Polymeric Mechanical Amplifiers of Tumor Cell Mechanotransduction and Cell Death
Michael J. Mitchell1, Robert Langer1
1Chemical Engineering and David H. Koch Institute for Integrative Cancer Research, MIT, Cambridge MA 02139
Introduction: It has become evident that tumor cells are responsive to mechanical forces in vivo, and prove critical to tumor cell proliferation and death. Recent work has shown that tumor cells exposed to fluid shear forces increase receptor-mediated signaling. We hypothesized that biocompatible, polymeric micro- and nanoparticles conjugated to the tumor cell surface via free amine coupling act as mechanical amplifiers in presence of fluid shear forces to increase mechanotransduction, and can be exploited to increase the efficacy of therapeutic ligands.
Materials and Methods: Polymeric particles ranging from 100 nm–6 μm in size were conjugated to free amines on tumor cells via NHS crosslinker chemistry (Fig. 1A). Nondegradable polystyrene and degradable (PLGA, PCL) particles bound to tumor cells were assessed using flow cytometry, brightfield, and confocal fluorescence microscopy. A cone-and-plate viscometer was used to apply a fluid shear force (2.0 dyn/cm2) to tumor cell suspensions and to amplify the force exerted by polymeric particles on tumor cells (Fig.1B). Tumor cells (COLO 205, PC-3) were treated with 1 μg/mL of a TNF-related apoptosis-inducing ligand (TRAIL) to assess amplified mechanotransduction and receptor-mediated apoptosis in the presence of polymeric particles. An annexin-V apoptosis assay was used to characterize the mode of cell death. Caspase colorimetric assays and inhibitors (Z-VAD-FMK) were utilized to assess caspase-dependence in the mechanotransduction response.
Figure 1: (A) Conjugation of polymer amplifiers to tumor cell surface via NHS crosslinker chemistry. (B) Polymeric particles amplify force exerted on the tumor cell surface under fluid forces to increase mechanotransduction and therapeutic efficacy. (C) Confocal and brightfield images of 1 μm polymeric particles conjugated to the surface of tumor cells. Scale bars: 10 μm. (D) Particle-functionalized tumor cell viability after TRAIL treatment in the presence of shear force.*P<0.05**P< 0.01. NS: not significant. (E,F) Annexin-V apoptosis assays of non-functionalized and functionalized tumor cells treated with TRAIL in the presence of shear forces. Lower left/right and upper left/right quadrants denote viable, early apoptotic, late apoptotic, and necrotic cells, respectively.
Results and Discussion: NHS crosslinker chemistry was successfully used to conjugate polymeric particles to the tumor cell surface (Fig. 1C). In the presence of fluid shear forces, it was found that polymeric particles act to mechanical amplify tumor cell mechanotransduction, as evidenced by increased receptor-mediated apoptosis and decreased tumor cell viability in the presence of the therapeutic ligand TRAIL (Fig. 1D). Additionally, amplification of TRAIL-mediated apoptosis was increased with particles of increasing size (Fig. 1D), demonstrating that increasing the force exerted on the cell surface with larger particles amplified the therapeutic response. Annexin-V apoptosis assays showed the addition of conjugated polymeric particles to the cell surfaces nearly doubled tumor cell apoptosis in the presence of TRAIL under shear forces (Fig.1E,F), and inhibition assays revealed the response to be caspase-dependent apoptosis.
Conclusions: These data demonstrate that polymeric particles, both degradable and non-degradable, act as mechanical amplifiers of tumor mechanotransduction in the presence of shear forces, and are exploited to increase therapeutic efficacy. Clinically, this approach shows that increased mechanical force applied to target tumor cells can increase sensitivity to therapeutic ligands.