hcd71 apc vio770  (Miltenyi Biotec)

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 Schematic representation of the macrophage loading with HFt and subsequent transfer to cancer cells. Created in BioRender. Taciak, B. (2024) https://BioRender.com/s50b255 . b Snapshots from the movie recorded under the confocal microscopy (Supplementary Video ) in 15 min where BMDM macrophages loaded with HFt-FITC transfer it to EMT6 cancer cell labeled with red CellTrace. c Representative flow cytometry scatter plot of macrophages with internalized HFt-AF488 and MDA-MB-231 breast cancer cells labeled with CellTrace (CellTrace Far Red—APC). d and ( e ) Flow cytometry quantification of HFt-AF488 transfer from hMDM and (loaded with HFt-AF488 at concentration 500 µg/ml for 1 h at 37 °C) to various human cancer cell lines at 24 h following co-culture at 2:1 ratio (macrophages: cancer cells). Data are presented as mean ± SEM from n = 3 different donors (hMDM). f Representative confocal microscopy images of HFt-AF488 (green) transfer from hMDM (loaded with HFt-AF488 at concentration 500 µg/ml for 1 h at 37 °C) to MDA‑MB‑231 breast cancer cells (blue) after 24 h of co-culture. Scale bar = 10 µm. Wheat Germ Agglutinin (WGA), Alexa Fluor-555 Conjugate (red) was used to visualize the cell membrane. g Comparative analysis of HFt-AF488 transfer from hMDM of different polarization states—M0, M1 (stimulated with LPS, 100 ng/ml), and M2 (stimulated with IL-4, 20 ng/ml)—to MDA-MB-231 breast cancer cells. hMDM were pre-loaded with HFt-AF488 (500 μg/ml) for 1 h at 37 °C, followed by co-culture with MDA-MB-231 cells for 4 and 24 h. Control conditions included co-cultures of M0, M1, and M2 hMDM with MDA-MB-231 cells without HFt-AF488. Data are presented as mean ± SEM from n = 3 different donors (hMDM). h and ( i ) Flow cytometry quantification of HFt-AF488 transfer from iPSC-derived macrophages (loaded with HFt-AF488 at concentration 500 µg/ml for 1 h at 37 °C) to various human cancer cell lines at 24 h following co-culture at 2:1 ratio (macrophages: cancer cells). Data are presented as mean ± SEM from n = 3 (A549, SK-OV-3) or n = 4 (DU145, MDA-MB-231, SW1353) independent replicates. j Representative confocal microscopy images of HFt-AF488 (green) transfer from iPSC-derived macrophages (loaded with HFt-AF-647 at concentration 500 µg/ml for 1 h at 37 °C) to MDA‑MB‑231 breast cancer cells (blue) after 24 h of co-culture. Scale bar = 10 µm. Wheat Germ Agglutinin (WGA), Alexa Fluor-555 Conjugate (red) was used to visualize the cell membrane. k Flow cytometry analysis of Tfn-AF647 transfer from RAW 264.7 and THP-1 macrophages (loaded with Tfn-AF-647 at concentration 0.2 mg/ml for 1 h at 37 °C) to cancer cells (EMT6 and MDA-MB-231, respectively) after 24 h of co-culture. Data are presented as mean ± SEM from n = 3 independent replicates. l Flow cytometry analysis of HFt-AF488 transfer from THP1-derived macrophages (loaded with HFt-AF488 at concentration 500 µg/ml for 1 h at 37 °C) to various cancer cell lines after 4 h co-culture. Data are presented as mean ± SEM from n = 3 independent replicates. m Correlation of the percentage of HFt-AF488-positive recipient cells from human cell lines shown in ( l ) to their CD71 (TfR1) receptor expression assessed by flow cytometry. n Western blot analysis showing TfR1 gene knockdown efficiency in MDA-MB-231 cells using two different siRNA sequences and scramble as a control. o–q Flow cytometry analysis of AF488 fluorescence in MDA-MB-231 cancer cells with TfR1 gene knockdown using two different siRNA sequences, cells transfected with negative control (Scramble) siRNA or untreated cells (Control). Effect on ( o ) Tfn-AF488 and ( p ) HFt-AF488 uptake from medium and ( q ) HFt-AF488 transfer from THP-1 macrophages in 4 h co-culture was calculated relative to Scramble. Data are presented as mean ± SEM from n = 3 independent replicates. r and ( s ) Flow cytometry quantification of HFt-AF488 transfer from RAW 264.7 or THP-1 macrophages (loaded with HFt-AF488 at concentration 500 µg/ml for 1 h at 37 °C) to ( r ) EMT6 or ( s ) MDA-MB-231 breast cancer cells (respectively) at 24 h following direct, or Transwell membrane-separated co-culture system (macrophages seeded on Transwell insets). Co-culture of macrophages without HFt-AF488 and cancer cells [co-culture HFt(−)] was used as a control. Data are presented as mean ± SEM from n = 3 independent replicates. Statistical analysis was performed using one-way ANOVA with post-hoc Tukey HSD test. For all panels, * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001. Source data are provided as a Source Data file.

Article Snippet: The cells were then incubated with one of the following antibodies: PE anti-human CD204/MSR1 antibody (1:20, 371904, BioLegend), APC anti-human CD71/TfR1 antibody (clone OKT9) (1:20, 17-0719-42, eBioscience), PE mouse IgG2a, κ isotype control antibody (1:20, 400214, BioLegend), APC mouse IgG1 κ isotype control antibody (P3.6.2.8.1) (1:20, 17-4714-82, eBioscience) for 1 h on ice in the dark.

Techniques: Confocal Microscopy, Labeling, Flow Cytometry, Concentration Assay, Co-Culture Assay, Membrane, Control, Derivative Assay, Expressing, Western Blot, Knockdown, Fluorescence, Transfection, Negative Control