Immunogenic Cell Death of Breast Cancer Stem Cells Induced by an Endoplasmic Reticulum‐Targeting Copper(II) Complex

Abstract Immunogenic cell death (ICD) offers a method of stimulating the immune system to attack and remove cancer cells. We report a copper(II) complex containing a Schiff base ligand and a polypyridyl ligand, 4, capable of inducing ICD in breast cancer stem cells (CSCs). Complex 4 kills both bulk breast cancer cells and breast CSCs at sub‐micromolar concentrations. Notably, 4 exhibits greater potency (one order of magnitude) towards breast CSCs than salinomycin (an established breast CSC‐potent agent) and cisplatin (a clinically approved anticancer drug). Epithelial spheroid studies show that 4 is able to selectively inhibit breast CSC‐enriched HMLER‐shEcad spheroid formation and viability over non‐tumorigenic breast MCF10 A spheroids. Mechanistic studies show that 4 operates as a Type II ICD inducer. Specifically, 4 readily enters the endoplasmic reticulum (ER) of breast CSCs, elevates intracellular reactive oxygen species (ROS) levels, induces ER stress, evokes damage‐associated molecular patterns (DAMPs), and promotes breast CSC phagocytosis by macrophages. As far as we are aware, 4 is the first metal complex to induce ICD in breast CSCs and promote their engulfment by immune cells.


Table of Content Experimental Details
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Chemical structures of the Schiff base ligand, L 1 and the copper(II) complex, [Cu(L 1 )Cl]. Figure S2.
High resolution ESI mass spectrum (positive mode) of 1. Figure S3.
High resolution ESI mass spectrum (positive mode) of 2. Figure S4.
High resolution ESI mass spectrum (positive mode) of 3. Figure S5.
High resolution ESI mass spectrum (positive mode) of 4. Figure S6.
Representative dose-response curves for the treatment of HMLER and HMLER-shEcad cells with 1 after 72 h incubation. Figure S10. Representative dose-response curves for the treatment of HMLER and HMLER-shEcad cells with 2 after 72 h incubation. Figure S11. Representative dose-response curves for the treatment of HMLER and HMLER-shEcad cells with 3 after 72 h incubation. Figure S12. Representative dose-response curves for the treatment of HMLER and HMLER-shEcad cells with 4 after 72 h incubation. Figure S13. Representative dose-response curves for the treatment of HMLER and HMLER-shEcad cells with [Cu(L 1 )Cl] after 72 h incubation. Figure S14. Quantification of spheroid formation with HMLER-shEcad cells untreated and treated with 1-4, [Cu(L 1 )Cl], or salinomycin at their respective IC 20 values for 5 days. Error bars = SD and Student t-test, * = p < 0.05. Figure S15. Representative bright-field images (× 20) of HMLER-shEcad spheroids in the absence and presence of 1-4 or salinomycin at their respective IC 20 values for 5 days. Figure S16. Representative bright-field images (× 20) of HMLER-shEcad spheroids in the absence and presence of [Cu(L 1 )Cl] at the respective IC 20 values for 5 days. Figure S17. Representative dose-response curves for the treatment of HMLER-shEcad spheroids with 1-4 and [Cu(L 1 )Cl]. Figure S18. Representative dose-response curve for the treatment of MCF10A spheroids with 4. Figure S19. Representative dose-response curve for the treatment of MCF10A spheroids with salinomycin. Figure S20. Copper content in whole cell and endoplasmic reticulum fractions isolated from HMLER-shEcad cells treated with 4 or [Cu(L 1 )Cl] (5 μM for 24 h at 37°C). Error bars = SD. Figure S21. Normalised ROS activity in untreated HMLER-shEcad cells (control) and HMLER-shEcad cells treated with 4 (IC 50 value for 1, 3, 6, 16, 24, and 48 h). Error bars = SD and Student t-test, * = p < 0.05. Figure S22. Representative dose-response curves for the treatment of HMLER-shEcad cells with 4 after 72 incubation in the presence of N-acetylcysteine (2.5 mM).   Figure S25. Immunoblotting analysis of proteins related to the unfolded protein response (UPR). Protein expression in HMLER-shEcad cells following treatment with thapsigargin (300 nM for 1 h). Figure S26. Immunoblotting analysis of high mobility group box 1 (HMGB-1). Protein expression in HMLER-shEcad cells following treatment with 4 (0.3 and 0.6 μM for 48 h) or cisplatin (150 μM for 48 h) and thapsigargin (7 μM for 48 h) or, cisplatin (150 μM for 48 h).

Measurement of water-octanol partition coefficient (LogP).
The LogP value for 1-4 was determined using the shake-flask method. The octanol used in this experiment was presaturated with water. An aqueous solution of 1-4 (500 μL, 100 μM) was incubated with octanol (500 μL) in a 1.5 mL tube. The tube was shaken at room temperature for 24 h. The two phases were separated by centrifugation and the 1-4 content in the aqueous phase was determined by inductively coupled plasma mass spectrometry (ICP-MS, PerkinElmer NexION 350D).  × 3), and harvested. The number of cells was counted at this stage, using a haemocytometer. This mitigates any cell death induced by 4 and [Cu(L 1 )Cl] at the administered concentration and experimental cell loss. The cells were centrifuged to form pellets. The cellular pellets were dissolved in 65% HNO 3 (250 μL) overnight. The cellular pellets were also used to determine the copper content in the endoplasmic reticulum (ER) fraction. The Sigma-Aldrich Endoplasmic Reticulum Isolation Kit was used to extract and separate the ER fraction. The fractions were dissolved in 65% HNO 3 overnight (250 μL final volume). All samples were diluted 5-fold with water and analysed using inductively coupled plasma mass spectrometry (ICP-MS, PerkinElmer NexION 350D). Copper levels are expressed as Cu (ppb) per million cells. Results are presented as the mean of five determinations for each data point.
Fluorescence Microscopy. HMLER-shEcad cells (1×10 4 ) were incubated with 4 (5 μM for 1 h) (using phenol-free MEGM cell media). The media was then removed and the cells were washed with additional media (2 mL × 2). After incubation of the cells with more media containing ER-Tracker Red (1.6 μM for 15 min), the media was removed, the cells were washed with additional media (2 mL × 2). Then the cells were incubated with more media containing the green-emitting ROS indicator, DCFDA (20 μM for 10 min), the media was removed, the cells were washed with additional media (2 mL × 2), and imaged using a fluorescent microscope. Fluorescence imaging experiments were performed using a Nikon Eclipse Ts2-FL microscope. The microscope was operated with the NIS Element software. The exposure time for acquisition of fluorescence images was kept constant for each series of images at each channel. The images were overlaid using ImageJ (version 1.45, NIH).
CRT cell surface exposure. Flow cytometry was used to analyse cell surface CRT exposure. HMLER-shEcad cells were seeded into a 6-well plate (at a density of 5 × 10 5 cells/ mL) and the cells were incubated at 37°C overnight. The cells were treated with 4 (IC 50 value) or cotreated with cisplatin (150 μM) with thapsigargin (7 μM) for 12 hours at 37°C. The cells were then harvested by trypsinization and collected by centrifugation. The pellet was suspended in PBS (500 μL), and after the addition of the Alexa Fluor® 488 nm labelled anti-CRT antibody (5 μl), the cells were incubated in the dark for 30 minutes. The cells were then washed with PBS (1 mL) and analysed using a FACSCanto II flow cytometer (BD Biosciences) (10,000 events per sample were acquired). The FL1 channel was used to assess CRT cell surface exposure. Cell populations were analysed using the FlowJo software (Tree Star).
ATP assay. HMLER-shEcad cells (5 × 10 3 cells /well) were seeded in a 96-well plate and incubated overnight. The cells were then treated with 4 (IC 50 value) or cisplatin (IC 50 value, positive control) for 24 hours at 37°C. The media was carefully extracted and transferred into a white-walled opaque 96-well plate, and a luciferin-based ENLITEN ATP Assay Kit (Promega) was used to measure the relative amount of ATP released into the supernatant.
Phagocytosis assay. HMLER-shEcad cells were seeded into a 6-well plate (at a density of 5 × 10 5 cells/ mL) and the cells were incubated at 37°C overnight. The cells were stained with CellTracker Green (30 min) and washed with MEGM media. The cells were then treated with 4 (5 μM), cisplatin (50 μM), or carboplatin (100 μM) for 4 h at 37°C. Then macrophages, obtained by differentiating THP-1 cells with phorbol 12-myristate 13-acetate (100 nM for 72 h) and pre-stained with CellTracker Orange for 30 min and washed with RPMI 1640 media, were added to the HMLER-shEcad cells (at a density of 1 × 10 5 cells/ mL). After 2 h, phagocytosis was assessed by fluorescence imaging experiments using a Nikon Eclipse Ts2-FL microscope. The microscope was operated with the NIS Element software. The exposure time for acquisition of fluorescence images was kept constant for each series of images at each channel. The images were overlaid using ImageJ (version 1.45, NIH).       Table S2. Selected bond lengths (Å) and angles (°) for 3.