Systemically targeting monocytic MDSCs using dendrimers and their cell-level biodistribution kinetics.

The focus of nanoparticles in vivo trafficking has been mostly on their tissue-level biodistribution and clearance. Recent progress in the nanomedicine field suggests that the targeting of nanoparticles to immune cells can be used to modulate the immune response and enhance therapeutic delivery to the diseased tissue. In the presence of tumor lesions, monocytic-myeloid-derived suppressor cells (M-MDSCs) expand significantly in the bone marrow, egress into peripheral blood, and traffic to the solid tumor, where they help maintain an immuno-suppressive tumor microenvironment. In this study, we investigated the interaction between PAMAM dendrimers and M-MDSCs in two murine models of glioblastoma, by examining the cell-level biodistribution kinetics of the systemically injected dendrimers. We found that M-MDSCs in the tumor and lymphoid organs can efficiently endocytose hydroxyl dendrimers. Interestingly, the trafficking of M-MDSCs from the bone marrow to the tumor contributed to the deposition of hydroxyl dendrimers in the tumor. M-MDSCs showed different capacities of endocytosing dendrimers of different functionalities in vivo. This differential uptake was mediated by the unique serum proteins associated with each dendrimer surface functionality. The results of this study set up the framework for developing dendrimer-based immunotherapy to target M-MDSCs for cancer treatment.

KR158 Spheres Harboring Slow-Cycling Cells Recapitulate High-Grade Glioma Features in an Immunocompetent System.

Glioblastoma (GBM) poses a significant challenge in clinical oncology due to its aggressive nature, heterogeneity, and resistance to therapies. Cancer stem cells (CSCs) play a critical role in GBM, particularly in treatment resistance and tumor relapse, emphasizing the need to comprehend the mechanisms regulating these cells. Also, their multifaceted contributions to the tumor microenvironment (TME) underline their significance, driven by their unique properties. This study aimed to characterize glioblastoma stem cells (GSCs), specifically slow-cycling cells (SCCs), in an immunocompetent murine GBM model to explore their similarities with their human counterparts. Using the KR158 mouse model, we confirmed that SCCs isolated from this model exhibited key traits and functional properties akin to human SCCs. KR158 murine SCCs, expanded in the gliomasphere assay, demonstrated sphere forming ability, self-renewing capacity, positive tumorigenicity, enhanced stemness and resistance to chemotherapy. Together, our findings validate the KR158 murine model as a framework to investigate GSCs and SCCs in GBM pathology, and explore specifically the SCC-immune system communications, understand their role in disease progression, and evaluate the effect of therapeutic strategies targeting these specific connections.

Glioma-derived M-CSF and IL-34 license M-MDSCs to suppress CD8+ T cells in a NOS-dependent manner.

Glioblastoma (GBM) is the most common malignant primary brain tumor, resulting in poor survival despite aggressive therapies. GBM is characterized by a highly heterogeneous and immunosuppressive tumor microenvironment (TME) made up predominantly of infiltrating peripheral immune cells. One significant immune cell type that contributes to glioma immune evasion is a population of immunosuppressive cells, termed myeloid-derived suppressor cells (MDSCs). Previous studies suggest that a subset of myeloid cells, expressing monocytic (M)-MDSC markers and dual expression of chemokine receptors CCR2 and CX3CR1, utilize CCR2 to infiltrate the TME. This study evaluated the mechanism of CCR2+/CX3CR1+ M-MDSC differentiation and T cell suppressive function in murine glioma models. We determined that bone marrow-derived CCR2+/CX3CR1+ cells adopt an immune suppressive cell phenotype when cultured with glioma-derived factors. Glioma secreted CSF1R ligands M-CSF and IL-34 were identified as key drivers of M-MDSC differentiation while adenosine and iNOS pathways were implicated in M-MDSC suppression of T cells. Mining a human GBM spatial RNAseq database revealed a variety of different pathways that M-MDSCs utilize to exert their suppressive function that are driven by complex niches within the microenvironment. These data provide a more comprehensive understanding of the mechanism of M-MDSCs in glioblastoma.

KR158 spheres harboring slow-cycling cells recapitulate GBM features in an immunocompetent system.

Glioblastoma (GBM) poses a significant challenge in clinical oncology due to its aggressive nature, heterogeneity, and resistance to therapies. Cancer stem cells (CSCs) play a critical role in GBM, particularly in treatment-resistance and tumor relapse, emphasizing the need to comprehend the mechanisms regulating these cells. Also, their multifaceted contributions to the tumor-microenvironment (TME) underline their significance, driven by their unique properties. This study aimed to characterize glioblastoma stem cells (GSCs), specifically slow-cycling cells (SCCs), in an immunocompetent murine GBM model to explore their similarities with their human counterparts. Using the KR158 mouse model, we confirmed that SCCs isolated from this model exhibited key traits and functional properties akin to human SCCs. KR158 murine SCCs, expanded in the gliomasphere assay, demonstrated sphere forming ability, self-renewing capacity, positive tumorigenicity, enhanced stemness and resistance to chemotherapy. Together, our findings validate the KR158 murine model as a framework to investigate GSCs and SCCs in GBM-pathology, and explore specifically the SCC-immune system communications, understand their role in disease progression, and evaluate the effect of therapeutic strategies targeting these specific connections.

DR5 disulfide bonding as a sensor and effector of protein folding stress.

New agents are needed that selectively kill cancer cells without harming normal tissues. The TRAIL ligand and its receptors, DR5 and DR4, exhibit cancer-selective toxicity, but TRAIL analogs or agonistic antibodies targeting these receptors have not received FDA approval for cancer therapy. Small molecules for activating DR5 or DR4 independently of protein ligands may bypass some of the pharmacological limitations of these protein drugs. Previously described Disulfide bond Disrupting Agents (DDAs) activate DR5 by altering its disulfide bonding through inhibition of the Protein Disulfide Isomerases (PDIs) ERp44, AGR2, and PDIA1. Work presented here extends these findings by showing that disruption of single DR5 disulfide bonds causes high-level DR5 expression, disulfide-mediated clustering, and activation of Caspase 8-Caspase 3 mediated pro-apoptotic signaling. Recognition of the extracellular domain of DR5 by various antibodies is strongly influenced by the pattern of DR5 disulfide bonding, which has important implications for the use of agonistic DR5 antibodies for cancer therapy. Disulfide-defective DR5 mutants do not activate the ER stress response or stimulate autophagy, indicating that these DDA-mediated responses are separable from DR5 activation and pro-apoptotic signaling. Importantly, other ER stressors, including Thapsigargin and Tunicamycin also alter DR5 disulfide bonding in various cancer cell lines and in some instances, DR5 mis-disulfide bonding is potentiated by overriding the Integrated Stress Response (ISR) with inhibitors of the PERK kinase or the ISR inhibitor ISRIB. These observations indicate that the pattern of DR5 disulfide bonding functions as a sensor of ER stress and serves as an effector of proteotoxic stress by driving extrinsic apoptosis independently of extracellular ligands.

Global stability and parameter analysis reinforce therapeutic targets of PD-L1-PD-1 and MDSCs for glioblastoma.

Glioblastoma (GBM) is an aggressive primary brain cancer that currently has minimally effective treatments. Like other cancers, immunosuppression by the PD-L1-PD-1 immune checkpoint complex is a prominent axis by which glioma cells evade the immune system. Myeloid-derived suppressor cells (MDSCs), which are recruited to the glioma microenviroment, also contribute to the immunosuppressed GBM microenvironment by suppressing T cell functions. In this paper, we propose a GBM-specific tumor-immune ordinary differential equations model of glioma cells, T cells, and MDSCs to provide theoretical insights into the interactions between these cells. Equilibrium and stability analysis indicates that there are unique tumorous and tumor-free equilibria which are locally stable under certain conditions. Further, the tumor-free equilibrium is globally stable when T cell activation and the tumor kill rate by T cells overcome tumor growth, T cell inhibition by PD-L1-PD-1 and MDSCs, and the T cell death rate. Bifurcation analysis suggests that a treatment plan that includes surgical resection and therapeutics targeting immune suppression caused by the PD-L1-PD1 complex and MDSCs results in the system tending to the tumor-free equilibrium. Using a set of preclinical experimental data, we implement the approximate Bayesian computation (ABC) rejection method to construct probability density distributions that estimate model parameters. These distributions inform an appropriate search curve for global sensitivity analysis using the extended fourier amplitude sensitivity test. Sensitivity results combined with the ABC method suggest that parameter interaction is occurring between the drivers of tumor burden, which are the tumor growth rate and carrying capacity as well as the tumor kill rate by T cells, and the two modeled forms of immunosuppression, PD-L1-PD-1 immune checkpoint and MDSC suppression of T cells. Thus, treatment with an immune checkpoint inhibitor in combination with a therapeutic targeting the inhibitory mechanisms of MDSCs should be explored.

Global stability and parameter analysis reinforce therapeutic targets of PD-L1-PD-1 and MDSCs for glioblastoma.

Glioblastoma (GBM) is an aggressive primary brain cancer that currently has minimally effective treatments. Like other cancers, immunosuppression by the PD-L1-PD-1 immune checkpoint complex is a prominent axis by which glioma cells evade the immune system. Myeloid-derived suppressor cells (MDSCs), which are recruited to the glioma microenviroment, also contribute to the immunosuppressed GBM microenvironment by suppressing T cell functions. In this paper, we propose a GBM-specific tumor-immune ordinary differential equations model of glioma cells, T cells, and MDSCs to provide theoretical insights into the interactions between these cells. Equilibrium and stability analysis indicates that there are unique tumorous and tumor-free equilibria which are locally stable under certain conditions. Further, the tumor-free equilibrium is globally stable when T cell activation and the tumor kill rate by T cells overcome tumor growth, T cell inhibition by PD-L1-PD-1 and MDSCs, and the T cell death rate. Bifurcation analysis suggests that a treatment plan that includes surgical resection and therapeutics targeting immune suppression caused by the PD-L1-PD1 complex and MDSCs results in the system tending to the tumor-free equilibrium. Using a set of preclinical experimental data, we implement the Approximate Bayesian Computation (ABC) rejection method to construct probability density distributions that estimate model parameters. These distributions inform an appropriate search curve for global sensitivity analysis using the extended Fourier Amplitude Sensitivity Test (eFAST). Sensitivity results combined with the ABC method suggest that parameter interaction is occurring between the drivers of tumor burden, which are the tumor growth rate and carrying capacity as well as the tumor kill rate by T cells, and the two modeled forms of immunosuppression, PD-L1-PD-1 immune checkpoint and MDSC suppression of T cells. Thus, treatment with an immune checkpoint inhibitor in combination with a therapeutic targeting the inhibitory mechanisms of MDSCs should be explored.

Business Versus Ethics? Thoughts on the Future of Business Ethics.

To commemorate 40 years since the founding of the Journal of Business Ethics, the editors in chief of the journal have invited the editors to provide commentaries on the future of business ethics. This essay comprises a selection of commentaries aimed at creating dialogue around the theme Business versus Ethics? (inspired by the title of the commentary by Jeffrey Harrison). The authors of these commentaries seek to transcend the age-old separation fallacy (Freeman in Bus Ethics Q 4(4):409-421, 1994) that juxtaposes business and ethics/society, posing a forced choice or trade off. Providing a contemporary take on the classical question "if it's legal is it ethical?", David Hess explores the role of the law in promoting or hindering stakeholder-oriented purpose and governance structure. Jeffrey Harrison encourages scholars to move beyond the presupposition that businesses are either strategic or ethical and explore important questions at the intersection of strategy and ethics. The proposition that business models might be inherently ethical or inherently unethical in their design is developed by Sheila Killian, who examines business systems, their morality, and who they serve. However, the conundrum that entrepreneurs are either lauded for their self-belief and risk-taking, or loathed for their self-belief and risk-taking, is discussed by M. Tina Dacin and Julia Roloff using the metaphor of taboos and totems. These commentaries seek to explore positions that advocate multiplicity and tensions in which business ethics is not either/or but both.

The Dynamics of Tumor-Infiltrating Myeloid Cell Activation and the Cytokine Expression Profile in a Glioma Resection Site during the Post-Surgical Period in Mice.

Glioblastoma is the most aggressive brain cancer and is highly infiltrated with cells of myeloid lineage (TIM) that support tumor growth and invasion. Tumor resection is the primary treatment for glioblastoma; however, the activation state of TIM at the site of tumor resection and its impact on glioma regrowth are poorly understood. Using the C57BL/6/GL261 mouse glioma implantation model, we investigated the state of TIM in the tumor resection area during the post-surgical period. TIM isolated from brain tissue at the resection site were analyzed at 0, 1, 4, 7, 14, and 21 days after tumor resection. An increase in expression of CD86 during the first 7 days after surgical resection and then upregulation of arginase 1 from the 14th to 21st days after resection were detected. Cytokine expression analysis combined with qRT-PCR revealed sustained upregulation of IL4, IL5, IL10, IL12, IL17, vascular endothelial growth factor (VEGF), and monocyte chemoattractant protein 1 (MCP1/CCL2) in TIM purified from regrown tumors compared with primary implanted tumors. Flow cytometry analysis revealed increased CD86+/CD206+ population in regrown tumors compared with primary implanted tumors. Overall, we found that TIM in primary implanted tumors and tumors regrown after resection exhibited different phenotypes and cytokine expression patterns.

Glioma-derived CCL2 and CCL7 mediate migration of immune suppressive CCR2+/CX3CR1+ M-MDSCs into the tumor microenvironment in a redundant manner.

Glioblastoma (GBM) is the most common and malignant primary brain tumor, resulting in poor survival despite aggressive therapies. GBM is characterized in part by a highly heterogeneous and immunosuppressive tumor microenvironment (TME) made up predominantly of infiltrating peripheral immune cells. One significant immune cell type that contributes to glioma immune evasion is a population of immunosuppressive, hematopoietic cells, termed myeloid-derived suppressor cells (MDSCs). Previous studies suggest that a potent subset of myeloid cells, expressing monocytic (M)-MDSC markers, distinguished by dual expression of chemokine receptors CCR2 and CX3CR1, utilize CCR2 to infiltrate into the TME. This study evaluated the T cell suppressive function and migratory properties of CCR2+/CX3CR1+ MDSCs. Bone marrow-derived CCR2+/CX3CR1+ cells adopt an immune suppressive cell phenotype when cultured with glioma-derived factors. Recombinant and glioma-derived CCL2 and CCL7 induce the migration of CCR2+/CX3CR1+ MDSCs with similar efficacy. KR158B-CCL2 and -CCL7 knockdown murine gliomas contain equivalent percentages of CCR2+/CX3CR1+ MDSCs compared to KR158B gliomas. Combined neutralization of CCL2 and CCL7 completely blocks CCR2-expressing cell migration to KR158B cell conditioned media. CCR2+/CX3CR1+ cells are also reduced within KR158B gliomas upon combination targeting of CCL2 and CCL7. High levels of CCL2 and CCL7 are also associated with negative prognostic outcomes in GBM patients. These data provide a more comprehensive understanding of the function of CCR2+/CX3CR1+ MDSCs and the role of CCL2 and CCL7 in the recruitment of these immune suppressive cells and further support the significance of targeting this chemokine axis in GBM.

Modulation of the chemokine/chemokine receptor axis as a novel approach for glioma therapy.

Chemokines are a large subfamily of cytokines known for their ability to facilitate cell migration, most notably leukocytes, throughout the body. Chemokines are necessary for a functioning immune system in both health and disease and have received considerable attention for their roles in orchestrating temporal-spatial regulation of immune cell populations in cancer. Gliomas comprise a group of common central nervous system (CNS) primary tumors that are extremely challenging to treat. Immunotherapy approaches for highly malignant brain tumors offer an exciting new avenue for therapeutic intervention but so far, have seen limited successful clinical outcomes. Herein we focus on important chemokine/chemokine receptor systems in the regulation of pro- and anti-tumor mechanisms, highlighting potential therapeutic advantages of modulating these systems in malignant gliomas and other cancers.

Adult immuno-oncology: using past failures to inform the future.

In oncology, "immunotherapy" is a broad term encompassing multiple means of utilizing the patient's immune system to combat malignancy. Prominent among these are immune checkpoint inhibitors, cellular therapies including chimeric antigen receptor T-cell therapy, vaccines, and oncolytic viruses. Immunotherapy for glioblastoma (GBM) has had mixed results in early trials. In this context, the past, present, and future of immune oncology for the treatment of GBM was discussed by clinical, research, and thought leaders as well as patient advocates at the first annual Remission Summit in 2019. The goal was to use current knowledge (published and unpublished) to identify possible causes of treatment failures and the best strategies to advance immunotherapy as a treatment modality for patients with GBM. The discussion focuses on past failures, current limitations, failure analyses, and proposed best practices moving forward.

CCR2 inhibition reduces tumor myeloid cells and unmasks a checkpoint inhibitor effect to slow progression of resistant murine gliomas.

Immunotherapy directed at the PD-L1/PD-1 axis has produced treatment advances in various human cancers. Unfortunately, progress has not extended to glioblastoma (GBM), with phase III clinical trials assessing anti-PD-1 monotherapy failing to show efficacy in newly diagnosed and recurrent tumors. Myeloid-derived suppressor cells (MDSCs), a subset of immunosuppressive myeloid derived cells, are known to infiltrate the tumor microenvironment of GBM. Growing evidence suggests the CCL2-CCR2 axis is important for this process. This study evaluated the combination of PD-1 blockade and CCR2 inhibition in anti-PD-1-resistant gliomas. CCR2 deficiency unmasked an anti-PD-1 survival benefit in KR158 glioma-bearing mice. CD11b+/Ly6Chi/PD-L1+ MDSCs within established gliomas decreased with a concomitant increase in overall CCR2+ cells and MDSCs within bone marrow of CCR2-deficient mice. The CCR2 antagonist CCX872 increased median survival as a monotherapy in KR158 glioma-bearing animals and further increased median and overall survival when combined with anti-PD-1. Additionally, combination of CCX872 and anti-PD-1 prolonged median survival time in 005 GSC GBM-bearing mice. In both models, CCX872 decreased tumor associated MDSCs and increased these cells within the bone marrow. Examination of tumor-infiltrating lymphocytes revealed an elevated population, increased IFNγ expression, indicating enhanced cytolytic activity, as well as decreased expression of exhaustion markers in CD4+ and CD8+ T cells following combination treatment. These data establish that combining CCR2 and PD-1 blockade extends survival in clinically relevant murine glioma models and provides the basis on which to advance this combinatorial treatment toward early-phase human trials.

Noncontact Imaging of Ion Dynamics in Polymer Electrolytes with Time-Resolved Electrostatic Force Microscopy.

Ionic-transport processes govern performance in many classic and emerging devices, ranging from battery storage to modern mixed-conduction organic electrochemical transistors (OECT). Here, we study local ion-transport dynamics in polymer films using time-resolved electrostatic force microscopy (trEFM). We establish a correspondence between local and macroscopic measurements using local trEFM and macroscopic electrical impedance spectroscopy (EIS). We use polymer films doped with lithium bis(trifluoromethane)sulfonimide (LiTFSI) as a model system where the polymer backbone has oxanorbornenedicarboximide repeat units with an oligomeric ethylene oxide side chain of length n. Our results show that the local polymer response measured in the time domain with trEFM follows stretched-exponential relaxation kinetics, consistent with the Havriliak-Negami relaxation we measure in the frequency-domain EIS data for macroscopic samples of the same polymers. Furthermore, we show that the trEFM results capture the same trends as the EIS results-changes in ion dynamics with increasing temperature, increasing salt concentration, and increasing volume fraction of ethylene oxide side chains in the polymer matrix evolve with the same trends in both measurement techniques. We conclude from this correlation that trEFM data reflect, at the nanoscale, the same ionic processes probed in conventional EIS at the device level. Finally, as an example application for emerging materials syntheses, we use trEFM and infrared photoinduced force microscopy (PiFM) to image a diblock copolymer electrolyte for next-generation solid-state energy storage applications.

Identifying Nanoscale Structure-Function Relationships Using Multimodal Atomic Force Microscopy, Dimensionality Reduction, and Regression Techniques.

Correlating nanoscale chemical specificity with operational physics is a long-standing goal of functional scanning probe microscopy (SPM). We employ a data analytic approach combining multiple microscopy modes using compositional information in infrared vibrational excitation maps acquired via photoinduced force microscopy (PiFM) with electrical information from conductive atomic force microscopy. We study a model polymer blend comprising insulating poly(methyl methacrylate) (PMMA) and semiconducting poly(3-hexylthiophene) (P3HT). We show that PiFM spectra are different from FTIR spectra but can still be used to identify local composition. We use principal component analysis to extract statistically significant principal components and principal component regression to predict local current and identify local polymer composition. In doing so, we observe evidence of semiconducting P3HT within PMMA aggregates. These methods are generalizable to correlated SPM data and provide a meaningful technique for extracting complex compositional information that is impossible to measure from any one technique.

Palliative Care: A Partnership Across the Continuum of Care.

Palliative care services are becoming more prevalent in the United States as greater portions of the population are requiring end-of-life services. Furthermore, recent policy changes and service foci have promoted more continuity and encompassing care. This study evaluates characteristics that distinguish hospitals with a palliative care program from hospitals without such a program in order to better define the markets and environments that promote the creation and usage of these programs. This study demonstrates that palliative care programs are more likely in communities with favorable economic factors and higher Medicare populations. Large hospitals with high occupancy rates and a high case mix index use palliative care programs to better meet patient needs and improve hospital efficiency. Managerial, nursing, and policy implications are discussed relating to further usage and implementation of palliative care programs.

Fast time-resolved electrostatic force microscopy: Achieving sub-cycle time resolution.

The ability to measure microsecond- and nanosecond-scale local dynamics below the diffraction limit with widely available atomic force microscopy hardware would enable new scientific studies in fields ranging from biology to semiconductor physics. However, commercially available scanning-probe instruments typically offer the ability to measure dynamics only on time scales of milliseconds to seconds. Here, we describe in detail the implementation of fast time-resolved electrostatic force microscopy using an oscillating cantilever as a means to measure fast local dynamics following a perturbation to a sample. We show how the phase of the oscillating cantilever relative to the perturbation event is critical to achieving reliable sub-cycle time resolution. We explore how noise affects the achievable time resolution and present empirical guidelines for reducing noise and optimizing experimental parameters. Specifically, we show that reducing the noise on the cantilever by using photothermal excitation instead of piezoacoustic excitation further improves time resolution. We demonstrate the discrimination of signal rise times with time constants as fast as 10 ns, and simultaneous data acquisition and analysis for dramatically improved image acquisition times.