Multiplex, high-throughput method to study cancer and immune cell mechanotransduction.

Studying cellular mechanoresponses during cancer metastasis is limited by sample variation or complex protocols that current techniques require. Metastasis is governed by mechanotransduction, whereby cells translate external stimuli, such as circulatory fluid shear stress (FSS), into biochemical cues. We present high-throughput, semi-automated methods to expose cells to FSS using the VIAFLO96 multichannel pipetting device custom-fitted with 22 G needles, increasing the maximum FSS 94-fold from the unmodified tips. Specifically, we develop protocols to semi-automatically stain live samples and to fix, permeabilize, and intracellularly process cells for flow cytometry analysis. Our first model system confirmed that the pro-apoptotic effects of TRAIL therapeutics in prostate cancer cells can be enhanced via FSS-induced Piezo1 activation. Our second system implements this multiplex methodology to show that FSS exposure (290 dyn cm-2) increases activation of murine bone marrow-derived dendritic cells. These methodologies greatly improve the mechanobiology workflow, offering a high-throughput, multiplex approach.

Spatial distribution of tumor-associated macrophages in an orthotopic prostate cancer mouse model.

Mounting evidence suggests that the immune landscape within prostate tumors influences progression, metastasis, treatment response, and patient outcomes. In this study, we investigated the spatial density of innate immune cell populations within NOD.SCID orthotopic prostate cancer xenografts following microinjection of human DU145 prostate cancer cells. Our laboratory has previously developed nanoscale liposomes that attach to leukocytes via conjugated E-selectin (ES) and kill cancer cells via TNF-related apoptosis inducing ligand (TRAIL). Immunohistochemistry (IHC) staining was performed on tumor samples to identify and quantify leukocyte infiltration for different periods of tumor growth and E-selectin/TRAIL (EST) liposome treatments. We examined the spatial-temporal dynamics of three different immune cell types infiltrating tumors using QuPath image analysis software. IHC staining revealed that F4/80+ tumor-associated macrophages (TAMs) were the most abundant immune cells in all groups, irrespective of time or treatment. The density of TAMs decreased over the course of tumor growth and decreased in response to EST liposome treatments. Intratumoral versus marginal analysis showed a greater presence of TAMs in the marginal regions at 3 weeks of tumor growth which became more evenly distributed over time and in tumors treated with EST liposomes. TUNEL staining indicated that EST liposomes significantly increased cell apoptosis in treated tumors. Additionally, confocal microscopy identified liposome-coated TAMs in both the core and periphery of tumors, highlighting the ability of liposomes to infiltrate tumors by "piggybacking" on macrophages. The results of this study indicate that TAMs represent the majority of innate immune cells within NOD.SCID orthotopic prostate tumors, and spatial density varies widely as a function of tumor size, duration of tumor growth, and treatment of EST liposomes.

Incorporation of ChatGPT and Other Large Language Models into a Graduate Level Computational Bioengineering Course.

The remarkable capabilities of generative artificial intelligence and large language models (LLMs) such as ChatGPT have delighted users around the world. Educators have regarded these tools as either a cause for great concern, an opportunity to educate students on cutting-edge technology, or often some combination of the two. Throughout the Fall 2023 semester, we explored the use of ChatGPT (and Bard, among other LLMs) in a graduate level numerical and statistical methods course for PhD-level bioengineers. In this article we share examples of this ChatGPT content, our observations on what worked best in our course, and speculate on how bioengineering students may be best served by this technology in the future.

Grand Challenges at the Interface of Engineering and Medicine.

Over the past two decades Biomedical Engineering has emerged as a major discipline that bridges societal needs of human health care with the development of novel technologies. Every medical institution is now equipped at varying degrees of sophistication with the ability to monitor human health in both non-invasive and invasive modes. The multiple scales at which human physiology can be interrogated provide a profound perspective on health and disease. We are at the nexus of creating "avatars" (herein defined as an extension of "digital twins") of human patho/physiology to serve as paradigms for interrogation and potential intervention. Motivated by the emergence of these new capabilities, the IEEE Engineering in Medicine and Biology Society, the Departments of Biomedical Engineering at Johns Hopkins University and Bioengineering at University of California at San Diego sponsored an interdisciplinary workshop to define the grand challenges that face biomedical engineering and the mechanisms to address these challenges. The Workshop identified five grand challenges with cross-cutting themes and provided a roadmap for new technologies, identified new training needs, and defined the types of interdisciplinary teams needed for addressing these challenges. The themes presented in this paper include: 1) accumedicine through creation of avatars of cells, tissues, organs and whole human; 2) development of smart and responsive devices for human function augmentation; 3) exocortical technologies to understand brain function and treat neuropathologies; 4) the development of approaches to harness the human immune system for health and wellness; and 5) new strategies to engineer genomes and cells.

Tuning of TRAIL clustering on the surface of nanoscale liposomes by phase separation.

Phase-separated liposomes were used to formulate tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a protein that selectively kills cancer cells while sparing most healthy ones. By controlling the average number of TRAIL molecules per liposome, we demonstrate the ability to tune the formation of TRAIL clusters and their resulting apoptotic activity.

Tumor nano-lysate activates dendritic cells to evoke a preventative immune response.

A tumor nano-lysate "TNL" vaccine comprised of sonicated 4T1 cells was developed, characterized and implemented for the prevention of triple-negative breast cancer. This study aimed to gain a better understanding of the immune response behind the success of the vaccine in vivo, through use of ex vivo and in vivo assays. Here, we analyze the activation of various immune cells isolated from healthy mouse spleens and find that antigen-presenting cells (APCs) such as dendritic cells (DCs) are being activated following 24 h incubation with 1:10 mg TNL/mg splenocytes. These cells were further explored to determine the pathway by which activation is occurring, and it was observed that TNL are phagocytosed by DCs to activate NF-kB and c-Fos pathways, resulting in enhanced cytokine release after 24 h. An in vivo temporal analysis was performed in mice to understand the immune response at 1, 3, 7 and 10 days after one 100 µL dose of TNL consisting of 105 sonicated 4T1 cells via cardiac puncture and splenocyte and peripheral blood mononuclear cell (PBMC) analysis. Changes were observed for up to one week. A multiple dose study was performed comparing mice that were vaccinated with one dose of TNL administered every ten days for 3 doses total, as well as a PBS vehicle control. Survival for TNL-vaccinated mice was enhanced compared to the PBS control, and there was an average delay of 10 days in the onset of metastasis. The differences between the groups at the end of the study demonstrate the potential for TNL as a preventative therapeutic.

The association of intraoperative opioid dose with postanesthesia care unit outcomes in children: a retrospective study.

PURPOSE: In children, the relationship between the dose of intraoperative opioid and postoperative outcomes is unclear. We examined the relationship between intraoperative opioid dose and postanesthesia care unit (PACU) pain scores and opioid and antiemetic administrations. METHODS: We performed a single-institution retrospective cohort study. Patients who were aged < 19 yr, had an American Society of Anesthesiologists Physical Status of I-III, were undergoing one of 11 procedures under general anesthesia and without regional anesthesia, and who were admitted to the PACU were included. Patients were analyzed by quartiles of total intraoperative opioid dose using multivariable regression, adjusting for confounders including procedure. An exploratory analysis of opioid-free anesthetics was also performed. RESULTS: Three thousand, seven hundred and thirty-three cases were included, and the mean age of included patients was 8.3 yr. After adjustment, there were no significant differences between the lowest and higher quartiles for first conscious pain score, mean pain score, PACU opioid dose, or PACU length of stay; in addition, estimated differences were small. Patients in higher quartiles were estimated to be more likely to receive antiemetics, significantly so for those in the second quartile. Patients in the lowest quartile received significantly more intraoperative nonopioid analgesics. In the exploratory analysis, no significant difference in PACU pain scores was found in cases without intraoperative opioids. CONCLUSIONS: Children who received lower doses of intraoperative opioids did not have worse PACU pain outcomes but required fewer antiemetics and received greater numbers of nonopioid analgesics intraoperatively. These findings suggest that lower doses of intraoperative opioids may be administered to children as long as other analgesics are used.

RéSUMé: OBJECTIF: Chez les enfants, la relation entre la dose peropératoire d'opioïdes et les issues postopératoires n'est pas claire. Nous avons examiné la relation entre la dose peropératoire d'opioïdes, les scores de douleur en salle de réveil, et les administrations d'opioïdes et d'antiémétiques. MéTHODE: Nous avons réalisé une étude de cohorte rétrospective dans un seul établissement. Nous avons inclus les patient·es âgé·es < 19 ans ayant un statut physique ASA de I-III et bénéficiant de l'une de 11 interventions sous anesthésie générale et sans anesthésie régionale, et qui avaient été admis·es en salle de réveil. Les patient·es ont été analysé·es par quartiles de la dose totale d'opioïdes peropératoires en utilisant une régression multivariée, en ajustant les données pour tenir compte des facteurs de confusion, notamment de l'intervention. Une analyse exploratoire des anesthésiques sans opioïdes a également été réalisée. RéSULTATS: Au total 3733 cas ont été inclus, et l'âge moyen des enfants était de 8,3 ans. Après ajustement, il n'y avait pas de différences significatives entre les quartiles inférieur et supérieur pour le premier score de douleur chez l'enfant conscient·e, le score de douleur moyen, la dose d'opioïdes en salle de réveil ou la durée du séjour en salle de réveil; de plus, les différences estimées étaient faibles. On a estimé que les patient·es des quartiles supérieurs étaient plus susceptibles de recevoir des antiémétiques et ce, de manière significative pour ceux et celles du deuxième quartile. Les patient·es du quartile inférieur ont reçu significativement plus d'analgésiques non opioïdes peropératoires. Dans l'analyse exploratoire, aucune différence significative dans les scores de douleur en salle de réveil n'a été trouvée dans les cas sans opioïdes peropératoires. CONCLUSION: Les enfants qui ont reçu des doses plus faibles d'opioïdes peropératoires n'ont pas eu de pires issues de douleur en salle de réveil, mais ont eu besoin de moins d'antiémétiques et ont reçu un plus grand nombre d'analgésiques non opioïdes en peropératoire. Ces résultats suggèrent que des doses plus faibles d'opioïdes peropératoires peuvent être administrées aux enfants tant que d'autres analgésiques sont utilisés.

TRAIL-conjugated liposomes that bind natural killer cells to induce colorectal cancer cell apoptosis.

Natural killer (NK) cell functionality is a strong indicator of favorable prognosis in cancer patients, making NK cells an appealing therapeutic target to prevent lymph node dissemination. We engineered liposomes that are conjugated with anti-CD335 antibodies for NK cell targeting, and the apoptotic ligand TRAIL to kill cancer cells. Liposomes were made using a thin film hydration method followed by extrusion to approximately 100 nm in diameter and conjugation of proteins via thiol-maleimide click chemistry. TRAIL/anti-CD335 liposomes successfully bound to isolated NK cells. Once piggybacked to the surface of NK cells, these "Super Natural Killer Cells" were able to more effectively kill oxaliplatin-resistant SW620 cells and metastatic COLO205 colorectal cancer cells via TRAIL-mediated apoptosis compared to NK cells alone. Importantly, Super NK cells were more effective under physiological levels of fluid shear stress found in the lymphatics. Liposome biodistribution after intravenous administration confirmed the sustained presence of liposomes within the spleen and tumor draining mesenteric lymph nodes for at least 4 days. These results demonstrate the enhanced apoptotic effects of NK cells armored with liposomal TRAIL against clinically relevant colorectal cancer cells, providing the groundwork for in vivo treatment studies in mouse models of colorectal cancer metastasis.

Activation of Dendritic Cells Isolated from the Blood of Patients with Prostate Cancer by Ex Vivo Fluid Shear Stress Stimulation.

Prostate cancer is one of the most common cancers among men in the United States and a leading cause of cancer-related death in men. Treatment options for patients with advanced prostate cancer include hormone therapies, chemotherapies, radioligand therapies, and immunotherapies. Provenge (sipuleucel-T) is an autologous cancer-vaccine-based immunotherapy approved for men with asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer (mCRPC). Administration of sipuleucel-T involves leukapheresis of patient blood to isolate antigen-presenting cells (APCs), including dendritic cells (DCs), and subsequent incubation of isolated APCs with both an antigen, prostatic acid phosphatase (PAP), and granulocyte macrophage-colony stimulating factor (GM-CSF) before their infusion back into the patient. Although sipuleucel-T has been shown to improve overall survival, other meaningful outcomes, such as prostate-specific antigen (PSA) levels and radiographic response, are inconsistent. This lack of robust response may be due to limited ex vivo activation of DCs using current protocols. Earlier studies have shown that many cell types can be activated ex vivo by external forces such as fluid shear stress (FSS). We hypothesize that novel fluid shear stress technologies and methods can be used to improve ex vivo efficacy of prostate cancer DC activation in prostate cancer. Herein, we report a new protocol for activating DCs from patients with prostate cancer using ex vivo fluid shear stress. Ultimately, the goal of these studies is to improve DC activation to expand the efficacy of therapies such as sipuleucel-T. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample collection and DC isolation Basic Protocol 2: Determination and application of fluid shear stress Basic Protocol 3: Flow cytometry analysis of DCs after FSS stimulation.

Dual Affinity Nanoparticles for the Transport of Therapeutics from Carrier Cells to Target Cells under Physiological Flow Conditions.

In this study, a novel two-stage nanoparticle delivery platform was developed based on the dual functionalization of a liposome with moieties that have fundamentally different strengths of adhesion and binding kinetics. The essential concept of this system is that the nanoparticles are designed to loosely bind to the carrier cell until they come into contact with the target cell, to which they bind with greater strength. This allows the nanoparticle to be transferred from one cell to another, circulating for longer periods of time in the blood and delivering the therapeutic agent to the target circulating tumor cell. Liposomes were prepared using the lipid cake and extrusion technique, then functionalized with E-selectin (ES), anti-cell surface vimentin antibody fragments, and TRAIL via click chemistry. The binding of dual affinity (DA) liposomes was confirmed with the neutrophil-like cell line PLB985, the colorectal cancer cell line HCT116, and healthy granulocytes isolated from peripheral whole blood under physiologically relevant fluid shear stress (FSS) in a cone-and-plate viscometer. Transfer of the DA liposomes from PLB985 to HCT116 cells under FSS was greater compared to all of the control liposome formulations. Additionally, DA liposomes demonstrated enhanced apoptotic effects on HCT116 cells in whole blood under FSS, surpassing the efficacy of the ES/TRAIL liposomes previously developed by the King Lab.

Fluid shear stress enhances dendritic cell activation.

Ex vivo activation of dendritic cells (DCs) has been widely explored for targeted therapies, although these treatments remain expensive. Reducing treatment costs while enhancing cell activation could help to make immunotherapies more accessible. Cells can be activated by both internal and external forces including fluid shear stress (FSS). FSS activates cells via opening of mechanosensitive ion channels. In this study, dendritic cells were activated by sustained exposure to circulatory levels of fluid shear stress using a cone-and-plate flow device and analyzed for activation markers. After 1 h of shear stress exposure, an increase in cytokine release was present in immortalized cells as well as phosphorylation of the proteins NF-κB and cFos in primary DCs. Changes in DC morphology, metabolism and proliferation were also observed. These compelling new findings point to the potential for using FSS to activate DCs for ex vivo therapeutics.

Ultrasound-Guided Occipital Nerve Blocks as Part of Multi-Modal Perioperative Analgesia in Pediatric Posterior Craniotomies: A Case Series.

Various regional anesthetics have been used for postoperative analgesia for pediatric craniotomy. In this case series, we report retrospectively collected data on postoperative pain and analgesic use in 44 patients who received ultrasound-guided occipital nerve blocks in addition to intravenous analgesic agents for posterior craniotomy procedures. In the immediate post-anesthesia care unit, pain was rated as zero or well controlled in 77% of patients, with only 43% requiring intravenous or demand patient-controlled analgesia opioids. There were no block-related complications. Occipital nerve blocks may constitute a safe and effective component of multimodal analgesia in this population.

Correction: Innovation and Entrepreneurship in Promotion and Tenure in Biomedical Engineering.

[This corrects the article DOI: 10.1007/s12195-023-00767-x.].

Innovation and Entrepreneurship in Promotion and Tenure in Biomedical Engineering: Communication from the Biomedical Engineering Society Long Range Planning Committee.

Promotion and tenure (P&T) remain the central tenets of academia. The criteria for P&T both create and reflect the mission of an institution. The discipline of biomedical engineering is built upon the invention and translation of tools to address unmet clinical needs. 'Broadening the bar' for P&T to include efforts in innovation, entrepreneurship, and technology-based transfer (I/E/T) will require establishing the criteria and communication of methodology for their evaluation. We surveyed the department chairs across the fields of biomedical and bioengineering to understand the state-of-the-art in incorporation, evaluation, and definition of I/E/T as applied to the P&T process. The survey results reflected a commitment to increasing and respecting I/E/T activities as part of the P&T criteria. This was balanced by an equally strong desire for improving the education and policy for evaluating I/E/T internally as well as externally. The potential for 'broadening the bar' for P&T to include I/E/T activities in biomedical engineering may serve as an example for other fields in engineering and applied sciences, and a template for potential inclusion of additional efforts such as diversity, equity, and inclusion (DEI) into the pillars of scholarship, education, and service.

Chemical Activation and Mechanical Sensitization of Piezo1 Enhance TRAIL-Mediated Apoptosis in Glioblastoma Cells.

Glioblastoma multiforme (GBM), the most common and aggressive type of primary brain tumor, has a mean survival of less than 15 months after standard treatment. Treatment with the current standard of care, temozolomide (TMZ), may be ineffective if damaged tumor cells undergo DNA repair or acquire mutations that inactivate transcription factor p53. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in multiple tumor types, while evading healthy cells, through a transcription-independent mechanism. GBM is particularly resistant to TRAIL, but studies have found that the mechanoreceptor Piezo1 can be activated under static conditions via Yoda1 agonist to induce TRAIL sensitization in other cancer cell lines. This study examines the effects and the mechanism of chemical and mechanical activation of Piezo1, via Yoda1 and fluid shear stress (FSS) stimulation, on TRAIL-mediated apoptosis in GBM cells. Here, we demonstrate that Yoda1 + TRAIL and FSS + TRAIL combination therapies significantly increase apoptosis in two GBM cell lines relative to controls. Further, cells known to be resistant to TMZ were found to have higher levels of Piezo1 expression and were more susceptible to TRAIL sensitization by Piezo1 activation. The combinatory Yoda1 + TRAIL treatment significantly decreased cell viability in TMZ-resistant GBM cells when compared to treatment with both low and high doses of TMZ. The results of this study suggest the potential of a highly specific and minimally invasive approach to overcome TMZ resistance in GBM by sensitizing cancer cells to TRAIL treatment via chemical or mechanical activation of Piezo1.

Neutralization of the new coronavirus by extracting their spikes using engineered liposomes.

The devastating COVID-19 pandemic motivates the development of safe and effective antivirals to reduce morbidity and mortality associated with infection. We developed nanoscale liposomes that are coated with the cell receptor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19. Lentiviral particles pseudotyped with the spike protein of SARS-CoV-2 were constructed and used to test the virus neutralization potential of the engineered liposomes. Under TEM, we observed for the first time a dissociation of spike proteins from the pseudovirus surface when the pseudovirus was purified. The liposomes potently inhibit viral entry into host cells by extracting the spike proteins from the pseudovirus surface. As the receptor on the liposome surface can be readily changed to target other viruses, the receptor-coated liposome represents a promising strategy for broad spectrum antiviral development.