Although immune checkpoint inhibitor (ICI) therapy has dramatically improved outcome for metastatic melanoma patients, many patients do not benefit. Since adverse events may be severe, biomarkers for ...resistance would be valuable, especially in the adjuvant setting. We performed high-plex digital spatial profiling (DSP) using the NanoString GeoMx® on 53 pre-treatment specimens from ICI-treated metastatic melanoma cases. We interrogated 77 targets simultaneously in four molecular compartments defined by S100B for tumor, CD68 for macrophages, CD45 for leukocytes, and nonimmune stromal cells defined as regions negative for all three compartment markers but positive for SYTO 13. For DSP validation, we confirmed the results obtained for some immune markers, such as CD8, CD4, CD20, CD68, CD45, and PD-L1, by quantitative immunofluorescence (QIF). In the univariable analysis, 38 variables were associated with outcome, 14 of which remained significant after multivariable adjustment. Among them, CD95 was further validated using multiplex immunofluorescence in the Discovery immunotherapy (ITX) Cohort and an independent validation cohort with similar characteristics, showing an association between high levels of CD95 and shorter progression-free survival. We found that CD95 in stroma was associated with resistance to ICI. With further validation, this biomarker could have value to select patients that will not benefit from immunotherapy.
BackgroundThe lung intratumor microbiome influences lung cancer tumorigenesis and treatment responses, but detailed data on the extent, location, and effects of microbes within lung tumors are ...missing, information needed for improved prognosis and treatment.MethodsTo address this gap, we developed a novel spatial meta-transcriptomic method simultaneously detecting the expression level of 1,811 host genes and 3 microbe targets (bacteria, fungi, and cytomegalovirus). After rigorous validation, we analyzed the spatial meta-transcriptomic profiles of tumor cells, T cells, macrophages, other immune cells, and stroma in surgically resected tumor samples from 12 patients with early-stage lung cancer.ResultsBacterial burden was significantly higher in tumor cells compared with T cells, macrophages, other immune cells, and stroma. This burden increased from tumor-adjacent normal lung and tertiary lymphoid structures to tumor cells to the airways, suggesting that lung intratumor bacteria derive from the latter route of entry. Expression of oncogenic β-catenin was strongly correlated with bacterial burden, as were tumor histological subtypes and environmental factors.ConclusionsIntratumor bacteria were enriched with tumor cells and associated with multiple oncogenic pathways, supporting a rationale for reducing the local intratumor microbiome in lung cancer for patient benefit.Trial registration numberNCT00242723, NCT02146170.
BackgroundRelapsed or refractory Acute Myeloid Leukemia (R-AML) is a deadly disease with an inadequate response rate to current treatments. Recent advances in immunotherapy shed light on R-AML, and ...several clinical trials have shown promising potential for combining immune checkpoint inhibitors (ICIs) with hypomethylating agents. A deeper understanding of the tumor-immune microenvironment in R-AML during combination ICI treatment is urgently needed for developing better therapeutics and stratifying treatment strategies.MethodsTo dissect the tumor-immune interactions in the bone marrow microenvironment, we employed nanoString GeoMx Digital Spatial Profiler (DSP) and performed a spatial-transcriptomic analysis of patients with R-AML who received pembrolizumab and decitabine. We compared the transcriptomic profiles and TCR clonalities of tumor-interacting T cells, bystander T cells, and other cells at baseline, post-pembrolizumab treatment, and post-decitabine, which enable us to identify R-AML’s suppressive immune microenvironment and immune cells’ responses to ICI and hypomethylating agent.ResultsWe obtained the spatial-transcriptomic profiles of T cells, stromal cells, and leukemia cells in patients with R-AML at different treatment points. Our TCR-specific probes were able to track T cell clonal changes during treatments.ConclusionsR-AML harbored a complex tumor immune microenvironment and diverse T cell clonality.AcknowledgementsThis research was supported in part by the Intramural Research Program of the NCI (the Center for Cancer Research), NHLBI, and NIH Clinical Center.Ethics ApprovalThis study is approved by NHLBI IRB.
A main impediment to effective development of new therapeutics for central nervous system disorders, and for the
testing of biological hypotheses in the brain, is the ability to rapidly measure the ...effect of novel agents and treatment combinations on the pathophysiology of native brain tissue. We have developed a miniaturized implantable microdevice (IMD) platform, optimized for direct stereotactic insertion into the brain, which enables the simultaneous measurement of multiple drug effects on the native brain tissue
. The IMD contains individual reservoirs which release microdoses of single agents or combinations into confined regions of the brain, with subsequent spatial analysis of phenotypic, transcriptomic or metabolomic effects. Using murine models of Alzheimer's disease (AD), we demonstrate that microdoses of various approved and investigational CNS drugs released from the IMD within a local brain region exhibit
phenotypes indicative of therapeutic responses, such as neuroprotection, reduction of hyperphosphorylation, immune cell modulation, and anti-inflammatory effects. We also show that local treatments with drugs affecting metabolism provide evidence for regulation of metabolite profiles and immune cell function in hMAPT AD mice. The platform should prove useful in facilitating the rapid testing of pharmacological or biological treatment hypotheses directly within native brain tissues (of various animal models and in patients) and help to confirm on-target effects,
pharmacodynamics and drug-induced microenvironment remodeling, much more efficiently than currently feasible.
Presented in this article are systematic microstructural and mechanical property data for anisotropic collagen scaffolds made by freeze casting. Three applied cooling rates (10 °C/min, 1 °C/min, ...0.1 °C/min) and two freezing directions (longitudinal and radial) were used during scaffold manufacture. Utilizing a semi-automated image analysis technique applied to confocal micrographs of fully hydrated scaffolds, pore area, long and short pore axes, and pore aspect ratio were determined. Compression testing was performed to determine scaffold modulus, yield strength, and toughness.
Despite considerable recent interest in micro- and nanofibrillated cellulose as constituents of lightweight structures and scaffolds for applications that range from thermal insulation to filtration, ...few systematic studies have been reported to date on structure-property-processing correlations in freeze-cast chitosan-nanocellulose composite scaffolds, in general, and their application in tissue regeneration, in particular. Reported in this study are the effects of the addition of plant-derived nanocellulose fibrils (CNF), crystals (CNCs), or a blend of the two (CNB) to the biopolymer chitosan on the structure and properties of the resulting composites. Chitosan-nanocellulose composite scaffolds were freeze-cast at 10 and 1 °C/min, and their microstructures were quantified in both the dry and fully hydrated states using scanning electron and confocal microscopy, respectively. The modulus, yield strength, and toughness (work to 60% strain) were determined in compression parallel and the modulus also perpendicular to the freezing direction to quantify anisotropy. Observed were the preferential alignments of CNCs and/or fibrils parallel to the freezing direction. Additionally, observed was the self-assembly of the nanocellulose into microstruts and microbridges between adjacent cell walls (lamellae), features that affected the mechanical properties of the scaffolds. When freeze-cast at 1 °C/min, chitosan-CNF scaffolds had the highest modulus, yield strength, toughness, and smallest anisotropy ratio, followed by chitosan and the composites made with the nanocellulose blend, and that with crystalline cellulose. These results illustrate that the nanocellulose additions homogenize the mechanical properties of the scaffold through cell-wall material self-assembly, on the one hand, and add architectural features such as bridges and pillars, on the other. The latter transfer loads and enable the scaffolds to resist deformation also perpendicular to the freezing direction. The observed property profile and the materials' proven biocompatibility highlight the promise of chitosan-nanocellulose composites for a large range of applications, including those for biomedical implants and devices.
COVID-19, which is caused by SARS-CoV-2, can result in acute respiratory distress syndrome and multiple organ failure
, but little is known about its pathophysiology. Here we generated single-cell ...atlases of 24 lung, 16 kidney, 16 liver and 19 heart autopsy tissue samples and spatial atlases of 14 lung samples from donors who died of COVID-19. Integrated computational analysis uncovered substantial remodelling in the lung epithelial, immune and stromal compartments, with evidence of multiple paths of failed tissue regeneration, including defective alveolar type 2 differentiation and expansion of fibroblasts and putative TP63
intrapulmonary basal-like progenitor cells. Viral RNAs were enriched in mononuclear phagocytic and endothelial lung cells, which induced specific host programs. Spatial analysis in lung distinguished inflammatory host responses in lung regions with and without viral RNA. Analysis of the other tissue atlases showed transcriptional alterations in multiple cell types in heart tissue from donors with COVID-19, and mapped cell types and genes implicated with disease severity based on COVID-19 genome-wide association studies. Our foundational dataset elucidates the biological effect of severe SARS-CoV-2 infection across the body, a key step towards new treatments.
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As a new strategy for improved urinary drainage, in parallel to the potential for additional functions such as drug release and self-removal, highly porous chitosan stents are ...manufactured by radial, bi-directional freeze-casting. Inserting the porous stent in to a silicone tube to emulate its placement in the ureter shows that it is shape conforming and remains safely positioned in place, also during flow tests, including those performed in a peristaltic pump. Cyclic compression tests on fully-hydrated porous stents reveal high stent resilience and close to full elastic recovery upon unloading. The drainage performance of the chitosan stent is evaluated, using effective viscosity in addition to volumetric flow and flux; the porous stent’s performance is compared to that of the straight portion of a commercial 8 Fr double-J stent which possesses, in its otherwise solid tube wall, regularly spaced holes along its length. Both the porous and the 8 Fr stent show higher effective viscosities, when tested in the silicone tube. The performance of the porous stent improves considerably more (47.5%) than that of the 8 Fr stent (30.6%) upon removal from the tube, illustrating the effectiveness of the radially aligned porosity for drainage. We conclude that the newly-developed porous chitosan ureteral stent merits further in vitro and in vivo assessment of its promise as an alternative and complement to currently available medical devices.
No papers, to date, report on porous ureteral stents, which we propose as a new strategy for improved urinary drainage. The highly porous chitosan stents of our study are manufactured by radial, bi-directional freeze casting. Cyclic compression tests on fully-hydrated porous stents revealed high stent resilience and close to full recovery upon unloading. The drainage performance of the chitosan is evaluated, using effective viscosity in addition to volumetric flow and flux, and compared to that of the straight portion of a commercial 8 Fr double-J stent. The performance of the porous stent improves considerably more (47.5%) than that of the 8 Fr stent (30.6%) upon removal from the tube, illustrating the effectiveness of the radially aligned porosity for drainage. While further studies are required to explore other potential benefits of the porous stent design such as antimicrobial behavior, drug release, and biodegradability, we conclude that the newly-developed porous chitosan ureteral stent has considerable potential as a medical device.
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