Journal: EMBO Reports

Article Title: Acquired resistance to PD-L1 inhibition enhances a type I IFN-regulated secretory program in tumors

doi: 10.1038/s44319-024-00333-0

Figure Lengend Snippet: ( A ) Schematic showing orthotopic breast EMT6 model of acquired resistance following PD-L1 inhibition. ( B ) Continuous αPD-L1 treatment in BALB/c mice ( n = 3–4 biological replicates) bearing orthotopically-implanted mouse mammary EMT6 cells (left) with tumor growth of selected P and PTR variants shown (right; circle indicates selection). PTR and P variants were maintained in vitro with respective αPD-L1 or IgG antibody (see Methods for details). ( C – F ) RNA sequencing analysis of EMT6-P and EMT6-PTR tumor tissues. n = 3. ( C ) Differentially expressed genes (Log [Fold Change] ≤ −2 or ≥2) in EMT6-PTR as summarized by dot plot (left) and heatmap (right). Red = upregulated; blue = downregulated. ( D ) Summary of stroma/tumor microenvironment, growth factor signaling, and cytokine signaling gene sets with significant positive enrichment (FDR ≤ 0.25) found in PTR tumors via GSEA of all canonical pathways (C2, Molecular Signatures Database Collection). Size of circles correspond to GSEA calculated FDR significance. See Appendix Table S for details. ( E ) Cytoscape GO analysis of significantly enriched biological processes in upregulated secretory genes, grouped by signaling categories. Size of circles correspond to p value significance of each process calculated via Fisher’s exact test and lines represent term-term interactions defined by Kappa score. ( F ) Bar graph representing Interferome Database secretory genes up- and down-regulated in EMT6-PTR (compared to EMT6-P). ( G , H ) GSEA of EMT6-PTR tumors (compared to P controls) showing lollipop plot representing ( G ) NES of published/Hallmark IFN gene sets, and ( H ), NES of an IFN-specific gene-set identified in αPD-L1-treated (durvalumab) NSCLC patients (described in Ref (Higgs et al, )). Size of circles correspond to FDR significance calculated via GSEA. See Appendix Table S for details. ( I ) Type I IFN-regulated ISGs in EMT6-PTR selected cell variants (qRTPCR). ┼ represent genes associated with secretory proteins. qRT-PCR, statistics performed via two-tailed t-test, n = 3–4. ( J ) Schematic showing orthotopic mouse kidney RENCA tumor model of innate resistance to αPD-L1 inhibition (top) and the BLI quantification in mice treated with αPD-L1 and IgG vehicle control (bottom; Balb/c mice; n = 3 biological replicates). ( K ) BLI of mice at endpoint (top) with representative images of mice shown (bottom). Circles indicate mice used to select P and PTR tumor variants which were selected from dissociated kidneys and maintained in vitro with IgG or αPD-L1 antibody, respectively (see Methods for details). ( L ) GSEA of RENCA-PDL1 tumor cells (compared to P controls) showing lollipop plot representing NES of published/Hallmark IFN gene sets. Size of circles correspond to FDR significance calculated via GSEA. See Appendix Table S for details. Data Information: Parental (P); αPD-L1 Treatment-Resistant (PTR); Control (Con); Gene Set Enrichment Analysis (GSEA); Gene Ontology (GO); interferon stimulated genes (ISGs); Counts per million (CPM); Bioluminescence imaging (BLI); normalized enrichment scores (NES); Non-small cell lung carcinoma (NSCLC); Standard Deviation (SD); Standard Error of the Mean (SEM). αPD-L1 (clone 80) and IgG/PBS were administered at 250 μg/mouse every 3 days until endpoint. Primary tumor burden was assessed by caliper measurement. Tumor growth line graph quantitative data represent mean ± SEM. Bar graphs show mean ± SD. Time to institutional endpoint was assessed by Kaplan–Meier. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, for exact p values see Fig. 1 Source data. All replicates shown represent technical replicates unless otherwise specified. .

Article Snippet: EMT6 (5 × 10 5 cells in 100 μl RPMI), RENCA (4 × 10 4 cells in 5 μl 1:1 RPMI:Matrigel) were implanted orthotopically into the right inguinal mammary fad pad or left kidney subcapsular space respectively in 6–8-week-old female NCI Balb/cAnNCr (Balb/c) mice (Charles River, USA).

Techniques: Inhibition, Selection, In Vitro, RNA Sequencing, Quantitative RT-PCR, Two Tailed Test, Control, Imaging, Standard Deviation

Journal: EMBO Reports

Article Title: Acquired resistance to PD-L1 inhibition enhances a type I IFN-regulated secretory program in tumors

doi: 10.1038/s44319-024-00333-0

Figure Lengend Snippet: ( A ) Generation of a PTIS signature comprised of 5 upregulated genes identified from transcriptome and proteomic analysis using EMT6-PTR cells. ( B ) Differentially expressed secretory genes (GO:0005576) (Log 2 [Fold Change] ≤ −2 or ≥2) in EMT6-PTR cells as summarized by dot plot (left) and heatmap (right). Red = upregulated; blue = downregulated. Embedded heatmap showing gene expression of PTIS factors. n = 3. ( C ) Blot images of cytokine protein array of EMT6-P and -PTR tumor cells before and after IFNAR1 knockdown. PTIS factors highlighted in red and shown as enlarged images (right). n = 1. ( D ) Cytokine protein array analysis of type I IFN regulated proteins in EMT6-PTR tumor cells compared to parental controls (left) and EMT6-PTR compared to EMT6-PTR IFNAR1 KD tumor cells. Circled proteins showing expression of PTIS factors. n = 1. ( E ) NOS2 and LCN2 levels in lysates of EMT6-P, EMT6-PTR, EMT6-P IFNAR1 KD , and EMT6-PTR IFNAR1 KD cells. Western blot, representative image n = 1. ( F ) Densitometry quantification of western blots shown in ( E ) representing LCN2 (left) and NOS2 (right) levels. n = 3, statistics performed via two-tailed t-test. ( G ) LCN2, IL6, and CCL2 levels in lysates of EMT6-P, EMT6-PTR, EMT6-P IFNAR1 KD , and EMT6-PTR IFNAR1 KD cells. ELISA, n = 5–6, statistics performed via two-tailed t-test. ( H ) Summary data of PTIS factor expression in EMT6-P and -PTR cells shown as relative to P controls and represented as bar graphs. qRT-PCR, n = 4–8 (1–2 biological replicates), statistics performed via two-tailed t-test. ( I ) Summary data of PTIS factor expression in EMT6-PTR-IFNAR1 KD cells shown as relative to PTR controls and represented as bar graphs. qRT-PCR, n = 4–20 (1–4 biological replicates), statistics performed via two-tailed t-test. ( J ) Heatmap summary of baseline transcriptomic and proteomic expression of PTIS factors showing comparisons of EMT6-P/PTR and IFNAR1 KD cells, statistics performed via two-tailed t-test. Data Information: αPD-L1 Treatment-Induced Secretome (PTIS); αPD-L1 Treatment-Resistant (PTR); Counts per million (CPM); IFNAR1 knockdown (IFNAR1 KD ); Control Knockdown (shCon); Not Tested (NT); Standard Deviation (SD). Bar graphs show mean ± SD. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, for exact p values see Fig. 2 Source data. All replicates shown represent technical replicates unless otherwise specified. .

Article Snippet: EMT6 (5 × 10 5 cells in 100 μl RPMI), RENCA (4 × 10 4 cells in 5 μl 1:1 RPMI:Matrigel) were implanted orthotopically into the right inguinal mammary fad pad or left kidney subcapsular space respectively in 6–8-week-old female NCI Balb/cAnNCr (Balb/c) mice (Charles River, USA).

Techniques: Gene Expression, Protein Array, Knockdown, Expressing, Western Blot, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Control, Standard Deviation

Journal: EMBO Reports

Article Title: Acquired resistance to PD-L1 inhibition enhances a type I IFN-regulated secretory program in tumors

doi: 10.1038/s44319-024-00333-0

Figure Lengend Snippet: ( A ) IFNAR1 expression in EMT6-P and -PTR before and after knockdown of IFNAR1 and respective vector controls. Flow cytometry, n = 3, statistics performed via two-tailed t-test. Bar graphs show mean ± SD. ( B ) Bar plot summary of cytokine protein array shown as EMT6-PTR compared to parental controls from Fig. . Bars representing mean value of two separate experiments (shown as dots), n = 2. ( C ) Heatmap summary of type I IFN regulated proteins in cytokine protein array. ( D ) Table summary of PTIS transcriptomic and proteomic confirmations shown in Fig. . Data Information: αPD-L1 Treatment-Induced Secretome (PTIS); αPD-L1 Treatment-Resistant (PTR);Counts per million (CPM); IFNAR1 knockdown (IFNAR1 KD ); Control Knockdown (shCon); Not Tested (NT). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, for exact p values see Fig. EV1 Source data. All replicates shown represent technical replicates unless otherwise specified. .

Article Snippet: EMT6 (5 × 10 5 cells in 100 μl RPMI), RENCA (4 × 10 4 cells in 5 μl 1:1 RPMI:Matrigel) were implanted orthotopically into the right inguinal mammary fad pad or left kidney subcapsular space respectively in 6–8-week-old female NCI Balb/cAnNCr (Balb/c) mice (Charles River, USA).

Techniques: Expressing, Knockdown, Plasmid Preparation, Flow Cytometry, Two Tailed Test, Protein Array, Control

Journal: EMBO Reports

Article Title: Acquired resistance to PD-L1 inhibition enhances a type I IFN-regulated secretory program in tumors

doi: 10.1038/s44319-024-00333-0

Figure Lengend Snippet: ( A ) Transcriptome and proteomic analysis were used to generate a signature comprised only of genes significantly down regulated (PTIS DOWN ) in EMT6-PTR cells. The 4-gene PTIS DOWN was comprised primarily of IFN-regulated genes (3 out of 4 total). ( B , C ) PTIS DOWN was measured in published preclinical datasets described in Fig. . In αPD-L1 treatment- sensitive and - insensitive tumor models, enrichment for the PTIS DOWN was variable, with no significant signature expression trends found (as measured by CPM). Only 1 of 5 models ( insensitive; RENCA-PDL1) showed significant negative GSEA enrichment of the PTIS DOWN signature. P values representative of statistics performed via two-tailed t-test. ( B ) Analysis of PTIS DOWN expression in αPD-L1 treatment- sensitive tumor models used data from (Lan et al, ; Sceneay et al, ; Efremova et al, ) (Data ref: Lan et al, ; Data ref: Sceneay et al, ; Data ref: Efremova et al, ). Data is compared to vehicle/IgG-treated controls. Central line depicts the median, n = 3–10 biological replicates. ( C ) Analysis of PTIS DOWN expression in αPD-L1 treatment- insensitive tumor models used data from (Sceneay et al, ) (Data ref: Sceneay et al, ) and RENCA-PDL1 model from this study. Central line depicts the median, n = 3–8 biological replicates. ( D , E ) Using published bulk and single cell RNAseq clinical datasets taken from tumor biopsies of αPD-L1 treatment- sensitive patients, PTIS DOWN expression was assessed by average CPM expression and GSEA comparisons. P values representative of statistics performed via two-tailed t-test. ( D ) Analysis of PTIS DOWN signature showed decreases in NSCLC after treatment significance not reached). Bulk RNAseq from (Gettinger et al, ) (Data ref: Gettinger et al, ) to compare Pre- and Post-treatment (Tx) tumor sample comparisons (Gray lines indicate matched Pre- and Post-tx samples). Central line depicts the median, n = 4–7 biological replicates. ( E ) Clustered analysis of MCC avelumab-treated samples showed variable, but significant PTIS DOWN changes in enriched tumor cell (decreased expression) and macrophage clusters (increased expression). Single-cell RNAseq data obtained from (Paulson et al, ) (Data ref: Paulson et al, ) represent untreated (No-Tx) or treated (avelumab, MCPyV-T cell, radiation) tumor samples ( n = 1) with tSNE plots (left) representing average log 2 CPM expression of PTIS in whole dataset, and bar graphs (right) representing clustered enrichment analysis populations identified by markers for tumors (CD45−), macrophages (CD68+), and T cells (CD3D+). Tumor sample that received No-Tx was compared to treated. Data Information: αPD-L1 Treatment-Induced Secretome involving only downregulated genes (PTIS DOWN ); αPD-L1 Treatment-Resistant (PTR); Counts per million (CPM); Gene set enrichment analysis (GSEA); False Discovery Rate (FDR); Gene Expression Omnibus (GEO); GEO Series records (GSE); database of Genotypes and Phenotypes (dbGaP); t-distributed stochastic neighbor embedding (tSNE); Treatment (Tx); non-small cell lung carcinoma (NSCLC); Merkel cell carcinoma (MCC). Bar graphs show mean ± SEM. Scatter dot plot central line show mean. Bolded numbers for GSEA represent FDR < 0.25 (see Methods). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, except for GSEA as indicated above, for exact p values see Fig. EV2 Source data. .

Article Snippet: EMT6 (5 × 10 5 cells in 100 μl RPMI), RENCA (4 × 10 4 cells in 5 μl 1:1 RPMI:Matrigel) were implanted orthotopically into the right inguinal mammary fad pad or left kidney subcapsular space respectively in 6–8-week-old female NCI Balb/cAnNCr (Balb/c) mice (Charles River, USA).

Techniques: Expressing, Two Tailed Test, Gene Expression

Journal: EMBO Reports

Article Title: Acquired resistance to PD-L1 inhibition enhances a type I IFN-regulated secretory program in tumors

doi: 10.1038/s44319-024-00333-0

Figure Lengend Snippet: ( A ) IFNα and IFNβ expression in EMT6-P and -PTR before and after knockdown of IFNAR1 KD . qRT-PCR, n = 14–20 (3–4 biological replicates), statistics performed via two-tailed t-test. ( B ) PTIS expression in EMT6-P and -PTR cells shown as relative to non-stimulated P controls after IFNα/β stimulation and represented as bar graph (top) and heatmap (bottom). qRT-PCR, n = 4, statistics performed via two-tailed t-test. ( C ) PTIS factor expression in EMT6-PTR-IFNAR1 KD cells shown as relative to PTR controls after IFNα/β stimulation and represented as bar graph (top) and heatmap (bottom). qRT-PCR, n = 4, statistics performed via two-tailed t-test. ( D ) PTIS factor expression in EMT6-PTR cells after IFNα/β stimulation shown as relative to non-stimulated PTR and represented as bar graph (top) and heatmap (bottom). qRT-PCR, n = 4, statistics performed via two-tailed t-test. ( E ) IL6 expression in EMT6-P and -PTR cell conditioned media. ELISA, n = 3, statistics performed via two-tailed t-test. ( F ) Phosphorylated and total levels of STAT1/3 in lysates of EMT6-P and -PTR before and after knockdown of IFNAR1 KD following IFNβ stimulation. Western Blot. ( G , H ) Densitometry quantification of western blots shown in ( F ) representing relative phosphorylated STAT1 compared to total STAT1 (top) and phosphorylated STAT3 levels compared to total STAT3 levels (bottom) at ( G ) baseline and ( H ) after IFNβ stimulation. n = 3, statistics performed via two-tailed t-test. ( I ) Antigen presentation machinery expression in EMT6-P and -PTR cells. qRT-PCR and flow cytometry as indicated n = 3–4, statistics performed via two-tailed t-test. ( J ) PD-L1 and MHC-I expression in EMT6-P and -PTR before and after knockdown of IFNAR1 KD , and after IFNβ stimulation. ELISA lysate and flow cytometry, respectively, n = 3, statistics performed via two-tailed t-test. ( K ) Heatmap summary of protein and gene expression analysis. Statistics comparing PTR versus P for each condition, statistics performed via two-tailed t-test. Data Information: Parental (P); αPD-L1 Treatment-Resistant (PTR); Conditioned Media (CM); Not Treated (NT); Mean Fluorescent Intensity (MFI); IFN stimulated genes (ISGs); IFNAR1 knockdown (IFNAR1 KD ); shRNA vector control (shCon; shown here as a ‘–’); Cells were treated with 10 ng/ml of IFNs and collected after 15 min (western blot shown in ( F )), and 5 days (IL6, PD-L1, MHC-I). Bar graphs show mean ± SD. Box plot indicate range between minimum and maximum, central line depicts the mean. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 indicate significance compared to untreated controls unless otherwise shown (lines), for exact p values see Fig. 4 Source data. #STAT activation measured by comparing phosphorylated STAT protein compared to total STAT protein levels. All replicates shown represent technical replicates unless otherwise specified. .

Article Snippet: EMT6 (5 × 10 5 cells in 100 μl RPMI), RENCA (4 × 10 4 cells in 5 μl 1:1 RPMI:Matrigel) were implanted orthotopically into the right inguinal mammary fad pad or left kidney subcapsular space respectively in 6–8-week-old female NCI Balb/cAnNCr (Balb/c) mice (Charles River, USA).

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Western Blot, Immunopeptidomics, Flow Cytometry, Gene Expression, shRNA, Plasmid Preparation, Control, Activation Assay

Journal: EMBO Reports

Article Title: Acquired resistance to PD-L1 inhibition enhances a type I IFN-regulated secretory program in tumors

doi: 10.1038/s44319-024-00333-0

Figure Lengend Snippet: ( A ) PTIS factor expression in EMT6-P and -PTR cells before and after knockdown of IFNAR1 KD , and after IFN α/β stimulation shown as bar plots. Full datasets for Fig. B, C, D. qRT-PCR, statistics performed via two-tailed t-test, n = 4. ( B ) Table summarizing two-tailed t-test statistical comparisons for qRT-PCR data shown in Fig. B, C, D. ( C ) ISG expression in EMT6-P and -PTR cells after IFN α/β stimulation shown as bar plots. qRT-PCR, statistics performed via two-tailed t-test, n = 3–7 (1–2 biological replicates). ( D ) EMT6-P and PTR were treated with IFNα (left), and IFNβ (right) for 5 days and proliferation measured daily by MTS, statistics performed via two-tailed t-test, n = 10. ( E , F ) Densitometry quantification of western blots shown in (Fig. ) representing relative (top) total STAT1 and (bottom) total STAT3 levels compared to total β-Actin levels ( E ) at baseline and ( F ) after IFNβ stimulation. Statistics performed via two-tailed t-test. Boxes indicate range between minimum and maximum, central line depicts the mean, n = 3. ( G ) pSTAT3 expression in lysates of EMT6-P and -PTR before and after knockdown of IFNAR1 KD , and after IFNβ stimulation. ELISA, statistics performed via two-tailed t-test. Boxes indicate range between minimum and maximum, central line depicts the mean, n = 6–9. ( H ) Antigen presentation machinery expression in EMT6-P and -PTR cells before and after knockdown of IFNAR1 KD shown as bar plots. Full dataset for Fig. . qRT-PCR and flow cytometry as indicated, statistics performed via two-tailed t-test, n = 3–4. ( I ) Table summarizing two-tailed t-test statistical comparisons for data shown in Figs. and EV3. Superscripts: a—ELISA, b—Flow cytometry, c—western comparing phosphorylated levels to total levels, d—western comparing total to β-Actin levels. Data Information: Parental (P); αPD-L1 Treatment-Resistant (PTR); Conditioned Media (CM); Mean Fluorescent Intensity (MFI); IFN stimulated genes (ISGs); IFNAR1 knockdown (IFNAR1 KD ); shRNA vector control (shCon); Cells were treated with 10 ng/ml of IFNs and collected after 15 min for STAT1/3 westerns and ELISA; for proliferation experiments cells were treated with 10 ng/ml of IFNs starting on day 0 and treatment and fresh media was replaced on day 3. Bar graphs and line graphs show mean ± SD. Box plots indicate range between minimum and maximum, central line depicts the mean. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 indicate significance compared untreated controls unless otherwise shown (lines), for exact p values see Fig. EV3 Source data. All replicates shown represent technical replicates unless otherwise specified. .

Article Snippet: EMT6 (5 × 10 5 cells in 100 μl RPMI), RENCA (4 × 10 4 cells in 5 μl 1:1 RPMI:Matrigel) were implanted orthotopically into the right inguinal mammary fad pad or left kidney subcapsular space respectively in 6–8-week-old female NCI Balb/cAnNCr (Balb/c) mice (Charles River, USA).

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Two Tailed Test, Western Blot, Enzyme-linked Immunosorbent Assay, Immunopeptidomics, Flow Cytometry, shRNA, Plasmid Preparation, Control

Journal: EMBO Reports

Article Title: Acquired resistance to PD-L1 inhibition enhances a type I IFN-regulated secretory program in tumors

doi: 10.1038/s44319-024-00333-0

Figure Lengend Snippet: ( A ) Schematic showing generation of EMT6-PTR VITRO cell variants following αPD-L1 treatment in vitro for >4 weeks (top box). Blot images of Cytokine protein array of EMT6-P VITRO and -PTR VITRO tumor cells with PTIS factors highlighted in red (bottom). n = 1. ( B ) Enlarged images of array shown in ( A ). n = 1. ( C ) Line graph summary of cytokine protein array showing type I IFN regulated proteins in EMT6-PTR VITRO tumor cells compared to EMT6–P VITRO shRNA controls. Circled proteins showing expression of PTIS factors. n = 1. ( D ) RNA sequencing analysis of EMT6 PTR VITRO cells before and after IFNβ stimulation shown as average CPM expression (top) and GSEA (bottom). n = 3, statistics performed via two-tailed t-test, bolded numbers indicate FDR < 0.25. ( E ) Heatmap summary of GSEA analysis of EMT6 PTR VITRO cells in (5D). n = 3, FDR calculated via GSEA and indicated by (*). ( F ) PTIS factor expression in EMT6-P VITRO and -PTR VITRO cells shown as relative to P VITRO controls before and after IFNβ stimulation represented as bar graphs. qRT-PCR, n = 7 (2 biological replicates). ( G ) Schematic showing generation of CT26-PTR VITRO cell variants following αPD-L1 treatment in vitro for >4 weeks (top). PTIS factor expression in CT26-P VITRO and -PTR VITRO cells shown as relative to P VITRO controls before and after IFNβ stimulation represented as bar graphs (bottom). qRT-PCR, n = 8 (2 biological replicates), statistics performed via two-tailed t-test. ( H ) Schematic showing generation of PDL1 KD cell variants following αPD-L1 treatment in vitro for >4 weeks (top). PTIS factor expression in EMT6-shCon and -PDL1 KD cells shown as relative to shCon before and after IFNβ stimulation represented as bar graphs (bottom). qRT-PCR, n = 11 (3 biological replicates), statistics performed via two-tailed t-test. ( I , J ) Heatmap summary of PTIS expression at baseline ( I ) and after IFNβ stimulation ( J ) in ( F – H ). Statistics performed via two-tailed t-test. Data Information: Parental (P); αPD-L1 Treatment-Resistant (PTR); IFNAR1 knockdown (IFNAR1 KD ); shRNA Vector Control (shCon); in vitro-derived Parental (P VITRO ); in vitro-derived PD-L1 Treatment Resistant (PTR VITRO ); Gene Set Enrichment Analysis (GSEA); αPD-L1 Treatment-Induced Secretome (PTIS); normalized enrichment scores (NES); false discovery rate (FDR); Conditioned media (CM); PD-L1 knockdown (PDL1-KD). Cells were treated with 10 ng/ml of IFNs and collected after 5 days for IL6 protein expression quantifications. Bar graphs show mean ± SD. Scatter dot plot central line shows mean. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 except for GSEA as indicated above, for exact p values see Fig. 5 Source data. All replicates shown represent technical replicates unless otherwise specified. .

Article Snippet: EMT6 (5 × 10 5 cells in 100 μl RPMI), RENCA (4 × 10 4 cells in 5 μl 1:1 RPMI:Matrigel) were implanted orthotopically into the right inguinal mammary fad pad or left kidney subcapsular space respectively in 6–8-week-old female NCI Balb/cAnNCr (Balb/c) mice (Charles River, USA).

Techniques: In Vitro, Protein Array, shRNA, Expressing, RNA Sequencing, Two Tailed Test, Quantitative RT-PCR, Knockdown, Plasmid Preparation, Control, Derivative Assay

Journal: EMBO Reports

Article Title: Acquired resistance to PD-L1 inhibition enhances a type I IFN-regulated secretory program in tumors

doi: 10.1038/s44319-024-00333-0

Figure Lengend Snippet: ( A ) Heatmap summary of PTIS factors in cytokine protein array for EMT6-PTR cells relative to -P controls (Fig. ) and EMT6-PTR VITRO cells relative to -P VITRO controls (Fig. ). ( B ) Heatmap summary of type I IFN regulated protein changes in cytokine protein array for EMT6-PTR cells relative to -P controls (Fig. ) and EMT6-PTR VITRO cells relative to -P VITRO controls (Fig. ). ( C ) PTIS and various ISG expression in EMT6-P VITRO and -PTR VITRO cells treated with IFNβ shown as relative to P controls and represented as bar graphs. Full dataset for Fig. . qRT-PCR, statistics performed via two-tailed t-test, n = 3–7 (1–2 biological replicates). ( D ) PTIS expression in CT26-P VITRO and -PTR VITRO cells treated with IFNβ shown as relative to P controls and represented as bar graphs. Full dataset for Fig. . qRT-PCR, statistics performed via two-tailed t-test, n = 8 (2 biological replicates). ( E ) PD-L1 expression in cell lysates of EMT6-P and EMT6–PD-L1 KD cells. ELISA, statistics performed via two-tailed t-test, n = 3. ( F ) PTIS and various ISG expression in EMT6-shCon and -PDL1 KD cells treated with IFNβ shown as relative to P controls and represented as bar graphs. Full dataset for Fig. . qRT-PCR, statistics performed via two-tailed t-test, n = 3–11 (1–3 biological replicates). ( G ) Tables summarizing two-tailed t-test statistical comparisons for qRT-PCR data shown in Fig. E, F, G, EV4C, EV4D, and EV4F. Data Information: Parental (P); αPD-L1 Treatment-Resistant (PTR); PD-L1 knockdown (PDL1 KD ) Bar graphs show mean ± SD. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 compared to vector controls unless noted otherwise, for exact p values see Fig. EV4 Source data. All replicates shown represent technical replicates unless otherwise specified. .

Article Snippet: EMT6 (5 × 10 5 cells in 100 μl RPMI), RENCA (4 × 10 4 cells in 5 μl 1:1 RPMI:Matrigel) were implanted orthotopically into the right inguinal mammary fad pad or left kidney subcapsular space respectively in 6–8-week-old female NCI Balb/cAnNCr (Balb/c) mice (Charles River, USA).

Techniques: Protein Array, Expressing, Quantitative RT-PCR, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Knockdown, Plasmid Preparation

Journal: EMBO Reports

Article Title: Acquired resistance to PD-L1 inhibition enhances a type I IFN-regulated secretory program in tumors

doi: 10.1038/s44319-024-00333-0

Figure Lengend Snippet: ( A ) Cibersort tissue deconvolution analysis of EMT6-P and -PTR RNAseq data using ImmuCC mouse signature with box-plot representing absolute total immune score. n = 3. ( B ) Heatmap representing log2 fold change of absolute scores of various immune signatures of results from ( A ), statistics performed via two-tailed t-test. ( C – E ) Apoptosis (Annexin V) and cell death (7-AAD) staining of CD45-gated tumor cells after co-incubation with splenocytes showing ( C ) representative contour plots of stimulated splenocyte groups, ( D ) EMT6-P and -PTR variants, and ( E ) EMT6-P and -PTR-IFNAR1 KD variants compared to controls. Flow cytometry, n = 3–6, statistics performed via two-tailed t-test. ( F ) Schematic of BALB/c-derived splenocyte proliferation and activation following incubation with EMT6-P and -PTR CM for experiments in ( G , H ). ( G ) CD8+ splenocyte division (CSFE dilution) after co-incubation with CM derived from EMT6-P and -PTR control and respective IFNAR1 KD variants. Flow cytometry, n = 3–6, statistics performed via two-tailed t-test. ( H ) CD8+ splenocyte activation marker expression (CD69) after co-incubation with CM derived from EMT6-P and -PTR control and respective IFNAR1 KD variants. Flow cytometry, n = 3–6, statistics performed via two-tailed t-test. ( I ) Schematic of Balb/c-derived splenocyte proliferation and activation following co-culture with EMT6-P and -PTR cells for experiments in ( J , K ). ( J ) CD8+ splenocyte division (CSFE dilution) after co-culture with EMT6-P and -PTR control and respective IFNAR1 KD variants. Flow cytometry, n = 3–6, statistics performed via two-tailed t-test. ( K ) CD8+ splenocyte activation marker expression (CD69) after co-culture with EMT6-P and -PTR control and respective IFNAR1 KD variants. Flow cytometry, n = 3–6, statistics performed via two-tailed t-test. ( L ) Heatmap summary of results from CM and co-culture experiments, statistics performed via two-tailed t-test. ( M ) Schematic summary of immune suppressive and stimulatory effects of PTR cells. Data Information: Parental (P); αPD-L1 Treatment-Resistant (PTR); IFNAR1 knockdown (IFNAR1 KD ); shRNA vector control (shCon); Conditioned Media (CM); Bar graphs show mean ± SD. Box plot indicates range between minimum and maximum, central line depicts the mean. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 compared to vector controls unless noted otherwise, for exact p values see Fig. 6 Source data. All replicates shown represent technical replicates unless otherwise specified. .

Article Snippet: EMT6 (5 × 10 5 cells in 100 μl RPMI), RENCA (4 × 10 4 cells in 5 μl 1:1 RPMI:Matrigel) were implanted orthotopically into the right inguinal mammary fad pad or left kidney subcapsular space respectively in 6–8-week-old female NCI Balb/cAnNCr (Balb/c) mice (Charles River, USA).

Techniques: Two Tailed Test, Staining, Incubation, Flow Cytometry, Derivative Assay, Activation Assay, Control, Marker, Expressing, Co-Culture Assay, Knockdown, shRNA, Plasmid Preparation

Journal: EMBO Reports

Article Title: Acquired resistance to PD-L1 inhibition enhances a type I IFN-regulated secretory program in tumors

doi: 10.1038/s44319-024-00333-0

Figure Lengend Snippet: ( A ) Schematic of Balb/c-derived splenocyte proliferation and activation following incubation EMT6-P and -PTR CM for experiments in ( B – E ). ( B , C ) CD8+ splenocyte activation marker ( B ) IFNγ and ( C ) Granzyme B after co-incubation with CM derived from EMT6-P and -PTR control and respective IFNAR1 KD variants. Flow cytometry, statistics performed via two-tailed t-test, n = 3–6. ( D ) Log 2 fold change analysis of CD8+ splenocyte activation markers (Granzyme B, IFNγ, CD69) after co-incubation with EMT6-P and -PTR-IFNAR1 KD variant CM compared to respective controls. Flow cytometry, statistics performed via two-tailed t-test, n = 3–6. ( E ) Schematic of Balb/c-derived splenocyte proliferation and activation following incubation EMT6-P and -PTR CM for experiments in ( F – H ). ( F , G ) CD8+ splenocyte activation marker ( F ) IFNγ ( G ) Granzyme B after co-culture with EMT6-P and -PTR control and respective IFNAR1 KD variants. Flow cytometry, statistics performed via two-tailed t-test, n = 3–6. ( H ) Log 2 fold change analysis of CD8+ splenocyte activation markers (Granzyme B, IFNγ, CD69) after co-culture with EMT6-P and -PTR-IFNAR1 KD variant compared to respective controls, statistics performed via two-tailed t-test, n = 4–6. Data Information: Parental (P); αPD-L1 Treatment-Resistant (PTR); IFNAR1 knockdown (IFNAR1 KD ); Conditioned Media (CM); Granzyme B (GZMB). Bar graphs show mean ± SD. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 compared to vector controls unless noted otherwise, for exact p values see Fig. EV5 Source data. All replicates shown represent technical replicates unless otherwise specified. .

Article Snippet: EMT6 (5 × 10 5 cells in 100 μl RPMI), RENCA (4 × 10 4 cells in 5 μl 1:1 RPMI:Matrigel) were implanted orthotopically into the right inguinal mammary fad pad or left kidney subcapsular space respectively in 6–8-week-old female NCI Balb/cAnNCr (Balb/c) mice (Charles River, USA).

Techniques: Derivative Assay, Activation Assay, Incubation, Marker, Control, Flow Cytometry, Two Tailed Test, Variant Assay, Co-Culture Assay, Knockdown, Plasmid Preparation

Journal: EMBO Reports

Article Title: Acquired resistance to PD-L1 inhibition enhances a type I IFN-regulated secretory program in tumors

doi: 10.1038/s44319-024-00333-0

Figure Lengend Snippet: ( A ) Orthotopic tumor growth of EMT6-P and EMT6–PTR, and respective IFNAR1 KD cell variants ( n = 5–8 biological replicates; Balb/c) with summary of AUC analysis (left-inset), statistics performed via two-tailed t-test. ( B ) Log 2 Fold Change analysis of data shown in ( A ) comparing IFNAR1 KD to respective controls. AUC analysis of comparisons shown (left-inset), statistics performed via two-tailed t-test. ( C ) Orthotopic tumor growth of EMT6-P and EMT6–PTR treated with αIL6 antibody ( n = 5–8 biological replicates; Balb/c) with summary of AUC analysis (left-inset), statistics performed via two-tailed t-test. ( D ) Log 2 Fold Change analysis of data shown in ( C ) comparing αIL6 antibody treatment to respective controls. AUC analysis of comparisons shown (left-inset), statistics performed via two-tailed t-test. ( E ) Metastasis and invasion of mice bearing EMT6-P and -PTR tumors (shown in A , B ) with invasion in the peritoneum wall after PBS, αIL6, Con., or IFNAR1 KD ( n = 5–8 biological replicates). ( F ) Schematic summarizing the effect of type I IFN secretome inhibition in P and PTR tumors. ( G ) Proposed model of IFN-signaling ‘rewired’ tumor cells following acquired resistance to PD-L1 inhibition. Data Information: Parental (P); αPD-L1 Treatment-Resistant (PTR); IFNAR1 knockdown (IFNAR1 KD ); shRNA Vector Control (shCon); Area under the curve (AUC) in vitro-derived Parental (P VITRO ); in vitro-derived PD-L1 Treatment Resistant (PTR VITRO ); Bar graphs show mean ± SD. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, for exact p values see Fig. 7 Source data. αIL6 was administered at 100 μg/mouse/3 days continuously, PBS was used as a control (Con.). Primary tumor burden was assessed by caliper measurement. Tumor growth line graph represent mean ± SEM. All replicates shown represent technical replicates unless otherwise specified. .

Article Snippet: EMT6 (5 × 10 5 cells in 100 μl RPMI), RENCA (4 × 10 4 cells in 5 μl 1:1 RPMI:Matrigel) were implanted orthotopically into the right inguinal mammary fad pad or left kidney subcapsular space respectively in 6–8-week-old female NCI Balb/cAnNCr (Balb/c) mice (Charles River, USA).

Techniques: Two Tailed Test, Inhibition, Knockdown, shRNA, Plasmid Preparation, Control, In Vitro, Derivative Assay

Journal: EMBO Reports

Article Title: Acquired resistance to PD-L1 inhibition enhances a type I IFN-regulated secretory program in tumors

doi: 10.1038/s44319-024-00333-0

Figure Lengend Snippet: Reagents and tools table

Article Snippet: EMT6 (5 × 10 5 cells in 100 μl RPMI), RENCA (4 × 10 4 cells in 5 μl 1:1 RPMI:Matrigel) were implanted orthotopically into the right inguinal mammary fad pad or left kidney subcapsular space respectively in 6–8-week-old female NCI Balb/cAnNCr (Balb/c) mice (Charles River, USA).

Techniques: Control, Purification, Recombinant, Red Blood Cell Lysis, Activation Assay, Membrane, Staining, Isolation, Plasmid Preparation, DC Protein Assay, Enzyme-linked Immunosorbent Assay, Gene Expression, shRNA, Amplification, Software, Imaging, Spectrophotometry, Lysis, Protease Inhibitor, Western Blot, SYBR Green Assay, cDNA Synthesis, Transfection

Journal: Cancer Immunology Research

Article Title: Trabectedin Enhances the Antitumor Effects of IL-12 in Triple-Negative Breast Cancer

doi: 10.1158/2326-6066.CIR-24-0775

Figure Lengend Snippet: Combination IL-12 and trabectedin effectively reduces TNBC tumor burden. A, General treatment schema for murine studies. PBS was used as the control. IL-12 (0.5 μg) was given intraperitoneally 3×/week and 0.15 mg/kg trabectedin was given intravenously 1×/week. B, BALB/c mice were inoculated with 4T1 cells and divided into one of four treatment groups once tumors reached ∼50 mm 3 [PBS control ( n = 11), IL-12 ( n = 10), trabectedin ( n = 10), and IL-12 plus trabectedin ( n = 12)]. Tumor growth was measured for 15 days. C, Individual tumor growth curves of mice treated with PBS control ( n = 11), IL-12 ( n = 10), trabectedin ( n = 10), and IL-12 plus trabectedin ( n = 12). D, Tumor growth curves of BALB/c mice inoculated with EMT6 cells and treated for 11 days, as described in ( A ) [PBS control ( n = 7), IL-12 ( n = 5), trabectedin ( n = 7), and IL-12+ trabectedin ( n = 6)]. E, Plasma IFN-γ levels in 4T1 tumor–bearing mice at day 15 ( n = 7–9/treatment group). F, Representative flow cytometry gating for M-MDSC and PMN-MDSC from splenocytes of control-treated and IL-12 and trabectedin–treated 4T1 tumor–bearing mice. G, Percent MDSC in the spleens of 4T1 tumor–bearing mice 15 days after treatment ( n = 3). H, Absolute counts of total MDSC and ( I ) PMN-MDSC and ( J ) M-MDSC subsets in the spleens of 4T1 tumor–bearing mice 15 days after treatment ( n = 3). For statistical analyses of tumor volumes, linear mixed modeling was used to model the longitudinal tumor volume for mice under each treatment. Comparisons were done at each time point and averaged across all time points using t-statistics. The Tukey–Kramer method was used for adjusting raw P values for multiple comparisons across treatment groups. An ordinary one-way ANOVA with the Tukey multiple comparisons test was used for statistical analysis of bar graphs. Data represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. SSC, side scatter; Trab, trabectedin.

Article Snippet: To establish orthotopic murine models of TNBC, 4- to 6-week-old female BALB/c mice (The Jackson Laboratory, RRID: MGI:2683685) were inoculated with 1 × 10 5 4T1 cells or 1 × 10 6 EMT6 cells in the mammary fat pad.

Techniques: Control, Clinical Proteomics, Flow Cytometry

Journal: mAbs

Article Title: Activity of murine surrogate antibodies for durvalumab and tremelimumab lacking effector function and the ability to deplete regulatory T cells in mouse models of cancer

doi: 10.1080/19420862.2020.1857100

Figure Lengend Snippet: Complete response rate of anti-mouse PD-L1 clone 80 and anti-mouse CTLA-4 mAbs across eight mouse syngeneic tumor models

Article Snippet: Syngeneic mouse colon cancer (CT26, MC38), breast cancer (EMT6, 4T1, EO771), melanoma (B16F10), renal cell cancer (RENCA), lung cancer (LL/2 lewis lung), or fibrosarcoma (MCA205) tumor cells were engrafted in 6- to 8-week-old female Balb/C (CT26, EMT6, 4T1, RENCA) or female C57BL/6 (B16F10, LL/2 lewis lung, MC38, EO771, MCA205) mice (Envigo).

Techniques:

Journal: mAbs

Article Title: Activity of murine surrogate antibodies for durvalumab and tremelimumab lacking effector function and the ability to deplete regulatory T cells in mouse models of cancer

doi: 10.1080/19420862.2020.1857100

Figure Lengend Snippet: Anti-tumor efficacy and pharmacodynamic profiling of mAbs. Shown are in vivo efficacy and pharmacodynamic effects of anti-mouse PD-L1 and CTLA-4 surrogates in mouse syngeneic tumor models that are sensitive to checkpoint inhibition. (a–c) Kaplan-Meier survival curves of mice engrafted with EMT6, CT26, or MCA205 tumors and treated with anti–PD-L1 or anti–CTLA-4 mAbs. (d–g) Pharmacodynamic activity of monotherapy or combination anti-PD–L1 plus anti–CTLA-4 therapy in EMT6, CT26, and MCA205 models as measured by CD8 or conventional CD4 T-cell proliferation (Ki67 positivity). (h–j) Proportion of FoxP3+ Tregs in tumors, tumor-draining lymph nodes, and spleens of mice engrafted with CT26 tumors

Article Snippet: Syngeneic mouse colon cancer (CT26, MC38), breast cancer (EMT6, 4T1, EO771), melanoma (B16F10), renal cell cancer (RENCA), lung cancer (LL/2 lewis lung), or fibrosarcoma (MCA205) tumor cells were engrafted in 6- to 8-week-old female Balb/C (CT26, EMT6, 4T1, RENCA) or female C57BL/6 (B16F10, LL/2 lewis lung, MC38, EO771, MCA205) mice (Envigo).

Techniques: In Vivo, Inhibition, Activity Assay