An in vitro CRISPR screen of cell-free DNA identifies apoptosis as the primary mediator of cell-free DNA release.

ABTRACT: Clinical circulating cell-free DNA (cfDNA) testing is now routine, however test accuracy remains limited. By understanding the life-cycle of cfDNA, we might identify opportunities to increase test performance. Here, we profile cfDNA release across a 24-cell line panel and utilize a cell-free CRISPR screen (cfCRISPR) to identify mediators of cfDNA release. Our panel outlines two distinct groups of cell lines: one which releases cfDNA fragmented similarly to clinical samples and purported as characteristic of apoptosis, and another which releases larger fragments associated with vesicular or necrotic DNA. Our cfCRISPR screens reveal that genes mediating cfDNA release are primarily involved with apoptosis, but also identify other subsets of genes such as RNA binding proteins as potential regulators of cfDNA release. We observe that both groups of cells lines identified primarily produce cfDNA through apoptosis. These results establish the utility of cfCRISPR, genetically validate apoptosis as a major mediator of DNA release in vitro, and implicate ways to improve cfDNA assays.

Correlates of Taxane-Induced Neuropathy, an Electronic Health Record Based Observational Study.

BACKGROUND: Chemotherapy-induced peripheral neuropathy (CIPN) is a common therapeutic complication affecting cancer patients' quality-of-life. We evaluated clinical characteristics, demographics, and lifestyle factors in association with CIPN following taxane treatment. METHODS: Data were extracted from the electronic health record of 3387 patients diagnosed with a primary cancer and receiving taxane (i.e., paclitaxel or docetaxel) at Vanderbilt University Medical Center. Neuropathy was assessed via a validated computer algorithm. Univariate and multivariate regression models were applied to evaluate odds ratios (ORs) and 95% confidence intervals (CIs) of CIPN-associated factors. RESULTS: Female sex (OR = 1.28, 95% CI = 1.01-1.62), high body-mass index (BMI) (OR = 1.31, 95% CI = 1.06-1.61 for overweight, and OR = 1.49, 95% CI = 1.21-1.83 for obesity), diabetes (OR = 1.66, 95% CI = 1.34-2.06), high mean taxane dose (OR = 1.05, 95% CI = 1.03-1.08 per 10 mg/m2), and more treatment cycles (1.12, 95% CI = 1.10-1.14) were positively associated with CIPN. Concurrent chemotherapy (OR = 0.74, 95% CI = 0.58-0.94) and concurrent radiotherapy (OR = 0.77, 95% CI = 0.59-1.00) were inversely associated with CIPN. Obesity and diabetes both had a stronger association with docetaxel CIPN compared to paclitaxel, although interaction was only significant for diabetes and taxane (p = 0.019). Increased BMI was associated with CIPN only among non-diabetic patients (OR:1.34 for overweight and 1.68 for obesity), while diabetes increased CIPN risk across all BMI strata (ORs were 2.65, 2.41, and 2.15 for normal weight, overweight, and obese, respectively) compared to normal-weight non-diabetic patients (p for interaction = 0.039). CONCLUSIONS: Female sex, obesity, and diabetes are significantly associated with taxine-induced CIPN. Further research is needed to identify clinical and pharmacologic strategies to prevent and mitigate CIPN in at-risk patient populations.

Patient-specific comorbidities as prognostic variables for survival in myelofibrosis.

Treatment decisions in primary myelofibrosis (PMF) are guided by numerous prognostic systems. Patient-specific comorbidities have influence on treatment-related survival and are considered in clinical contexts but have not been routinely incorporated into current prognostic models. We hypothesized that patient-specific comorbidities would inform prognosis and could be incorporated into a quantitative score. All patients with PMF or secondary myelofibrosis with available DNA and comprehensive electronic health record (EHR) data treated at Vanderbilt University Medical Center between 1995 and 2016 were identified within Vanderbilt's Synthetic Derivative and BioVU Biobank. We recapitulated established PMF risk scores (eg, Dynamic International Prognostic Scoring System [DIPSS], DIPSS plus, Genetics-Based Prognostic Scoring System, Mutation-Enhanced International Prognostic Scoring System 70+) and comorbidities through EHR chart extraction and next-generation sequencing on biobanked peripheral blood DNA. The impact of comorbidities was assessed via DIPSS-adjusted overall survival using Bonferroni correction. Comorbidities associated with inferior survival include renal failure/dysfunction (hazard ratio [HR], 4.3; 95% confidence interval [95% CI], 2.1-8.9; P = .0001), intracranial hemorrhage (HR, 28.7; 95% CI, 7.0-116.8; P = 2.83e-06), invasive fungal infection (HR, 41.2; 95% CI, 7.2-235.2; P = 2.90e-05), and chronic encephalopathy (HR, 15.1; 95% CI, 3.8-59.4; P = .0001). The extended DIPSS model including all 4 significant comorbidities showed a significantly higher discriminating power (C-index 0.81; 95% CI, 0.78-0.84) than the original DIPSS model (C-index 0.73; 95% CI, 0.70-0.77). In summary, we repurposed an institutional biobank to identify and risk-classify an uncommon hematologic malignancy by established (eg, DIPSS) and other clinical and pathologic factors (eg, comorbidities) in an unbiased fashion. The inclusion of comorbidities into risk evaluation may augment prognostic capability of future genetics-based scoring systems.

Insights From Dynamic Neuro-Immune Imaging on Murine Immune Responses to CNS Damage.

Evolving technologies and increasing understanding of human physiology over the past century have afforded our ability to intervene on human diseases using implantable bio-materials. These bio-electronic devices present a unique challenge through the creation of an interface between the native tissue and implantable bio-materials: the generation of host immune response surrounding such devices. While recent developments in cancer immunology seek to stimulate the immune system against cancer, successful long-term application of implantable bio-material devices need to durably minimize reactive immune processes at involved anatomical sites. Peripheral immune system response has been studied extensively for implanted bio-materials at various body sites. Examples include tooth composites (Gitalis et al., 2019), inguinal hernia repair (Heymann et al., 2019), and cardiac stents and pacemaker leads (Slee et al., 2016). Studies have also been extended to less well-studied immune reactivity in response to CNS neural-electronic implant devices. Recent technological advances in 2-Photon Laser Scanning Microscopy (2P-LSM) have allowed novel insights into in vivo immune response in a variety of tissue microenvironments. While imaging of peripheral tissues has provided an abundance of data with regards to immune cell dynamics, central nervous system (CNS) imaging is comparatively complicated by tissue accessibility and manipulation. Despite these challenges, the results of dynamic intravital neuro-immune imaging thus far have provided foundational insights into basic CNS biology. Utilizing a combination of intravital and ex vivo 2P-LSM, we have observed novel pathways allowing immune cells, stromal cells, cancer cells and proteins to communicate between the CNS parenchyma and peripheral vasculature. Similar to what has been reported in the intestinal tract, we have visualized myeloid cells extend dendritic processes across the blood brain barrier (BBB) into pial blood vessels. Furthermore, transient vessel leaks seen during systemic inflammation provide opportunities for cellular protein to be exchanged between the periphery and CNS. These insights provide new, visual information regarding immune surveillance and antigen presentation within the CNS. Furthermore, when combining intravital 2P-LSM and microfluidic devices complexed with mathematical modeling, we are gaining new insights into the intravascular behavior of circulating immune cells. This new knowledge into the basic mechanisms by which cells migrate to and interact with the CNS provide important considerations for the design of neuro-electronic biomaterials that have the potential to connect the peripheral-neural microenvironments into a unique, artificial interface.

Biomimetic post-capillary venule expansions for leukocyte adhesion studies.

Leukocyte adhesion and extravasation are maximal near the transition from capillary to post-capillary venule, and are strongly influenced by a confluence of scale-dependent physical effects. Mimicking the scale of physiological vessels using in vitro microfluidic systems allows the capture of these effects on leukocyte adhesion assays, but imposes practical limits on reproducibility and reliable quantification. Here we present a microfluidic platform that provides multiple (54-512) technical replicates within a 15-minute sample collection time, coupled with an automated computer vision analysis pipeline that captures leukocyte adhesion probabilities as a function of shear and extensional stresses. We report that in post-capillary channels of physiological scale, efficient leukocyte adhesion requires erythrocytes forcing leukocytes against the wall, a phenomenon that is promoted by the transitional flow in post-capillary venule expansions and dependent on the adhesion molecule ICAM-1.

Cdk5 disruption attenuates tumor PD-L1 expression and promotes antitumor immunity.

Cancers often evade immune surveillance by adopting peripheral tissue- tolerance mechanisms, such as the expression of programmed cell death ligand 1 (PD-L1), the inhibition of which results in potent antitumor immunity. Here, we show that cyclin-dependent kinase 5 (Cdk5), a serine-threonine kinase that is highly active in postmitotic neurons and in many cancers, allows medulloblastoma (MB) to evade immune elimination. Interferon-γ (IFN-γ)-induced PD-L1 up-regulation on MB requires Cdk5, and disruption of Cdk5 expression in a mouse model of MB results in potent CD4(+) T cell-mediated tumor rejection. Loss of Cdk5 results in persistent expression of the PD-L1 transcriptional repressors, the interferon regulatory factors IRF2 and IRF2BP2, which likely leads to reduced PD-L1 expression on tumors. Our finding highlights a central role for Cdk5 in immune checkpoint regulation by tumor cells.

Transient Surface CCR5 Expression by Naive CD8+ T Cells within Inflamed Lymph Nodes Is Dependent on High Endothelial Venule Interaction and Augments Th Cell-Dependent Memory Response.

In inflamed lymph nodes, Ag-specific CD4(+) and CD8(+) T cells encounter Ag-bearing dendritic cells and, together, this complex enhances the release of CCL3 and CCL4, which facilitate additional interaction with naive CD8(+) T cells. Although blocking CCL3 and CCL4 has no effect on primary CD8(+) T cell responses, it dramatically impairs the development of memory CD8(+) T cells upon Ag rechallenge. Despite the absence of detectable surface CCR5 expression on circulating native CD8(+) T cells, these data imply that naive CD8(+) T cells are capable of expressing surface CCR5 prior to cognate Ag-induced TCR signaling in inflamed lymph nodes; however, the molecular mechanisms have not been characterized to date. In this study, we show that CCR5, the receptor for CCL3 and CCL4, can be transiently upregulated on a subset of naive CD8(+) T cells and that this upregulation is dependent on direct contact with the high endothelial venule in inflamed lymph node. Binding of CD62L and CD11a on T cells to their ligands CD34 and CD54 on the high endothelial venule can be enhanced during inflammation. This enhanced binding and subsequent signaling promote the translocation of CCR5 molecules from intracellular vesicles to the surface of the CD8(+) T cell. The upregulation of CCR5 on the surface of the CD8(+) T cells increases the number of contacts with Ag-bearing dendritic cells, which ultimately results in increased CD8(+) T cell response to Ag rechallenge.

Multiple Administrations of Viral Nanoparticles Alter in Vivo Behavior-Insights from Intravital Microscopy.

Multiple administrations of nanoparticle-based formulations are often a clinical requirement for drug delivery and diagnostic imaging applications. Steady pharmacokinetics of nanoparticles is desirable to achieve efficient therapeutic or diagnostic outcomes over such repeat administrations. While clearance through mononuclear phagocytic system is a key determinant of nanoparticle persistence in vivo, multiple administrations could potentially result in altered pharmacokinetics by evoking innate or adaptive immune responses. Plant viral nanoparticles (VNPs) represent an emerging class of programmable nanoparticle platform technologies that offer a highly organized proteinaceous architecture and multivalency for delivery of large payloads of drugs and molecular contrast agents. These very structural features also render them susceptible to immune recognition and subsequent accelerated systemic clearance that could potentially affect overall efficiency. While the biodistribution and pharmacokinetics of VNPs have been reported, the biological response following repeat administrations remains an understudied area of investigation. Here, we demonstrate that weekly administration of filamentous plant viruses results in the generation of increasing levels of circulating, carrier-specific IgM and IgG antibodies. Furthermore, PVX specific immunoglobulins from the serum of immunized animals quickly form aggregates when incubated with PVX in vitro. Such aggregates of VNP-immune complexes are also observed in the mouse vasculature in vivo following repeat injections when imaged in real time using intravital two-photon laser scanning microscopy (2P-LSM). The size of aggregates diminishes at later time points, coinciding with antibody class switching from IgM to IgG. Together, our results highlight the need for careful in vivo assessment of (viral) nanoparticle-based platform technologies, especially in studying their performance after repeat administration. We also demonstrate the utility of intravital microscopy to aid in this evaluation.

Comparison of intravital thinned skull and cranial window approaches to study CNS immunobiology in the mouse cortex.

Fluorescent imaging coupled with high-resolution femto-second pulsed infrared lasers allows for interrogation of cellular interactions deeper in living tissues than ever imagined. Intra-vital imaging of the central nervous system (CNS) has provided insights into neuronal development, synaptic transmission, and even immune interactions. In this review we will discuss the two most common intravital approaches for studying the cerebral cortex in the live mouse brain for pre-clinical studies, the thinned skull and cranial window techniques, and focus on the advantages and drawbacks of each approach. In addition, we will discuss the use of neuronal physiologic parameters as determinants of successful surgical and imaging preparation.