Integrin Subtypes and Nanoscale Ligand Presentation Influence Drug Sensitivity in Cancer Cells

Cancer cell–matrix interactions have been shown to enhance cancer cell survival via the activation of pro-survival signaling pathways. These pathways are initiated at the site of interaction, i.e., integrins, and thus, their inhibition has been the target of therapeutic strategies. Individual roles for fibronectin-binding integrin subtypes αvβ3 and α5β1 have been shown for various cellular processes; however, a systematic comparison of their function in adhesion-dependent chemoresistance is lacking. Here, we utilize integrin subtype-specific peptidomimetics for αvβ3 and α5β1, both as blocking agents on fibronectin-coated surfaces and as surface-immobilized adhesion sites, in order to parse out their role in breast cancer cell survival. Block copolymer micelle nanolithography is utilized to immobilize peptidomimetics onto highly ordered gold nanoparticle arrays with biologically relevant interparticle spacings (35, 50, or 70 nm), thereby providing a platform for ascertaining the dependence of ligand spacing in chemoprotection. We show that several cellular properties—morphology, focal adhesion formation, and migration—are intricately linked to both the integrin subtype and their nanospacing. Importantly, we show that chemotherapeutic drug sensitivity is highly dependent on both parameters, with smaller ligand spacing generally hindering survival. Furthermore, we identify ligand type-specific patterns of drug sensitivity, with enhanced chemosurvival when cells engage αvβ3 vs α5β1 on fibronectin; however, this is heavily reliant on nanoscale spacing, as the opposite is observed when ligands are spaced at 70 nm. These data imply that even nanoscale alterations in extracellular matrix properties have profound effects on cancer cell survival and can thus inform future therapies and drug testing platforms.


Materials and Methods
Protein functionalized surfaces. White-walled tissue culture treated plastic 96-well plates (Greiner Bio-One) were coated with 10 µg/mL human plasma fibronectin (Roche) or human plasma vitronectin (Promega) in sterile 1X PBS overnight at 4°C with gentle shaking to ensure a uniform layer was deposited. After incubation, plates were rinsed two times with sterile 1X PBS. 1% BSA (Serva) dissolved in sterile 1X PBS was added for 15 min at 37°C in order to prevent non-specific cell binding to any potentially remaining exposed surface. Plates were rinsed again three times with sterile 1X PBS then sterilized under UV for 30 min before cell plating.
Peptide functionalization of AuNP array-coated substrates. In order to avoid potential protein adsorption and cell attachment onto the glass surface between the AuNPs, surfaces were activated with O2 plasma for 10 min at 150 W and 0.4 mbar and directly passivated by adding 0.25 mg/mL PLL(20)-g[3.5]-PEG(2) (SuSoS Surface Technology, Switzerland) dissolved in HEPES buffer (10 mM, pH 7.4) for 45 min, then rinsed in MiliQ H2O and dried under N2 flow. Under sterile conditions, 16-well ProPlate Ò molds (Grace Bio-labs, USA) were mounted onto the AuNP glass slides, while coverslips were placed in a petri dish, then all samples were rinsed 3X with sterile MiliQ H2O. 50µL of 25 µM peptidomimetics dissolved in sterile MiliQ H2O were added to each well/coverslip for 2 hr at RT. The wells were then rinsed 2X with water, then 2X with sterile 1X PBS before cell plating. Controls either without AuNPs or bare AuNPs were also included in each experiment in order to confirm successful passivation (in the former case) or peptidomimeticspecific binding of cells (in the latter case).
Characterization of AuNP substrates. After plasma treatment, samples were characterized via scanning electron microscopy (SEM) for spacing vs. spin speed and organization of nanoparticles. SEM samples were sputter-coated with ~ 7 nm of carbon (Low Vacuum Coater EM ACE200, Leica) and imaged in an Ultra 55FE-SEM mounted with a Gemini column (Carl Zeiss) using an in-lens detector at an accelerating voltage of 5 kV and working distance of ~ 6mm. For each sample, at least three images distributed across the surface were taken and were subsequently quantified for both interparticle spacing and hexagonal order (k = 6) via calculation of the k-nearest neighbors as described previously. A custom script in Fiji was utilized, with each image containing ~ 600 -2,500 particles, depending on spacing. Samples with AuNP spacing of 35, 50 and 70 nm with uniform, hexagonal arrangement determined by the 6-fold bond orientational order parameter, as previously described 2 , were utilized.
Cell culture. MDA-MB-231s (ATCC) were maintained in DMEM (Gibco) supplemented with 10 % fetal bovine serum (FBS, Sigma), 1 % non-essential amino acids (Gibco) and 1 % Penicillin-Streptomycin (Gibco) in a sterile, humidified incubator at 37°C and 5 % CO2. For initial integrin blocking and cell plating, media with only 1% FBS was used in order to avoid non-specific interactions. After trypsinization (0.05% Tryspin-EDTA, Gibco; 4 min at 37°C), cells were counted using a Z2 Coulter Particle Count and Size Analyzer (Beckman Coulter, USA), and then plated at a density of 150 cells/mm 2 . For blocking experiments, cells were re-suspended in 1/5 of the final plating volume required and peptidomimetics were added at 50 µM for 10 min on ice. Warm media was then added up to the final volume such that the peptidomimetics were at 10 µM and the cells were plated. Cells were allowed to adhere for 3 hr, at which point media was exchanged for 10 % FBS media with or without drugs. 5-Fluorouracil (5-FU; Sigma-Aldrich) was added at 100 µM and Paclitaxel (Acros Organics) was added at 50 nM to elicit ~ 50 % cell death (Fig. S5). Negative controls were prepared according to the solution in which the drugs were dissolved, i.e. PBS for 5-FU and DMSO for Paclitaxel. If peptidomimetics were used, they were also added into the media exchange at a concentration of 10 µM.
Immunofluorescence microscopy. In order to examine cell morphology, focal adhesion (FA) area, and confirm integrin-specific binding, immunofluorescence was performed. Cells were fixed in 3.7 % paraformaldehyde in PBS for 20 min, rinsed 3X in PBS, and permeabilized with 1 % Triton-X 100 in PBS for 10 min. Cells were rinsed in 1 % PBS-T (1X PBS with 1 % Tween) 3X and then stained with the following primary antibodies depending on the experiment at a dilution of 1:250 in 1 % BSA in PBS-T at 4°C overnight: Anti-paxillin [Y113] rabbit monoclonal antibody (Abcam ab32084, UK); Anti-integrin alpha V beta 3 mouse monoclonal antibody [LM609] (Abcam ab190147, UK); Anti-integrin alpha 5 rabbit monoclonal antibody [EPR7854] (Abcam ab150361, UK). Following 3X rinse in PBS-T, samples were incubated with the following secondary antibodies: Chicken anti-rabbit Alexa Fluor Ò 488 (Life Technologies), Goat anti-mouse Alexa Fluor Ò 647 (Life Technologies), and Alexa Fluor Ò 568 phalloidin (for actin, Invitrogen) at 1:1000 at RT for 2 hr. Samples were then rinsed 3X in MiliQ H2O and mounted in Fluoromount-G Ò w/DAPI (Southern Biotech) and sealed with a #1 coverslip. Imaging was performed on a Zeiss Axiovert 200M (Carl Zeiss AG, Germany) using an oil-immersion 63X objective for FA visualization and a 10X objective for cell morphology visualization, captured with AxioVision Rel. 4.8 software.
Live cell microscopy. After plating cells on functionalized substrates, plates were moved to an Axio Observer.Z1 (Carl Zeiss AG, Germany) microscope fitted with a custom-built cell incubation chamber so that imaging could proceed in a humidified, 37°C, and 5 % CO2 environment. Phase contrast images were taken at 3 -4 points per sample every 10 minutes for 72 hr using AxioVision Rel. 4.8 software. Movies of cells migrating were analyzed in Imaris (Bitplane) software.
Survival and proliferation assay. Cells treated with chemotherapeutic drugs were examined for survival using the CellTiter-Glo ® Luminescent Cell Viability Assay (Promega) in which the measured luminescent signal is proportional to the amount of ATP present, which is directly proportional to the number of cells in culture. After drug treatment, cell culture plates were stabilized to room temperature for 30 min, the CellTiter-Glo ® Reagent was added at a 1:1 ratio to media (i.e. 100 µL media + 100 µL reagent), the plate was mixed to induce cell lysis for 2 min, the luminescent signal was stabilized for 10 minutes at room temperature, and luminescence was recorded with an integration time of 1 second per well. Control wells (media alone, with and without drugs, plus reagent) were also prepared to account for any experiment-specific background. The concentration of drugs used, i.e. 5-Fluorouracil at 100 µM and Paclitaxel at 50 nM, was shown to elicit ~ 50 % cell death from a drug dose experiment (Fig. S5). Proliferation based on integrin subtype engagement on fibronectin (via performing blocking experiments) at 3 and 48 hr was also assessed (Fig. S4).
Image and statistical analyses. Immunofluorescent 63X images were analyzed in Fiji 3 for FA morphology and 10X images were analyzed in CellProfiler 4 for cell morphology. Cell numbers analyzed indicated in corresponding figure captions. For live cell experiments, after acquiring time lapse videos, cells were tracked and analyzed using Imaris (Bitplane) software for xy speed (µm/min). Roseplots representing 17 hrs of culture (in order to better visualize tracks) were created using the XTension Pack for Advanced Object Movement Analysis. All statistical analyses were performed using one-way or two-way ANOVA or student's t-test, as indicated, using Prism (GraphPad) Software. Statistical differences among groups were assessed to identify the interaction between spacing, drug treatment, and integrin subtype-specific peptidomimetics when p < 0.05. All data is presented as mean ± 95% confidence interval (CI) or standard error of the mean (sem) as indicated from triplicate biological experiments with at least 2 technical replicates per sample, unless otherwise indicated. All significance comparisons from Figures 1 C-F and 2 C-F are displayed in Tables S1 and S2, respectively.

Supporting Experiments
Peptidomimetic specificity. While the integrin-specificity of our synthesized peptidomimetics has been confirmed previously 5 , we ensured functionality in our system by performing immunofluorescence staining of integrins αvβ3 or α5β1 to observe integrin expression patterns, i.e. when engaging α5β1, the presence of αvβ3 was greatly reduced and vice versa (Fig. S2A). As an additional control, cells were plated on vitronectin (Vn), in which αvβ3 is the major binding integrin, and treated with the αvβ3 peptidomimetic. We observed that cell attachment was greatly hindered, with no detectable focal complex formation, thereby demonstrating the potency of the molecule at the concentration utilized (Fig. S1B-E). Proper integrin engagement was further confirmed on our peptidomimetic-functionalized surfaces (Fig. S2B).
Cell proliferation based on integrin subtype. In order to ensure differential cell survival mediated by engagement of specific integrin subtypes was not due to integrin subtype specific-mediated proliferation effects, we monitored cell proliferation over the time course of the experiment and observed no differences between cells on Fn engaging integrin α5β1, integrin αvβ3, or both, compared to initial seeding (Fig. S4). . Cells were stained for actin (red), paxillin (green), and nucleus (blue). Insets show zoomed in focal adhesions Scale bar: 50 µm. Cell morphology in terms of cell area (B) and form factor (C) was quantified for no blocking (i.e. αvβ3 engagement), αvβ3 blocking (i.e. no integrin engagement), and α5β1 blocking (i.e. αvβ3 engagement). Focal adhesion (FA) morphology in terms of area (D) and major axis length (E) was quantified for all conditions as in (B, C). Blocking αvβ3 engagement resulted in very few cells attached to the surface as indicated by the gray bar; FA area could not be calculated. ncells (no blocking) = 246; ncells (αvβ3 blocking) = 20; ncells (α5β1 blocking) = 312. nFAs > 620. Data is mean ± 95% CI. ns = not significant by two-tailed t-test.

Tables of Significance
All significance comparisons by one-way ANOVA are displayed for experiments in Figure 1C-F (Table S1) and Figure 2C-F (Table S2).