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Breast cancer is a heterogenous disease with variability in tumor cells and in the surrounding tumor microenvironment (TME). Understanding the molecular diversity in breast cancer is critical for improving prediction of therapeutic response and prognostication. High-plex spatial profiling of tumors enables characterization of heterogeneity in the breast TME, which can holistically illuminate the biology of tumor growth, dissemination and, ultimately, response to therapy. The GeoMx Digital Spatial Profiler (DSP) enables researchers to spatially resolve and quantify proteins and RNA transcripts from tissue sections. The platform is compatible with both formalin-fixed paraffin-embedded and frozen tissues. RNA profiling was developed at the whole transcriptome level for human and mouse samples and protein profiling of 100-plex for human samples. Tissue can be optically segmented for analysis of regions of interest or cell populations to study biology-directed tissue characterization. The GeoMx Breast Cancer Consortium (GBCC) is composed of breast cancer researchers who are developing innovative approaches for spatial profiling to accelerate biomarker discovery. Here, the GBCC presents best practices for GeoMx profiling to promote the collection of high-quality data, optimization of data analysis and integration of datasets to advance collaboration and meta-analyses. Although the capabilities of the platform are presented in the context of breast cancer research, they can be generalized to a variety of other tumor types that are characterized by high heterogeneity.

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 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 fullyhydrated 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.

A novel freeze-cast porous chitosan conduit for peripheral nerve repair with highly-aligned, double layered porosity, which provides the ideal mechanical and chemical properties was designed, manufactured, and assessed in vivo. Efficacies of the conduit and the control inverted nerve autograft were evaluated in bridging 10-mm Lewis rat sciatic nerve gap at 12 weeks post-implantation. Biocompatibility and regenerative efficacy of the porous chitosan conduit were evaluated through the histomorphometric analysis of longitudinal and transverse sections. The porous chitosan conduit was found to have promising regenerative characteristics, promoting the desired neovascularization, and axonal ingrowth and alignment through a combination of structural, mechanical and chemical cues.

The SARS-CoV-2 pandemic has caused over 1 million deaths globally, mostly due to acute lung injury and acute respiratory distress syndrome, or direct complications resulting in multiple-organ failures. Little is known about the host tissue immune and cellular responses associated with COVID-19 infection, symptoms, and lethality. To address this, we collected tissues from 11 organs during the clinical autopsy of 17 individuals who succumbed to COVID-19, resulting in a tissue bank of approximately 420 specimens. We generated comprehensive cellular maps capturing COVID-19 biology related to patients demise through single-cell and single-nucleus RNA-Seq of lung, kidney, liver and heart tissues, and further contextualized our findings through spatial RNA profiling of distinct lung regions. We developed a computational framework that incorporates removal of ambient RNA and automated cell type annotation to facilitate comparison with other healthy and diseased tissue atlases. In the lung, we uncovered significantly altered transcriptional programs within the epithelial, immune, and stromal compartments and cell intrinsic changes in multiple cell types relative to lung tissue from healthy controls. We observed evidence of: alveolar type 2 (AT2) differentiation replacing depleted alveolar type 1 (AT1) lung epithelial cells, as previously seen in fibrosis; a concomitant increase in myofibroblasts reflective of defective tissue repair; and, putative TP63+ intrapulmonary basal-like progenitor (IPBLP) cells, similar to cells identified in H1N1 influenza, that may serve as an emergency cellular reserve for severely damaged alveoli. Together, these findings suggest the activation and failure of multiple avenues for regeneration of the epithelium in these terminal lungs. SARS-CoV-2 RNA reads were enriched in lung mononuclear phagocytic cells and endothelial cells, and these cells expressed distinct host response transcriptional programs. We corroborated the compositional and transcriptional changes in lung tissue through spatial analysis of RNA profiles in situ and distinguished unique tissue host responses between regions with and without viral RNA, and in COVID-19 donor tissues relative to healthy lung. Finally, we analyzed genetic regions implicated in COVID-19 GWAS with transcriptomic data to implicate specific cell types and genes associated with disease severity. Overall, our COVID-19 cell atlas is a foundational dataset to better understand the biological impact of SARS-CoV-2 infection across the human body and empowers the identification of new therapeutic interventions and prevention strategies.

Few systematic structure-property-processing correlations for directionally freeze-cast biopolymer scaffolds are reported. Such correlations are critical to enable scaffold design with attractive structural and mechanical cues in vivo. This study focuses on freeze-cast collagen scaffolds with three different applied cooling rates (10, 1, and 0.1°C/min) and two freezing directions (longitudinal and radial). A semi-automated approach for structural characterization of fully hydrated scaffolds by confocal microscopy is developed to facilitate an objective quantification and comparison of structural features. Additionally, scanning electron microscopy, and compression testing are performed longitudinally and transversely. Structural and mechanical properties are determined on dry and fully hydrated scaffolds. Longitudinally-frozen scaffold have aligned and regular pores while those in radially-frozen ones exhibit greater variations in pore geometry and alignment. Lamellar spacing, pore area, and cell wall thickness increase with decreasing cooling rate: in longitudinally-frozen scaffolds from 25 μm to 83.5 μm, 814 μm 2 to 8,452 μm 2 , and 4.21 μm to 10.4 μm, and in radially-frozen ones, from 69 μm to 116 μm, 7,679 μm 2 to 25,670 μm 2 , and 6.18 μm to 13.6 μm, respectively. Both longitudinally-and radially-frozen scaffolds possess higher mechanical property values, when loaded parallel rather than perpendicular to the ice-crystal growth direction. Modulus and yield strength range from 779-4,700 kPa and 38-137 kPa, respectively, as a function of cooling rate and freezing direction. Collated, the correlations obtained in this study enable the custom-design of freeze-cast collagen scaffolds, which are ideally suited for a large variety of tissue regeneration applications.