Journal: Nature Communications

Article Title: Harnessing macrophage-drug conjugates for allogeneic cell-based therapy of solid tumors via the TRAIN mechanism

doi: 10.1038/s41467-025-56637-9

Figure Lengend Snippet: a Representative confocal microscopy images of colocalization of HFt with AcLDL in human macrophages. hMDM were incubated simultaneously with HFt-AF488 (50 μg/ml) (green) and AcLDL-AF594 (5 μg/ml) (red) for 15 min at 37 °C. Nuclei were stained with Hoechst 33342 (blue). Merged fluorescence images show colocalization of HFt and AcLDL (yellow foci) for hMDM. Scale bar = 20 μm (10 µm in zoomed region). b Flow cytometry analysis of internalized HFt-AF488 (5 μg/ml) by hMDM in the absence or presence of AcLDL at indicated concentrations within 30 min at 37 °C. Flow cytometry data are presented as the mean fluorescence intensity (MFI) of HFt-AF488. Data are presented as mean ± SEM from n = 3 different donors. c Representative confocal microscopy images of internalized HFt-AF488 (5 μg/ml) (green) by hMDM in the absence or presence of AcLDL (100 μg/ml) within 30 min at 37 °C. Nuclei were stained with Hoechst 33342 (blue). Scale bar = 20 μm. d Flow cytometry analysis of internalized AcLDL-AF488 (1 μg/ml) by hMDM in the absence or presence of HFt at indicated concentrations within 30 min at 37 °C. Data are presented as mean fluorescence intensity (MFI) of AcLDL-AF488. Data are presented as mean ± SEM from n = 3 different donors. e Representative confocal microscopy images of internalized AcLDL-AF488 (1 μg/ml) (green) by hMDM in the absence or presence of HFt (2,5 mg/ml) within 30 min at 37 °C. Nuclei were stained with Hoechst 33342 (blue). Scale bar = 20 μm. f–h Flow cytometry analysis of internalized HFt-AF488 (50 μg/ml) within 30 min at 37 °C by hMDM untreated or pre-treated for 30 min at 37 °C with indicated concentrations of various ligands of class A scavenger receptors or structurally related ligands that do not bind to this group of receptors (negative controls): poly(I), poly(G) and poly(C) (control) ( f ), fucoidan and mannan (control) ( g ) and dextran sulfate or chondroitin sulfate (control) ( h ) Flow cytometry data are presented as % of HFt-AF488 uptake in untreated, control cells. Data are presented as mean ± SEM from n = 3 ( f , h ) or 6 ( g ) different donors (hMDM). i–k Representative confocal microscopy images of internalized HFt-AF488 (50 μg/ml) (green) within 30 min at 37 °C by hMDM untreated or pre-treated for 30 min at 37 °C with indicated concentrations of various ligands of class A scavenger receptors or structurally related ligands that do not bind to this group of receptors (negative controls): poly(I) and poly(C) (control) ( i ), fucoidan and mannan (control) ( j ) and dextran sulfate or chondroitin sulfate (control) ( k ). Nuclei were stained with Hoechst 33342 (blue). Scale bar = 20 μm. l Quantitative real-time PCR analysis of MSR1, SCARA5, and MARCO mRNA expression in hMDM. Data are presented as mean ± SEM from n = 3 different donors (hMDM). m Western blot analysis of MSR1 protein expression in monocytes and hMDM. Representative western blot images are shown. n Flow cytometry analysis of MSR1 cell surface staining in hMDM upon stimulation with HFt (200 µg/ml) for indicated time points at 37 °C. For comparison, untreated cells were used (Control). Data are presented as mean ± SEM % of MSR1 expression in control cells, n = 3 independent donors (hMDM). o Western blot analysis of MSR1 protein expression in hMDM that were untreated (Control) or treated with cycloheximide (CHX) (20 µg/ml) for 1 h prior to HFt stimulation (200 µg/ml) (CHX+HFt) for the indicated time points at 37 °C. For comparison, cells treated only with CHX were used. Representative western blot images are shown. p Quantitative analysis of relative MSR1 expression in hMDM shown in ( o ). Data are presented as mean ± SEM from n = 3 independent replicates. q Western blot analysis showing MSR1 expression in either untransfected hMDM (Control) or cells transfected with one of the following siRNA sequences: scramble siRNA (siScr), no. 1 siRNA targeting MSR1 (siMSR1-1), no. 2 siRNA targeting MSR1 (siMSR1-2) at 72 h after transfection. r Quantitative analysis of western blot for MSR1 expression shown in ( q ). Data are presented as mean ± SEM % of MSR1 expression in control cells (Control), n = 3 different donors (hMDM). s and ( t ) Flow cytometry analysis of internalized AcLDL-AF488 (5 µg/ml) ( s ) or HFt-AF488 (100 μg/ml) ( t ) by hMDM after MSR1 gene-knockdown within 30 min at 37 °C. For comparison, untreated cells (Control) and cells treated with a negative, scramble control siRNA (siScr) were used. Flow cytometry data are presented as mean ± SEM % of ligand uptake in control cells (Control), n = 3 different donors. u and ( v ) Representative confocal microscopy images of internalized AcLDL-AF488 (5 μg/ml) (green) ( u ) and HFt-AF488 (50 μg/ml) (green) ( v ) within 30 min at 37 °C by THP-1 macrophages after MSR1 gene-knockdown. Afterwards, cells were fixed and stained with Hoechst 33342 (blue). Scale bar = 20 μm. w Western blot analysis of MSR1 protein expression in hMDM macrophages polarized to M1 or M2 phenotypes. Representative western blot images are shown. x Relative expression of MSR1 in M1 and M2 hMDM macrophages quantified based on western blot analysis shown in ( w ). Data are presented as mean ± SEM from n = 4 different donors. y Flow cytometry analysis of internalized HFt-AF488 by M1 and M2 hMDM macrophages given at 25 µg/ml within 20 or 60 min at 37 °C. Flow cytometry data are presented as the mean fluorescence intensity (MFI) of HFt-AF488. Data are presented as mean ± SEM from n = 4 different donors. The one-way ANOVA and Dunnett’s post-hoc test were used for statistical analysis in panels b , d , r – t . The two-way ANOVA and Tukey’s post-hoc tests were used for statistical analysis in panels f – h . The two-way ANOVA followed by Sidak’s multiple comparisons post hoc test was used for statistical analysis in panels ( p , y ). The Student’s t -test was used for statistical analysis in panel ( x ). For all panels, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Source data are provided as a Source Data file.

Article Snippet: DNA plasmid transfection of CHO-K1 and HEK293 cell lines was performed using the negative control vector (pCMV3-SP-N-His, Sino Biological, CV023) and the plasmid encoding human MSR1 (CD204/MSR1 cDNA ORF Clone, N-His tag expression plasmid (Sino Biological, HG10427-NH).

Techniques: Confocal Microscopy, Incubation, Staining, Fluorescence, Flow Cytometry, Control, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Comparison, Transfection, Knockdown

Journal: Nature Communications

Article Title: Harnessing macrophage-drug conjugates for allogeneic cell-based therapy of solid tumors via the TRAIN mechanism

doi: 10.1038/s41467-025-56637-9

Figure Lengend Snippet: a Western blot analysis of MSR1 protein expression in HEK293 cells at 24 h after transfection with the control plasmid (p-mCherry2) or the plasmid encoding the MSR1 gene (p-MSR1-mCherry2). Representative western blot images are shown. b and ( c ) Flow cytometry analysis of cell surface expression of MSR1 in HEK293 cells at 24 h after transfection with the control plasmid (p-mCherry2) or the plasmid encoding the MSR1 gene (p-MSR1-mCherry2). Flow cytometry data are presented as the mean fluorescence intensity (MFI) of BV421. Data are presented as mean ± SEM from n = 3 independent replicates. d Flow cytometry analysis of internalized AcLDL-AF488 (5 μg/ml) and HFt-AF488 (50 μg/ml) within 30 min at 37 °C by HEK293 cells, transfected with either p-mCherry2 or p-MSR1-mCherry2. Flow cytometry data are presented as mean fluorescence intensity (MFI) of fluorescently labeled ligands. Data are presented as mean ± SEM from n = 3 independent replicates. e , f Representative confocal microscopy images of internalized HFt-AF488 (50 μg/ml) ( e ) and AcLDL-AF488 (5 μg/ml) ( f ) (green) within 30 min at 37 °C by HEK293 cells, transfected with either p-mCherry2 or p-MSR1-mCherry2 (red). Afterwards, cells were fixed and stained with Hoechst 33342 (blue). Scale bar = 20 μm. g Optimization of AlphaScreen assay conditions by cross-titration of His-MSR1 (0.41–100 nM, 3-fold serial dilution) and Biotin-HFt (0.022–50 nM, 3-fold serial dilution) at fixed 10 μg/ml concentration of both nickel chelated acceptor and streptavidin donor beads. Data shown are from a single pilot run conducted for assay optimization. h Competitive effect of unlabeled HFt on AlphaScreen signal generated by His-MSR1 and Biotin-HFt interaction. Fixed concentrations of 4 nM His-MSR1 and 2 nM Biotin-HFt were used below the hook point of the bead assay ( g ) in the presence of increasing concentration of unlabeled HFt (0.076–1500 nM, 3-fold serial dilution). IC50 potency of unlabeled HFt in displacing Biotin-HFt from interaction with His-MSR1 was calculated by fitting it to a four-parameter nonlinear regression. Data presented as mean ± SEM from n = 3 independent replicates. i Spectral shift dose–response curve between unlabeled HFt and His-MSR1 conjugated with the RED-tris-NTA dye. Data was analyzed using software provided by the manufacturer and is presented as the emission fluorescence ratio 670/650 vs. log(ligand concentration). K d value was calculated by fitting data with a 1:1 binding model. Data presented as mean ± SEM from n = 5 independent replicates. j Schematic representation of the full-length MSR1 and SRCR domain deletion mutant of MSR1. Created in BioRender. Taciak, B. (2024) https://BioRender.com/a34q680 . k Western blot analysis of MSR1 protein expression in HEK293 cells at 24 h after transfection with the control plasmid (p-mCherry2), the plasmid encoding the full-length MSR1 gene (p-MSR1-mCherry2) or the plasmid encoding SRCR domain deletion mutant of MSR1 gene (p-ΔMSR1-mCherry2). Representative western blot images are shown. l and ( m ) Flow cytometry analysis of internalized HFt-AF488 (50 μg/ml) within 30 min at 37 °C by HEK293 cells, transfected with the control plasmid (p-mCherry2), the plasmid encoding the full-length MSR1 gene (p-MSR1-mCherry2) or the plasmid encoding SRCR domain deletion mutant of MSR1 gene (p-ΔMSR1-mCherry2). Flow cytometry data are presented as the mean fluorescence intensity (MFI) of HFt-AF488. Data are presented as mean ± SEM from n = 4 independent replicates. n Representative confocal microscopy images of internalized HFt-AF488 (50 μg/ml) within 30 min at 37 °C by HEK293 cells, transfected with the control plasmid (p-mCherry2), the plasmid encoding the full-length MSR1 gene (p-MSR1-mCherry2) or the plasmid encoding SRCR domain deletion mutant of MSR1 gene ( p -ΔMSR1-mCherry2) (red). Nuclei were stained with Hoechst 33342 (blue). Scale bar = 20 μm. The two-way ANOVA followed by Sidak’s multiple comparisons post hoc test was used for statistical analysis. For all panels, **** P ≤ 0.0001. Source data are provided as a Source Data file.

Article Snippet: DNA plasmid transfection of CHO-K1 and HEK293 cell lines was performed using the negative control vector (pCMV3-SP-N-His, Sino Biological, CV023) and the plasmid encoding human MSR1 (CD204/MSR1 cDNA ORF Clone, N-His tag expression plasmid (Sino Biological, HG10427-NH).

Techniques: Western Blot, Expressing, Transfection, Control, Plasmid Preparation, Flow Cytometry, Fluorescence, Labeling, Confocal Microscopy, Staining, Amplified Luminescent Proximity Homogenous Assay, Titration, Serial Dilution, Concentration Assay, Generated, Software, Binding Assay, Mutagenesis

Journal: Nature Communications

Article Title: Harnessing macrophage-drug conjugates for allogeneic cell-based therapy of solid tumors via the TRAIN mechanism

doi: 10.1038/s41467-025-56637-9

Figure Lengend Snippet: a Representative confocal microscopy images of colocalization of the 735 drug with HFt-735 in human macrophages. The hMDM were incubated with HFt-AF488 (50 μg/ml) or HFt-735-AF488 for 15 min at 37 °C, fixed, and stained with the anti-735 antibody and Hoechst 33342 (blue). Merged fluorescence images show colocalization of HFt and 735 drug (yellow foci). Scale bar = 20 μm. b and ( c ) Flow cytometry analysis of internalized HFt-AF488 and HFt-735-AF488 (5 μg/ml) by hMDM ( b ) or THP-1 macrophages ( c ) in the absence or presence of AcLDL within 30 min at 37 °C. Flow cytometry data are presented as mean fluorescence intensity (MFI) of HFt-AF488 or HFt-735-AF488. Data presented as mean ± SEM from n = 3 different donors ( b , hMDM) or n = 3 independent replicates ( c , THP-1). d Flow cytometry analysis of internalized HFt-AF488 and HFt-735-AF488 (50 μg/ml) within 30 min at 37 °C by hMDM untreated or pre-treated for 30 min at 37 °C with poly(G) binding to scavenger receptor and poly(C) (control). Flow cytometry data are presented as % of HFt-AF488 and HFt-735-AF488 uptake in untreated, control cells. Data presented as mean ± SEM from n = 3 different donors (hMDM). e Representative confocal microscopy images of internalized HFt-AF488 and HFt-735-AF488 (50 μg/ml) (green) within 30 min at 37 °C by hMDM untreated or pre-treated for 30 min at 37 °C ligand of class A scavenger receptor or structurally related ligand that does not bind to this group of receptors (negative control): poly(G) and poly(C) (control). Afterwards cells were fixed and stained with Hoechst 33342 (blue) and anti-735 drug antibody. Scale bar = 20 μm. f Flow cytometry analysis of internalized HFt-AF488 and HFt-735-AF488 (50 μg/ml) within 30 min at 37 °C by THP-1 cells untreated or pre-treated for 30 min at 37 °C with poly(G) binding to scavenger receptor and poly(C) (control). Flow cytometry data are presented as % of HFt-AF488 and HFt-735-AF488 uptake in untreated, control cells. Data presented as mean ± SEM from n = 3 (HFt-AF488) and n = 3 (HFt-735-AF488) independent replicates. g Representative confocal microscopy images of internalized HFt-AF488 and HFt-735-AF488 (50 μg/ml) (green) within 30 min at 37 °C by THP-1 cells untreated or pre-treated for 30 min at 37 °C ligand of class A scavenger receptor or structurally related ligand that does not bind to this group of receptors (negative control): poly(G) and poly(C) (control). Afterwards cells were fixed and stained with Hoechst 33342 (blue) and anti-735 drug antibody. Scale bar = 20 μm. h Flow cytometry analysis of internalized HFt-AF488 (50 μg/ml) and HFt-735-AF488 by hMDM after MSR1 gene-knockdown within 30 min at 37 °C. For comparison, untreated cells (Control) and cells treated with a negative, scramble control siRNA (siScr) were used. Flow cytometry data are presented as % of ligand uptake in control cells (Control). Data presented as mean ± SEM from n = 3 different donors (hMDM). i Optimization of AlphaScreen assay conditions by cross-titration of His-MSR1 (0.41–100 nM, 3-fold serial dilution) and Biotin-HFt (0.008–18 nM, 3-fold serial dilution) at fixed 10 μg/ml concentration of both nickel chelated acceptor and streptavidin donor beads. Data shown are from a single pilot run conducted for assay optimization. j Competitive effect of HFt-735 on AlphaScreen signal generated by His-MSR1 & Biotin-HFt interaction. Fixed concentrations of 8 nM His-MSR1 and 2 nM Biotin-HFt were used below the hook point of the bead assay ( i ) in the presence of increasing concentration of HFt-735 (0.006–1000 nM, 3-fold serial dilution). IC 50 potency of HFt-735 in displacing Biotin-HFt from interaction with His-MSR1 was calculated by fitting it to a four-parameter nonlinear regression. Data presented as mean ± SEM from n = 3 independent replicates. k In vitro evaluation of cancer cell killing by MDC-735 against MDA-MB-231, BxPC-3, SK-OV-3 and A549 cancer cells in co-culture. Macrophages were incubated with 10, 100, 1000 μmol of HFt-735 complex or plain medium (0). Data are presented as mean ± SEM of n = 3 independent replicates. l Representative confocal microscopy images captured after 24 h co-culture of MDC-735 (pre-labeled with CellTrace Violet CT) with SK-OV-3 cancer cells (pre-labeled with CellTrace Far Red). Cells fixed after 24 h were additionally stained with anti-735 (green) and anti-HFt (red) antibodies. Co-localization of green and red signals both in macrophages and in cancer cells are pointed out by the arrowheads. Scale bar = 10 µM. The one-way ANOVA with Dunnett’s post-hoc test was used for statistical analysis in panels b – d , f , h . The two-way ANOVA and Tukey’s post-hoc tests were used for statistical analysis in panel k . For all panels, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Source data are provided as a Source Data file.

Article Snippet: DNA plasmid transfection of CHO-K1 and HEK293 cell lines was performed using the negative control vector (pCMV3-SP-N-His, Sino Biological, CV023) and the plasmid encoding human MSR1 (CD204/MSR1 cDNA ORF Clone, N-His tag expression plasmid (Sino Biological, HG10427-NH).

Techniques: Confocal Microscopy, Incubation, Staining, Fluorescence, Flow Cytometry, Binding Assay, Control, Negative Control, Knockdown, Comparison, Amplified Luminescent Proximity Homogenous Assay, Titration, Serial Dilution, Concentration Assay, Generated, In Vitro, Co-Culture Assay, Labeling

Journal: Nature Communications

Article Title: Harnessing macrophage-drug conjugates for allogeneic cell-based therapy of solid tumors via the TRAIN mechanism

doi: 10.1038/s41467-025-56637-9

Figure Lengend Snippet: a Representative confocal microscopy images of colocalization of HFt with AcLDL in human macrophages. hMDM were incubated simultaneously with HFt-AF488 (50 μg/ml) (green) and AcLDL-AF594 (5 μg/ml) (red) for 15 min at 37 °C. Nuclei were stained with Hoechst 33342 (blue). Merged fluorescence images show colocalization of HFt and AcLDL (yellow foci) for hMDM. Scale bar = 20 μm (10 µm in zoomed region). b Flow cytometry analysis of internalized HFt-AF488 (5 μg/ml) by hMDM in the absence or presence of AcLDL at indicated concentrations within 30 min at 37 °C. Flow cytometry data are presented as the mean fluorescence intensity (MFI) of HFt-AF488. Data are presented as mean ± SEM from n = 3 different donors. c Representative confocal microscopy images of internalized HFt-AF488 (5 μg/ml) (green) by hMDM in the absence or presence of AcLDL (100 μg/ml) within 30 min at 37 °C. Nuclei were stained with Hoechst 33342 (blue). Scale bar = 20 μm. d Flow cytometry analysis of internalized AcLDL-AF488 (1 μg/ml) by hMDM in the absence or presence of HFt at indicated concentrations within 30 min at 37 °C. Data are presented as mean fluorescence intensity (MFI) of AcLDL-AF488. Data are presented as mean ± SEM from n = 3 different donors. e Representative confocal microscopy images of internalized AcLDL-AF488 (1 μg/ml) (green) by hMDM in the absence or presence of HFt (2,5 mg/ml) within 30 min at 37 °C. Nuclei were stained with Hoechst 33342 (blue). Scale bar = 20 μm. f–h Flow cytometry analysis of internalized HFt-AF488 (50 μg/ml) within 30 min at 37 °C by hMDM untreated or pre-treated for 30 min at 37 °C with indicated concentrations of various ligands of class A scavenger receptors or structurally related ligands that do not bind to this group of receptors (negative controls): poly(I), poly(G) and poly(C) (control) ( f ), fucoidan and mannan (control) ( g ) and dextran sulfate or chondroitin sulfate (control) ( h ) Flow cytometry data are presented as % of HFt-AF488 uptake in untreated, control cells. Data are presented as mean ± SEM from n = 3 ( f , h ) or 6 ( g ) different donors (hMDM). i–k Representative confocal microscopy images of internalized HFt-AF488 (50 μg/ml) (green) within 30 min at 37 °C by hMDM untreated or pre-treated for 30 min at 37 °C with indicated concentrations of various ligands of class A scavenger receptors or structurally related ligands that do not bind to this group of receptors (negative controls): poly(I) and poly(C) (control) ( i ), fucoidan and mannan (control) ( j ) and dextran sulfate or chondroitin sulfate (control) ( k ). Nuclei were stained with Hoechst 33342 (blue). Scale bar = 20 μm. l Quantitative real-time PCR analysis of MSR1, SCARA5, and MARCO mRNA expression in hMDM. Data are presented as mean ± SEM from n = 3 different donors (hMDM). m Western blot analysis of MSR1 protein expression in monocytes and hMDM. Representative western blot images are shown. n Flow cytometry analysis of MSR1 cell surface staining in hMDM upon stimulation with HFt (200 µg/ml) for indicated time points at 37 °C. For comparison, untreated cells were used (Control). Data are presented as mean ± SEM % of MSR1 expression in control cells, n = 3 independent donors (hMDM). o Western blot analysis of MSR1 protein expression in hMDM that were untreated (Control) or treated with cycloheximide (CHX) (20 µg/ml) for 1 h prior to HFt stimulation (200 µg/ml) (CHX+HFt) for the indicated time points at 37 °C. For comparison, cells treated only with CHX were used. Representative western blot images are shown. p Quantitative analysis of relative MSR1 expression in hMDM shown in ( o ). Data are presented as mean ± SEM from n = 3 independent replicates. q Western blot analysis showing MSR1 expression in either untransfected hMDM (Control) or cells transfected with one of the following siRNA sequences: scramble siRNA (siScr), no. 1 siRNA targeting MSR1 (siMSR1-1), no. 2 siRNA targeting MSR1 (siMSR1-2) at 72 h after transfection. r Quantitative analysis of western blot for MSR1 expression shown in ( q ). Data are presented as mean ± SEM % of MSR1 expression in control cells (Control), n = 3 different donors (hMDM). s and ( t ) Flow cytometry analysis of internalized AcLDL-AF488 (5 µg/ml) ( s ) or HFt-AF488 (100 μg/ml) ( t ) by hMDM after MSR1 gene-knockdown within 30 min at 37 °C. For comparison, untreated cells (Control) and cells treated with a negative, scramble control siRNA (siScr) were used. Flow cytometry data are presented as mean ± SEM % of ligand uptake in control cells (Control), n = 3 different donors. u and ( v ) Representative confocal microscopy images of internalized AcLDL-AF488 (5 μg/ml) (green) ( u ) and HFt-AF488 (50 μg/ml) (green) ( v ) within 30 min at 37 °C by THP-1 macrophages after MSR1 gene-knockdown. Afterwards, cells were fixed and stained with Hoechst 33342 (blue). Scale bar = 20 μm. w Western blot analysis of MSR1 protein expression in hMDM macrophages polarized to M1 or M2 phenotypes. Representative western blot images are shown. x Relative expression of MSR1 in M1 and M2 hMDM macrophages quantified based on western blot analysis shown in ( w ). Data are presented as mean ± SEM from n = 4 different donors. y Flow cytometry analysis of internalized HFt-AF488 by M1 and M2 hMDM macrophages given at 25 µg/ml within 20 or 60 min at 37 °C. Flow cytometry data are presented as the mean fluorescence intensity (MFI) of HFt-AF488. Data are presented as mean ± SEM from n = 4 different donors. The one-way ANOVA and Dunnett’s post-hoc test were used for statistical analysis in panels b , d , r – t . The two-way ANOVA and Tukey’s post-hoc tests were used for statistical analysis in panels f – h . The two-way ANOVA followed by Sidak’s multiple comparisons post hoc test was used for statistical analysis in panels ( p , y ). The Student’s t -test was used for statistical analysis in panel ( x ). For all panels, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Source data are provided as a Source Data file.

Article Snippet: Biotin-HFt, His-tagged MSR1 (His-MSR1, R&D Systems, 2708-MS-050), nickel chelated acceptor and streptavidin-coated donor beads (PerkinElmer, 6760619C) were diluted in assay buffer consisting of PBS (Biowest, L06151000), 1 mg/ml BSA (Sigma-Aldrich, A3294) and 0.05% Tween 20 (Sigma-Aldrich, P1379).

Techniques: Confocal Microscopy, Incubation, Staining, Fluorescence, Flow Cytometry, Control, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Comparison, Transfection, Knockdown

Journal: Nature Communications

Article Title: Harnessing macrophage-drug conjugates for allogeneic cell-based therapy of solid tumors via the TRAIN mechanism

doi: 10.1038/s41467-025-56637-9

Figure Lengend Snippet: a Western blot analysis of MSR1 protein expression in HEK293 cells at 24 h after transfection with the control plasmid (p-mCherry2) or the plasmid encoding the MSR1 gene (p-MSR1-mCherry2). Representative western blot images are shown. b and ( c ) Flow cytometry analysis of cell surface expression of MSR1 in HEK293 cells at 24 h after transfection with the control plasmid (p-mCherry2) or the plasmid encoding the MSR1 gene (p-MSR1-mCherry2). Flow cytometry data are presented as the mean fluorescence intensity (MFI) of BV421. Data are presented as mean ± SEM from n = 3 independent replicates. d Flow cytometry analysis of internalized AcLDL-AF488 (5 μg/ml) and HFt-AF488 (50 μg/ml) within 30 min at 37 °C by HEK293 cells, transfected with either p-mCherry2 or p-MSR1-mCherry2. Flow cytometry data are presented as mean fluorescence intensity (MFI) of fluorescently labeled ligands. Data are presented as mean ± SEM from n = 3 independent replicates. e , f Representative confocal microscopy images of internalized HFt-AF488 (50 μg/ml) ( e ) and AcLDL-AF488 (5 μg/ml) ( f ) (green) within 30 min at 37 °C by HEK293 cells, transfected with either p-mCherry2 or p-MSR1-mCherry2 (red). Afterwards, cells were fixed and stained with Hoechst 33342 (blue). Scale bar = 20 μm. g Optimization of AlphaScreen assay conditions by cross-titration of His-MSR1 (0.41–100 nM, 3-fold serial dilution) and Biotin-HFt (0.022–50 nM, 3-fold serial dilution) at fixed 10 μg/ml concentration of both nickel chelated acceptor and streptavidin donor beads. Data shown are from a single pilot run conducted for assay optimization. h Competitive effect of unlabeled HFt on AlphaScreen signal generated by His-MSR1 and Biotin-HFt interaction. Fixed concentrations of 4 nM His-MSR1 and 2 nM Biotin-HFt were used below the hook point of the bead assay ( g ) in the presence of increasing concentration of unlabeled HFt (0.076–1500 nM, 3-fold serial dilution). IC50 potency of unlabeled HFt in displacing Biotin-HFt from interaction with His-MSR1 was calculated by fitting it to a four-parameter nonlinear regression. Data presented as mean ± SEM from n = 3 independent replicates. i Spectral shift dose–response curve between unlabeled HFt and His-MSR1 conjugated with the RED-tris-NTA dye. Data was analyzed using software provided by the manufacturer and is presented as the emission fluorescence ratio 670/650 vs. log(ligand concentration). K d value was calculated by fitting data with a 1:1 binding model. Data presented as mean ± SEM from n = 5 independent replicates. j Schematic representation of the full-length MSR1 and SRCR domain deletion mutant of MSR1. Created in BioRender. Taciak, B. (2024) https://BioRender.com/a34q680 . k Western blot analysis of MSR1 protein expression in HEK293 cells at 24 h after transfection with the control plasmid (p-mCherry2), the plasmid encoding the full-length MSR1 gene (p-MSR1-mCherry2) or the plasmid encoding SRCR domain deletion mutant of MSR1 gene (p-ΔMSR1-mCherry2). Representative western blot images are shown. l and ( m ) Flow cytometry analysis of internalized HFt-AF488 (50 μg/ml) within 30 min at 37 °C by HEK293 cells, transfected with the control plasmid (p-mCherry2), the plasmid encoding the full-length MSR1 gene (p-MSR1-mCherry2) or the plasmid encoding SRCR domain deletion mutant of MSR1 gene (p-ΔMSR1-mCherry2). Flow cytometry data are presented as the mean fluorescence intensity (MFI) of HFt-AF488. Data are presented as mean ± SEM from n = 4 independent replicates. n Representative confocal microscopy images of internalized HFt-AF488 (50 μg/ml) within 30 min at 37 °C by HEK293 cells, transfected with the control plasmid (p-mCherry2), the plasmid encoding the full-length MSR1 gene (p-MSR1-mCherry2) or the plasmid encoding SRCR domain deletion mutant of MSR1 gene ( p -ΔMSR1-mCherry2) (red). Nuclei were stained with Hoechst 33342 (blue). Scale bar = 20 μm. The two-way ANOVA followed by Sidak’s multiple comparisons post hoc test was used for statistical analysis. For all panels, **** P ≤ 0.0001. Source data are provided as a Source Data file.

Article Snippet: Biotin-HFt, His-tagged MSR1 (His-MSR1, R&D Systems, 2708-MS-050), nickel chelated acceptor and streptavidin-coated donor beads (PerkinElmer, 6760619C) were diluted in assay buffer consisting of PBS (Biowest, L06151000), 1 mg/ml BSA (Sigma-Aldrich, A3294) and 0.05% Tween 20 (Sigma-Aldrich, P1379).

Techniques: Western Blot, Expressing, Transfection, Control, Plasmid Preparation, Flow Cytometry, Fluorescence, Labeling, Confocal Microscopy, Staining, Amplified Luminescent Proximity Homogenous Assay, Titration, Serial Dilution, Concentration Assay, Generated, Software, Binding Assay, Mutagenesis

Journal: Nature Communications

Article Title: Harnessing macrophage-drug conjugates for allogeneic cell-based therapy of solid tumors via the TRAIN mechanism

doi: 10.1038/s41467-025-56637-9

Figure Lengend Snippet: a Representative confocal microscopy images of colocalization of the 735 drug with HFt-735 in human macrophages. The hMDM were incubated with HFt-AF488 (50 μg/ml) or HFt-735-AF488 for 15 min at 37 °C, fixed, and stained with the anti-735 antibody and Hoechst 33342 (blue). Merged fluorescence images show colocalization of HFt and 735 drug (yellow foci). Scale bar = 20 μm. b and ( c ) Flow cytometry analysis of internalized HFt-AF488 and HFt-735-AF488 (5 μg/ml) by hMDM ( b ) or THP-1 macrophages ( c ) in the absence or presence of AcLDL within 30 min at 37 °C. Flow cytometry data are presented as mean fluorescence intensity (MFI) of HFt-AF488 or HFt-735-AF488. Data presented as mean ± SEM from n = 3 different donors ( b , hMDM) or n = 3 independent replicates ( c , THP-1). d Flow cytometry analysis of internalized HFt-AF488 and HFt-735-AF488 (50 μg/ml) within 30 min at 37 °C by hMDM untreated or pre-treated for 30 min at 37 °C with poly(G) binding to scavenger receptor and poly(C) (control). Flow cytometry data are presented as % of HFt-AF488 and HFt-735-AF488 uptake in untreated, control cells. Data presented as mean ± SEM from n = 3 different donors (hMDM). e Representative confocal microscopy images of internalized HFt-AF488 and HFt-735-AF488 (50 μg/ml) (green) within 30 min at 37 °C by hMDM untreated or pre-treated for 30 min at 37 °C ligand of class A scavenger receptor or structurally related ligand that does not bind to this group of receptors (negative control): poly(G) and poly(C) (control). Afterwards cells were fixed and stained with Hoechst 33342 (blue) and anti-735 drug antibody. Scale bar = 20 μm. f Flow cytometry analysis of internalized HFt-AF488 and HFt-735-AF488 (50 μg/ml) within 30 min at 37 °C by THP-1 cells untreated or pre-treated for 30 min at 37 °C with poly(G) binding to scavenger receptor and poly(C) (control). Flow cytometry data are presented as % of HFt-AF488 and HFt-735-AF488 uptake in untreated, control cells. Data presented as mean ± SEM from n = 3 (HFt-AF488) and n = 3 (HFt-735-AF488) independent replicates. g Representative confocal microscopy images of internalized HFt-AF488 and HFt-735-AF488 (50 μg/ml) (green) within 30 min at 37 °C by THP-1 cells untreated or pre-treated for 30 min at 37 °C ligand of class A scavenger receptor or structurally related ligand that does not bind to this group of receptors (negative control): poly(G) and poly(C) (control). Afterwards cells were fixed and stained with Hoechst 33342 (blue) and anti-735 drug antibody. Scale bar = 20 μm. h Flow cytometry analysis of internalized HFt-AF488 (50 μg/ml) and HFt-735-AF488 by hMDM after MSR1 gene-knockdown within 30 min at 37 °C. For comparison, untreated cells (Control) and cells treated with a negative, scramble control siRNA (siScr) were used. Flow cytometry data are presented as % of ligand uptake in control cells (Control). Data presented as mean ± SEM from n = 3 different donors (hMDM). i Optimization of AlphaScreen assay conditions by cross-titration of His-MSR1 (0.41–100 nM, 3-fold serial dilution) and Biotin-HFt (0.008–18 nM, 3-fold serial dilution) at fixed 10 μg/ml concentration of both nickel chelated acceptor and streptavidin donor beads. Data shown are from a single pilot run conducted for assay optimization. j Competitive effect of HFt-735 on AlphaScreen signal generated by His-MSR1 & Biotin-HFt interaction. Fixed concentrations of 8 nM His-MSR1 and 2 nM Biotin-HFt were used below the hook point of the bead assay ( i ) in the presence of increasing concentration of HFt-735 (0.006–1000 nM, 3-fold serial dilution). IC 50 potency of HFt-735 in displacing Biotin-HFt from interaction with His-MSR1 was calculated by fitting it to a four-parameter nonlinear regression. Data presented as mean ± SEM from n = 3 independent replicates. k In vitro evaluation of cancer cell killing by MDC-735 against MDA-MB-231, BxPC-3, SK-OV-3 and A549 cancer cells in co-culture. Macrophages were incubated with 10, 100, 1000 μmol of HFt-735 complex or plain medium (0). Data are presented as mean ± SEM of n = 3 independent replicates. l Representative confocal microscopy images captured after 24 h co-culture of MDC-735 (pre-labeled with CellTrace Violet CT) with SK-OV-3 cancer cells (pre-labeled with CellTrace Far Red). Cells fixed after 24 h were additionally stained with anti-735 (green) and anti-HFt (red) antibodies. Co-localization of green and red signals both in macrophages and in cancer cells are pointed out by the arrowheads. Scale bar = 10 µM. The one-way ANOVA with Dunnett’s post-hoc test was used for statistical analysis in panels b – d , f , h . The two-way ANOVA and Tukey’s post-hoc tests were used for statistical analysis in panel k . For all panels, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Source data are provided as a Source Data file.

Article Snippet: Biotin-HFt, His-tagged MSR1 (His-MSR1, R&D Systems, 2708-MS-050), nickel chelated acceptor and streptavidin-coated donor beads (PerkinElmer, 6760619C) were diluted in assay buffer consisting of PBS (Biowest, L06151000), 1 mg/ml BSA (Sigma-Aldrich, A3294) and 0.05% Tween 20 (Sigma-Aldrich, P1379).

Techniques: Confocal Microscopy, Incubation, Staining, Fluorescence, Flow Cytometry, Binding Assay, Control, Negative Control, Knockdown, Comparison, Amplified Luminescent Proximity Homogenous Assay, Titration, Serial Dilution, Concentration Assay, Generated, In Vitro, Co-Culture Assay, Labeling