Overcoming Multidrug Resistance via Photodestruction of ABCG2-Rich Extracellular Vesicles Sequestering Photosensitive Chemotherapeutics

Multidrug resistance (MDR) remains a dominant impediment to curative cancer chemotherapy. Efflux transporters of the ATP-binding cassette (ABC) superfamily including ABCG2, ABCB1 and ABCC1 mediate MDR to multiple structurally and functionally distinct antitumor agents. Recently we identified a novel mechanism of MDR in which ABCG2-rich extracellular vesicles (EVs) form in between attached neighbor breast cancer cells and highly concentrate various chemotherapeutics in an ABCG2-dependent manner, thereby sequestering them away from their intracellular targets. Hence, development of novel strategies to overcome MDR modalities is a major goal of cancer research. Towards this end, we here developed a novel approach to selectively target and kill MDR cancer cells. We show that illumination of EVs that accumulated photosensitive cytotoxic drugs including imidazoacridinones (IAs) and topotecan resulted in intravesicular formation of reactive oxygen species (ROS) and severe damage to the EVs membrane that is shared by EVs-forming cells, thereby leading to tumor cell lysis and the overcoming of MDR. Furthermore, consistent with the weak base nature of IAs, MDR cells that are devoid of EVs but contained an increased number of lysosomes, highly accumulated IAs in lysosomes and upon photosensitization were efficiently killed via ROS-dependent lysosomal rupture. Combining targeted lysis of IAs-loaded EVs and lysosomes elicited a synergistic cytotoxic effect resulting in MDR reversal. In contrast, topotecan, a bona fide transport substrate of ABCG2, accumulated exclusively in EVs of MDR cells but was neither detected in lysosomes of normal breast epithelial cells nor in non-MDR breast cancer cells. This exclusive accumulation in EVs enhanced the selectivity of the cytotoxic effect exerted by photodynamic therapy to MDR cells without harming normal cells. Moreover, lysosomal alkalinization with bafilomycin A1 abrogated lysosomal accumulation of IAs, consequently preventing lysosomal photodestruction of normal breast epithelial cells. Thus, MDR modalities including ABCG2-dependent drug sequestration within EVs can be rationally converted to a pharmacologically lethal Trojan horse to selectively eradicate MDR cancer cells

Structure and Function of ABCG2-Rich Extracellular Vesicles Mediating Multidrug Resistance

Multidrug resistance (MDR) is a major impediment to curative cancer chemotherapy. The ATP-Binding Cassette transporters ABCG2, ABCB1 and ABCC2 form a unique defense network against multiple structurally and functionally distinct chemotherapeutics, thereby resulting in MDR. Thus, deciphering novel mechanisms of MDR and their overcoming is a major goal of cancer research. Recently we have shown that overexpression of ABCG2 in the membrane of novel extracellular vesicles (EVs) in breast cancer cells results in mitoxantrone resistance due to its dramatic sequestration in EVs. However, nothing is known about EVs structure, biogenesis and their ability to concentrate multiple antitumor agents. To this end, we here found that EVs are structural and functional homologues of bile canaliculi, are apically localized, sealed structures reinforced by an actin-based cytoskeleton and secluded from the extracellular milieu by the tight junction proteins occludin and ZO-1. Apart from ABCG2, ABCB1 and ABCC2 were also selectively targeted to the membrane of EVs. Moreover, Ezrin-Radixin-Moesin protein complex selectively localized to the border of the EVs membrane, suggesting a key role for the tethering of MDR pumps to the actin cytoskeleton. The ability of EVs to concentrate and sequester different antitumor drugs was also explored. Taking advantage of the endogenous fluorescence of anticancer drugs, we found that EVs-forming breast cancer cells display high level resistance to topotecan, imidazoacridinones and methotrexate via efficient intravesicular drug concentration hence sequestering them away from their cellular targets. Thus, we identified a new modality of anticancer drug compartmentalization and resistance in which multiple chemotherapeutics are actively pumped from the cytoplasm and highly concentrated within the lumen of EVs via a network of MDR transporters differentially targeted to the EVs membrane. We propose a composite model for the structure and function of MDR pump-rich EVs in cancer cells and their ability to confer multiple anticancer drug resistance

Photosensitization of C-1371-accumulating EVs results in destruction of EVs in MCF-7/MR cells.

<p>(A) MCF-7/MR cells were grown as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035487#pone-0035487-g001" target="_blank">Figure 1</a> legend and exposed to C-1371 (10 µM) for 24 h at 37°C to allow for its accumulation within EVs. Then, random fields were photographed using a Zeiss inverted Cell-Observer microscope at an ×630 magnification. Next, these selected fields were continuously exposed to blue light using the GFP+DsRED longpass filter (excitation 470±27; emission 512±20 and 630±98) for 10 min and re-photographed using the same parameters. Continuous arrows point at the location of EVs, whereas dashed arrows point at the nuclei of EVs-forming cells. (B) Following illumination, cells were analyzed by Z-stack sections creating five 1 µm-thick optical slices. The black arrows point at the MSIS formed following photosensitization.</p

Immunofluorescence studies of ABCG2 and ZO-1 localization following illumination of C-1371-accumulating EVs formed in MCF-7/MR cells.

<p>(A) A merged image of bright field and C-1371 green fluorescence in live MCF-7/MR cells before illumination. Immediately after illumination, both the illuminated (B, C and D, a–d) and the non-illuminated (D, e–h) MCF-7/MR cells were fixed in 4% formaldehyde and co-reacted with monoclonal antibodies to ABCG2 (BXP-53) and ZO-1. (B) Illuminated MCF-7/MR cells were stained with ZO-1 (green), ABCG2 (red) and DAPI (blue), at a ×630 magnification. (C)The bright field image underlines the structures of illuminated EVs. (D) An enlarged image of the area is indicated by a white square in panel B. The green nuclear fluorescence in panel C is due to the intercalation of IA into DNA after the rupture of the membrane of EVs due to photosensitization. C-1371-loaded, non-illuminated MCF-7/MR cells served as a control (e–h). Since immunofluorescent staining includes membrane permeabilization, no accumulation of IAs was detected in EVs. Continuous arrows point at EVs whereas dashed arrows point at the nuclei.</p

Treatment with bafilomycin A1 abolishes lysosomal accumulation of C-1371, but has no impact on its accumulation in EVs.

<p>Cells were grown as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035487#pone-0035487-g007" target="_blank">Figure 7</a> legend. MCF-7/MR (A) and MCF-10A (B) cells were exposed to C-1371(10 µM) for 24 h or 5 h, respectively, in the absence or presence of bafilomycin A. Lysosomes and nuclei were visualized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035487#pone-0035487-g007" target="_blank">Figure 7</a> legend. Cells were then photographed using the Cell-Observer microscope at an ×630 magnification. Cells exposed solely to bafilomycin A1 served as negative controls (i–l in A; g–I in B). Continuous arrows point at the EVs accumulation of C-1371 (green), whereas the dashed arrows point at the lysosomes (red).</p

Inhibition of cell growth by IAs and their accumulation in EVs.

<p>(A) MCF-7/MR cells were seeded in 6-well dishes (2×10⁴cells/well; 2 ml medium/well) containing glass bottom and grown for 7 days to allow for the formation of EVs. Before adding IAs, cells were provided with riboflavin-deficient medium for at least 48 h to avoid riboflavin autofluorescence. Then, cells were exposed to 5 µM of various IAs for 24 h at 37°C. (B) Cells were pre-treated for one hour with FTC (10 µM) following co-incubation with C-1371 as in panel A. Control cells were cultured in FTC-free medium in the presence of C-1371. Arrows indicate the localization of EVs that lack or contain IAs accumulation. (C) MCF-7 and MCF-7/MR cells were exposed to various concentrations of C-1371 for 72 h in the presence or absence of FTC (10 µM), following which viable cell numbers were determined. Shown are the means of three independent experiments, each performed in triplicates ± SD. The IC₅₀ values of C-1371 in MCF-7 and MCF-7/MR cells were 4.3±0.1 and 27.8±3.7, respectively. Throughout the entire study, the bar denotes10 µm.</p

Sub-cellular localization of IAs that are non-ABCG2 substrates and the impact of illumination on cell viability in C-1266-accumulating cells.

<p>(A) MCF-7/MR cells were incubated with C-1266 (10 µM), which is not an ABCG2 transport substrate, for 5 h at 37°C (a–c). Selected fields were continuously exposed to blue light for 1–2 min using the Cell-Observer microscope at a ×630 magnification (d–f). Continuous arrows denote the location of EVs that are devoid of IAs (i.e. empty EVs), whereas dashed arrows point at the lysosomal or the nuclear localization of C-1266. (B) MCF-7 and MCF-7/MR cells were exposed to various concentrations of C-1266 for 72 h in the presence or absence of Ko143 (0.7 µM), following which viable cell numbers were determined. Shown are the means of three independent experiments, each performed in triplicates ± SD.</p

Immunofluorescence studies of ERM and ABCG2 localization following illumination of C-1371-accumulating EVs formed in MCF-7/MR cells.

<p>MCF-7/MR cells accumulating C-1371 (10 µM) were either illuminated (excitation 470±27 emission 512±20 and 630±98) for 10 min at a ×400 magnification to allow for photodestruction of EVs (A–D) or not illuminated (E–H). Cells were then immediately fixed and co-reacted with monoclonal antibodies to ABCG2 (BXP-21) and ERM. The green nuclear staining in panel B is due to IA intercalation into nuclear DNA, following EVs photodestruction. Continuous arrows point at EVs whereas dashed arrows point at nuclei.</p

The effect of illumination on the viability and morphology of IAs-accumulating MCF-7 and MCF-7/MR monolayers.

<p>(A) Confluent MCF-7/MR (a–f) and MCF-7 (g–h) monolayers were loaded with the indicated IAs (20 µM): 24 h incubation in the case of ABCG2-substrates (i.e. C-1492 and C-1371), and 3 h incubation in the case of non-ABCG2 substrate C-1266. Then, cells were exposed to constant light: 10 min for ABCG2-substrates (a–d) and 2 min for non-ABCG2 substrates (e–f) or while using MCF-7 cells (g–h). The illuminated foci were lysed thus appeared as round, blank areas with cells debris and residual damaged cytoskeletal structures (see arrows). Cells that were not exposed to light surround the illuminated areas and retained their intact morphology. Following illumination, cells were shifted to a fresh medium and incubated for an additional 48 h at 37°C. At the end of the incubation period, cells were fixed with methanol, stained with Crystal Violet, to improve visibility, and analyzed using a Leica binocular under a ×6.4 magnification (a,c,e and g). Bar denotes 100 µm. Enlarged images of the areas indicated by a bold white square, on the left column, were photographed at a ×25.6 magnification and depicted on the right (b,d,f and h). Bar denotes 25 µm. Arrowheads point at the EVs surrounded by cell debris. Confluent MCF-7 (B) and MCF-7/MR (C) monolayers were exposed to the indicated IAs (3 µM) for 72 h, following which cells were exposed to 10 min illumination, whereas control cells were not. Then, growth inhibition was determined as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035487#s4" target="_blank">Materials & Methods</a>.</p