Fig 1: Human macrophages utilize the same receptor for HFt and HFt-drug complex uptake.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). IC50 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.