda/iota in cutaneous angiosarcoma

doi: 10.1111/cas.13981

Figure Lengend Snippet: Pharmacological inhibition of atypical protein kinase C lambda/iota ( aPKC λ) suppresses programmed cell death ligand 1 ( PD ‐L1) expression in HUVEC s and AS ‐Ms. A, mRNA relative expression of CD 274 reduced after treatment with 100 μmol/L sodium aurothiomalate hydrate ( ATM ) in HUVEC s, analyzed by real‐time RT ‐ PCR (n = 3). B, Decreased protein levels of PD ‐L1 in HUVECs were also identified by western blot analysis (n = 3). C, Treatment with 100 μmol/L ATM caused decreased mRNA relative expression levels of CD 274 in AS ‐M cells (n = 3). D, PD ‐L1 expression analyzed by western blotting also became significantly lower after 100 μmol/L ATM treatment (n = 3). Data represent mean ± SEM by 2‐tailed unpaired t test. * P < .05

Article Snippet: Formaldehyde‐fixed paraffin‐embedded angiosarcoma tissue samples were deparaffinized, and antigen retrieval was carried out by boiling the slides in EDTA buffer (pH 8.0) for 15 minutes, blocked with 5% BSA/5% FBS/0.1% Tween‐20 for 30 minutes, and treated with rabbit anti‐human PD‐L1 Ab (1:100 dilution; Cell Signaling Technology, Danvers, MA, USA), mouse anti‐human PD‐1 Ab (1:100 dilution; Cell Signaling Technology), and goat anti‐human vascular endothelial (VE) ‐cadherin Ab (1:100 dilution; R&D Systems, Minneapolis, MN, USA) at 4°C overnight.

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Western Blot

Journal: Cancer Science

Article Title: Regulation of programmed cell death ligand 1 expression by atypical protein kinase C lambda/iota in cutaneous angiosarcoma

doi: 10.1111/cas.13981

Figure Lengend Snippet: Schematic figure. Atypical protein kinase C ( aPKC ) phosphorylates DNA ‐binding domain of Forkhead box protein O1 (FoxO1) to suppress its DNA ‐binding ability. As a result, c‐Myc expression is increased by modulating microRNA (miR)‐34c abundance. In addition to controlling cell proliferation, this aPKC / pS er218 FoxO1 signaling axis regulates programmed cell death ligand 1 ( PD ‐L1) expression. Inhibition of aPKC would be promising treatment for malignant tumors such as angiosarcoma

Article Snippet: Formaldehyde‐fixed paraffin‐embedded angiosarcoma tissue samples were deparaffinized, and antigen retrieval was carried out by boiling the slides in EDTA buffer (pH 8.0) for 15 minutes, blocked with 5% BSA/5% FBS/0.1% Tween‐20 for 30 minutes, and treated with rabbit anti‐human PD‐L1 Ab (1:100 dilution; Cell Signaling Technology, Danvers, MA, USA), mouse anti‐human PD‐1 Ab (1:100 dilution; Cell Signaling Technology), and goat anti‐human vascular endothelial (VE) ‐cadherin Ab (1:100 dilution; R&D Systems, Minneapolis, MN, USA) at 4°C overnight.

Techniques: Binding Assay, Expressing, Inhibition

Journal: Journal for Immunotherapy of Cancer

Article Title: Spatially resolved analysis of the T cell immune contexture in lung cancer-associated brain metastases

doi: 10.1136/jitc-2021-002684

Figure Lengend Snippet: Immune cell density and activity within primary lung tumors and BrMs. (A) Representative images of primary lung tumors and BrMs. H&E images are provided for reference in addition to fluorescence panels showing immune cell subsets. (B) Immune cell infiltration was significantly lower in BrMs compared with primary lung tumors as measured by visual assessment (TIL) or QIF of CD3 + T cells ( p <0.0001), CD4 + helper cells ( p =0.0416), CD8 + effector cells ( p =0.0003), CD20 + B cells ( p =0.0058), and FOXP3 + CD4 + regulatory T cells ( p =0.0002). Expression is displayed as ratios normalized to the average expression in the lung. (C) GZB expression in CD3+ T cells was also significantly lower in BrMs compared with primary lung ( p =0.019), as measured by QIF. Ki67 expression, a measure of proliferation, was not significantly different between primary lung tumors or BrMs ( p =0.944). (D) High CD3 + T cells in BrMs were associated with longer survival ( p <0.0001). The median cutpoint was used to define high/low expression. * p <0.05. BrM, brain metastasis; GZB, granzyme B; QIF, quantitative immunofluorescence; TIL, tumor-infiltrating lymphocyte.

Article Snippet: Panels of primary antibodies included CD4 (clone SP35, Spring Bioscience), CD8 (clone C8/144B, DAKO), and CD20 (clone L26, DAKO) for the TIL panel; CD4, CD8, and FOXP3 (clone D2W8E, Cell Signaling Technology) for the regulatory T cell panel; CD3 (polyclonal, DAKO; clone SP7, Novus), PD-1 (clone EH33, Cell Signaling Technology), TIM-3 (clone D5D5R, Cell Signaling Technology), and LAG-3 (clone 11E3, Abcam) for the co-inhibitory receptor panel; CD3, granzyme B (clone 4E6, Abcam), and Ki67 (clone MIB-1, DAKO) for the activation marker panel.

Techniques: Activity Assay, Fluorescence, Expressing, Immunofluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: Spatially resolved analysis of the T cell immune contexture in lung cancer-associated brain metastases

doi: 10.1136/jitc-2021-002684

Figure Lengend Snippet: CD3 + T cell expression of the coinhibitory receptors in primary lung tumors and BrMs. (A) Representative fluorescence images showing the simultaneous staining of DAPI (blue), CD3 (red), PD-1 (yellow), TIM-3 (purple), and LAG-3 (green) in primary lung tumors and BrMs. (B) CD3+ T cells within BrMs have lower expression of PD-1 ( p =0.013), Tim-3 ( p =0.021), and LAG-3 ( p =0.008) compared with primary lung tumors, as measured by QIF. Expression is displayed as ratios normalized to the average expression in the lung. (C) High TIM-3 ( p =0.041) and LAG-3 ( p =0.035) expression in CD3+ T cells in BrMs were associated with longer survival. The median cutpoint was used to define high/low expression. BrM, brain metastasis; LAG-3, lymphocyte activation gene 3; PD-1, programmed cell death 1; QIF, quantitative immunofluorescence; TIM-3, T cell immunoglobulin mucin receptor 3. Asterix (*) indicates p <0.05.

Article Snippet: Panels of primary antibodies included CD4 (clone SP35, Spring Bioscience), CD8 (clone C8/144B, DAKO), and CD20 (clone L26, DAKO) for the TIL panel; CD4, CD8, and FOXP3 (clone D2W8E, Cell Signaling Technology) for the regulatory T cell panel; CD3 (polyclonal, DAKO; clone SP7, Novus), PD-1 (clone EH33, Cell Signaling Technology), TIM-3 (clone D5D5R, Cell Signaling Technology), and LAG-3 (clone 11E3, Abcam) for the co-inhibitory receptor panel; CD3, granzyme B (clone 4E6, Abcam), and Ki67 (clone MIB-1, DAKO) for the activation marker panel.

Techniques: Expressing, Fluorescence, Staining, Activation Assay, Immunofluorescence

Journal: Journal of Cancer

Article Title: Circulating Lymphocytes, PD-L1 Expression on Tumor-infiltrating Lymphocytes, and Survival of Colorectal Cancer Patients with Different Mismatch Repair Gene Status

doi: 10.7150/jca.25187

Figure Lengend Snippet: PD-1/PD-L1 expression pattern between dMMR and pMMR CRC patients. (a-b). Distribution of PD-1 (a) or PD-L1 (b) expression for dMMR cohorts (n=168) versus pMMR cohorts (n=169) in CRC patients. (c-d). Group comparison combined the level of TIL PD-L1 and AJCC stages in dMMR (c) or pMMR patients (d). (e-f). Group comparison combined the level of stroma PD-L1 (non-TIL and non-tumor) and AJCC stages in dMMR (e) or pMMR patients (f). * P <0.05, N.S, not significant.

Article Snippet: The primary antibodies were used as follows: anti-PD-1 (CST Corp, #10084, Massachusetts, USA), anti-PD-L1 (CST Corp, #10084, Massachusetts, USA), and DAKO Envision kit (DAKO Corp, Carpinteria, CA, USA).

Techniques: Expressing