Fluid shear increases immune cytokine-mediated apoptosis

To examine how physical forces affect TRAIL-mediated tumour cell apoptosis, tumour cells in suspension were treated with TRAIL in vitro and exposed to physiologically relevant fluid shear stress (Supplementary Fig. 1a). In the presence of fluid shear stress, significant increases in tumour cell killing were observed in TRAIL-treated human colon, prostate and breast tumour cells as compared with those treated under static conditions (Supplementary Fig. 1b–d). Increased tumour cell killing in the presence of fluid forces was observed in both TRAIL-sensitive (COLO 205) and TRAIL-resistant (MCF7) tumour cells (Supplementary Fig. 1b–d). Normal cells with negligible TRAIL sensitivity, including human peripheral blood mononuclear leukocytes and human endothelial cell monolayers, were not sensitized to TRAIL-mediated killing upon shear stress exposure (Supplementary Fig. 1h,i). Across a range of fluid shear forces characteristic of those in soft tissues and in the vascular microenvironment, it was evident that increased shear force enhanced TRAIL-mediated tumour cell killing (Supplementary Fig. 1e–g). We then assessed whether shear force exposure increased TRAIL-mediated apoptosis via caspase-mediated signalling, which is triggered upon TRAIL binding to death receptors DR4 and DR5 (ref. 24). Indeed, treatment with the general caspase inhibitor Z-VAD-FMK abolished TRAIL-mediated tumour cell killing in the presence of fluid shear stress (Supplementary Fig. 1j). These data suggest that physiological forces exerted on tumour cells enhance the therapeutic effect of TRAIL. Building upon previous work, which suggested that shear forces increase the killing of TRAIL-sensitive tumour cells in vitro25, this work indicates that mechanical forces increased both TRAIL-sensitive and TRAIL-resistant tumour cell killing, with negligible toxic effects on normal cells.

Polymeric mechanical amplifiers

Although our data suggest that mechanical forces sensitize tumour cells to receptor-mediated apoptosis, fluid shear forces are highly variable in vivo. Thus, we investigated whether biocompatible polymeric particles conjugated to the tumour cell surface act to locally amplify the forces exerted on the tumour cell membrane. By locally increasing the physical force exerted on the cell surface, it is possible to amplify the apoptotic effect of TRAIL on target tumour cells without altering the fluid forces of the surrounding area. To accomplish this, we stably conjugated polymeric particles across a range of sizes (diameter: 100–1,000 nm) to the tumour cell surface via free amine coupling using N-hydroxysuccinimide (NHS)–PEG 12 –biotin heterobifunctional linkers (Fig. 1a,b). For this study, we chose nondegradable polystyrene (PS) and degradable poly(lactic-co-glycolic acid) (PLGA) particles given their biocompatibility, biodegradability and current use in in vivo clinical applications26,27,28. Particles were stably bound to the surface of colon and prostate tumour cells (Fig. 1c), with minimal internalization observed in the overall cell population after treatment and 4 h post treatment (Fig. 1d). Although some polymeric particles adsorbed to the tumour cell surface without PEG linkers in a nonspecific manner, these particles were easily removed from ∼95% of the overall cell population during mild cell washing steps (Fig. 1e,g). However, polymeric particles conjugated to the cell surface using PEG linkers remained bound to >99% of the overall cell population after exposure to identical washing steps (Fig. 1f,g). Hundreds of polymeric particles were stably conjugated to the tumour cell surface using this technique, with negligible effects on cell viability (Supplementary Fig. 3). Fluorescence readings indicated that a negligible amount of fluorescent particles remained in suspension after functionalization as compared with controls (Supplementary Fig. 4a). In addition, flow cytometry results showed a normal Gaussian distribution of fluorescent cells post functionalization, indicating that the majority of the tumour cell population was uniformly functionalized with particles (Supplementary Fig. 4b). Furthermore, conjugation of particles to the tumour cell surface did not significantly interfere with the ability of TRAIL to interact with death receptors DR4 and DR5, as no significant differences in cell viability were observed post treatment (Supplementary Fig. 5). This finding suggests that PEG linkers enable stable conjugation of polymeric particles to the tumour cell surface with minimal internalization, and has negligible effects on both cell viability and TRAIL-mediated signalling under static conditions.

Figure 1: Functionalization of the tumour cell surface with polymeric particles. (a,b) NHS–PEG–biotin linkers (a) were used to conjugate a range of streptavidin-functionalized polymeric particles to the tumour cell surface (b). (c) Brightfield (top) and confocal (bottom) micrographs of polymeric polystyrene (PS) particles conjugated to the surface of colon (COLO 205; left) and prostate (PC-3; right) tumour cell lines. The 500 nm diameter PS particles bound to tumour cells in brightfield micrographs. Brightfield micrographs show 500 nm diameter PS particles bound to tumour cells. The 200 nm diameter PS particles bound to tumour cells in confocal micrographs. Confocal micrographs shown 200 nm diameter PS particles bound to tumour cells. Green indicates polymeric PS particles and blue indicates nucleus. Scale bar, 10 μm. (d) Percentage of tumour cells with internalized fluorescent PS particles, immediately after and 4 h post functionalization. Internalized fluorescent PS particles quantified using a Trypan blue fluorescence quenching assay and confocal microscopy. N=5 per treatment. (e,f) Flow cytometry plots of epithelial cell adhesion molecule (EpCAM)+ tumour cells functionalized with fluorescent PS particles in the absence (e) and presence (f) of a PEG linker. N=5 per treatment. (g) Percentage of fluorescent PS particle+ tumour cells after functionalization with a PEG linker. Data are reported as the mean±s.e. Different treatment groups were compared for statistical significance using Student’s two-tailed t-test. N=5 per treatment. NHS, N-hydroxysuccinimide; NS, not significant; SA, streptavidin. ***P<0.001. Full size image

Mechanical amplification of tumour cell apoptosis in vitro

Given that fluid shear stress enhanced TRAIL-mediated tumour cell killing (Supplementary Fig. 1), we then investigated whether polymeric particles conjugated to the tumour cell surface amplify the apoptotic effect of TRAIL in the presence of shear forces (Fig. 2a). Under both shear and static conditions, particle conjugation to the tumour cell surface had no effect on viability in the absence of TRAIL (Supplementary Fig. 6). Furthermore, conjugation of particles across a range of sizes (100–1,000 nm) to the cell surface had no effect on TRAIL-mediated killing under static conditions (Supplementary Fig. 7). However, particles tethered to the cell surface amplified TRAIL-mediated colon and prostate cancer cell killing in the presence of fluid shear stress (Fig. 2b–d). Specifically, conjugation of particles of increased size had a pronounced effect on TRAIL-mediated tumour cell killing in the presence of fluid shear stress as compared with treatment in the absence of particles. Increased tumour cell killing in the presence of larger particles could be due to greater compressive forces exerted on the tumour cell membrane in the presence of fluid shear stress. Two spherical particles will experience a compressive force (F c ) between them when colliding in a linear shear flow that scales as F c ∼μGab, where μ is the fluid viscosity, G is the shear rate, and a and b are the radii of the smaller and larger sphere, respectively29. Therefore, a 10 μm diameter tumour cell colliding with a 100 nm particle will experience 10 times the compressive force of a 10 nm particle colliding with a tumour cell. Similar effects were not observed in normal cells, as particle conjugation to both human peripheral blood mononuclear cells and human endothelial cell monolayers had no significant effect on TRAIL-mediated killing (Supplementary Fig. 8). This finding suggests that polymeric particles can be utilized to amplify TRAIL-mediated tumour cell killing in the presence of fluid shear stress while sparing normal cells.

Figure 2: Polymeric particles conjugated to tumour cell surface amplify TRAIL-mediated apoptosis in the presence of fluid shear stress. (a) Schematic of polymeric particles acting as mechanical amplifiers by increasing TRAIL-mediated tumour cell apoptosis in presence of a fluid shear force. (b) Brightfield micrographs of particle-functionalized COLO 205 tumour cells after 30 min of exposure to various treatment conditions. TRAIL-treated samples incubated with 0.1 μg ml−1 TRAIL for 30 min. Sheared samples were exposed to a fluid shear stress of 4.0 dyn cm−2. Tumour cells were treated with 240 PS (500 nm diameter) particles per tumour cell before exposure to TRAIL and fluid shear stress. Insets denote viable and apoptotic tumour cells after treatment conditions. Scale bar, 30 μm. (c,d) Viability of particle-functionalized COLO 205 (c) and PC-3 (d) tumour cells after treatment with TRAIL (0.1 μg ml−1) in the presence of fluid shear stress. N=4 per treatment. (e) Viability of particle-functionalized, TRAIL-resistant HT29 tumour cells after treatment with TRAIL (0.1 μg ml−1) in the presence of fluid shear stress for 6 h. For combination therapies, cells were treated with 15 μM piperlongumine (PL) in addition to TRAIL (0.1 μg ml−1). N=4 per treatment. (f) Viability of particle-functionalized COLO 205 tumour cells after treatment with doxorubicin (DOX; concentration: 20 μM) in the presence of fluid shear stress. N=4 per treatment. Particle diameter: 500 nm. (g,h) Annexin-V/propidium iodide (PI) flow cytometry plots of nonfunctionalized (g) and particle (500 nm)-functionalized (h) PC-3 tumour cells after treatment with TRAIL (0.1 μg ml−1) in the presence of static conditions and fluid shear stress, respectively. Particle diameter: 500 nm. N=5 per treatment. (i,j) Percentage of annexin-V+, particle-functionalized COLO 205 (i) and PC-3 (j) tumour cells after treatment with TRAIL (0.1 μg ml−1) in the presence of fluid shear stress. N=5 per treatment. (k) Viability of biodegradable PLGA particle-functionalized COLO 205 tumour cells after treatment with TRAIL (0.1 μg ml−1) in the presence of fluid shear stress. Data are reported as the mean±s.e. Different treatment groups were compared for statistical significance using Student’s two-tailed t-test. Particle diameter: 500 nm. N=5 per treatment. *P<0.05, **P<0.01 and ***P<0.001. NS, not significant. Full size image

TRAIL resistance in tumour cell types is a major challenge to its broad use in cancer therapy22,30. To examine the potential of our approach to overcome resistance, we conducted experiments using HT29 colon cancer cells that have been shown to be TRAIL resistant31,32,33,34. Our results confirm that TRAIL treatment under static conditions has minimal effects on HT29 cell viability (Fig. 2e). However, a significant decrease in HT29 cell viability was measured when cells were functionalized with particles and then treated with TRAIL in the presence of fluid shear stress (Fig. 2e). We then assessed whether our particle-based approach in combination with piperlongumine, a natural alkaloid shown to sensitize tumour cells to TRAIL via upregulation of DR5 expression, can further increase tumour cell killing35. Indeed, combining piperlongumine with our approach induced a fivefold greater tumour cell death than TRAIL treatment alone. These results suggest that our particle-based approach can act in combination with chemical sensitizers to increase TRAIL-resistant tumour cell killing in the presence of physical forces. Given that TRAIL combination therapies with various classes of sensitizers are being explored in preclinical and clinical trials22,30,36, we envision that our nontoxic, particle-based approach can act in combination with sensitizers to exert synergistic apoptotic effects on TRAIL-resistant tumour cells.

We then compared mechanical amplification of tumour cell killing with treatment with doxorubicin. Doxorubicin also triggers tumour cell apoptosis, but through intracellular mechanisms including inhibition of topo-isomerase II and DNA intercalation37. Although polymeric particles amplified the effect of TRAIL in the presence of fluid shear stress (Fig. 2c–e), they did not enhance the therapeutic effect of doxorubicin (Fig. 2f), demonstrating that such an approach is specific to receptor-mediated therapeutics. Upon analysing the mode of tumour cell death using an annexin-V/propidium iodide (PI) assay, increased tumour cell killing using our approach occurred almost entirely via increased apoptosis (Fig. 2g–j). Conjugation of particles to the cell surface nearly doubled the number of apoptotic prostate and colon cancer cells in the presence of fluid shear stress as compared with treatment under static conditions (Fig. 2i,j). Furthermore, no significant differences in tumour cell necrosis were observed (Supplementary Fig. 9). Given that TRAIL primarily triggers cell death via apoptosis rather than necrosis, the annexin-V/PI assay further suggests that mechanical amplification of apoptosis using polymeric particles is TRAIL specific and does not exert separate cytotoxic effects.

In addition to nondegradable particles, we sought to utilize biodegradable polymeric PLGA particles to mechanically amplify TRAIL-mediated apoptosis. Biodegradable particles such as PLGA are particularly advantageous for in vivo administration, where the material can degrade in a safe manner38,39. PLGA particles functionalized with an epithelial cell adhesion molecule (EpCAM)-targeting antibody, which binds to EpCAM expressed on tumour cells of epithelial origin40, were bound to the tumour cell surface with minimal effects on cell viability under shear and static conditions (Supplementary Fig. 10a). Furthermore, PLGA particles did not hinder TRAIL-mediated killing under static conditions (Supplementary Fig. 10b). Similar to nondegradable particles (Fig. 2b–e), biodegradable particles amplified the apoptotic effect of TRAIL in the presence of fluid shear stress as compared with samples in the absence of particles (Fig. 2k). This finding suggests that biodegradable polymeric particles can also be utilized to amplify TRAIL-mediated apoptosis in the presence of fluid shear stress.

Our results with larger polymeric particles conjugated to the tumour cell surface showed increased TRAIL-mediated tumour cell killing under shear, suggesting that greater force exerted by larger particles increased the effect of TRAIL (Fig. 2). To assess the effects of both shear stress exposure and particle size, tumour cells were conjugated with polymeric particles across a range of sizes (200–1,000 nm) before TRAIL treatment under varied fluid shear stress (1.0–12.0 dyn cm−2; Fig. 3a). Higher shear stress values were not examined in vitro because of cell delamination and reduced cell recovery from the viscometer device (Supplementary Fig. 11). Particles across the range of sizes increased TRAIL-mediated tumour cell killing with increasing fluid shear stress exposure (Fig. 3a). In addition, increased TRAIL-mediated tumour cell killing was observed when cells were bound to larger particles at a given fluid shear stress as compared with smaller particles (Fig. 3a). For example, although a 200 nm particle had minimal effect on TRAIL-mediated tumour cell killing at a low fluid shear stress (1.0 dyn cm−2), functionalization with larger particles (1,000 nm) significantly increased tumour cell killing (Fig. 3a). These results suggest that particle size and shear stress exposure both act to modulate TRAIL-mediated tumour cell killing. In addition, we treated tumour cells with varying numbers of particles (concentration: 0–480 particles per cell) before TRAIL treatment under shear exposure (Fig. 3b). In the absence of TRAIL, increasing numbers of particles had no significant effect on cell viability under shear or static conditions (Supplementary Fig. 12). In the presence of TRAIL, increased particle conjugation increased tumour cell killing under shear conditions (Fig. 3c,d). Increased particle conjugation did not affect tumour cell killing under static conditions (Fig. 3c,d), providing further evidence that the apoptotic effect of TRAIL is amplified by particles specifically in the presence of fluid shear stress. Furthermore, the apoptotic effect of TRAIL increased with greater numbers of particles conjugated to the cell surface (Fig. 3e,f), whereas no significant differences in cellular necrosis were measured (Fig. 3g,h). In addition, particle functionalization had no effect on tumour cell apoptosis and necrosis in the absence of TRAIL, regardless of the number of particles (Supplementary Fig. 13). These data provide further evidence that mechanical amplification of TRAIL-mediated tumour cell killing occurs via apoptosis, and can be modulated by (1) altering particle size, (2) increasing shear force exposure and (3) increasing the number of particles tethered to the tumour cell surface.

Figure 3: Increased shear stress, particle diameter and number of particles conjugated to tumour cell surface enhance TRAIL-mediated apoptosis. (a) Viability of particle-functionalized PC-3 tumour cells treated with TRAIL in the presence of a range of shear forces (1.0–12.0 dyn cm−2) across a range of particle sizes (diameter: 200–1,000 nm). (b) COLO 205 tumour cells treated with 0–120 PS particles per cell in suspension. Scale bar, 10 μm. (c,d) Viability of particle-functionalized COLO 205 (c) and PC-3 tumour cells (d) treated with TRAIL in the presence of fluid shear stress. N=5 per treatment. (e,f) Percentage of annexin-V+ particle-functionalized COLO 205 (e) and PC-3 cells (f) treated with TRAIL in the presence of fluid shear stress. N=5 per treatment. (g,h) Percentage of annexin-V-/PI+ particle-functionalized COLO 205 (g) and PC-3 cells (h) treated with TRAIL in the presence of fluid shear stress (shear stress: 4.0 dyn cm−2). All tumour cells were incubated with 0–480 PS particles (500 nm diameter) per cell before all treatments. N=5 per treatment. TRAIL concentration: 0.1 μg ml−1 for all TRAIL-treated samples. Shear stress: 4.0 dyn cm−2 for all samples exposed to shear. Data are reported as mean±s.e. Different treatment groups were compared for statistical significance using Student’s two-tailed t-test. NS, not significant. *P<0.05 and **P<0.01. Full size image

Mechanical amplification of caspase signalling

To assess whether amplification of receptor-mediated apoptosis in the presence of fluid shear stress is dependent on caspase signalling, we treated particle-functionalized tumour cells with the general caspase inhibitor Z-VAD-FMK. Minimal membrane blebbing, indicative of reduced TRAIL-mediated apoptosis, was observed in samples treated with Z-VAD-FMK (Fig. 4a). No significant decreases in viability were measured in Z-VAD-FMK-treated samples as compared with viable controls (Fig. 4b). Given that TRAIL primarily induces extrinsic apoptosis via caspase-8, cells were also treated with the caspase-8 inhibitor Z-IETD-FMK, and similar results were observed compared with general caspase inhibition (Fig. 4b). Furthermore, Annexin-V/PI assays showed reduced apoptosis when treated with Z-VAD-FMK or Z-IETD-FMK (Fig. 4c–e). We then labelled for biomarkers of caspase-8 activation using a caspase-8 activity assay. TRAIL treatment alone induced a 3-fold increase in caspase-8 activity, while TRAIL treatment of particle-functionalized cells induced over a 4.5-fold increase in caspase-8 activity in the presence of fluid shear stress as compared with controls (Fig. 4f). These results suggest that polymeric particles amplify TRAIL-mediated apoptosis in the presence of shear forces via increased caspase-8 activity.

Figure 4: Amplification of TRAIL apoptotic effect via polymeric particles conjugation is caspase dependent and increases death receptor expression. (a) Brightfield micrographs of particle (diameter: 500 nm)-functionalized COLO 205 tumour cells treated with 0.1 μg ml−1 TRAIL under fluid shear stress exposure (shear stress: 4.0 dyn cm−2) for 1 h in the absence and presence of 50 μM pan caspase inhibitor Z-VAD-FMK. (b) Viability of particle-functionalized COLO 205 tumour cells treated with 0.1 μg ml−1 TRAIL under fluid shear stress exposure for 1 h in the presence of 50 μM pan caspase inhibitor Z-VAD-FMK, 50 μM caspase-8 inhibitor Z-IETD-FMK or 50 μM caspase negative control inhibitor Z-FA-FMK. N=4 for all treatments. (c,d) Annexin-V/propidium iodide (PI) flow cytometry plots of particle-functionalized COLO 205 tumour cells treated with 0.1 μg ml−1 TRAIL in the presence of a fluid shear stress for 1 h without and with Z-VAD-FMK treatment. N=4 for all treatments. (e) Annexin-V quantification of particle-functionalized COLO 205 tumour cells treated with 0.1 μg ml−1 TRAIL under fluid shear stress exposure for 1 h in the presence of 50 μM pan caspase inhibitor Z-VAD-FMK, 50 μM caspase-8 inhibitor Z-IETD-FMK or 50 μM caspase negative control inhibitor Z-FA-FMK. N=4 for all treatments. (f) Caspase-8 activity of particle-functionalized COLO 205 tumour cells after treatment with TRAIL (0.1 μg ml−1) in the presence and absence of fluid shear stress for 1 h. N=4 for all treatments. (g) TRAIL death receptor (DR) 4 and 5 expression after treatment of particle-functionalized COLO 205 tumour cells with TRAIL (0.1 μg ml−1) in the presence and absence of fluid shear stress for 1 h. N=5 for all treatments. Data are reported as mean±s.e. Different treatment groups were compared for statistical significance using Student’s two-tailed t-test for two conditions and one-way analysis of variance (ANOVA) for multiple comparisons. Percent of max represents the number of events normalized according to FlowJo algorithms. *P<0.05 and **P<0.01. NS, not significant. Full size image

To assess whether mechanical amplification of TRAIL-mediated apoptosis alters death receptor (DR4, DR5) expression on the cell surface, we measured receptor expression of particle-functionalized tumour cells after exposure to TRAIL and fluid shear stress via flow cytometry. TRAIL binding to DR4 and DR5 on the tumour cell surface engages the caspase signalling cascade and triggers apoptosis41. In addition, TRAIL treatment alone increases death receptor expression in malignant epithelial cells in a process mediated by nuclear factor-κB activation42. Therefore, increased DR4 and DR5 expression on particle-functionalized tumour cells in the presence of shear could explain increased TRAIL-mediated apoptosis due of increased receptor–ligand interactions and subsequent apoptotic signalling. Indeed, flow cytometry analysis showed increased death receptor expression on TRAIL-treated, particle-functionalized tumour cells in the presence of fluid shear stress (Fig. 4g). Shear stress exposure in the absence of TRAIL did not have an effect on death receptor expression, consistent with previous work25. Thus, our data suggest that mechanical amplification of TRAIL-mediated apoptosis using polymeric particles is dependent on caspase signalling and could be due to increased death receptor expression. The enhanced apoptotic effect of TRAIL could be because of increased compressive forces exerted by particles conjugated to the tumour cell surface in the presence of fluid shear stress. Such forces act to flatten the biologically inert glycocalyx expressed on the surface of cells, and the physics of force-induced flattening and penetration of cell glycocalyx have been shown to facilitate receptor–ligand interactions43,44. Given that the glycocalyx is overexpressed on many tumour cell types, increased compressive forces and glycocalyx flattening can potentially increase TRAIL–death receptor interactions, thus increasing both death receptor expression and apoptosis.

Amplification of tumour cell death in vivo

We then assessed whether a targeted particle formulation can amplify TRAIL-mediated apoptosis of tumour cells in the bloodstream in vivo, where physical force exposure is highly variable. GFP- and luciferase-expressing COLO 205 tumour cells were injected systemically into the tail vein of nu/nu mice, followed 15 min later by an injection of EpCAM-targeted, PEG-functionalized PLGA particles (Fig. 5a). Given that EpCAM is expressed on tumour cells of epithelial origin with negligible expression on blood cells in the vascular microenvironment, this particle platform enables targeting of EpCAM+ tumour cells in the vasculature. At 30 min post particle treatment, mice were treated with soluble TRAIL via systemic administration (Fig. 5a), enabling the therapeutic to interact with particle-functionalized tumour cells under in vivo fluid shear stress exposure. Viable tumour cells in the bloodstream were measured by collecting 200 μl blood samples from mice 90 min post injection via submandibular bleeding (Fig. 5a). Flow cytometry analysis showed a significant decrease in the number of targeted particle-bound tumour cells subsequently treated with soluble TRAIL in the circulation as compared with control mice (Fig. 5b). Using flow cytometry, we measured ∼40,000 tumour cells per ml of blood for mice injected with tumour cells alone, and mice injected with tumour cells followed by targeted or non-targeted particle administration (Fig. 5c). We measured ∼14,000 tumour cells per ml of blood for mice injected with soluble TRAIL as compared with <3,000 tumour cells per ml for mice injected with EpCAM-targeted particles followed by treatment with soluble TRAIL (Fig. 5c). Upon injection with nontargeted particles, we measured similar levels of tumour cells in blood compared with tumour cells treated with soluble TRAIL alone, indicating that EpCAM targeting enables particle binding to the tumour cell surface and is necessary for amplifying the apoptotic effect of TRAIL (Fig. 5c). In addition to reduced tumour cells in the circulation, annexin-V analysis showed that ∼65% of the particle-bound tumour cell population in circulation was apoptotic after TRAIL treatment, whereas ∼45% of the tumour cell population treated with TRAIL in the absence of particles were apoptotic (Supplementary Fig. 14). Given that tumour cells administered via tail vein injection can lodge within the vasculature >2 h post injection45, we utilized whole-body bioluminescence imaging (BLI) to track the remaining tumour cell burden 7 and 14 days post injection (Fig. 5a). Tumour cell burden was readily apparent within control nu/nu mice treated with tumour cells 7 days post injection, along with tumour cells treated with targeted or nontargeted particles in the absence of TRAIL treatment (Fig. 5d). Administration of soluble TRAIL alone reduced the number of tumour cells in vivo, as measured via BLI imaging (Fig. 5d). The apoptotic effect of soluble TRAIL on tumour cells was significantly increased in vivo after pretreatment with EpCAM-targeted particles, as BLI signals were significantly reduced 7 days post injection. Quantitative analysis showed a >90% reduction in BLI signal for tumour cells treated with EpCAM-targeted particles followed by soluble TRAIL administration in vivo as compared with controls (Fig. 5e).

Figure 5: Polymeric particles targeted to tumor cell surface amplify immune cytokinemediated apoptosis in vivo. (a) Schematic of epithelial cell adhesion molecule (EpCAM)-targeted particle delivery to COLO 205 tumour cells in nude (nu/nu) mice in vivo, followed by treatment with TRAIL. Mice were inoculated with COLO 205 tumour cells via tail vein injection (2 × 106 cells), followed by injection of nontargeted and EpCAM-targeted PLGA particles (500 nm diameter; ∼500 particles per tumour cell) 15 min post tumour cell injection. At 30 min post particle injection, mice were treated with TRAIL (0.1 μg ml−1 plasma concentration). Tumour cells in blood were collected via submandibular bleed 90 min post TRAIL injection. Tumour cells were detected in vivo via whole-body bioluminescent imaging (BLI) at 7 and 14 days post injection. (b) Representative flow cytometry plots of GFP+ COLO 205 tumour cells removed after delivery of nontargeted particles (Particles) and EpCAM-targeted particles (t-Particles) followed by TRAIL. FSC, forward scatter; SSC, side scatter. (c) Number of viable GFP+ COLO 205 tumour cells per ml mouse blood 90 min post TRAIL treatment of tumour cells in vivo under various conditions. Cells only denotes mice treated with tumour cells followed by PBS via tail vein injection. N=5 mice for all treatments. (d) Representative whole-body BLI images of COLO 205 tumour cells in mice 7 days post injection of particles and targeted particles followed by TRAIL. (e) COLO 205 BLI signals in mice 7 and 14 days post injection of COLO 205 tumour cells under various conditions. N=5 mice for all treatments. (f) PC-3 tumour growth curves after intravenous injections of targeted particles (40 mg kg−1) followed by TRAIL (15 mg kg−1) 3 h post particle injection. For combination therapies, tumour-bearing nu/nu mice were also treated with the TRAIL-sensitizer resveratrol (30 mg kg−1). After tumour formation (100 mm3), mice began treatment regimen and tumour volume was measured every 3 days. Blue arrows indicate days where mice were treated with targeted particles, followed 3 h later by TRAIL treatment. Green arrows indicate days where mice were treated with resveratrol via oral gavage. N=5 mice for all treatments. Data are reported as the mean±s.e. Different treatment groups were compared for statistical significance using Student’s two-tailed t-test for two conditions and one-way analysis of variance (ANOVA) for multiple comparisons. *P<0.05, **P<0.01 and ***P<0.001. NS, not significant. Full size image

In addition to tumour cells in the circulation, we assessed whether our approach could amplify TRAIL-mediated apoptosis in solid tumour models, where tumour cells are exposed to a variety of physical forces including blood flow from leaky tumour vasculature, interstitial flows between blood and lymphatic circulation as well as interstitial fluid pressures generated within solid tumours10,46,47,48. Using a prostate cancer, EpCAM+ PC-3 xenograft model, we first confirmed that EpCAM-targeted PLGA particles localized within the tumour 3 h post injection using in vivo fluorescence imaging (Supplementary Fig. 15a). Harvesting of mouse organs showed that particles mainly accumulated within the tumour and liver, with some particle clearance to the kidneys (Supplementary Fig. 15b). Over a 21-day treatment period, we measured a significant decrease in PC-3 tumour growth in mice treated with targeted particles followed by TRAIL administration (3 h post particle injection), compared with TRAIL treatment alone (Fig. 5f). Given the benefits of combining TRAIL with natural products to enhance tumour cell apoptosis in vitro in our current study (Fig. 2e), along with results by others22, we assessed whether administration of the natural product resveratrol could combine with our approach to further increase TRAIL-mediated tumour apoptosis in vivo. Resveratrol is a naturally occurring polyphenol that exhibits numerous health benefits including anti-inflammatory, antioxidant and antitumour activities, and has previously been shown to increase TRAIL-mediated apoptosis in solid tumours49. In combination with resveratrol, amplification of TRAIL apoptosis using our particle-based approach reduced tumour growth by over 80% compared with control mice (Fig. 5f). These combined results suggest that the targeted polymeric particles can be utilized to mechanically amplify TRAIL-mediated apoptosis in solid tumour models, and can be combined with natural products to further reduce tumour growth.

Toxicity results showed that treated mice exhibited no evidence of elevated liver enzymes in serum (Fig. 6a,b), no significant differences in haematocrit relative to untreated mice (Fig. 6c), no loss of appetite or body weight or behavioural distress compared with untreated mice (Fig. 6d) and no enlarged kidney mass (Fig. 6e). It is important to note that the TRAIL dosage used to target tumour cells in the bloodstream is approximately two orders of magnitude less than the concentrations shown to be well tolerated in previous animal and human trials with soluble TRAIL22. Collectively, these results suggest that targeted polymeric particles delivered and bound to tumour cells in vivo can leverage physical forces to increase the apoptotic effect of TRAIL, with negligible off-target toxicity.