Journal: Nature Cancer

Article Title: Efferocytosis reprograms the tumor microenvironment to promote pancreatic cancer liver metastasis

doi: 10.1038/s43018-024-00731-2

Figure Lengend Snippet: a , Schematic of bulk RNA sequencing on fresh liver metastasis biopsies from chemotherapy-naive patients with PDAC ( n = 5) (top) and heat map showing scores (normalized enrichment score (NES); single-sample gene set enrichment analysis (ssGSEA)) for immune signatures (bottom). NK, natural killer. b – d , Representative immunofluorescence images ( b ) and quantification of CD8 + T cells ( c ) and macrophages (CD68 + ) ( d ) in the tumor margin and core of human PDAC liver metastasis ( n = 3 patients). Cancer cells were indicated by CK19 + staining. Arrowheads indicate CD8 + T cells. Scale bars, 50 µm. Error bars, mean ± s.e.m. P values, two-tailed unpaired t -test. DAPI, 4,6-diamidino-2-phenylindole. e , Uniform Manifold Approximation and Projection (UMAP) plot identifying ten clusters within macrophages (F4/80 + ) isolated by flow cytometry from healthy liver, early metastatic livers (d5) and advanced metastatic livers (d10) induced by intra-portal implantation of KPC-derived cells into mice with established orthotopic PDAC tumors ( n = 3 mice per group). f , g , UMAP plots ( f ) and violin plots ( g ) depicting expression of common markers of KCs ( Clec4f , Vsig4 and Timd4 ) and MoMs ( Ccr2 ) in the scRNA-seq dataset. h , i , UMAP plots ( h ) and bar chart ( i ) depicting distribution of different macrophage clusters in healthy livers, early metastatic livers (d5) and advanced metastatic livers (d10). j , Heat map depicting relative average expression of the top upregulated differentially expressed genes in each macrophage cluster compared to all other clusters in the scRNA-seq dataset. Representative genes are labeled for each cluster. k , Enriched Gene Ontology (GO) biological processes (BP) in major MAM clusters derived from KCs (cluster 1 and 4) and monocytes (cluster 2 and 3). Statistical enrichment analyses were performed using Fisher’s exact test on g:Profiler. LPS, lipopolysaccharide; pp, processing and presentation; MHC, major histocompatibility complex; IL, interleukin. l , Heat map showing signature scores (NES; ssGSEA) of major KC-MAM and Mo-MAM clusters in human PDAC liver metastasis samples ( n = 5 patients).

Article Snippet: For the CD8 + T cell depletion study, 100 μg of anti-mouse CD8α (clone 2.43, BioXcell) or IgG2b isotype control (BioXcell) was administered via intraperitoneal injection every 3 d. For induction of liver injury, a single dose (100 mg kg −1 ) of N -acetyl-para-aminophenol (APAP, Sigma) was administered via intraperitoneal injection 2 d before induction of liver metastasis.

Techniques: RNA Sequencing, Immunofluorescence, Staining, Two Tailed Test, Isolation, Flow Cytometry, Derivative Assay, Expressing, Labeling, Immunopeptidomics

Journal: Nature Cancer

Article Title: Efferocytosis reprograms the tumor microenvironment to promote pancreatic cancer liver metastasis

doi: 10.1038/s43018-024-00731-2

Figure Lengend Snippet: (a-b) Representative immunofluorescence images (a) and quantification (b) of cytotoxic GzmB + CD8 + T cells in human PDAC liver metastasis (n = 3 patients). Cancer cells were indicated by CK19 + staining. Scale bars, 50 µm. Error bars, mean ± SEM. P -value, two-tailed unpaired t-test. (c) Schematic of the scRNA sequencing approach combined with a spatial in-situ labeling strategy. MAMs=metastasis associated macrophages. (d,e) Representative immunofluorescence images of healthy livers (d) and advanced stage (d10) (e) of liver metastasis following in situ labeling with FITC-conjugated F4/80 antibody (n = 3 mice/group, from one experiment) via retrograde perfusion, followed by ex vivo staining with pan-macrophage marker CD68. Scale bars, 50 µm. (f) Quantification of double-labeled macrophages (F4/80 FITC+ /CD68 + ) in healthy and tumor bearing livers (healthy n = 4 mice, metastasis n = 3 mice) as shown in (d, e) . Error bars, mean ± SEM. P -value, two-tailed unpaired t-test. (g) FACS gating strategy for sorting of proximal (pMAMs; F4/80 APC+ F4/80 FITC− ) and distal metastasis-associated macrophages (dMAMs; F4/80 APC+ F4/80 FITC+ ). (h) Heatmap depicting expressions of the most enriched genes in KC vs MoM clusters in the scRNAseq dataset. (i) Proportions of each macrophage cluster identified in the scRNAseq in healthy liver, early metastasis, and advanced metastasis. (j) Expression values of the genes in the cluster 1-4 signatures from scRNAseq analyses versus all genes in the human samples (cluster 1 n = 751 genes; cluster 4 n = 291 genes; cluster 2 n = 255 genes; cluster 3 n = 310 genes). Boxplots indicate the median, first and third quartiles (hinges), and outlier points extend beyond 1.5× interquartile ranges from either hinge (whiskers).

Article Snippet: For the CD8 + T cell depletion study, 100 μg of anti-mouse CD8α (clone 2.43, BioXcell) or IgG2b isotype control (BioXcell) was administered via intraperitoneal injection every 3 d. For induction of liver injury, a single dose (100 mg kg −1 ) of N -acetyl-para-aminophenol (APAP, Sigma) was administered via intraperitoneal injection 2 d before induction of liver metastasis.

Techniques: Immunofluorescence, Staining, Two Tailed Test, Sequencing, In Situ, Labeling, Ex Vivo, Marker, Expressing

Journal: Nature Cancer

Article Title: Efferocytosis reprograms the tumor microenvironment to promote pancreatic cancer liver metastasis

doi: 10.1038/s43018-024-00731-2

Figure Lengend Snippet: a , UMAP plots showing distribution of pMAMs and dMAMs in advanced metastatic tumors (d10) based on in situ labeling. b , Violin plots depicting expression levels of KC ( Vsig4 , Clec4f and Timd4 ) and MoM ( Ccr2 ) genes in pMAMs and dMAMs. c , Representative immunofluorescent images showing distributions of KCs (CD68 + VSIG4 + ) and MoMs (CD68 + VSIG4 − ) in tumor core (top) and margin areas (bottom, dashed line) of liver metastasis derived from patients with PDAC ( n = 3 patients). Metastatic cancer cells were indicated by CK19 + staining. Scale bar, 50 µm. d , Quantification of KCs and MoMs among intralesional/core macrophages as shown in c . Error bars, mean ± s.e.m. P value, two-tailed unpaired t -test. e , UMAP plot identifying six clusters of MoMs derived from cluster 2, 3 and 7 in the original UMAP (Fig. ). f , Heat map depicting relative expression of upregulated DEGs in each MoM cluster compared to all other MoM clusters in the RNA-seq dataset. Representative genes are labeled for each cluster. g , UMAP plots depicting distribution of MoM clusters in early (d5) and advanced metastatic livers (d10). h , Diagram showing distribution of MoM clusters in early (d5) and advanced metastatic livers (d10). i – k , Representative immunofluorescent images of different macrophages (F4/80 + ) expressing antigen presentation marker CD74 or M2 marker YM-1 in early ( i ) and advanced ( j ) liver metastasis derived from experimental intrasplenic model ( n = 3 mice per group, from one experiment). Scale bars, 50 µm. Quantification of staining showing percentages of intralesional macrophages resembling major MoM clusters: cluster C-like (Cl.C) (CD74 − YM-1 − ), cluster B-like (Cl.B) (CD74 + YM-1 − ) and cluster A-like (Cl.A) (CD74 − YM-1 + ) ( k ). Error bars, mean ± s.e.m. l , Relative CD8 + T cell activation measured by percentages of interferon (IFN)γ + CD8 + T cells following stimulation with anti-CD3/CD28-coupled Dynabeads and co-culture with FACS-sorted early or advanced MoMs (F4/80 + TIM4 − ) from an experimental intrasplenic model compared to a Dynabead-only control (ctrl) ( n = 3 biological replicates per group from one experiment). Error bars, mean ± s.e.m. P values, one-way analysis of variance (ANOVA) with Sidak’s post-test.

Article Snippet: For the CD8 + T cell depletion study, 100 μg of anti-mouse CD8α (clone 2.43, BioXcell) or IgG2b isotype control (BioXcell) was administered via intraperitoneal injection every 3 d. For induction of liver injury, a single dose (100 mg kg −1 ) of N -acetyl-para-aminophenol (APAP, Sigma) was administered via intraperitoneal injection 2 d before induction of liver metastasis.

Techniques: In Situ, Labeling, Expressing, Derivative Assay, Staining, Two Tailed Test, RNA Sequencing, Immunopeptidomics, Marker, Activation Assay, Co-Culture Assay, Control

Journal: Nature Cancer

Article Title: Efferocytosis reprograms the tumor microenvironment to promote pancreatic cancer liver metastasis

doi: 10.1038/s43018-024-00731-2

Figure Lengend Snippet: (a) Representative H&E image of dead hepatocytes in pre-metastatic liver at d10 post pancreatic implantation of KPC-derived cells (n = 3 mice, from one experiment). Scale bar, 50 µm. (b) Heatmap depicting relative expression of T cell-stimulatory (red) or repressive genes (blue) in MoM clusters. (c) UMAP plots depicting Arg1 expression levels in MoMs isolated from pre-metastatic and spontaneous advanced metastatic livers. (d) Schematic of efferocytosis assay in primary bone marrow-derived macrophages (BMMs). (e) FACS plot (left) and quantification (right) of apoptotic thymocytes post staurosporine treatment (n = 3 biological replicates/group). Error bars, mean ± SEM. (f) qPCR analysis of ARG1 in human primary MoMs (n = 3 biological replicates/group). Error bars, mean ± SEM. P -value, two-tailed unpaired t-test. (g,h) Relative activation levels of CD8 + T cell, measured as percentages of IFNγ + (g) or GzmB + (h) cells, stimulated with anti-CD3/CD28-coupled Dynabeads and co-cultured with MoMs from advanced metastatic livers compared to Dynabeads-only control (n = 3 biological replicates/group from one experiment). Error bars, mean ± SEM. P -values, one-way ANOVA with Sidak’s post-test. (i) Percentages of apoptotic (Apotracker + ) CD8 + T-cells, following stimulation with anti-CD3/CD28-coupled Dynabeads and co-culture with BMMs (n = 3 biological replicates/group). Error bars, mean ± SEM. P -values, one-way ANOVA with Sidak’s post-test.

Article Snippet: For the CD8 + T cell depletion study, 100 μg of anti-mouse CD8α (clone 2.43, BioXcell) or IgG2b isotype control (BioXcell) was administered via intraperitoneal injection every 3 d. For induction of liver injury, a single dose (100 mg kg −1 ) of N -acetyl-para-aminophenol (APAP, Sigma) was administered via intraperitoneal injection 2 d before induction of liver metastasis.

Techniques: Derivative Assay, Expressing, Isolation, Two Tailed Test, Activation Assay, Cell Culture, Control, Co-Culture Assay

Journal: Nature Cancer

Article Title: Efferocytosis reprograms the tumor microenvironment to promote pancreatic cancer liver metastasis

doi: 10.1038/s43018-024-00731-2

Figure Lengend Snippet: a , Representative hematoxylin and eosin (H&E) images of hepatic necroses in autochthonous KPC mice with pre-metastatic PDAC (left) and 48 h post-intrasplenic implantation of KPC cells in WT mice (right). Dotted lines demarcate necrotic areas (N, necrotic; H, healthy) ( n = 3 mice per group, from one experiment). Scale bars, 50 µm. b , c , Mice were given KPC TCM or control DMEM once daily for 3 d. Livers were collected 24 h after the last injection. Representative H&E images of livers ( b ) and quantification of hepatic necroses areas ( c ) in the livers ( n = 3 mice per group, from one experiment). Dotted line demarcates the necrotic area. Scale bars, 50 µm. Error bars, mean ± s.e.m. P value, two-tailed unpaired t -test. d , Schematic of chimeric mice generation via transplantation of non-labeled (tdT − ) donor BM cells into tdT + hosts ( n = 3 mice). e , Representative immunofluorescent images of efferocytic MoMs (arrowheads, tdT + debris-containing F4/80 + tdT − cells) in healing necrotic areas ( n = 3 mice, from one experiment). Scale bar, 50 µm. f , g , qPCR analysis of Arg1 in BMMs co-cultured for 3 h with apoptotic thymocytes at 1 h ( f ) or 16 h ( g ) after washout. BMM, bone marrow-derived macrophages. Error bars, mean ± s.e.m. ( n = 3 biological replicates per group). P value, two-tailed unpaired t -test. h , Representative western blot image of arginase 1 and loading control cofilin levels in BMMs (3 + 16 h, experiment was performed three times with similar results). i , j , Relative activation levels of CD8 + T cells, measured as percentages of IFNγ + ( i ) or granzyme B (GzmB) + ( j ), stimulated with anti-CD3/CD28-coupled Dynabeads and co-cultured with CD74 −/low MoMs (F4/80 + TIM4 − ) from d5 livers compared to Dynabead-only control ( n = 3 biological replicates per group from one experiment). Error bars, mean ± s.e.m. P values, one-way ANOVA with Sidak’s post-test. k , l , Relative activation levels of CD8 + T cells, measured as percentages of IFNγ + ( k ) or granzyme B (GzmB) + cells ( l ), stimulated with anti-CD3/CD28-coupled Dynabeads and co-cultured with BMMs compared to Dynabead-only control ( n = 3 biological replicates per group). Error bars, mean ± s.e.m. P values, one-way ANOVA with Sidak’s post-test.

Article Snippet: For the CD8 + T cell depletion study, 100 μg of anti-mouse CD8α (clone 2.43, BioXcell) or IgG2b isotype control (BioXcell) was administered via intraperitoneal injection every 3 d. For induction of liver injury, a single dose (100 mg kg −1 ) of N -acetyl-para-aminophenol (APAP, Sigma) was administered via intraperitoneal injection 2 d before induction of liver metastasis.

Techniques: Control, Injection, Two Tailed Test, Transplantation Assay, Labeling, Cell Culture, Derivative Assay, Western Blot, Activation Assay

Journal: Nature Cancer

Article Title: Efferocytosis reprograms the tumor microenvironment to promote pancreatic cancer liver metastasis

doi: 10.1038/s43018-024-00731-2

Figure Lengend Snippet: a , b , Representative fluorescence image ( a ) and quantification of engulfed CSFE/pHrodo-labeled apoptotic thymocytes in BMMs ( b ) (vehicle n = 65 cells; MerTKi n = 112 cells, experiment was performed twice with similar results). CFSE, carboxyfluorescein succinimidyl ester. Error bars, mean ± s.e.m. P value, two-tailed unpaired t -test. c , qPCR analysis of Arg1 in BMMs (20 h). Error bars, mean ± s.e.m. (control/vehicle n = 3, control/MerTKi n = 4, efferocytic/vehicle n = 4, efferocytic/MerTKi n = 4 biological replicates). P values, two-way ANOVA with Sidak’s post-test. d , Relative activation level of CD8 + T cell, measured as percentages of IFNγ + cells, stimulated with anti-CD3/CD28-coupled Dynabeads and co-cultured BMMs compared to Dynabead-only control ( n = 3 biological replicates per group). Error bars, mean ± s.e.m. P values, two-way ANOVA with Sidak’s post-test. e , Schematic illustrating the MerTKi experiment timeline. f , g , Representative bioluminescence imaging (BLI) images (left) and relative tumor burden (right) of d5 ( n = 7 mice per group, from one experiment) ( f ) or d14 livers ( g ) (control n = 7 mice, MerTKi n = 6 mice, from two experiments). Error bars, mean ± s.e.m. P values, two-tailed unpaired t -test. h , qPCR analysis of Arg1 in MoMs (F4/80 + TIM4 − ) from d5 livers ( n = 4 mice per group). Error bars, mean ± s.e.m. P value, two-tailed unpaired t -test. i – k , Representative immunofluorescence images ( i ) and quantification of total ( j ) and cytotoxic GzmB + ( k ) CD8 + T cells in d5 livers. Arrowheads indicate CD8 + T cells ( n = 3 mice per group, from one experiment). Scale bars, 50 µm. Error bars, mean ± s.e.m. P values, two-tailed unpaired t -test. l , Schematic illustrating the CD8 + T cell depletion experiment timeline. m , Representative ex vivo BLI images (left) and relative tumor burden (right) of d14 livers (vehicle/IgG n = 4 mice, vehicle/anti-CD8 n = 5 mice, MerTKi/IgG n = 4 mice, MerTKi/anti-CD8 n = 4 mice, from one experiment). Error bars, mean ± s.e.m. P values, two-way ANOVA with Sidak’s post-test. n , Schematic diagram illustrating the MerTKi experiment in spontaneous liver metastasis model. o , Representative liver photographs (left) and d40 tumor burden from indicated treatment groups (control n = 5 mice, MerTKi n = 4 mice, from one experiment). Error bars, mean ± s.e.m. P value, two-tailed unpaired t -test. p , qPCR analysis of Arg1 in MoMs (F4/80 + TIM4 − ) from d40 livers ( n = 3 mice per group). Error bars, mean ± s.e.m. P value, two-tailed unpaired t -test.

Article Snippet: For the CD8 + T cell depletion study, 100 μg of anti-mouse CD8α (clone 2.43, BioXcell) or IgG2b isotype control (BioXcell) was administered via intraperitoneal injection every 3 d. For induction of liver injury, a single dose (100 mg kg −1 ) of N -acetyl-para-aminophenol (APAP, Sigma) was administered via intraperitoneal injection 2 d before induction of liver metastasis.

Techniques: Fluorescence, Labeling, Two Tailed Test, Control, Activation Assay, Cell Culture, Imaging, Immunofluorescence, Ex Vivo

Journal: Nature Cancer

Article Title: Efferocytosis reprograms the tumor microenvironment to promote pancreatic cancer liver metastasis

doi: 10.1038/s43018-024-00731-2

Figure Lengend Snippet: (a ) Representative Western blot image of Arginase 1 and loading control Cofilin levels in BMMs (experiment was performed three times with similar results). (b) Relative activation levels of CD8 + T cell, measured as percentages of IFNγ + cells, stimulated with anti-CD3/CD28-coupled Dynabeads and co-cultured with d5 liver MoMs (F4/80 + TIM4 – ) compared to Dynabeads-only control (n = 3 biological replicates/group from one experiment). Error bars, mean ± SEM. P -values, one-way ANOVA with Sidak’s post-test. (c,d) Representative H&E images (c) and quantification (d) of necroses in d5 livers (n = 3 mice/group, from one experiment). Scale bars, 50 µm. Error bars, mean ± SEM. P -value, two-tailed unpaired t-test. (e) Flow cytometry analysis of MerTK + cells among cancer cells (CD45 – ZsGreen+) or macrophages (CD45 + F4/80 + ) in d14 livers (n = 6 mice/group). Error bars, mean ± SEM. (f-h) Representative immunofluorescence images (f) and quantification of total (g) and CD74 − and/or YM-1-expressing (h) macrophages (F4/80 + ) in d5 livers (n = 3 mice/group, from one experiment). Scale bars, 50 µm. Error bars, mean ± SEM. P -value, two-tailed unpaired t-test. (i ) qPCR analysis of Ifnb in MoMs (F4/80 + TIM4 – ) from d5 livers (n = 3 mice/group). Error bars, mean ± SEM. P -value, two-tailed unpaired t-test. (j) Relative activation levels of CD8 + T cells, measured as percentages of IFNγ + cells, stimulated with anti-CD3/CD28-coupled Dynabeads and co-cultured with d5 liver MoMs (F4/80 + TIM4 – ) compared to Dynabeads-only control (n = 3 biological replicates/group from one experiment). Error bars, mean ± SEM. P -values, one-way ANOVA with Sidak’s post-test. (k) Flow cytometry analysis of activated (CD69 + ) CD8 + T cells in d5 livers (n = 6 mice/group). Error bars, mean ± SEM. P -value, two-tailed unpaired t-test. (l) qPCR analysis of Arg1 in MoMs (F4/80 + TIM4 – ) isolated from d14 livers (n = 3 mice/group). Error bars, mean ± SEM. P -value, two-tailed unpaired t-test. (m-o) Representative immunofluorescence images (m) and quantification of total (n) and YM-1 + (o) macrophages (F4/80 + ) in d14 livers. Arrowheads indicate YM-1 + macrophages (n = 3 mice/group, from one experiment). Scale bars, 50 µm. Error bars, mean ± SEM. P -value, two-tailed unpaired t-test. (p) Organoid-derived primary tumor burden at d40. Error bars, mean ± SEM. P -value, two-tailed unpaired t-test. (q) Flow cytometry analysis of MerTK + cells among cancer cells (CD45 – Epcam+) or macrophages (CD45 + F4/80 + ) in d40 livers (n = 6 mice/group). Error bars, mean ± SEM.

Article Snippet: For the CD8 + T cell depletion study, 100 μg of anti-mouse CD8α (clone 2.43, BioXcell) or IgG2b isotype control (BioXcell) was administered via intraperitoneal injection every 3 d. For induction of liver injury, a single dose (100 mg kg −1 ) of N -acetyl-para-aminophenol (APAP, Sigma) was administered via intraperitoneal injection 2 d before induction of liver metastasis.

Techniques: Western Blot, Control, Activation Assay, Cell Culture, Two Tailed Test, Flow Cytometry, Immunofluorescence, Expressing, Isolation, Derivative Assay

Journal: Nature Cancer

Article Title: Efferocytosis reprograms the tumor microenvironment to promote pancreatic cancer liver metastasis

doi: 10.1038/s43018-024-00731-2

Figure Lengend Snippet: (a) Schematic diagram illustrating the APAP experiment timeline. (b) Representative H&E images of hepatic necroses at 24 hours post vehicle or APAP injection (n = 3 mice/group, from one experiment). Scale bars, 50 µm. (c,d) Representative ex vivo bioluminescence imaging (BLI) images (left) and relative tumor burden (right) of d5 (c) and d14 (d) livers (d5 n = 3 mice/group, from one experiment; d14 control n = 6 mice; APAP n = 5 mice, from two experiments). Error bars, mean ± SEM. P -value, two-tailed unpaired t-test. (e-g) Representative immunofluorescence images (e) and quantification of total ( f ) and CD74 − and/or YM-1-expressing (g) macrophages (F4/80 + ) in d5 livers (n = 3 mice/group). Scale bars, 50 µm. Error bars, mean ± SEM. P -value, two-tailed unpaired t-test. (h) qPCR analysis of Arg1 in MoMs (F4/80 + TIM4 – ) from d5 livers (n = 3 mice/group). Error bars, mean ± SEM. P -value, two-tailed unpaired t-test. (i-k) Representative immunofluorescence images (i) and quantification of total (j) and cytotoxic granzyme B + (k) CD8 + T-cells in d5 livers (n = 3 mice/group). Arrowheads indicate CD8 + T cells. Scale bars, 50 µm. Error bars, mean ± SEM. P -value, two-tailed unpaired t-test. (l) Flow cytometry analysis of activated (CD69 + ) CD8 + T cells in d5 livers (n = 3 mice/group). Error bars, mean ± SEM. P -value, two-tailed unpaired t-test. (m) qPCR analysis of Arg1 in MoMs (F4/80 + TIM4 – ) from d14 livers (n = 3 mice/group). Error bars, mean ± SEM. P -value, two-tailed unpaired t-test. (n-p) Representative immunofluorescence images (n) and quantification of total (o) and YM-1 + (p) macrophages (F4/80 + ) (n = 3 mice/group). Arrowheads indicate YM-1 + macrophages. Scale bars, 50 µm. Error bars, mean ± SEM. P -value, two-tailed unpaired t-test.

Article Snippet: For the CD8 + T cell depletion study, 100 μg of anti-mouse CD8α (clone 2.43, BioXcell) or IgG2b isotype control (BioXcell) was administered via intraperitoneal injection every 3 d. For induction of liver injury, a single dose (100 mg kg −1 ) of N -acetyl-para-aminophenol (APAP, Sigma) was administered via intraperitoneal injection 2 d before induction of liver metastasis.

Techniques: Injection, Ex Vivo, Imaging, Control, Two Tailed Test, Immunofluorescence, Expressing, Flow Cytometry

Journal: Nature Cancer

Article Title: Efferocytosis reprograms the tumor microenvironment to promote pancreatic cancer liver metastasis

doi: 10.1038/s43018-024-00731-2

Figure Lengend Snippet: (a) Violin plot depicting expression of Grn in MoM clusters from the scRNAseq dataset. (b) qPCR analysis of GRN in THP-1-derived macrophages (20hrs). Error bars, mean ± SEM (n = 3 independent experiments). P -value, two-tailed unpaired t-test. (c) qPCR analysis of Grn in murine primary BMMs (20hrs). Error bars, mean ± SEM (n = 4 biological replicates/group). P -value, two-tailed unpaired t-test. (d) qPCR analysis of Grn gene copy number in MoMs (F4/80 + TIM4 – ) (n = 3 mice/group). Error bars, mean ± SEM. P -value, two-tailed unpaired t-test. (e) qPCR analysis of Arg1 in MoMs (F4/80 + TIM4 – ) from d14 livers (n = 3 mice/group). Error bars, mean ± SEM. P -value, two-tailed unpaired t-test. (f-h) Representative immunofluorescence images (f) and quantification of total (g) and YM-1 + (h) macrophages (F4/80 + ) in d14 livers. Arrowheads indicate YM-1 + macrophages (n = 3 mice/group, from one experiment). Scale bars, 50 µm. Error bars, mean ± SEM. P -value, two-tailed unpaired t-test. (i) Flow cytometry analysis of (CD69 + ) CD8 + T-cells in d5 livers (n = 3 mice/group). Error bars, mean ± SEM. P -value, two-tailed unpaired t-test. (j) Organoid-derived primary tumor burden at d40. Error bars, mean ± SEM. P -value, two-tailed unpaired t-test.

Article Snippet: For the CD8 + T cell depletion study, 100 μg of anti-mouse CD8α (clone 2.43, BioXcell) or IgG2b isotype control (BioXcell) was administered via intraperitoneal injection every 3 d. For induction of liver injury, a single dose (100 mg kg −1 ) of N -acetyl-para-aminophenol (APAP, Sigma) was administered via intraperitoneal injection 2 d before induction of liver metastasis.

Techniques: Expressing, Derivative Assay, Two Tailed Test, Immunofluorescence, Flow Cytometry

Journal: Nature Cancer

Article Title: Efferocytosis reprograms the tumor microenvironment to promote pancreatic cancer liver metastasis

doi: 10.1038/s43018-024-00731-2

Figure Lengend Snippet: a , Schematic illustrating experiment to track progranulin localization in efferocytic mCherry-PGRN-expressing macrophages. PGRN, progranulin. b , Representative immunofluorescence image of mCherry-PGRN in efferosomes (asterisks) of murine BMMs ( n = 33 cells, experiment was performed twice with similar results). Scale bar, 50 µm. c , Representative fluorescence image of mCherry-PGRN in efferosome (arrowhead) of human MoMs over time as assessed by live-cell imaging (experiment was performed twice with similar results). d , qPCR analysis of Arg1 in BMMs (20 h, n = 4 biological replicates per group). Error bars, mean ± s.e.m. P values, two-way ANOVA with Sidak’s post-test. e , Representative western blot image of arginase 1 and loading control cofilin levels in BMMs (3 + 16 h, experiment was performed three times with similar results). f , g , Relative activation of CD8 + T cell, measured by percentages of IFNγ + ( f ) or granzyme B (GzmB) + ( g ) T cells, stimulated with anti-CD3/CD28-coupled Dynabeads and co-cultured with BMMs compared to Dynabead-only control ( n = 3 biological replicates per group from one experiment). Error bars, mean ± s.e.m. P values, one-way ANOVA with Sidak’s post-test. h , Schematic illustrating the Grn KO experiment timeline. i , j , Representative ex vivo BLI images (left) and relative tumor burden (right) of d5 ( n = 8 mice per group, from two experiments) ( i ) and d14 (WT, n = 6 mice; KO, n = 8 mice, from two experiments) ( j ) livers. Error bars, mean ± s.e.m. P values, two-tailed unpaired t -test. k , qPCR analysis of Arg1 in MoMs (F4/80 + TIM4 − ) from d5 livers ( n = 4 mice per group). Error bars, mean ± s.e.m. P values, two-tailed unpaired t -test. l – n , Representative immunofluorescence images ( l ) and quantification of total ( m ) and cytotoxic GzmB + ( n ) CD8 + T cells in d5 livers ( n = 3 mice per group, from one experiment). Arrowheads indicate CD8 + T cells. Scale bars, 50 µm. Error bars, mean ± s.e.m. P values, two-tailed unpaired t -test. o , Schematic illustrating the Grn KO experiment in a spontaneous liver metastasis model. p , Representative liver photographs (left) and tumor burden (WT, n = 6 mice; KO, n = 4 mice, from one experiment). Error bars, mean ± s.e.m. P values, two-tailed unpaired t -test. q , qPCR analysis of Arg1 in MoMs (F4/80 + TIM4 − ) from d5 livers ( n = 3 mice per group). Error bars, mean ± s.e.m. P values, two-tailed unpaired t -test.

Article Snippet: For the CD8 + T cell depletion study, 100 μg of anti-mouse CD8α (clone 2.43, BioXcell) or IgG2b isotype control (BioXcell) was administered via intraperitoneal injection every 3 d. For induction of liver injury, a single dose (100 mg kg −1 ) of N -acetyl-para-aminophenol (APAP, Sigma) was administered via intraperitoneal injection 2 d before induction of liver metastasis.

Techniques: Expressing, Immunofluorescence, Fluorescence, Live Cell Imaging, Western Blot, Control, Activation Assay, Cell Culture, Ex Vivo, Two Tailed Test

Journal: Nature Cancer

Article Title: Efferocytosis reprograms the tumor microenvironment to promote pancreatic cancer liver metastasis

doi: 10.1038/s43018-024-00731-2

Figure Lengend Snippet: (a) Schematic of proximity ligation assay (PLA) for mCherry and CFTR (left) and representative fluorescence images (right) of PLA probe-bound fluorophore (PLA, green) in BMMs transduced with mCherry-tagged PGRN (experiment was performed three times with similar results). (b) qPCR analysis of Cftr in BMMs (20hrs). Error bars, mean ± SEM (n = 3 biological replicates/group). P-values, two-way ANOVA with Sidak’s post-test. (c) Representative fluorescence images of LysoSensor-labeled BMMs (n = 38-44 cells/group, experiment was performed twice with similar results) incubated with CellTrace-labeled apoptotic thymocytes (ApopT). (d) Quantification of LysoSensor intensity as depicted in (c) . Error bars, mean ± SEM from one experiment. P-value, two-tailed unpaired t-test. (e) Representative Western blot image of Arginase 1 and loading control Cofilin levels in BMMs (3+16hrs, experiment was performed three times with similar results). (f) Relative activation level of CD8 + T cells, measured as percentages of GzmB+ cells, stimulated with anti-CD3/CD28-coupled Dynabeads and co-cultured with BMMs compared to Dynabeads-only control (n = 3 biological replicates/group from one experiment). Error bars, mean ± SEM. P-values, one-way ANOVA with Sidak’s post-test. (g) Flow cytometry analysis of activated (CD69+) CD8 + T-cells in d5 livers (n = 4 mice/group). Error bars, mean ± SEM. P-value, two-tailed unpaired t-test. (h) qPCR analysis of Arg1 in MoMs (F4/80 + TIM4–) from d14 livers (n = 3 mice/group). Error bars, mean ± SEM. P-value, two-tailed unpaired t-test. (i-k) Representative immunofluorescence images (i) and quantification of total (j) and YM-1+ (k) macrophages (F4/80+) in d14 livers. Arrowheads indicate YM-1+ macrophages (n = 3 mice/group, from one experiment). Scale bars, 50 µm. Error bars, mean ± SEM. P-value, two-tailed unpaired t-test.

Article Snippet: For the CD8 + T cell depletion study, 100 μg of anti-mouse CD8α (clone 2.43, BioXcell) or IgG2b isotype control (BioXcell) was administered via intraperitoneal injection every 3 d. For induction of liver injury, a single dose (100 mg kg −1 ) of N -acetyl-para-aminophenol (APAP, Sigma) was administered via intraperitoneal injection 2 d before induction of liver metastasis.

Techniques: Proximity Ligation Assay, Fluorescence, Transduction, Labeling, Incubation, Two Tailed Test, Western Blot, Control, Activation Assay, Cell Culture, Flow Cytometry, Immunofluorescence

Journal: Nature Cancer

Article Title: Efferocytosis reprograms the tumor microenvironment to promote pancreatic cancer liver metastasis

doi: 10.1038/s43018-024-00731-2

Figure Lengend Snippet: a , Representative immunofluorescence images of CFTR in efferocytic BMMs (experiment was performed twice with similar results). Asterisks indicate engulfed apoptotic thymocytes. b , qPCR analysis of Arg1 in efferocytic BMMs (20 h). Error bars, mean ± s.e.m. ( n = 3 biological replicates/group). P values, two-way ANOVA with Sidak’s post-test. c , Relative activation level of CD8 + T cells, measured as percentages of IFNγ + cells, stimulated with anti-CD3/CD28-coupled Dynabeads and co-cultured with BMMs compared to Dynabead-only control ( n = 3 biological replicates per group from one experiment). Error bars, mean ± s.e.m. P values, one-way ANOVA with Sidak’s post-test. d , Schematic illustrating the CFTRi experiment timeline. e , f , Representative ex vivo BLI images (left) and relative tumor burden (right) of d5 ( n = 5 mice per group, from one experiment) ( e ) and d14 ( f ) livers ( n = 5 mice per group, from two experiments). Error bars, mean ± s.e.m. P values, two-tailed unpaired t-test. g , qPCR analysis of Arg1 in MoMs (F4/80 + TIM4 − ) from d5 livers ( n = 4 mice per group). Error bars, mean ± s.e.m. P value, two-tailed unpaired t -test. h – j , Representative immunofluorescence images ( h ) and quantification of total ( i ) and cytotoxic GzmB + ( j ) CD8 + T cells in d5 livers ( n = 3 mice per group, from one experiment). Arrowheads indicate CD8 + T cell. Scale bars, 50 µm. Error bars, mean ± s.e.m. P value, two-tailed unpaired t -test. k , Graphical schematic summarizing the role of efferocytic macrophages in PDAC liver metastasis. During early stage of metastasis, seeding of cancer cells induces liver injury leading to clearance of dead cell debris by monocyte-derived macrophages (MoMs) via receptor MerTK. Engulfed dead cells are degraded in acidic phagolysosome lumen, a process that is dependent on lysosomal acidification by progranulin (PGRN) and CFTR. Following efficient lysosomal degradation of the dead cell cargo, LXRα is activated and induces expression of the T cell inhibitory factor, arginase 1. Arginase 1-mediated reduction in T cell numbers and activation eventually facilitates metastatic outgrowth. Impairment in these processes and suppression of tumor growth can be achieved via depletion of progranulin or blockade of MerTK or CFTR functions.

Article Snippet: For the CD8 + T cell depletion study, 100 μg of anti-mouse CD8α (clone 2.43, BioXcell) or IgG2b isotype control (BioXcell) was administered via intraperitoneal injection every 3 d. For induction of liver injury, a single dose (100 mg kg −1 ) of N -acetyl-para-aminophenol (APAP, Sigma) was administered via intraperitoneal injection 2 d before induction of liver metastasis.

Techniques: Immunofluorescence, Activation Assay, Cell Culture, Control, Ex Vivo, Two Tailed Test, Derivative Assay, Expressing

Journal: Blood

Article Title: CD8 + T cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia

doi: 10.1182/blood-2015-03-635417

Figure Lengend Snippet: Depletion of CD8+ T cells results in more severe thrombocytopenia and impairs responsiveness to steroid therapy in vivo. For the active model, WT mice were transplanted with immunized β3−/− splenocytes with or without depletion of CD8+ T cells. DEX treatment began at day 6. (A) Thrombocytopenia was more severe in mice given CD8+ T cell–depleted splenocytes compared with those transplanted with nondepleted splenocytes as indicated by &&&P < .001. Mice transplanted with CD8+ T cell–depleted immunized β3−/− splenocytes were less responsive to oral DEX compared with mice transplanted with nondepleted splenocytes as indicated by ###P < .001. ***P < .001, nondepleted splenocytes vs nondepleted splenocytes + DEX. N = 8. In our passive mouse model, CD8+ T cells were depleted from WT mice by injection of anti-CD8 mAb (400 μg intravenously) before inducing passive ITP with anti-β3 mAb (9D2, 1 μg intraperitoneally). Platelet counts were not significantly affected by anti-CD8 mAb depletion of CD8+ T cells. DEX was administered (10 mg/kg/day, intraperitoneally, daily) beginning at 4 hours after anti-β3 mAb injection. (B) Thrombocytopenia was more severe in CD8+ T cell–depleted mice compared with those given the anti-β3 integrin mAb alone. N = 6. (C) CD8+ T cell–depleted thrombocytopenic mice were less responsive to DEX compared with mice with normal levels of CD8+ T cells. N = 6. ***P < .001. Mean ± SD.

Article Snippet: In vivo CD8 + T-cell depletion Anti-CD8 mAb (400 μg; clone 2.43; ATCC) was injected intravenously to deplete CD8 + T cells from recipient mice before induction of thrombocytopenia.

Techniques: In Vivo, Injection

Journal: Blood

Article Title: CD8 + T cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia

doi: 10.1182/blood-2015-03-635417

Figure Lengend Snippet: CD8+ T-cell transfusion therapeutically attenuated platelet clearance and enhanced response to DEX in passive murine model of ITP. In the passive model of ITP, mice were injected with anti-β3 integrin mAb (9D2, 1 μg, intraperitoneally) at day 0 and transfused with 106 CD8+ T cells from either WT, naïve β3−/−, or immunized β3−/− mice. Control cells are remaining splenocytes following CD8+ purification. (A) CD8+ T cells significantly increased platelet count in the absence of any DEX treatment. N = 6. *P < .05, **P < .01, ***P < .001 vs PBS. (B) DEX (10 mg/kg) was administered at 4 hours after mAb injection and CD8+ T-cell transfusion. N = 6. *P < .05, **P < .01, ***P < .001 vs PBS or indicated groups. Mean ± SD.

Article Snippet: In vivo CD8 + T-cell depletion Anti-CD8 mAb (400 μg; clone 2.43; ATCC) was injected intravenously to deplete CD8 + T cells from recipient mice before induction of thrombocytopenia.

Techniques: Injection, Control, Purification

Journal: Blood

Article Title: CD8 + T cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia

doi: 10.1182/blood-2015-03-635417

Figure Lengend Snippet: Steroid treatment selectively increased CD8+ Tregs in both the passive and active models of ITP. (A) In the passive model of ITP, mice were injected with anti-β3 mAb (9D2, 1 μg, intraperitoneally) at day 0. In some mice, 10 mg/kg DEX was administered via intraperitoneal injection 4 hours after antibody injection. On indicated days, mice were bled, and different CD8+ subsets were detected via flow cytometry. N = 8 to 10. (B) In the active model, mice were transfused with immunized β3−/− splenocytes on day 0. DEX treatment (oral, 10 mg/kg) or control water was initiated on day 6. Mice were bled on the indicated days, and CD8+ subsets were detected as above. N = 6, *P < .05, **P < .01, ***P < .001. Mean ± SD.

Article Snippet: In vivo CD8 + T-cell depletion Anti-CD8 mAb (400 μg; clone 2.43; ATCC) was injected intravenously to deplete CD8 + T cells from recipient mice before induction of thrombocytopenia.

Techniques: Injection, Flow Cytometry, Control

Journal: Blood

Article Title: CD8 + T cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia

doi: 10.1182/blood-2015-03-635417

Figure Lengend Snippet: CD8+ T cells suppressed platelet apoptosis and clearance in vitro. (A-B) CD8+ T cells from immunized β3−/− mice were added to CD8+-depleted splenocytes and then cocultured with WT platelets. Control has no CD8+ T cells added. (A) After 72 hours, platelets remaining in culture were identified by forward and side scatter characteristics and gated on 7-AAD− and assessed for apoptosis with caspase 3/7 and Annexin V. N = 4. (B) CD8+ T cells from immunized β3−/− mice were cocultured with platelets for 72 hours, and then the remaining platelets in solution were counted, with flow cytometry as events per second. More platelets remained in the coculture system after incubation with CD8+ T cells from immunized β3−/− mice compared with control or naïve β3−/− CD8+ T cells. N = 4. (C) Platelets were labeled with 9D2 primary antibody and FITC anti-mouse IgG. Platelets were then cocultured with macrophages with or without CD8+ T cells. After 24 hours, FITC-positive macrophages were detected by flow cytometry. CD8+, CD8+CD25+, CD8+CD122+, and CD8+CD103+ T cells from immunized β3−/− and WT mice significantly suppressed phagocytosis of platelets by macrophages. Furthermore, the CD8+ T cells from immunized β3−/− mice suppressed phagocytosis more effectively (P < .05). This inhibitory function was also dose dependent (supplemental Figure 4) *P < .05, **P < .01. Mean ± SEM.

Article Snippet: In vivo CD8 + T-cell depletion Anti-CD8 mAb (400 μg; clone 2.43; ATCC) was injected intravenously to deplete CD8 + T cells from recipient mice before induction of thrombocytopenia.

Techniques: In Vitro, Control, Flow Cytometry, Incubation, Labeling

Journal: Blood

Article Title: CD8 + T cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia

doi: 10.1182/blood-2015-03-635417

Figure Lengend Snippet: CD8+ Tregs from immunized β3−/− mice inhibit the adaptive antiplatelet immune response in vitro. For proliferation assays, purified splenic CD8+ T cells or CD8+ Tregs from WT, naïve β3−/−, or WT platelet immunized β3−/− mice were added to CD8+ T cell–depleted splenocytes in the presence of 1 × 107 WT platelets per well to induce T- or B-cell proliferation. Control cells were CD8+-depleted splenocytes with PBS or platelets (PLT) as indicated. Both immunized β3−/− CD8+ T cells and CD8+ Tregs inhibited (A,E) CD4+ T-cell proliferation, (B,F) CD19+ B-cell proliferation, and (C,G) platelet-associated IgG production. (D) Immunized β3−/− CD8+ T cells also inhibited IL-10 cytokine production dose dependently. N = 8. Imm, immunized. *P < .05, **P < .01. Mean ± SEM.

Article Snippet: In vivo CD8 + T-cell depletion Anti-CD8 mAb (400 μg; clone 2.43; ATCC) was injected intravenously to deplete CD8 + T cells from recipient mice before induction of thrombocytopenia.

Techniques: In Vitro, Purification, Control

Journal: Blood

Article Title: CD8 + T cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia

doi: 10.1182/blood-2015-03-635417

Figure Lengend Snippet: CD8+ Tregs from immunized β3−/− mice inhibit CD8+ cytotoxic activity against platelets. Platelets (5 × 106) were cultured with purified 1 × 105 cytotoxic CD8+ T cells (CD8+CD178+) with or without 5 × 105 CD8+ Treg cells (according to the original ratio) for 24 hours. Platelet cytotoxicity was measured with (A) remaining platelet number in supernatant, determined as number of platelets per second counted by flow cytometry69 and (B) caspase 3/7 expression. N = 4. PLT, platelet; CTL, cytotoxic CD8+ T cells. *P < .05, **P < .01. Mean ± SEM.

Article Snippet: In vivo CD8 + T-cell depletion Anti-CD8 mAb (400 μg; clone 2.43; ATCC) was injected intravenously to deplete CD8 + T cells from recipient mice before induction of thrombocytopenia.

Techniques: Activity Assay, Cell Culture, Purification, Expressing

Journal: Scientific Reports

Article Title: CD8 + T-cells restrict the development of peritoneal metastasis and support the efficacy of hyperthermic intraperitoneal chemotherapy (HIPEC)

doi: 10.1038/s41598-024-72826-w

Figure Lengend Snippet: Comparison of patient characteristics between CD8 + T-cell high and CD8 + T-cell low . The data is.

Article Snippet: CD-4 + and CD8+-T-cell depletion in mice was achieved by the intraperitoneal injection of 100ug CD4 or CD8a depletion antibody (BioXCell, USA; clone GK 1.5 for CD4 + T-cells and clone YTS 169.4 was used for CD8 + T-cells) 1 day prior to tumor cells injection and 1 day prior to the treatment.

Techniques: Comparison, Cell Differentiation, Mutagenesis, Adjuvant

Journal: Scientific Reports

Article Title: CD8 + T-cells restrict the development of peritoneal metastasis and support the efficacy of hyperthermic intraperitoneal chemotherapy (HIPEC)

doi: 10.1038/s41598-024-72826-w

Figure Lengend Snippet: Assessment of CD8 + T cells in patient samples. ( a and b ) analysis of CD8 + T cells in stroma and epithelium of the primary tumor of 19 patients ( a ) and the PM lesions of 37 patients. The graphs illustrate the number of CD8 + T-cells normalized to the corresponding area of stroma or epithelium. ( c ).The bar graph shows the distribution of intraepithelial CD8 + T-cells normalized by area in PM-lesions among the 37 patients. The dotted line indicates the median and divide the cohort into CD8 + T-cell high and low. ( d ) The scanned histological slide of PM lesions. The upper left picture presents an example with high CD8 + T-cell infiltration and the corresponding HALO classified picture below. The left upper example is CD8 + T-cell low. The classified pictures show the different areas of the tumor (green: stroma, violet: necrosis, red: intraepithelial, yellow: white space). DFS ( e ) and OS ( f ) based on intraepithelial CD8 + T-cell counts of PM lesions. 18 patients belong to the CD8 + T-cell high group and 19 patients to the CD8 + T-cell low group. DFS ( g ) and OS ( h ) based on the stroma content and CD8 + T-cell distribution. 15 patients with a CD8 + T-cell high PM lesion were associated with low stromal content (continuous line), 9 patients with a CD8 + T-cell low PM-lesion had a rich stroma (fine dotted line),10 patients with a CD8 + T-cell low PMlesion had a poor stroma (bold dotted line) and 3 patients with a CD8 + T-cell high PM-lesion were stroma rich (dotted line). Error bars represent the median and the lines the interquartile range. Each dot represents a patient. **** = p ≤ 0.0001, *** = p ≤ 0.001, ** = = p ≤ 0.01, * = p ≤ 0.05, ns = p > 0.05.

Article Snippet: CD-4 + and CD8+-T-cell depletion in mice was achieved by the intraperitoneal injection of 100ug CD4 or CD8a depletion antibody (BioXCell, USA; clone GK 1.5 for CD4 + T-cells and clone YTS 169.4 was used for CD8 + T-cells) 1 day prior to tumor cells injection and 1 day prior to the treatment.

Techniques:

Journal: Scientific Reports

Article Title: CD8 + T-cells restrict the development of peritoneal metastasis and support the efficacy of hyperthermic intraperitoneal chemotherapy (HIPEC)

doi: 10.1038/s41598-024-72826-w

Figure Lengend Snippet: Impact of HIPEC treatment in PM mouse model. ( a ) measurement of peritoneal tumor load as PCI. mice were treated with PBS ( n = 9) or heated PBS ( n = 7) or with M/D (n = 10) or with heated M/D (n = 11). ( b ) Staining of tumor tissues for the presence of CD8 + T cells, Granzyme B + cells and macrophages. ( c and d ) Quantification of CD8 + T cells and GZMB + cells. ( e ) PCI of the treated mice with and without CD8 + T-cells. Each dot represents one mouse. Error bars show the mean +/-SD. **** = p ≤ 0.0001, *** = p ≤ 0.001, ** = = p ≤ 0.01,* = p ≤ 0.05, ns = p > 0.05.

Article Snippet: CD-4 + and CD8+-T-cell depletion in mice was achieved by the intraperitoneal injection of 100ug CD4 or CD8a depletion antibody (BioXCell, USA; clone GK 1.5 for CD4 + T-cells and clone YTS 169.4 was used for CD8 + T-cells) 1 day prior to tumor cells injection and 1 day prior to the treatment.

Techniques: Staining

Journal: Scientific Reports

Article Title: CD8 + T-cells restrict the development of peritoneal metastasis and support the efficacy of hyperthermic intraperitoneal chemotherapy (HIPEC)

doi: 10.1038/s41598-024-72826-w

Figure Lengend Snippet: Induction of antigen-specific CD8 + T cells via heated chemotherapy. ( a )Time-line of the experiment. ( b ) flow cytometry data on the maturation state of Mo-DC`s depending on the cancer-cell treatment. ( c )Time-line of the experiment with additional co-culture of CD8 + T-cells. ( d ) Shows flow cytometry data of CD8 + T-cells and their IFN-γ production depending on the cancer-cell treatment. ( e ) Shows the time-line of a similar experiment using splenocytes from OT-I mice, which have a specific TCR for the ovalbumin. ( f ) Presents the ratio of IFN-γ positive CD8 + T-cells after co-culturing with PBS, or M/D or heated M/D treated MC-38-Ova cancer cells with splenocytes from a OT-I mouse. Cancer cell killing by specific CD8 + T-cells. ( g ) time-line of the co-culture experiment. The % of cancer cell killing by the treatment with or without specific CD8 + T-cells is shown in ( h ).The experiments were performed in triplicates. Error bars show the mean +/-SD. **** = p ≤ 0.0001, *** = p ≤ 0.001, ** == p ≤ 0.01, * = p ≤ 0.05, ns = p > 0.05.

Article Snippet: CD-4 + and CD8+-T-cell depletion in mice was achieved by the intraperitoneal injection of 100ug CD4 or CD8a depletion antibody (BioXCell, USA; clone GK 1.5 for CD4 + T-cells and clone YTS 169.4 was used for CD8 + T-cells) 1 day prior to tumor cells injection and 1 day prior to the treatment.

Techniques: Flow Cytometry, Co-Culture Assay

Journal: Scientific Reports

Article Title: CD8 + T-cells restrict the development of peritoneal metastasis and support the efficacy of hyperthermic intraperitoneal chemotherapy (HIPEC)

doi: 10.1038/s41598-024-72826-w

Figure Lengend Snippet: Intraepithelial CD8 + T-cell number after HIPEC in 2 patients. ( a ) Numbers of intraepithelial CD8 + T-cells before first HIPEC and after the first HIPEC treatment of 2 different patient and the DFS and OS of these two patients ( b ).

Article Snippet: CD-4 + and CD8+-T-cell depletion in mice was achieved by the intraperitoneal injection of 100ug CD4 or CD8a depletion antibody (BioXCell, USA; clone GK 1.5 for CD4 + T-cells and clone YTS 169.4 was used for CD8 + T-cells) 1 day prior to tumor cells injection and 1 day prior to the treatment.

Techniques:

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: Iron overload‐associated ZDHHC12 primarily participates in maintaining the activation of CD8+T cells in renal cell carcinoma. (a) UMAP plot showing the subpopulation classification of CD8+ T cells ( GSE121636 ). (b) UMAP visualization of ZDHHC12 expression across different CD8+ T cell subsets. (c) Violin plot depicting ZDHHC12 expression patterns in various CD8+ T cell subpopulations. (d) BALB/c mice fed either a high‐iron‐load diet or a normal diet were subcutaneously inoculated with Renca tumor cells on the dorsum, and starting from the day of inoculation, 200 µg of anti‐CD8 depletion antibody (αCD8) or an isotype control antibody was intraperitoneally administered every three days; tumor volume was measured from day 7 post‐inoculation. Data were presented as mean ± SD. e and f, Western blot (e) and RT‐qPCR (f) analyses were performed on CD8+ T cells sorted from renal cancer tissues of both the normal diet and high‐iron diet groups. (g–i), The figure illustrates schematic diagrams of different treatment groups (g). Specifically, tumor cells were isolated and sorted from the tumor tissues of the renal cancer model. Subsequently, the collected tumor cells were subcutaneously injected into the dorsum of either Zdhhc12 wild‑type or knockout mice. Tumor size was measured regularly, and growth curves were plotted (h). On day 19, the mice were sacrificed, and tumors were excised and weighed (i). (j–m), Portions of tumor tissues were processed for cell extraction, staining, and flow cytometry analysis to evaluate CD8+ T cell function in Zdhhc12 wild‐type and KO mice. Specifically, the proportions of CD3+/CD45+ (j), CD8+/CD3+ (k), GZMB+/CD8+ (l), and IFN‐γ+/CD8+ (m) T cells within tumor masses were quantified. (n,o) Portions of tumor tissues were embedded, sectioned, stained, and subjected to immunofluorescence analysis to assess CD8+ T cell function in Zdhhc12 wild‐type and KO mice. Specifically, multiplex staining was performed for DAPI/CD3/CD8/GZMB, and the proportions of positive cells were quantified (n); similarly, multiplex staining for DAPI/CD3/CD8/IFN‐γ was conducted with subsequent quantification of positive cell ratios (o).

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Activation Assay, Expressing, Control, Western Blot, Quantitative RT-PCR, Isolation, Injection, Knock-Out, Extraction, Staining, Flow Cytometry, Cell Function Assay, Immunofluorescence, Multiplex Assay

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: Iron overload promotes the ubiquitin‐proteasome pathway degradation of ZDHHC12 by enhancing its binding with TRIM28. (a) Cells were collected for Western blot analysis after treating sorted CD8+ T cells with FAC at the indicated concentrations for 48 h. (b) Cells were collected for Western blot analysis after treating sorted CD8+ T cells with FAC at the indicated concentration gradients for 48 h. (c) Cells were collected for Western blot analysis after treating 293T cells with FAC at the indicated concentrations for 48 h. (d) Cells were collected for Western blot analysis after treating 293T cells with FAC at the indicated concentration gradients for 48 h. (e) The degradation of ZDHHC12 in 293T cells was assessed by CHX chase assay after treatment with or without FAC (20 µg/mL) for 48 h. (f) HA‐ZDHHC12 was transfected into 293T cells, and co‐immunoprecipitation was performed using an anti‐HA‐ZDHHC12 antibody, followed by mass spectrometry to identify potential ZDHHC12‐interacting proteins. The peptide spectrum of TRIM28 is shown. (g) The degradation of ZDHHC12 in 293T cells was assessed by CHX chase assay with or without TRIM28 knockout. (h) Schematic diagrams depicting the protein domain architecture of TRIM28 and the TRIM28 △RING truncation mutant. (i) Sorted CD8+ T cells were stably transfected with the plasmids illustrated in the figure, followed by cell collection for Western blot analysis and quantitative assessment. (j) After transfection with the indicated plasmids, 293T cells were treated with FAC, followed by MG132 treatment, and then collected for Co‐IP and Western blot analysis. (k) After treatment with FAC (0, 20, 40 µg/mL), 293T cells were treated with MG132, collected, and subjected to Co‐IP and Western blot analysis. (l) After transfection with the indicated plasmids for 24 h, 293T cells were collected for Co‐IP and Western blot analysis.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Ubiquitin Proteomics, Binding Assay, Western Blot, Concentration Assay, Transfection, Immunoprecipitation, Mass Spectrometry, Knock-Out, Mutagenesis, Stable Transfection, Co-Immunoprecipitation Assay

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: ZDHHC12 inhibits cuproptosis in CD8+T cells through interaction with FDX1. (a) We reviewed the mass spectrometry results and found that FDX1 may interact with ZDHHC12, and the peptide spectrum of FDX1 is shown in the figure. (b) HA‐ZDHHC12 and Flag‐FDX1 were transfected into 293T cells, followed by cell lysis and co‐immunoprecipitation using Flag or HA antibodies, and then analyzed by western blot. (c) 293T cells were lysed and subjected to co‐immunoprecipitation with ZDHHC12 or FDX1 antibodies, followed by western blot detection. (d) Immunofluorescence staining was performed in 293T cells using ZDHHC12 and FDX1 antibodies, and images were acquired and analyzed by confocal microscopy. (e) GST pull‐down assay was performed using the recombinant protein of ZDHHC12. (f) GST pull‐down assay was performed using the recombinant protein of FDX1. (g) Protein structure prediction and molecular docking were conducted for ZDHHC12 and FDX1. (h,i) Western blot (h) and RT‐qPCR (i) were performed in 293T cells after transfection with the indicated plasmids. (j,k) Western blot (j) and RT‐qPCR (k) were performed in 293T cells after transfection with the indicated plasmids. (l) Western blot analysis was conducted in 293T cells 72 h after transfection with the indicated plasmids. (m) Western blot analysis was performed in 293T cells after transfection with the indicated plasmids. (n) CD8+ T cells were isolated from renal cancer tissues of Zdhhc12 WT and Zdhhc12 KO mice and subjected to western blot analysis. (o) After successful transfection with the indicated plasmids in 293T cells, cells were collected and treated with different concentrations of elesclomol‐Cu or disulfiram‐Cu as shown in the figure for 48 h, followed by CCK‐8 assay to measure and quantify cell viability. (p,q) Following successful transfection with the indicated plasmids in 293T cells, cells were collected and immunostained with DLAT and Mito‐tracker, then imaged by confocal microscopy (p) to quantify (q) DLAT oligomerization. (r) After successful transfection with the indicated plasmids in 293T cells, cells were treated with different concentrations of elesclomol‐Cu or disulfiram‐Cu for 48 h in the presence or absence of tetrathiomolybdate (TTM) (1 µ m ) as indicated, followed by CCK‐8 assay to measure and quantify cell viability. (s,t) Following successful transfection with the indicated plasmids in 293T cells, cells were collected after treatment with or without tetrathiomolybdate (TTM) (1 µ m ), then immunostained with DLAT and Mito‐tracker and imaged by confocal microscopy (s) to quantify (t) DLAT oligomerization. (u) After successful transfection with the indicated plasmids in CD8+ T cells, cells were treated with or without tetrathiomolybdate (TTM) (1 µ m ), then collected for western blot analysis. (v) Following successful transfection with the indicated plasmids in 293T cells, cells were collected and treated with different concentrations of elesclomol‐Cu or disulfiram‐Cu as shown in the figure for 48 h, followed by CCK‐8 assay to measure and quantify cell viability. (w) After successful transfection with the indicated plasmids in sorted CD8+ T cells, cells were collected for western blot analysis.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Mass Spectrometry, Transfection, Lysis, Immunoprecipitation, Western Blot, Immunofluorescence, Staining, Confocal Microscopy, Pull Down Assay, Recombinant, Quantitative RT-PCR, Isolation, CCK-8 Assay

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: ZDHHC12‐mediated palmitoylation of FDX1 at Cys152/155 promotes its degradation. (a) In 293T and CD8+T cells, FDX1 was immunoprecipitated using an anti‐FDX1 antibody, followed by the acyl‐biotin exchange (ABE) assay performed either in the presence or absence of hydroxylamine (HAM) treatment, with subsequent streptavidin‐HRP pulldown of biotin‐conjugated proteins to specifically detect palmitoylated FDX1. (b) In 293T cells, FDX1 was immunoprecipitated using an anti‐ FDX1 antibody, followed by the acyl‐biotin exchange (ABE) assay performed with or without hydroxylamine (HAM) treatment, and subsequently enriched with streptavidin‐HRP to isolate biotin‐conjugated proteins, thereby enabling the detection of FDX1 palmitoylation levels in both 2‐BP (25 µ m , 24 h)‐treated and untreated conditions. (c) 293T cells treated with or without palmitic acid azide were collected for Click‐IT reaction and streptavidin pulldown. (d) 293T cells were treated with different concentrations of 2‐BP (+, 20 µ m ; ++, 40 µ m ) for 24 h, then collected for western blot analysis. (e) 293T cells treated with or without 2‐BP (25 µ m , 24 h) were exposed to CHX and collected at different time points for western blot analysis. (f) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to ABE assay and western blot analysis with or without HAM treatment. (g) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to ABE assay and western blot analysis with or without HAM treatment. (h) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to ABE assay and western blot analysis with or without HAM treatment. (i) 293T cells transfected with the indicated plasmids were treated with or without 2‐BP (25 µ m , 24 h) after 24 h, then collected for western blot analysis. (j) The peptide spectrum for FDX1 palmitoylation site identification. (k) Conservation sequences of FDX1 C152 and FDX1 C155 sites across different species. (l) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to Click‐iT pull‐down assay and western blot analysis with or without palmitic acid azide treatment. m, AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to ABE assay and western blot analysis with or without HAM treatment. (n) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to Click‐iT pull‐down assay and western blot analysis with or without palmitic acid azide treatment, followed by quantitative analysis of FDX1 palmitoylation levels. (o) CD8+ T cells were transfected with the indicated plasmids, then treated with PBS or FAC (20 µg/mL), collected, and stained with the indicated fluorescent dyes for flow cytometry analysis. (p) CD8+ T cells were transfected with the indicated plasmids, then collected and stained with the indicated fluorescent dyes for flow cytometry analysis. (q) CD8+ T cells were transfected with the indicated plasmids, then treated with PBS or FAC (20 µg/mL), collected, and stained with the indicated fluorescent dyes for flow cytometry analysis.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Immunoprecipitation, Western Blot, Knock-Out, Transfection, Pull Down Assay, Staining, Flow Cytometry

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: Development of lipid nanoparticles encapsulating Zdhhc12 mRNA (Zdhhc12‐LNP). (a) Schematic diagram of the LNP synthesis process; (b) The average zeta potential and the average particle size; (c) The average PDI and the average encapsulation efficiency; (d) Representative cryo‐EM image showing the morphology of synthesized LNPs. (e) Spectrum showing the purity of the sample. (f) Representative chromatogram showing mRNA capping efficiency. (g) Western blot analysis of Zdhhc12 expression in CD8+ T cells isolated from mice and treated with control, blank LNP, or Zdhhc12‐encapsulated LNP, following a 24 h pre‐stimulation with 2 µg/mL anti‐CD3/CD28 and subsequent 24 h incubation with LNPs. (h) ABE assay and Western blot analysis were performed on CD8+ T cells isolated from mice to assess the palmitoylation level of Fdx1, where cells were first cultured for 24 h post‐infection without anti‐CD3/CD28, restimulated with 2 µg/mL anti‐CD3/CD28 for 24 h, incubated with LNPs for 24 h, and then harvested for analysis. (i) Renca cells were subcutaneously injected into the backs of BALB/c mice, LNPs were administered via intratumoral injection, and when tumors reached an appropriate size, tumors were harvested for sorting of CD8+ T cells, followed by Western blot analysis to assess Zdhhc12 expression level in CD8+ T cells. (j–m) Renca cells were subcutaneously injected into the dorsal region of BALB/c mice. After tumor establishment, mice received intratumoral injection of LNPs or no injection, and serum levels of ALT (j), AST (k), CRE (l), and BUN (m) were measured at appropriate time points. Ns, not significant. (n,o) BALB/c mice were subcutaneously injected with Renca cells, intratumorally administered Zdhhc12‐LNP or control, and intraperitoneally injected with 200 µg anti‐CD8 (αCD8) or control; tumor growth was recorded starting from day 7 post‐tumor inoculation (n), and flow cytometry analysis of CD8+ T cell content in the peripheral blood of mice (n = 5) was performed at the end of the experiment (o), with data presented as mean ± SD.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Zeta Potential Analyzer, Encapsulation, Cryo-EM Sample Prep, Synthesized, Western Blot, Expressing, Isolation, Control, Incubation, Cell Culture, Infection, Injection, Flow Cytometry

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: Under normal iron conditions, ZDHHC12‐mediated palmitoylation of FDX1 promotes FDX1 degradation by the mitochondrial matrix protease AFG3L2, ultimately suppressing cuproptosis in CD8+ T cells and maintaining their immune clearance function. Under iron overload conditions, increased binding between ZDHHC12 and TRIM28 leads to ZDHHC12 degradation, consequently inducing cuproptosis in CD8+ T cells and facilitating immune escape.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Binding Assay