Journal: Science Advances

Article Title: Efficient blockade of locally reciprocated tumor-macrophage signaling using a TAM-avid nanotherapy

doi: 10.1126/sciadv.aaz8521

Figure Lengend Snippet: ( A ) YUMMER1.7 melanoma tumors were excised and analyzed by flow cytometry for TAMs (GFP − CD45 + CD11b + F4/80 + ), 24 hours after three daily doses of dabrafenib (30 mg/kg) and trametinib (0.3 mg/kg) (+D/T; n ≥ 3). ( B to D ) Representative images (left) and quantification (right) of dextran-NP + TAMs (cyan) within GFP-labeled (magenta) (B) YUMMER1.7, (C) intraperitoneally disseminated ES2 OVCA, or (D) intraperitoneally disseminated PtD OVCA tumors. At least one tumor each from n = 3 nu/nu mice per group was excised 24 hours after three daily doses of MAPKi (+T trametinib alone; two-tailed t test). Ctrl, control. Scale bars, 50 μm. ( E to H ) Schematic depicting daily imaging and trametinib treatment schedule for mice bearing ES2 xenograft tumors implanted in dorsal skin-fold window chambers (top). Representative intravital microscopy of OVCA and TAMs (bottom) (E) and their quantification (F) to (H) are shown ( n ≥ 4 nu/nu mice per group, two-tailed t test). Scale bar, 250 μm. Tumor size and fluorescence were compared at 0 and 96 hours. Data are means ± SEM for all.

Article Snippet: Under conditions where cells were treated with trametinib (Selleck Chem), a 100 nM dose was used, unless otherwise specified.

Techniques: Flow Cytometry, Labeling, Two Tailed Test, Control, Imaging, Intravital Microscopy, Fluorescence

Journal: Science Advances

Article Title: Efficient blockade of locally reciprocated tumor-macrophage signaling using a TAM-avid nanotherapy

doi: 10.1126/sciadv.aaz8521

Figure Lengend Snippet: ( A ) Schematic of M2-MΦ conditioned medium experiments (left) and data showing ES2 cell count following 72-hour trametinib ± conditioned medium from M2-MΦ that were pretreated with 24-hour trametinib (right) ( n = 3). ( B ) OVCA viability was measured by propidium iodide and annexin V staining for three cell lines treated for 48 hours with 100 nM trametinib ± transwell coculture with M2-MΦ (averaged across five donors with n = 3 reps per donor, two-tailed t test). ( C ) Heat map of supernatant and lysate proteins from PBMC-derived M1-MΦ and M2-MΦ. Analyte levels of MΦs treated with 24-hour trametinib are plotted relative to their respective no-treatment controls ( n = 3, * P < 0.05, two-tailed Mann-Whitney U test with false discovery rate correction). GM-CSF, granulocyte-MΦ CSF; TGFα, transforming growth factor–α; TNFα, tumor necrosis factor–α; p-NFκB, phosphorylation of nuclear factor κB. ( D and E ) Loading (D) and score (E) plots generated from principal components analysis (PCA) of data from (C). Red shadings represent M2-MΦ, blue shadings represent M1-MΦ, and gray shading superimposed on each represents control conditions. ( F ) MΦ uptake of trametinib-treated ES2 cell debris following pretreatment of M2-MΦ with (+T) trametinib ( n = 3, two-tailed t test). ( G ) RTK levels of U937-derived M2-MΦ, 24 hours after trametinib ( n = 3 and n = 6 for pooled MERTK two-tailed t test). Data are means ± SEM for all.

Article Snippet: Under conditions where cells were treated with trametinib (Selleck Chem), a 100 nM dose was used, unless otherwise specified.

Techniques: Cell Counting, Staining, Two Tailed Test, Derivative Assay, MANN-WHITNEY, Phospho-proteomics, Generated, Control

Journal: Science Advances

Article Title: Efficient blockade of locally reciprocated tumor-macrophage signaling using a TAM-avid nanotherapy

doi: 10.1126/sciadv.aaz8521

Figure Lengend Snippet: ( A ) ES2 cell count, 72 hours after treatment with 100 nM trametinib ± AXL-Fc ligand trap, HGF-neutralizing antibody (HGF NAb), and conditioned medium from M2-MΦ treated with 24-hour trametinib (M2-MΦ CM) ( n = 3, two-tailed t test). ( B ) Illustrative dose-response surfaces for two model drugs with increasing degrees of the Loewe synergy parameter α. ( C ) α was calculated from ES2 cells treated for 72 hours with MEKi + RTKi across n = 24 combinations of concentrations and n = 2 replicates (thick line, median). ( D to F ) α was calculated as in (C) for trametinib + foretinib, but with ES2 cultured in recombinant growth factor (100 ng/ml) (D) or AXL-Fc (1 μg/ml), GAS6 (400 ng/ml), and/or M2-MΦ-CM (E and F) ( n = 2, two-tailed t test). ( G and H ) p-RTK levels were measured in ES2 (G) and RTK and p-RTK levels in U937-derived M2-MΦ (H) 24 hours after treatment with trametinib (T), foretinib (F), or the combination ( n = 3, two-tailed t test). For all except (C), data are means ± SEM.

Article Snippet: Under conditions where cells were treated with trametinib (Selleck Chem), a 100 nM dose was used, unless otherwise specified.

Techniques: Cell Counting, Two Tailed Test, Cell Culture, Recombinant, Derivative Assay

Journal: Science Advances

Article Title: Efficient blockade of locally reciprocated tumor-macrophage signaling using a TAM-avid nanotherapy

doi: 10.1126/sciadv.aaz8521

Figure Lengend Snippet: ( A ) Levels of 11 intracellular phosphoproteins were correlated with three p-RTK (p-AXL/p-MERTK/p-MET) levels across three OVCA cell lines, three MΦ coculture conditions, and ±24-hour 100 nM trametinib. A total of n = 756 total Luminex measurements were considered (see figs. S4B and S5A). ( B ) Phosphoproteins were measured in ES2 cells, 24 hours after trametinib ± transwell M2-MΦ coculture (CC) ( n = 3, two-tailed t test). ( C ) Cytoplasmic-to-nuclear (C/N) ratios from ERK and JNK kinase translocation reporter (KTRs) were measured in ES2 cells 24 hours after treatment with trametinib ± transwell coculture with M2-MΦ ( n = 60 cells across six wells, * P < 0.05, two-tailed t test). ( D and E ) Representative confocal microscopy (D) of disseminated intraperitoneal tumors, showing ES2 cells expressing ERK and JNK KTRs near and far from TAMs, from nu/nu xenografts 24 hours after treatment (trametinib, 0.3 mg/kg, by mouth), quantified in (E) ( n = 120 cells across n = 12 tumors and three mice per group, two-tailed t test). Scale bars, 50 μm or 10 μm as indicated). ( F to H ) Time-lapse intravital microscopy of ES2 xenografts imaged following intravenous (i.v.) trametinib treatment. Representative images (F) and quantification (G and H) are shown. Scale bar, 10 μm. In (G), thin and thick lines denote single cells and their mean, and in (H), data points denote single cells at 60 min after treatment ( n = 20 cells across two tumors, Spearman’s exact test). Data are means ± SEM for (B), (C), and (E). n.s., not significant.

Article Snippet: Under conditions where cells were treated with trametinib (Selleck Chem), a 100 nM dose was used, unless otherwise specified.

Techniques: Luminex, Two Tailed Test, Translocation Assay, Confocal Microscopy, Expressing, Intravital Microscopy

Journal: Science Advances

Article Title: Efficient blockade of locally reciprocated tumor-macrophage signaling using a TAM-avid nanotherapy

doi: 10.1126/sciadv.aaz8521

Figure Lengend Snippet: ( A ) Schematic of foretinib nanoparticle encapsulation (NanoFore). PDI, polydispersity index. ( B ) GSEA of a genome-wide CRISPR screen, showing that RAS/RAF/MEK/ERK pathway silencing is enriched for enhanced phagocytic activity in U937 cells (GSEA permutation test). ( C and D ) NanoFore accumulation was measured 24 hours after treatment with trametinib (T) or dabrafenib + trametinib (D/T) in ES2 (C) and YUMMER1.7 (“Y1.7”) (D) tumors by fluorescence reflectance imaging (FRI), 24 hours after MAPKi and maraviroc ( n ≥ 3). ( E ) Corresponding with (D), representative confocal microscopy (left) and quantification (right) measured colocalization between NanoFore and TAMs in YUMMER1.7 tumors ( n ≥ 3). Scale bar, 50 μm. a.u., arbitrary units. ( F ) Flow cytometry of YUMMER1.7 tumors quantified NanoFore uptake, CSF1R, and MERTK levels in dextran-NP + TAMs ( n ≥ 3). ( G ) Nu/nu mice bearing intraperitoneal ES2 tumors were treated with trametinib (daily starting on day 3; gray arrow), NanoFore (every 4 days; blue arrows), or the combination and were monitored for reaching the experimental end point (top; n ≥ 5, two-tailed log-rank test) and imaged by FRI (bottom; n ≥ 3, two-tailed t test). ( H ) Corresponding to (G), single-cell ERK and JNK activities were measured at time of experimental end point for individual subjects ( n = 120 cells across 12 tumors, two-tailed t test). For (C) to (E) and G, data are means ± SEM (two-tailed t test). ( I ) Summary of schematic. Untreated tumor cells with constitutive RAS/RAF/MEK/ERK signaling produce soluble AXL and MET RTK ectodomains; TAMs express MERTK and CSF1R and accumulate in MAPK-driven tumors (left). MAPKi using dabrafenib and/or trametinib increases TAMs in resistant tumors, reduces AXL and MET ectodomain shedding in cancer cells, leads to AXL and MET accumulation on the cancer cell surface, enhances production of GAS6 by TAMs and cancer cells, enhances HGF production by TAMs, and stimulates cancer cell production of the TAM-recruiting cytokine RANTES/CCL5. TAM-proximal cancer cells exhibit elevated JNK/cJUN phosphosignaling (center), but this can be inhibited by NanoFore, which is efficiently taken up by TAMs (right).

Article Snippet: Under conditions where cells were treated with trametinib (Selleck Chem), a 100 nM dose was used, unless otherwise specified.

Techniques: Encapsulation, Genome Wide, CRISPR, Activity Assay, Fluorescence, Imaging, Confocal Microscopy, Flow Cytometry, Two Tailed Test

Journal: Science Advances

Article Title: Efficient blockade of locally reciprocated tumor-macrophage signaling using a TAM-avid nanotherapy

doi: 10.1126/sciadv.aaz8521

Figure Lengend Snippet: ( A ) Schematic of foretinib nanoparticle encapsulation (NanoFore). PDI, polydispersity index. ( B ) GSEA of a genome-wide CRISPR screen, showing that RAS/RAF/MEK/ERK pathway silencing is enriched for enhanced phagocytic activity in U937 cells (GSEA permutation test). ( C and D ) NanoFore accumulation was measured 24 hours after treatment with trametinib (T) or dabrafenib + trametinib (D/T) in ES2 (C) and YUMMER1.7 (“Y1.7”) (D) tumors by fluorescence reflectance imaging (FRI), 24 hours after MAPKi and maraviroc ( n ≥ 3). ( E ) Corresponding with (D), representative confocal microscopy (left) and quantification (right) measured colocalization between NanoFore and TAMs in YUMMER1.7 tumors ( n ≥ 3). Scale bar, 50 μm. a.u., arbitrary units. ( F ) Flow cytometry of YUMMER1.7 tumors quantified NanoFore uptake, CSF1R, and MERTK levels in dextran-NP + TAMs ( n ≥ 3). ( G ) Nu/nu mice bearing intraperitoneal ES2 tumors were treated with trametinib (daily starting on day 3; gray arrow), NanoFore (every 4 days; blue arrows), or the combination and were monitored for reaching the experimental end point (top; n ≥ 5, two-tailed log-rank test) and imaged by FRI (bottom; n ≥ 3, two-tailed t test). ( H ) Corresponding to (G), single-cell ERK and JNK activities were measured at time of experimental end point for individual subjects ( n = 120 cells across 12 tumors, two-tailed t test). For (C) to (E) and G, data are means ± SEM (two-tailed t test). ( I ) Summary of schematic. Untreated tumor cells with constitutive RAS/RAF/MEK/ERK signaling produce soluble AXL and MET RTK ectodomains; TAMs express MERTK and CSF1R and accumulate in MAPK-driven tumors (left). MAPKi using dabrafenib and/or trametinib increases TAMs in resistant tumors, reduces AXL and MET ectodomain shedding in cancer cells, leads to AXL and MET accumulation on the cancer cell surface, enhances production of GAS6 by TAMs and cancer cells, enhances HGF production by TAMs, and stimulates cancer cell production of the TAM-recruiting cytokine RANTES/CCL5. TAM-proximal cancer cells exhibit elevated JNK/cJUN phosphosignaling (center), but this can be inhibited by NanoFore, which is efficiently taken up by TAMs (right).

Article Snippet: Where indicated, mice were treated with trametinib (0.3 mg/kg; 1% DMSO, 0.5% methylcellulose, and 0.2% Tween-80 in water; Selleck Chem), dabrafenib (30 mg/kg; 1% DMSO, 0.5% methylcellulose, and 0.2% Tween-80 in water; Selleck Chem), and/or maraviroc (30 mg/kg; 5% DMSO, 0.5% 0.1 N HCl in water; Selleck Chem) via oral gavage ( , ).

Techniques: Encapsulation, Genome Wide, CRISPR, Activity Assay, Fluorescence, Imaging, Confocal Microscopy, Flow Cytometry, Two Tailed Test

Journal: Science Advances

Article Title: Efficient blockade of locally reciprocated tumor-macrophage signaling using a TAM-avid nanotherapy

doi: 10.1126/sciadv.aaz8521

Figure Lengend Snippet: ( A ) YUMMER1.7 melanoma tumors were excised and analyzed by flow cytometry for TAMs (GFP − CD45 + CD11b + F4/80 + ), 24 hours after three daily doses of dabrafenib (30 mg/kg) and trametinib (0.3 mg/kg) (+D/T; n ≥ 3). ( B to D ) Representative images (left) and quantification (right) of dextran-NP + TAMs (cyan) within GFP-labeled (magenta) (B) YUMMER1.7, (C) intraperitoneally disseminated ES2 OVCA, or (D) intraperitoneally disseminated PtD OVCA tumors. At least one tumor each from n = 3 nu/nu mice per group was excised 24 hours after three daily doses of MAPKi (+T trametinib alone; two-tailed t test). Ctrl, control. Scale bars, 50 μm. ( E to H ) Schematic depicting daily imaging and trametinib treatment schedule for mice bearing ES2 xenograft tumors implanted in dorsal skin-fold window chambers (top). Representative intravital microscopy of OVCA and TAMs (bottom) (E) and their quantification (F) to (H) are shown ( n ≥ 4 nu/nu mice per group, two-tailed t test). Scale bar, 250 μm. Tumor size and fluorescence were compared at 0 and 96 hours. Data are means ± SEM for all.

Article Snippet: Where indicated, mice were treated with trametinib (0.3 mg/kg; 1% DMSO, 0.5% methylcellulose, and 0.2% Tween-80 in water; Selleck Chem), dabrafenib (30 mg/kg; 1% DMSO, 0.5% methylcellulose, and 0.2% Tween-80 in water; Selleck Chem), and/or maraviroc (30 mg/kg; 5% DMSO, 0.5% 0.1 N HCl in water; Selleck Chem) via oral gavage ( , ).

Techniques: Flow Cytometry, Labeling, Two Tailed Test, Control, Imaging, Intravital Microscopy, Fluorescence

Journal: Science Advances

Article Title: Efficient blockade of locally reciprocated tumor-macrophage signaling using a TAM-avid nanotherapy

doi: 10.1126/sciadv.aaz8521

Figure Lengend Snippet: ( A ) Schematic of foretinib nanoparticle encapsulation (NanoFore). PDI, polydispersity index. ( B ) GSEA of a genome-wide CRISPR screen, showing that RAS/RAF/MEK/ERK pathway silencing is enriched for enhanced phagocytic activity in U937 cells (GSEA permutation test). ( C and D ) NanoFore accumulation was measured 24 hours after treatment with trametinib (T) or dabrafenib + trametinib (D/T) in ES2 (C) and YUMMER1.7 (“Y1.7”) (D) tumors by fluorescence reflectance imaging (FRI), 24 hours after MAPKi and maraviroc ( n ≥ 3). ( E ) Corresponding with (D), representative confocal microscopy (left) and quantification (right) measured colocalization between NanoFore and TAMs in YUMMER1.7 tumors ( n ≥ 3). Scale bar, 50 μm. a.u., arbitrary units. ( F ) Flow cytometry of YUMMER1.7 tumors quantified NanoFore uptake, CSF1R, and MERTK levels in dextran-NP + TAMs ( n ≥ 3). ( G ) Nu/nu mice bearing intraperitoneal ES2 tumors were treated with trametinib (daily starting on day 3; gray arrow), NanoFore (every 4 days; blue arrows), or the combination and were monitored for reaching the experimental end point (top; n ≥ 5, two-tailed log-rank test) and imaged by FRI (bottom; n ≥ 3, two-tailed t test). ( H ) Corresponding to (G), single-cell ERK and JNK activities were measured at time of experimental end point for individual subjects ( n = 120 cells across 12 tumors, two-tailed t test). For (C) to (E) and G, data are means ± SEM (two-tailed t test). ( I ) Summary of schematic. Untreated tumor cells with constitutive RAS/RAF/MEK/ERK signaling produce soluble AXL and MET RTK ectodomains; TAMs express MERTK and CSF1R and accumulate in MAPK-driven tumors (left). MAPKi using dabrafenib and/or trametinib increases TAMs in resistant tumors, reduces AXL and MET ectodomain shedding in cancer cells, leads to AXL and MET accumulation on the cancer cell surface, enhances production of GAS6 by TAMs and cancer cells, enhances HGF production by TAMs, and stimulates cancer cell production of the TAM-recruiting cytokine RANTES/CCL5. TAM-proximal cancer cells exhibit elevated JNK/cJUN phosphosignaling (center), but this can be inhibited by NanoFore, which is efficiently taken up by TAMs (right).

Article Snippet: Where indicated, mice were treated with trametinib (0.3 mg/kg; 1% DMSO, 0.5% methylcellulose, and 0.2% Tween-80 in water; Selleck Chem), dabrafenib (30 mg/kg; 1% DMSO, 0.5% methylcellulose, and 0.2% Tween-80 in water; Selleck Chem), and/or maraviroc (30 mg/kg; 5% DMSO, 0.5% 0.1 N HCl in water; Selleck Chem) via oral gavage ( , ).

Techniques: Encapsulation, Genome Wide, CRISPR, Activity Assay, Fluorescence, Imaging, Confocal Microscopy, Flow Cytometry, Two Tailed Test