Association of Tumor-informed Circulating Tumor DNA Detectability Before and After Radical Cystectomy with Disease-free Survival in Patients with Bladder Cancer.

BACKGROUND AND OBJECTIVE: Despite curative-intent radical cystectomy (RC), patients with muscle-invasive bladder cancer (MIBC) are at high risk of recurrence. Biomarkers are urgently needed to refine prognostication and selection of appropriate perioperative systemic therapies. Our aim was to evaluate the prognostic and predictive value of tumor-informed circulating tumor DNA (ctDNA) results in a multicenter cohort of patients with bladder cancer who underwent RC. METHODS: We performed a retrospective analysis of real-world data for a commercial ctDNA test (Signatera; Natera, Austin, TX, USA) performed in 167 patients (852 plasma samples) before RC and during molecular residual disease (MRD; adjuvant decision) and surveillance windows. We assessed the correlation between recurrence and ctDNA status before and after RC using Cox regression analysis. RESULTS AND LIMITATIONS: During study-defined postoperative MRD and surveillance windows, detectable ctDNA was associated with shorter disease-free survival (DFS) when compared to undetectable ctDNA (MRD: hazard ratio 6.93; p < 0.001; surveillance: hazard ratio 23.02; p < 0.001). Of note, patients with undetectable ctDNA did not appear to benefit from adjuvant therapy (p = 0.34). Detectable ctDNA in the pre-RC (p = 0.045), MRD (p = 0.002), and surveillance (p < 0.001) windows was the only risk factor independently associated with shorter DFS. Limitations include the retrospective and nonrandomized nature of the study. CONCLUSIONS: ctDNA testing in patients with bladder cancer undergoing RC was prognostic and potentially predictive. Identification of patients at high risk of recurrence may aid in patient counseling and decision-making. PATIENT SUMMARY: We found that outcomes for patients with muscle-invasive bladder cancer are strongly linked to detection of tumor DNA in blood samples. The results show the value of tumor-informed testing for tumor DNA in blood for decisions on the best treatment for each individual patient.

Longitudinal Monitoring of Circulating Tumor DNA to Assess the Efficacy of Immune Checkpoint Inhibitors in Patients With Advanced Genitourinary Malignancies.

PURPOSE: Circulating tumor DNA (ctDNA) detection in blood has emerged as a prognostic and predictive biomarker demonstrating improved assessment of treatment response in patients receiving immune checkpoint inhibitors (ICIs). Here, we performed a pilot study to support the role of ctDNA for longitudinal treatment response monitoring in patients with advanced genitourinary (GU) malignancies receiving ICIs. MATERIALS AND METHODS: Patients with histologically confirmed advanced GU malignancies were prospectively enrolled. All eligible patients received ICI treatment for at least 12 weeks, followed by serial collection of blood samples every 6-8 weeks and conventional scans approximately every 12 weeks until disease progression. ctDNA analysis was performed using Signatera, a tumor-informed multiplex-polymerase chain reaction next-generation sequencing assay. Overall, the objective response rate (ORR) was reported and its association with ctDNA status was evaluated. Concordance rate between ctDNA dynamics and conventional imaging was also assessed. RESULTS: ctDNA analysis was performed on 98 banked plasma samples from 20 patients (15 renal, four urothelial, and one prostate). The median follow-up from the time of initiation of ICI to progressive disease (PD) or data cutoff was 67.7 weeks (range, 19.6-169.6). The ORR was 70% (14/20). Eight patients ultimately developed PD. The overall concordance between ctDNA dynamics and radiographic response was observed in 83% (15/18) of patients. Among the three patients with discordant results, two developed CNS metastases and one progressed with extracranial systemic disease while ctDNA remained undetectable. CONCLUSION: In this pilot study, longitudinal ctDNA analysis for monitoring response to ICI in patients with advanced GU tumors was feasible. Larger prospective studies are warranted to validate the utility of ctDNA as an ICI response monitoring tool in patients with advanced GU malignancies.

Evaluation of M2-like macrophage enrichment after diffuse traumatic brain injury through transient interleukin-4 expression from engineered mesenchymal stromal cells.

BACKGROUND: Appropriately modulating inflammation after traumatic brain injury (TBI) may prevent disabilities for the millions of those inflicted annually. In TBI, cellular mediators of inflammation, including macrophages and microglia, possess a range of phenotypes relevant for an immunomodulatory therapeutic approach. It is thought that early phenotypic modulation of these cells will have a cascading healing effect. In fact, an anti-inflammatory, "M2-like" macrophage phenotype after TBI has been associated with neurogenesis, axonal regeneration, and improved white matter integrity (WMI). There already exist clinical trials seeking an M2-like bias through mesenchymal stem/stromal cells (MSCs). However, MSCs do not endogenously synthesize key signals that induce robust M2-like phenotypes such as interleukin-4 (IL-4). METHODS: To enrich M2-like macrophages in a clinically relevant manner, we augmented MSCs with synthetic IL-4 mRNA to transiently express IL-4. These IL-4 expressing MSCs (IL-4 MSCs) were characterized for expression and functionality and then delivered in a modified mouse TBI model of closed head injury. Groups were assessed for functional deficits and MR imaging. Brain tissue was analyzed through flow cytometry, multi-plex ELISA, qPCR, histology, and RNA sequencing. RESULTS: We observed that IL-4 MSCs indeed induce a robust M2-like macrophage phenotype and promote anti-inflammatory gene expression after TBI. However, here we demonstrate that acute enrichment of M2-like macrophages did not translate to improved functional or histological outcomes, or improvements in WMI on MR imaging. To further understand whether dysfunctional pathways underlie the lack of therapeutic effect, we report transcriptomic analysis of injured and treated brains. Through this, we discovered that inflammation persists despite acute enrichment of M2-like macrophages in the brain. CONCLUSION: The results demonstrate that MSCs can be engineered to induce a stronger M2-like macrophage response in vivo. However, they also suggest that acute enrichment of only M2-like macrophages after diffuse TBI cannot orchestrate neurogenesis, axonal regeneration, or improve WMI. Here, we also discuss our modified TBI model and methods to assess severity, behavioral studies, and propose that IL-4 expressing MSCs may also have relevance in other cavitary diseases or in improving biomaterial integration into tissues.

MicroRNA-mRNA Interactions at Low Levels of Compressive Solid Stress Implicate mir-548 in Increased Glioblastoma Cell Motility.

Glioblastoma (GBM) is an astrocytic brain tumor with median survival times of <15 months, primarily as a result of high infiltrative potential and development of resistance to therapy (i.e., surgical resection, chemoradiotherapy). A prominent feature of the GBM microenvironment is compressive solid stress (CSS) caused by uninhibited tumor growth within the confined skull. Here, we utilized a mechanical compression model to apply CSS (<115 Pa) to well-characterized LN229 and U251 GBM cell lines and measured their motility, morphology, and transcriptomic response. Whereas both cell lines displayed a peak in migration at 23 Pa, cells displayed differential response to CSS with either minimal (i.e., U251) or large changes in motility (i.e., LN229). Increased migration of LN229 cells was also correlated to increased cell elongation. These changes were tied to epigenetic signaling associated with increased migration and decreases in proliferation predicted via Ingenuity® Pathway Analysis (IPA), characteristics associated with tumor aggressiveness. miRNA-mRNA interaction analysis revealed strong influence of the miR548 family (i.e., mir-548aj, mir-548az, mir-548t) on differential signaling induced by CSS, suggesting potential targets for pharmaceutical intervention that may improve patient outcomes.

Effect of Electrospun Fiber Mat Thickness and Support Method on Cell Morphology.

Electrospun fiber mats (EFMs) are highly versatile biomaterials used in a myriad of biomedical applications. Whereas some facets of EFMs are well studied and can be highly tuned (e.g., pore size, fiber diameter, etc.), other features are under characterized. For example, although substrate mechanics have been explored by several groups, most studies rely on Young's modulus alone as a characterization variable. The influence of fiber mat thickness and the effect of supports are variables that are often not considered when evaluating cell-mechanical response. To assay the role of these features in EFM scaffold design and to improve understanding of scaffold mechanical properties, we designed EFM scaffolds with varying thickness (50-200 µm) and supporting methodologies. EFM scaffolds were comprised of polycaprolactone and were either electrospun directly onto a support, suspended across an annulus (3 or 10 mm inner diameter), or "tension-released" and then suspended across an annulus. Then, single cell spreading (i.e., Feret diameter) was measured in the presence of these different features. Cells were sensitive to EFM thickness and suspended gap diameter. Overall, cell spreading was greatest for 50 µm thick EFMs suspended over a 3 mm gap, which was the smallest thickness and gap investigated. These results are counterintuitive to conventional understanding in mechanobiology, which suggests that stiffer materials, such as thicker, supported EFMs, should elicit greater cell polarization. Additional experiments with 50 µm thick EFMs on polystyrene and polydimethylsiloxane (PDMS) supports demonstrated that cells can "feel" the support underlying the EFM if it is rigid, similar to previous results in hydrogels. These results also suggest that EFM curvature may play a role in cell response, separate from Young's modulus, possibly because of internal tension generated. These parameters are not often considered in EFM design and could improve scaffold performance and ultimately patient outcomes.

Beyond Linear Elastic Modulus: Viscoelastic Models for Brain and Brain Mimetic Hydrogels.

With their high degree of specificity and investigator control, in vitro disease models provide a natural complement to in vivo models. Especially in organs such as the brain, where anatomical limitations make in vivo experiments challenging, in vitro models have been increasingly used to mimic disease pathology. However, brain mimetic models may not fully replicate the mechanical environment in vivo, which has been shown to influence a variety of cell behaviors. Specifically, many disease models consider only the linear elastic modulus of brain, which describes the stiffness of a material with the assumption that mechanical behavior is independent of loading rate. Here, we characterized porcine brain tissue using a modified stress relaxation test, and across a panel of viscoelastic models, showed that stiffness depends on loading rate. As such, the linear elastic modulus does not accurately reflect the viscoelastic properties of native brain. Among viscoelastic models, the Maxwell model was selected for further analysis because of its simplicity and excellent curve fit (R2 = 0.99 ± 0.0006). Thus, mechanical response of native brain and hydrogel mimetic models was analyzed using the Maxwell model and the linear elastic model to evaluate the effects of strain rate, time post mortem, region, tissue type (i.e., bulk brain vs white matter), and in brain mimetic models, hydrogel composition, on observed mechanical properties. In comparing the Maxwell and linear elastic models, linear elastic modulus is consistently lower than the Maxwell elastic modulus across all brain regions. Additionally, the Maxwell model is sensitive to changes in viscosity and small changes in elasticity, demonstrating improved fidelity. These findings demonstrate the insufficiency of linear elastic modulus as a primary mechanical characterization for brain mimetic materials and provide quantitative information toward the future design of materials that more closely mimic mechanical features of brain.

Micelle-templated, poly(lactic-co-glycolic acid) nanoparticles for hydrophobic drug delivery.

PURPOSE: Poly(lactic-co-glycolic acid) (PLGA) is widely used for drug delivery because of its biocompatibility, ability to solubilize a wide variety of drugs, and tunable degradation. However, achieving sub-100 nm nanoparticles (NPs), as might be desired for delivery via the enhanced permeability and retention effect, is extremely difficult via typical top-down emulsion approaches. METHODS: Here, we present a bottom-up synthesis method yielding PLGA/block copolymer hybrids (ie, "PolyDots"), consisting of hydrophobic PLGA chains entrapped within self-assembling poly(styrene-b-ethylene oxide) (PS-b-PEO) micelles. RESULTS: PolyDots exhibit average diameters <50 nm and lower polydispersity than conventional PLGA NPs. Drug encapsulation efficiencies of PolyDots match conventional PLGA NPs (ie, ~30%) and are greater than those obtained from PS-b-PEO micelles (ie, ~7%). Increasing the PLGA:PS-b-PEO weight ratio alters the drug release mechanism from chain relaxation to erosion controlled. PolyDots are taken up by model glioma cells via endocytotic mechanisms within 24 hours, providing a potential means for delivery to cytoplasm. PolyDots can be lyophilized with minimal change in morphology and encapsulant functionality, and can be produced at scale using electrospray. CONCLUSION: Encapsulation of PLGA within micelles provides a bottom-up route for the synthesis of sub-100 nm PLGA-based nanocarriers with enhanced stability and drug-loading capacity, and tunable drug release, suitable for potential clinical applications.

Hydrogels That Allow and Facilitate Bone Repair, Remodeling, and Regeneration.

Bone defects can originate from a variety of causes, including trauma, cancer, congenital deformity, and surgical reconstruction. Success of the current "gold standard" treatment (i.e., autologous bone grafts) is greatly influenced by insufficient or inappropriate bone stock. There is thus a critical need for the development of new, engineered materials for bone repair. This review describes the use of natural and synthetic hydrogels as scaffolds for bone tissue engineering. We discuss many of the advantages that hydrogels offer as bone repair materials, including their potential for osteoconductivity, biodegradability, controlled growth factor release, and cell encapsulation. We also discuss the use of hydrogels in composite devices with metals, ceramics, or polymers. These composites are useful because of the low mechanical moduli of hydrogels. Finally, the potential for thermosetting and photo-cross-linked hydrogels as three-dimensionally (3D) printed, patient-specific devices is highlighted. Three-dimensional printing enables controlled spatial distribution of scaffold materials, cells, and growth factors. Hydrogels, especially natural hydrogels present in bone matrix, have great potential to augment existing bone tissue engineering devices for the treatment of critical size bone defects.

Effects of hydrophobicity and mat thickness on release from hydrogel-electrospun fiber mat composites.

Poly(ethylene glycol) (PEG)-based hydrogel-electrospun fiber mat (EFM) composites are a promising new controlled release system for hydrophilic drugs, providing longer and more linear release characteristics accompanied by a smaller initial burst than traditional hydrogel systems. However, the effect of EFM properties on release characteristics has not yet been examined. Here, we investigated the influence of EFM thickness and hydrophobicity on swelling and release behavior using bovine serum albumin as a model hydrophilic protein. EFMs investigated were comprised of poly(ε-caprolactone) (PCL) at thicknesses of 300, 800, or 1100 µm. Hydrophobicity was adjusted through surface modification: fluorinated PCL, core/shell PCL/PEGPCL, and acrylic acid (AAc)-treated PCL EFMs were examined. EFMs comprised of the external composite surface, forming a sandwich around PEG-poly(lactic acid) (PEGPLA) hydrogels, and significantly restrained hydrogel swelling in the radial direction while increasing swelling in the axial direction. Incorporation of EFMs also reduced initial hydrophilic protein release rates and extended the duration of release. Increased EFM thickness and hydrophobicity were equally correlated with longer and more linear release profiles. Increased thickness most likely increases the diffusional path length, whereas increased hydrophobicity hinders hydrophilic drug diffusion. These composites form a promising new class of tunable release materials having properties superior to those of unmodified hydrogels.