Preclinical Efficacy of Cabazitaxel Loaded Poly(2-alkyl cyanoacrylate) Nanoparticle Variants.

Background: Biodegradable poly(alkyl cyanoacrylate) (PACA) nanoparticles (NPs) are receiving increasing attention in anti-cancer nanomedicine development not only for targeted cancer chemotherapy, but also for modulation of the tumor microenvironment. We previously reported promising results with cabazitaxel (CBZ) loaded poly(2-ethylbutyl cyanoacrylate) NPs (PEBCA-CBZ NPs) in a patient derived xenograft (PDX) model of triple-negative breast cancer, and this was associated with a decrease in M2 macrophages. The present study aims at comparing two endotoxin-free PACA NP variants (PEBCA and poly(2-ethylhexyl cyanoacrylate); PEHCA), loaded with CBZ and test whether conjugation with folate would improve their effect. Methods: Cytotoxicity assays and cellular uptake of NPs by flow cytometry were performed in different breast cancer cells. Biodistribution and efficacy studies were performed in PDX models of breast cancer. Tumor associated immune cells were analyzed by multiparametric flow cytometry. Results: In vitro studies showed similar NP-induced cytotoxicity patterns despite difference in early NP internalization. On intravenous injection, the liver cleared the majority of NPs. Efficacy studies in the HBCx39 PDX model demonstrated an enhanced effect of drug-loaded PEBCA variants compared with free drug and PEHCA NPs. Furthermore, the folate conjugated PEBCA variant did not show any enhanced effects compared with the unconjugated counterpart which might be due to unfavorable orientation of folate on the NPs. Finally, analyses of the immune cell populations in tumors revealed that treatment with drug loaded PEBCA variants affected the myeloid cells, especially macrophages, contributing to an inflammatory, immune activated tumor microenvironment. Conclusion: We report for the first time, comparative efficacy of PEBCA and PEHCA NP variants in triple negative breast cancer models and show that CBZ-loaded PEBCA NPs exhibit a combined effect on tumor cells and on the tumor associated myeloid compartment, which may boost the anti-tumor response.

Liver X receptors induce antiproliferative effects in basal-like breast cancer.

Liver X receptors (LXRs) are nuclear transcription factors important in the regulation of cholesterol transport, and glucose and fatty acid metabolism. The antiproliferative role of LXRs has been studied in a variety of malignancies and may represent a therapeutic opportunity in cancers lacking targeted therapies, such as triple-negative breast cancer. In this study, we investigated the impact of LXR agonists alone and in combination with carboplatin in preclinical models of breast cancer. In vitro experiments revealed a dose-dependent decrease in tumor cell proliferation in estrogen receptor-positive breast cancer cells, whereas LXR activation in vivo resulted in an increased growth inhibitory effect in a basal-like breast cancer model (in combination with carboplatin). Functional proteomic analysis identified differences in protein expression between responding and nonresponding models related to Akt activity, cell-cycle progression, and DNA repair. Furthermore, pathway analysis suggested that the LXR agonist in combination with carboplatin inhibits the activity of targets of E2F transcription factors and affects cholesterol homeostasis in basal-like breast cancer.

Biodistribution of Poly(alkyl cyanoacrylate) Nanoparticles in Mice and Effect on Tumor Infiltration of Macrophages into a Patient-Derived Breast Cancer Xenograft.

We have investigated the biodistribution and tumor macrophage infiltration after intravenous injection of the poly(alkyl cyanoacrylate) nanoparticles (NPs): PEBCA (poly(2-ethyl-butyl cyanoacrylate), PBCA (poly(n-butyl cyanoacrylate), and POCA (poly(octyl cyanoacrylate), in mice. These NPs are structurally similar, have similar PEGylation, and have previously been shown to give large variations in cellular responses in vitro. The PEBCA NPs had the highest uptake both in the patient-derived breast cancer xenograft MAS98.12 and in lymph nodes, and therefore, they are the most promising of these NPs for delivery of cancer drugs. High-resolution magic angle spinning magnetic resonance (HR MAS MR) spectroscopy did not reveal any differences in the metabolic profiles of tumors following injection of the NPs, but the PEBCA NPs resulted in higher tumor infiltration of the anti-tumorigenic M1 macrophages than obtained with the two other NPs. The PEBCA NPs also increased the ratio of M1/M2 (anti-tumorigenic/pro-tumorigenic) macrophages in the tumors, suggesting that these NPs might be used both as a vehicle for drug delivery and to modulate the immune response in favor of enhanced therapeutic effects.

Mechanism of cellular uptake and cytotoxicity of paclitaxel loaded lipid nanocapsules in breast cancer cells.

Lipid nanocapsules (LNCs) have proven their efficacy in delivering different drugs to various cancers, but no studies have yet described their uptake mechanisms, paclitaxel (PTX) delivery or resulting cytotoxicity towards breast cancer cells. Herein, we report results concerning cellular uptake of LNCs and cytotoxicity studies of PTX-loaded LNCs (LNCs-PTX) on the three breast cancer cell lines MCF-7, MDA-MB-231 and MDA-MB-468. LNCs-PTX of sizes 50 ± 2 nm, 90 ± 3 nm and 120 ± 4 nm were developed by the phase inversion method. Fluorescence microscopy and flow cytometry were used to observe the uptake of fluorescently labeled LNCs and cellular uptake of LNCs-PTX was measured using HPLC analyses of cell samples. These studies revealed a higher uptake of LNCs-PTX in MDA-MB-468 cells than in the other two cell lines. Moreover, free PTX and LNCs-PTX exhibited a similar pattern of toxicity towards each cell line, but MDA-MB-468 cells appeared to be more sensitive than the other two cell lines, as evaluated by the MTT cytotoxicity assay and a cell proliferation assay based upon [3H]thymidine incorporation. Studies with inhibitors of endocytosis indicate that the cellular uptake is mainly via the Cdc42/GRAF-dependent endocytosis as well as by macropinocytosis, whereas dynamin-dependent processes are not required. Furthermore, our results indicate that endocytosis of LNCs-PTX is important for the toxic effect on cells. Western blot analysis revealed that LNCs-PTX induce cytotoxicity by means of apoptosis in all the three cell lines. Altogether, the results demonstrate that LNCs-PTX exploit different mechanisms of endocytosis in a cell-type dependent manner, and subsequently induce apoptotic cell death in the breast cancer cells here studied. The article also describes biodistribution studies following intravenous injection of fluorescently labeled LNCs in mice.

Physicochemical Characterization, Toxicity and In Vivo Biodistribution Studies of a Discoidal, Lipid-Based Drug Delivery Vehicle: Lipodisq Nanoparticles Containing Doxorubicin.

Many promising pharmaceutically active compounds have low solubility in aqueous environments and their encapsulation into efficient drug delivery vehicles is crucial to increase their bioavailability. Lipodisq nanoparticles are approximately 10 nm in diameter and consist of a circular phospholipid bilayer, stabilized by an annulus of SMA (a hydrolysed copolymer of styrene and maleic anhydride). SMA is used extensively in structural biology to extract and stabilize integral membrane proteins for biophysical studies. Here, we assess the potential of these nanoparticles as drug delivery vehicles, determining their cytotoxicity and the in vivo excretion pathways of their polymer and lipid components. Doxorubicin-loaded Lipodisqs were cytotoxic across a panel of cancer cell lines, whereas nanoparticles without the drug had no effect on cell proliferation. Intracellular doxorubicin release from Lipodisqs in HeLa cells occurred in the low-pH environment of the endolysosomal system, consistent with the breakdown of the discoidal structure as the carboxylate groups of the SMA polymer become protonated. Biodistribution studies in mice showed that, unlike other nanoparticles injected intravenously, most of the Lipodisq components were recovered in the colon, consistent with rapid uptake by hepatocytes and excretion into bile. These data suggest that Lipodisqs have the potential to act as delivery vehicles for drugs and contrast agents.

An entropy-based approach to detect and localize intraoperative bleeding during minimally invasive surgery.

BACKGROUND: During minimally invasive surgery (either robotic or traditional laparoscopic), vascular injuries may occur because of inadvertent surgical tool movements or actions. These vascular injuries can lead to arterial or venous bleeding with varying degrees of severity that may be life-threatening. MATERIALS AND METHODS: Given that a bloody spot is characterized by homogenous and uniform texture, our algorithm automatically scans the entire surgical video frame- by- frame using a local entropy filter to segment each image into different regions sequence. By comparing changes in entropy in the frames sequences, the algorithm detects the moment of bleeding occurrence and its pixel location. We preliminarily tested the algorithm using ten minimally invasive-surgery videos., each of which contains one surgical-tool-induced bleeding. RESULTS: Our results show that the algorithm can detect bleeding within 0.635 s, on average, after their occurrences and locate the bleeding sources within, on average, 2.5% of discrepancy in pixels from their origins. CONCLUSION: In this study, we present a novel and promising local-entropy-based image processing algorithm that detects spurts of blood and locate their source in real-time.

Drug-Loaded Photosensitizer-Chitosan Nanoparticles for Combinatorial Chemo- and Photodynamic-Therapy of Cancer.

In this study we have developed biodegradable polymeric nanoparticles (NPs) containing the cytostatic drugs mertansine (MRT) or cabazitaxel (CBZ). The NPs are based on chitosan (CS) conjugate polymers synthesized with different amounts of the photosensitizer tetraphenylchlorin (TPC). These TPC-CS NPs have high loading capacity and strong drug retention due to π-π stacking interactions between the drugs and the aromatic photosensitizer groups of the polymers. CS polymers with 10% of the side chains containing TPC were found to be optimal in terms of drug loading capacity and NP stability. The TPC-CS NPs loaded with MRT or CBZ displayed higher cytotoxicity than the free form of these drugs in the breast cancer cell lines MDA-MB-231 and MDA-MB-468. Furthermore, light-induced photochemical activation of the NPs elicited a strong photodynamic therapy effect on these breast cancer cells. Biodistribution studies in mice showed that most of the TPC-CS NPs accumulated in liver and lungs, but they were also found to be localized in tumors derived from HCT-116 cells. These data suggest that the drug-loaded TPC-CS NPs have a potential in combinatory anticancer therapy and as contrast agents.

A Brief Survey of Telerobotic Time Delay Mitigation.

There is a substantial number of telerobotics and teleoperation applications ranging from space operations, ground/aerial robotics, drive-by-wire systems to medical interventions. Major obstacles for such applications include latency, channel corruptions, and bandwidth which limit teleoperation efficacy. This survey reviews the time delay problem in teleoperation systems. We briefly review different solutions from early approaches which consist of control-theory-based models and user interface designs and focus on newer approaches developed since 2014. Future solutions to the time delay problem will likely be hybrid solutions which include modeling of user intent, prediction of robot movements, and time delay prediction all potentially using time series prediction methods. Hence, we examine methods that are primarily based on time series prediction. Recent prediction approaches take advantage of advances in nonlinear statistical models as well as machine learning and neural network techniques. We review Recurrent Neural Networks, Long Short-Term Memory, Sequence to Sequence, and Generative Adversarial Network models and examine each of these approaches for addressing time delay. As time delay is still an unsolved problem, we suggest some possible future research directions from information-theory-based modeling, which may lead to promising new approaches to advancing the field.

Development and evaluation of an autonomous camera control algorithm on the da Vinci Surgical System.

BACKGROUND: Manual control of the camera arm in telerobotic surgical systems requires the surgeon to repeatedly interrupt the flow of the surgery. During surgery, there are instances when one or even both tools can drift out of the field of view. These issues may lead to increased workload and potential errors. METHODS: We performed a 20-participant subject study (including four surgeons) to compare different methods of camera control on a customized da Vinci Surgical System. We tested (a) an autonomous camera algorithm, (b) standard clutched control, and (c) an experienced camera operator using a joystick. RESULTS: The automated algorithm surpassed the traditional method of clutched camera control in measures of userperceived workload, efficiency, and progress. Additionally, it was consistently able to generate more centered and appropriately zoomed viewpoints than the other methods while keeping both tools safely inside the camera's field of view. CONCLUSIONS: Clinical systems of the future should consider automating the camera control aspects of robotic surgery.

Paclitaxel-loaded biodegradable ROS-sensitive nanoparticles for cancer therapy.

BACKGROUND: Reactive oxygen species (ROS), such as hydrogen peroxide and superoxide, trigger biodegradation of polymer-based nanoparticles (NPs) bearing pinacol-type boronic ester groups. These NPs may selectively release their cargo, in this case paclitaxel (PTX), at the high levels of ROS present in the intracellular environment of inflamed tissues and most tumors. PURPOSE: The main objective was to determine anti-tumor efficacy of PTX-loaded ROS-sensitive NPs and to examine whether macrophage infiltration had any impact on treatment efficacy. METHODS: NPs were synthesized and their characteristics in the presence of H2O2 were demonstrated. Both confocal microscopy as well as flow cytometry approaches were used to determine degradation of ROS-sensitive NPs. HeLa cells were cultured in vitro and used to establish tumor xenografts in nude mice. In vivo experiments were performed to understand toxicity, biodistribution and anti-tumor efficacy of the NPs. Moreover, we performed immunohistochemistry on tumor sections to study infiltration of M1 and M2 subsets of macrophages. RESULTS: We demonstrated that PTX delivered in NPs containing a ROS-sensitive polymer exhibits a better anti-tumor efficacy than PTX in NPs containing ROS-non-sensitive polymer, free PTX or Abraxane® (nab-PTX). The biodistribution revealed that ROS-sensitive NPs exhibit retention in liver, spleen and lungs, suggesting a potential to target cancer metastasizing to these organs. Finally, we demonstrated a correlation between infiltrated macrophage subsets and treatment efficacy, possibly contributing to the efficient anti-tumor effects. CONCLUSION: Treatment with ROS-sensitive NPs containing PTX gave an improved therapeutic effect in HeLa xenografts than their counterpart, free PTX or nab-PTX. Our data revealed a correlation between macrophage infiltration and efficiency of the different antitumor treatments, as the most effective NPs resulted in the highest infiltration of the anti-tumorigenic M1 macrophages.

Cabazitaxel-loaded Poly(2-ethylbutyl cyanoacrylate) nanoparticles improve treatment efficacy in a patient derived breast cancer xenograft.

The effect of poly(2-ethyl-butyl cyanoacrylate) nanoparticles containing the cytotoxic drug cabazitaxel was studied in three breast cancer cell lines and one basal-like patient-derived xenograft model grown in the mammary fat pad of immunodeficient mice. Nanoparticle-encapsulated cabazitaxel had a much better efficacy than similar concentrations of free drug in the basal-like patient-derived xenograft and resulted in complete remission of 6 out of 8 tumors, whereas free drug gave complete remission only with 2 out of 9 tumors. To investigate the different efficacies obtained with nanoparticle-encapsulated versus free cabazitaxel, mass spectrometry quantification of cabazitaxel was performed in mice plasma and selected tissue samples. Nanoparticle-encapsulated drug had a longer circulation time in blood. There was approximately a three times higher drug concentration in tumor tissue 24 h after injection, and two times higher 96 h after injection of nanoparticles with drug compared to the free drug. The tissue biodistribution obtained after 24 h using mass spectrometry analyses correlates well with biodistribution data obtained using IVIS® Spectrum in vivo imaging of nanoparticles labeled with the fluorescent substance NR668, indicating that these data also are representative for the nanoparticle distribution. Furthermore, immunohistochemistry was used to estimate infiltration of macrophages into the tumor tissue following injection of nanoparticle-encapsulated and free cabazitaxel. The higher infiltration of anti-tumorigenic versus pro-tumorigenic macrophages in tumors treated with the nanoparticles might also contribute to the improved effect obtained with the nanoparticle-encapsulated drug. Tumor infiltration of pro-tumorigenic macrophages was four times lower when using nanoparticles containing cabazitaxel than when using particles without drug, and we speculate that the very good therapeutic efficacy obtained with our cabazitaxel-containing particles may be due to their ability to reduce the level of pro-tumorigenic macrophages in the tumor. In summary, encapsulation of cabazitaxel in poly(2-ethyl-butyl cyanoacrylate) nanoparticles seems promising for treatment of breast cancer.

Basal-like breast cancer engages tumor-supportive macrophages via secreted factors induced by extracellular S100A4.

The tumor microenvironment (TME) may influence both cancer progression and therapeutic response. In breast cancer, particularly in the aggressive triple-negative/basal-like subgroup, patient outcome is strongly associated with the tumor's inflammatory profile. Tumor-associated macrophages (TAMs) are among the most abundant immune cells in the TME, shown to be linked to poor prognosis and therapeutic resistance. In this study, we investigated the effect of the metastasis- and inflammation-associated microenvironmental factor S100A4 on breast cancer cells (BCCs) of different subtypes and explored their further interactions with myeloid cells. We demonstrated that extracellular S100A4 activates BCCs, particularly the basal-like subtype, to elevate secretion of pro-inflammatory cytokines. The secreted factors promoted conversion of monocytes to TAM-like cells that exhibited protumorigenic activities: stimulated epithelial-mesenchymal transition, proliferation, chemoresistance, and motility in cancer cells. In conclusion, we have shown that extracellular S100A4 instigates a tumor-supportive microenvironment, involving a network of cytokines and TAM-like cells, which was particularly characteristic for basal-like BCCs and potentiated their aggressive properties. The S100A4-BCC-TAM interaction cascade could be an important contributor to the aggressive behavior of this subtype and should be further explored for therapeutic targeting.

Automated robot-assisted surgical skill evaluation: Predictive analytics approach.

BACKGROUND: Surgical skill assessment has predominantly been a subjective task. Recently, technological advances such as robot-assisted surgery have created great opportunities for objective surgical evaluation. In this paper, we introduce a predictive framework for objective skill assessment based on movement trajectory data. Our aim is to build a classification framework to automatically evaluate the performance of surgeons with different levels of expertise. METHODS: Eight global movement features are extracted from movement trajectory data captured by a da Vinci robot for surgeons with two levels of expertise - novice and expert. Three classification methods - k-nearest neighbours, logistic regression and support vector machines - are applied. RESULTS: The result shows that the proposed framework can classify surgeons' expertise as novice or expert with an accuracy of 82.3% for knot tying and 89.9% for a suturing task. CONCLUSION: This study demonstrates and evaluates the ability of machine learning methods to automatically classify expert and novice surgeons using global movement features.

Distance-based time series classification approach for task recognition with application in surgical robot autonomy.

BACKGROUND: Robotic-assisted surgery allows surgeons to perform many types of complex operations with greater precision than is possible with conventional surgery. Despite these advantages, in current systems, a surgeon should communicate with the device directly and manually. To allow the robot to adjust parameters such as camera position, the system needs to know automatically what task the surgeon is performing. METHODS: A distance-based time series classification framework has been developed which measures dynamic time warping distance between temporal trajectory data of robot arms and classifies surgical tasks and gestures using a k-nearest neighbor algorithm. RESULTS: Results on real robotic surgery data show that the proposed framework outperformed state-of-the-art methods by up to 9% across three tasks and by 8% across gestures. CONCLUSION: The proposed framework is robust and accurate. Therefore, it can be used to develop adaptive control systems that will be more responsive to surgeons' needs by identifying next movements of the surgeon. Copyright © 2016 John Wiley & Sons, Ltd.

Towards the Implementation of an Autonomous Camera Algorithm on the da Vinci Platform.

Camera positioning is critical for all telerobotic surgical systems. Inadequate visualization of the remote site can lead to serious errors that can jeopardize the patient. An autonomous camera algorithm has been developed on a medical robot (da Vinci) simulator. It is found to be robust in key scenarios of operation. This system behaves with predictable and expected actions for the camera arm with respect to the tool positions. The implementation of this system is described herein. The simulation closely models the methodology needed to implement autonomous camera control in a real hardware system. The camera control algorithm follows three rules: (1) keep the view centered on the tools, (2) keep the zoom level optimized such that the tools never leave the field of view, and (3) avoid unnecessary movement of the camera that may distract/disorient the surgeon. Our future work will apply this algorithm to the real da Vinci hardware.

Tube formation in the first trimester placental trophoblast cells: Differential effects of angiogenic growth factors and fatty acids.

The study aims to investigate whether cytosolic fatty acid-binding protein-4 (FABP4) is involved in angiogenic growth factors- and fatty acid-induced tube formation in first trimester placental trophoblast cells, HTR8/SVneo. We determined the tube formation both at basal as well as stimulated levels in the absence and presence of inhibitors of FABP4 and VEGF signaling pathways. Basal level of tube formation was maximally reduced in the presence of 50 µM of FABP4 inhibitor compared with those by VEGF signaling pathway inhibitors (rapamycin, L-NAME, and p38 MAP kinase inhibitor). Whereas docosahexaenoic acid, 22:6n-3 (DHA)-, and VEGF-induced tube formation was maximally inhibited by p38 MAP kinase inhibitor (63.7 and 34.5%, respectively), however, leptin-induced tube formation was inhibited maximally by FABP4 inhibitor (50.7%). ANGPTL4 and oleic acid (OA)-induced tube formation was not blocked by any of these inhibitors. The FABP4 inhibitor inhibited cell growth stimulated by DHA, leptin, VEGF, and OA (P < 0.05) but was not affected by ANGPTL4. VEGF, leptin, and OA also increased FABP4 protein level in these cells, though the uptake of fatty acids by these cells was not affected by the presence of FABP4 inhibitor. Our data demonstrate that FABP4 may be involved in part in the basal level, and stimulated tube formation by VEGF, DHA, and leptin, whereas it has little or no effect in ANGPTL4- and OA-induced tube formation in these cells. Thus, FABP4 may play a differential role in fatty acids and angiogenic growth factors-mediated tube formation in the first trimester trophoblast cells in vitro.

Task analysis of laparoscopic camera control schemes.

BACKGROUND: Minimally invasive surgeries rely on laparoscopic camera views to guide the procedure. Traditionally, an expert surgical assistant operates the camera. In some cases, a robotic system is used to help position the camera, but the surgeon is required to direct all movements of the system. Some prior research has focused on developing automated robotic camera control systems, but that work has been limited to rudimentary control schemes due to a lack of understanding of how the camera should be moved for different surgical tasks. METHODS: This research used task analysis with a sample of eight expert surgeons to discover and document several salient methods of camera control and their related task contexts. RESULTS: Desired camera placements and behaviours were established for two common surgical subtasks (suturing and knot tying). CONCLUSION: The results can be used to develop better robotic control algorithms that will be more responsive to surgeons' needs. Copyright © 2015 John Wiley & Sons, Ltd.

Voice-Activated Lightweight Reacher to Assist with Upper Extremity Movement Limitations: A Case Study.

The focus of this research was to design a functional and user-friendly reacher for people with spinal cord injuries (SCIs). Engineering advancements have taken assistive robotics to new dimensions. Technologies such as wheelchair robotics and myo-electronically controlled systems have opened up a wide range of new applications to assist people with physical disabilities. Similarly, exo-skeletal limbs and body suits have provided new foundations from which technologies can aid function. Unfortunately, these devices have issues of usability, weight, and discomfort with donning. The Smart Assistive Reacher Arm (SARA) system, developed in this research, is a voice-activated, lightweight, mobile device that can be used when needed. SARA was built to help overcome daily reach challenges faced by individuals with limited arm and hand movement capability, such as people with cervical level 5-6 (C5-6) SCI. This article shows that a functional reacher arm with voice control can be beneficial for this population. Comparison study with healthy participants and an SCI participant shows that, when using SARA, a person with SCI can perform simple reach and grasp tasks independently, without someone else's help. This suggests that the interface is intuitive and can be easily used to a high level of proficiency by a SCI individual.

Towards an autonomous robot for camera control during laparoscopic surgery.

INTRODUCTION: During laparoscopic surgery, the surgeon currently must instruct a human camera operator or a robotic arm to move the camera. This process is distracting, and the camera is not always placed in an ideal location. To mitigate these problems, we have developed a test platform that tracks laparoscopic instruments and automatically moves a camera with no explicit human direction. MATERIALS AND METHODS: The test platform is designed to mimic a typical laparoscopic working environment, where two hand-operated tools are manipulated through small ports. A pan-tilt-zoom camera is positioned over the tools, which emulates the positioning capabilities of a straight (0°) scope placed through a trocar. A camera control algorithm automatically keeps the tools in the view. In addition, two test tasks that require camera movement have been developed to aid in future evaluation of the system. RESULTS: The system was found to successfully track the laparoscopic instruments in the camera view as intended. The camera is moved and zoomed to follow the instruments in a smooth and consistent fashion. CONCLUSIONS: This technology shows that it is possible to create an autonomous camera system that cooperates with a surgeon without requiring any explicit user input. However, the currently implemented camera control behaviors are not ideal or sufficient for many surgical tasks. Future work will be performed to develop, test, and refine more complex behaviors that are optimized for different kinds of surgical tasks. In addition, portions of the test platform will be redesigned to enable its use in actual laparoscopic procedures.