Journal: Journal for Immunotherapy of Cancer

Article Title: Single-cell RNA sequencing reveals distinct cellular factors for response to immunotherapy targeting CD73 and PD-1 in colorectal cancer

doi: 10.1136/jitc-2021-002503

Figure Lengend Snippet: Differential cellular composition and lineage of AB680 treated, and programmed cell death protein 1 (PD-1) blocker treated and untreated tumor-infiltrating lymphocytes (TILs). (A) Workflow of the study design. Single-cell RNA sequencing (scRNA-seq) was conducted with CD45 + TIL cells extracted from three untreated controls, AB680-treated and PD-1 blocking antibody-treated tumors. Eight tumors per group were used for fluorescence-activated cell sorting (FACS) analysis. (B) Five days after inoculation of CT26 cells (1×10 6 cells/mouse), AB680 (20 mg/kg) or the PD-1 blocking antibody (20 mg/kg) was administered from day 0 to day 15 after initiation of treatment via the intraperitoneal route, as described in the Materials and methods section (n=9 mice). Tumor sizes were measured at day 3. (C) At day 15, mice were sacrificed and the extracted tumors were weighted. (D) Uniform manifold approximation and projection (UMAP) plot for clusters including all samples. (E) Proportion of clusters in each sample. (F) Heatmap representing cluster-specific gene expressions. The yellow color represents high expression, and purple represents low expression. (G) Proportions of clusters among the controls, AB680-treated, and PD-1 blocker treated TILs. The error bar denotes SEM (t-test, *p<0.05, **p<0.05, ***p=0.001). NS, non-significant.

Article Snippet: AB680 (10 mg/kg) (MedChemExpress) was administered from day 0 to day 21 after the end of third cycle of DSS treatment via the intraperitoneal route.

Techniques: RNA Sequencing Assay, Blocking Assay, Fluorescence, FACS, Expressing

Journal: Journal for Immunotherapy of Cancer

Article Title: Single-cell RNA sequencing reveals distinct cellular factors for response to immunotherapy targeting CD73 and PD-1 in colorectal cancer

doi: 10.1136/jitc-2021-002503

Figure Lengend Snippet: Distinct T cell subclusters of AB680 treated, and programmed cell death protein 1 (PD-1) blocker treated and untreated tumor-infiltrating lymphocytes (TILs). (A) Uniform manifold approximation and projection (UMAP) plot for T cell subclusters. (B) UMAP plots for the group-specific distribution of T cells. (C) Heatmap representing cluster-specific gene expressions. (D) Heatmap for the expression profile of marker genes (z-score of log 2 (CPM+1)) used to identify the cell type of each subcluster and to determine the degree of depletion. Red and blue colors denote upregulated and downregulated genes, respectively. (E) Proportion of clusters in each sample. (F) Box-and-whisker plot comparing the proportion of cells within a T cell subcluster effector CD8 + T cell (C9) between the controls and the PD-2 blocker (t-test, *p < 0.05), within MALAT1 high Treg T cells (C2) between the controls and the PD-2 blocker (t-test, **p = 0.01), and within CD4 + exhausted T cells (C11) between the controls and PD-2 blocker (t-test, ***p = 0.001). NS, non-significant.

Article Snippet: AB680 (10 mg/kg) (MedChemExpress) was administered from day 0 to day 21 after the end of third cycle of DSS treatment via the intraperitoneal route.

Techniques: Expressing, Marker, Whisker Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Single-cell RNA sequencing reveals distinct cellular factors for response to immunotherapy targeting CD73 and PD-1 in colorectal cancer

doi: 10.1136/jitc-2021-002503

Figure Lengend Snippet: T cell receptor (TCR) diversity and distinct transcriptional profiles related to the expression of NT5E, ENTPD1, and PDCD1 in T cells. Fold changes of TCR diversity of (A) Nt5e-negative to Nt5e-positive T cells. (B) Entpd1-negative to Entpd1-positive T cells, and (C) Pdcd1-negative to Pdcd1-positive T cell in the control, AB680-treated and programmed cell death protein 1 (PD-1) blockade-treated intratumoral T cells.(D) TCR diversity among the control, AB680-treated, and PD-1 blocker-treated intratumoral T cells. (E) Uniform manifold approximation and projection (UMAP) plot for the expression of Nt5e in T cells. (F) Volcano plots for the differentially expressed genes (DEGs) of Nt5e-positive T cells compared with negative T cells. Red and blue dots denote upregulated and downregulated DEGs, respectively, with a p value <0.05 and a |ln(FC: fold change)|>0.25. (G) UMAP plot for the expression of Entpd1 in T cells. (H) Volcano plots for the DEGs of Entpd1-positive T cells compared with the negative T cells. Red and blue dots denote upregulated and downregulated DEGs, respectively, with a p value <0.05 and a |ln(FC: fold change)|>0.25. (I) UMAP plot for the expression of Pdcd1 in T cells. (J) Volcano plots for the DEGs of Pdcd1-positive T cells compared with negative T cells. Red and blue dots denote upregulated and downregulated DEGs, respectively, with a p value <0.05 and a |ln(FC: fold change)|>0.25. (K) Kaplan-Meier plot for overall survival rates between Nt5e high and low expression colorectal cancer (CRC) patients. (L) Kaplan-Meier plot for overall survival rates between Entpd1 high and low expression CRC patients. (M) Kaplan-Meier plot for overall survival rates between Pdcd1 high and low expression CRC patients. NS, non-significant.

Article Snippet: AB680 (10 mg/kg) (MedChemExpress) was administered from day 0 to day 21 after the end of third cycle of DSS treatment via the intraperitoneal route.

Techniques: Expressing, Control

Journal: Journal for Immunotherapy of Cancer

Article Title: Single-cell RNA sequencing reveals distinct cellular factors for response to immunotherapy targeting CD73 and PD-1 in colorectal cancer

doi: 10.1136/jitc-2021-002503

Figure Lengend Snippet: Treg and CD4 + exhausted T cell subclusters. (A) Uniform manifold approximation and projection (UMAP) plot for the expression of the marker gene, Foxp3 , which characterizes Treg cell subclusters. (B, C) Volcano plots for the differentially expressed genes (DEGs) of Treg cell subclusters 2 and 4, compared with the other clusters. Red and blue dots denote upregulated and downregulated DEGs, respectively, with a p value <0.05 and a |ln(FC: fold change)|>0.25. (D) Heatmap for the expression profile of marker genes (z-score of log 2 (CPM+1)) used to identify the cell type of Treg subcluster (C2) and to determine the degree of changes in the gene expression caused by the AB680 and programmed cell death protein 1 (PD-1) blocker treatment. Red and blue colors denote upregulated and downregulated genes, respectively. (E) Representative dot plots show the percentage (%) of CD25 + , Foxp3 + , and Treg cells from CD4 + T cells extracted from the control, AB680-treated, and the PD-1 blocker-treated tumor-infiltrating lymphocytes (TILs) through fluorescence-activated cell sorting (FACS) analysis. (F) Graphical representation of the percentages (%) of Treg cells from CD4 + T cells extracted from eight different control TILs, AB680-treated TILs, and PD-1 blocker-treated TILs. Short horizontal lines indicate the means (t-test, NS, non-significant; *p<0.05; **p<0.01). (G) UMAP plot for the expression of marker genes, IKZF2 and TOX , to characterize exhausted T-cell subclusters. (H) Heatmap for the expressions of marker genes used to identify the cell type of each subcluster. Red and blue colors denote upregulated and downregulated genes, respectively, which were used to determine the degree of changes by the AB680 and PD-1 blocker treatment. (I) Volcano plots for DEGs of exhausted CD4 + T cell subcluster (C11) compared with those of the other clusters. NS, non-significant.

Article Snippet: AB680 (10 mg/kg) (MedChemExpress) was administered from day 0 to day 21 after the end of third cycle of DSS treatment via the intraperitoneal route.

Techniques: Expressing, Marker, Control, Fluorescence, FACS

Journal: Journal for Immunotherapy of Cancer

Article Title: Single-cell RNA sequencing reveals distinct cellular factors for response to immunotherapy targeting CD73 and PD-1 in colorectal cancer

doi: 10.1136/jitc-2021-002503

Figure Lengend Snippet: CD8 + exhausted T cell and effector CD8 + T cell subclusters. (A) Uniform manifold approximation and projection (UMAP) plot for the expression of the marker gene, CX3CR1 , characterizing CD8 + -exhausted T cell subclusters. (B) Heatmap for the expression profile of marker genes (z-score of log 2 (CPM+1)) used to identify the cell type of CD8+-exhausted T cells (C0, 1, 5, 6, 8, 10) and to determine the degree of changes in gene expression by the AB680 and programmed cell death protein 1 (PD-1) blocker treatment. Red and blue colors denote upregulated and downregulated genes, respectively. (C) Representative dot plots show the percentage (%) of CX3CR1 + CD8 + T cells from T cells and their expressions of Lag3 and Havcr2 extracted from control tumor-infiltrating lymphocytes (TILs) through fluorescence-activated cell sorting (FACS) analysis. (D) Graphical representation of the percentages (%) of CX3CR1 + Lag + CD8 + T cells, GzB + CX3CR1 + CD8 + T cells, and IFNγ + CX3CR1 + CD8 + T cells extracted from control TILs, AB680-treated TILs, and PD-1 blocker-treated TILs. Short horizontal lines indicate the means (t-test, NS, non-significant; *p<0.05; ***p<0.001). (E) UMAP plot for the expression of marker genes, Gzmb and CD69 , characterizing effector CD8 + T cell subclusters. (F) Volcano plots for the DEGs of the effector CD8 + T cell subcluster (C9) compared with other clusters. Red and blue dots denote upregulated and downregulated DEGs, respectively, with a p value <0.05 and a |ln(FC: fold change)|>0.25. (G) Heatmap for the expression profiles of marker genes (z-score of log 2 (CPM+1)) used to identify the cell type of the effector CD8 + T cell subcluster (C9) and to determine the degree of changes in the AB680 and PD-1 blocker treatments. Red and blue colors denote upregulated and downregulated genes, respectively. (H) Representative histograms show the Mean fluorescence of intensity(MFI) for CD69 expressed in CD8 + T cells extracted from the control, AB680-treated, and PD-1 blocker treated TILs through FACS analysis. (I) Graphical representation of the percentages (%) of CD69 + CD8 + T cells extracted from control TILs, AB680-treated TILs and PD-1 blocker-treated TILs. Short horizontal lines indicate the means (t-test, NS, non-significant; *p<0.05; **p<0.01).

Article Snippet: AB680 (10 mg/kg) (MedChemExpress) was administered from day 0 to day 21 after the end of third cycle of DSS treatment via the intraperitoneal route.

Techniques: Expressing, Marker, Control, Fluorescence, FACS

Journal: Journal for Immunotherapy of Cancer

Article Title: Single-cell RNA sequencing reveals distinct cellular factors for response to immunotherapy targeting CD73 and PD-1 in colorectal cancer

doi: 10.1136/jitc-2021-002503

Figure Lengend Snippet: Myeloid cell subclusters. (A) Uniform manifold approximation and projection (UMAP) plot for myeloid cell subclusters. (B) Proportion of clusters in each sample. (C) Heatmap representing cluster-specific gene expressions. (D) Heatmap for the expression profile of marker genes (z-score of log 2 (CPM+1)) used to identify the cell type of each subcluster and to determine the degree of depletion. Red and blue colors denote upregulated and downregulated genes, respectively. (E) Box-and-whisker plot comparing the proportion of cells within the myeloid cell M1 macrophages (C0) with the controls and AB680, between the controls and the PD-2 blocker (t-test, *p = 0.05). (F) Box-and-whisker plot comparing the proportion of cells within M2 macrophages (C3) between the controls and the PD-2 blocker (t-test, *p = 0.05). NS, non-significant; PD-1, programmed cell death protein 1.

Article Snippet: AB680 (10 mg/kg) (MedChemExpress) was administered from day 0 to day 21 after the end of third cycle of DSS treatment via the intraperitoneal route.

Techniques: Expressing, Marker, Whisker Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Single-cell RNA sequencing reveals distinct cellular factors for response to immunotherapy targeting CD73 and PD-1 in colorectal cancer

doi: 10.1136/jitc-2021-002503

Figure Lengend Snippet: The effect of AB680 and programmed cell death protein 1 (PD-1) blockade on the in vivo AOM-DSS colorectal cancer (CRC) model. After sacrifice of mice as described in the Materials and methods section, (A) representative images for tumors in large intestines of mice for the AOM/DSS-induced control (n=8), AOM/DSS+AB680 (10 mg/kg) treated (n=8), AOM/DSS+PD-1 blockade (10 mg/kg) treated (n=8) and AOM/DSS+AB680 (10 mg/kg)+PD-1 blockade (10 mg/kg) treated group (n=8). (B) The diameters of all tumors in large intestines of mice for each group. (C) The number of tumors per mouse were measured for each group. Data are presented as mean ± SEM (t-test, NS, non-significant; *p<0.05; **p<0.01). (D) Percentages of CD4 + T cells and CD8 + T cells and the percentages of CD4 + Foxp3 + Tregs of large intestinal tissues from healthy and AOM-DSS murine models with or without AB680 or PD-1 blocker treatment. These results are representative from all independent experiments (n=8 per each group). The graphical results show (E) the percentages (%) of CD4 + Foxp3 + Treg per total T cells and (F) the percentages (%) of GzB + CD8 + T cells of large intestinal tissues. Data are presented as mean ± SEM (t-test, NS, non-significant; ***p<0.001). (G) The histograms show the proliferating CD8 + T cells in the in vitro culture with or without ATP (20 µM) and AB680 (0.5 and 20 µM). These results are representative from three independent experiments. (H) The representative histograms show the mean fluorescence of intensity (MFI) for CD73 expression level of CD8 + T cells in the in vitro culture with or without ATP (20 µM) and AB680 (20 µM). These results are representative from three independent experiments. (I) The graphical result shows the percentages (%) of IFNγ + CD8 + T cells after the in vitro culture with or without ATP (20 µM) and AB680 (0.5 and 20 µM) (n=6). Data are presented as mean±SEM (t-test, **p<0.01; ***p<0.001).

Article Snippet: AB680 (10 mg/kg) (MedChemExpress) was administered from day 0 to day 21 after the end of third cycle of DSS treatment via the intraperitoneal route.

Techniques: In Vivo, Control, In Vitro, Fluorescence, Expressing