Spatially Resolved Single-Cell Assessment of Pancreatic Cancer Expression Subtypes Reveals Co-expressor Phenotypes and Extensive Intratumoral Heterogeneity

Pancreatic ductal adenocarcinoma (PDAC) has been classified into classical and basal-like transcriptional subtypes by bulk RNA measurements. However, recent work has uncovered greater complexity to transcriptional subtypes than was initially appreciated using bulk RNA expression profiling. To provide a deeper understanding of PDAC subtypes, we developed a multiplex immunofluorescence (mIF) pipeline that quantifies protein expression of six PDAC subtype markers (CLDN18.2, TFF1, GATA6, KRT17, KRT5, and S100A2) and permits spatially resolved, single-cell interrogation of pancreatic tumors from resection specimens and core needle biopsies. Both primary and metastatic tumors displayed striking intratumoral subtype heterogeneity that was associated with patient outcomes, existed at the scale of individual glands, and was significantly reduced in patient-derived organoid cultures. Tumor cells co-expressing classical and basal markers were present in \u3e 90% of tumors, existed on a basal-classical polarization continuum, and were enriched in tumors containing a greater admixture of basal and classical cell populations. Cell-cell neighbor analyses within tumor glands further suggested that co-expressor cells may represent an intermediate state between expression subtype poles. The extensive intratumoral heterogeneity identified through this clinically applicable mIF pipeline may inform prognosis and treatment selection for patients with PDAC

Microenvironment drives cell state, plasticity, and drug response in pancreatic cancer

Prognostically relevant RNA expression states exist in pancreatic ductal adenocarcinoma (PDAC), but our understanding of their drivers, stability, and relationship to therapeutic response is limited. To examine these attributes systematically, we profiled metastatic biopsies and matched organoid models at single-cell resolution. In vivo, we identify a new intermediate PDAC transcriptional cell state and uncover distinct site- and state-specific tumor microenvironments (TMEs). Benchmarking models against this reference map, we reveal strong culture-specific biases in cancer cell transcriptional state representation driven by altered TME signals. We restore expression state heterogeneity by adding back in vivo-relevant factors and show plasticity in culture models. Further, we prove that non-genetic modulation of cell state can strongly influence drug responses, uncovering state-specific vulnerabilities. This work provides a broadly applicable framework for aligning cell states across in vivo and ex vivo settings, identifying drivers of transcriptional plasticity and manipulating cell state to target associated vulnerabilities

Oxygen regulation of tumor perfusion by S-nitrosohemoglobin reveals a pressor activity of nitric oxide.

In erythrocytes, S-nitrosohemoglobin (SNO-Hb) arises from S-nitrosylation of oxygenated hemoglobin (Hb). It has been shown that SNO-Hb behaves as a nitric oxide (NO) donor at low oxygen tensions. This property, in combination with oxygen transport capacity, suggests that SNO-Hb may have unique potential to reoxygenate hypoxic tissues. The present study was designed to test the idea that the allosteric properties of SNO-Hb could be manipulated to enhance oxygen delivery in a hypoxic tumor. Using Laser Doppler flowmetry, we showed that SNO-Hb infusion to animals breathing 21% O2 reduced tumor perfusion without affecting blood pressure and heart rate. Raising the pO2 (100% O2) slowed the release of NO bioactivity from SNO-Hb (ie, prolonged the plasma half-life of the SNO in Hb), preserved tumor perfusion, and raised the blood pressure. In contrast, native Hb reduced both tumor perfusion and heart rate independently of the oxygen concentration of the inhaled gas, and did not elicit hypertensive effects. Window chamber (to image tumor arteriolar reactivity in vivo) and hemodynamic measurements indicated that the preservation of tissue perfusion by micromolar concentrations of SNO-Hb is a composite effect created by reduced peripheral vascular resistance and direct inhibition of the baroreceptor reflex, leading to increased blood pressure. Overall, these results indicate that the properties of SNO-Hb are attributable to allosteric control of NO release by oxygen in central as well as peripheral issues

Live cell tagging tracking and isolation for spatial transcriptomics using photoactivatable cell dyes

AbstractA cell’s phenotype and function are influenced by dynamic interactions with its microenvironment. To examine cellular spatiotemporal activity, we developed SPACECAT—Spatially PhotoActivatable Color Encoded Cell Address Tags—to annotate, track, and isolate cells while preserving viability. In SPACECAT, samples are stained with photocaged fluorescent molecules, and cells are labeled by uncaging those molecules with user-patterned near-UV light. SPACECAT offers single-cell precision and temporal stability across diverse cell and tissue types. Illustratively, we target crypt-like regions in patient-derived intestinal organoids to enrich for stem-like and actively mitotic cells, matching literature expectations. Moreover, we apply SPACECAT to ex vivo tissue sections from four healthy organs and an autochthonous lung tumor model. Lastly, we provide a computational framework to identify spatially-biased transcriptome patterns and enriched phenotypes. This minimally perturbative and broadly applicable method links cellular spatiotemporal and/or behavioral phenotypes with diverse downstream assays, enabling insights into the connections between tissue microenvironments and (dys)function.</jats:p

The technocratic barrier to wage policy: theoretical insights from the Chilean Concertación

During the Latin American left turn, most governments rapidly understood the importance of committing to macroeconomic equilibriums, successfully managing to combine this goal with a wide array of social policies. Wage policy proved to be a conflictive arena coming from a period of harsh austerity measures. This article provides unique insights, from the Chilean Concertación governments (1990-2010) about the importance intra-left conflicts had in the advancement of labor collective rights. The working hypothesis is that the conflict between party leaders and technocrats alongside a perceived trade-off between growth and distribution is a prime factor for understanding wage reform outcomes. The analysis relies on a mixed-methods approach combining regression analysis and process-tracing. Chile’s labor reform attempts during the Concertación governments, with feeble societal linkages, provides relevant theoretical insights for the understanding of how the abovementioned perceived trade-off may have played in other cases, not only in Latin America but also in other regions of the developing world. The analysis is novel in bringing intra-left conflict back in as an important driver for labor relations reforms and improves our understanding of the political economy of intra-left conflicts during the post-neoliberal period

Enteric Coronavirus Infection and Treatment Modeled With an Immunocompetent Human Intestine-On-A-Chip

Many patients infected with coronaviruses, such as SARS-CoV-2 and NL63 that use ACE2 receptors to infect cells, exhibit gastrointestinal symptoms and viral proteins are found in the human gastrointestinal tract, yet little is known about the inflammatory and pathological effects of coronavirus infection on the human intestine. Here, we used a human intestine-on-a-chip (Intestine Chip) microfluidic culture device lined by patient organoid-derived intestinal epithelium interfaced with human vascular endothelium to study host cellular and inflammatory responses to infection with NL63 coronavirus. These organoid-derived intestinal epithelial cells dramatically increased their ACE2 protein levels when cultured under flow in the presence of peristalsis-like mechanical deformations in the Intestine Chips compared to when cultured statically as organoids or in Transwell inserts. Infection of the intestinal epithelium with NL63 on-chip led to inflammation of the endothelium as demonstrated by loss of barrier function, increased cytokine production, and recruitment of circulating peripheral blood mononuclear cells (PBMCs). Treatment of NL63 infected chips with the approved protease inhibitor drug, nafamostat, inhibited viral entry and resulted in a reduction in both viral load and cytokine secretion, whereas remdesivir, one of the few drugs approved for COVID19 patients, was not found to be effective and it also was toxic to the endothelium. This model of intestinal infection was also used to test the effects of other drugs that have been proposed for potential repurposing against SARS-CoV-2. Taken together, these data suggest that the human Intestine Chip might be useful as a human preclinical model for studying coronavirus related pathology as well as for testing of potential anti-viral or anti-inflammatory therapeutics.</jats:p

Impaired local intrinsic immunity to SARS-CoV-2 infection in severe COVID-19

SARS-CoV-2 infection can cause severe respiratory COVID-19. However, many individuals present with isolated upper respiratory symptoms, suggesting potential to constrain viral pathology to the nasopharynx. Which cells SARS-CoV-2 primarily targets and how infection influences the respiratory epithelium remains incompletely understood. We performed scRNA-seq on nasopharyngeal swabs from 58 healthy and COVID-19 participants. During COVID-19, we observe expansion of secretory, loss of ciliated, and epithelial cell repopulation via deuterosomal cell expansion. In mild and moderate COVID-19, epithelial cells express anti-viral/interferon-responsive genes, while cells in severe COVID-19 have muted anti-viral responses despite equivalent viral loads. SARS-CoV-2 RNA+ host-target cells are highly heterogenous, including developing ciliated, interferon-responsive ciliated, AZGP1high goblet, and KRT13+ “hillock”-like cells, and we identify genes associated with susceptibility, resistance, or infection response. Our study defines protective and detrimental responses to SARS-CoV-2, the direct viral targets of infection, and suggests that failed nasal epithelial anti-viral immunity may underlie and precede severe COVID-19

Genomic and transcriptomic correlates of immunotherapy response within the tumor microenvironment of leptomeningeal metastases

AbstractLeptomeningeal disease (LMD) is a devastating complication of solid tumor malignancies, with dire prognosis and no effective systemic treatment options. Over the past decade, the incidence of LMD has steadily increased due to therapeutics that have extended the survival of cancer patients, highlighting the need for new interventions. To examine the efficacy of immune checkpoint inhibitors (ICI) in patients with LMD, we completed two phase II clinical trials. Here, we investigate the cellular and molecular features underpinning observed patient trajectories in these trials by applying single-cell RNA and cell-free DNA profiling to longitudinal cerebrospinal fluid (CSF) draws from enrolled patients. We recover immune and malignant cell types in the CSF, characterize cell behavior changes following ICI, and identify genomic features associated with relevant clinical phenomena. Overall, our study describes the liquid LMD tumor microenvironment prior to and following ICI treatment and demonstrates clinical utility of cell-free and single-cell genomic measurements for LMD research.</jats:p

Impact of Endoluminal Flow Diverter Number on Aneurysm Treatment Outcomes: A Multicenter Study

Background The purpose of this study is to evaluate the impact multiple overlapping flow‐diverting stents have on aneurysm occlusion rates and iatrogenic complications relative to single flow‐diverting stents. Methods A retrospective review of a multicenter aneurysm database from 2012 to 2020 was performed to identify saccular aneurysms treated initially with single and multiple flow‐diverting stents with ≥12‐month angiographic and clinical follow‐up. Aneurysm occlusion rates as a function of stent number served as a primary outcome measure with iatrogenic complications serving as a secondary outcome measure. Results A total of 250 patients were initially treated with a single Pipeline embolization device (PED), and 48 patients were initially treated with multiple PEDs. There was no significant difference in aneurysm size, morphology, or dual‐antiplatelet therapy regimen used between groups. There was no significant difference in the aneurysm occlusion (single, 83.6%, versus multiple, 83.4%; P=0.65) or retreatment rates (single, 8.0%, versus multiple, 10.4%; P=0.58) between groups. There was no significant difference in the number of procedure‐related complications between groups (single, 8.0%, versus multiple, 4.2%; P=0.42), with 0.8% of patients treated with a single PED and 2.1% of patients treated with multiple PEDs experiencing a procedure‐related ischemic stroke. Conclusions There is no significant difference in overall aneurysm occlusion rates between aneurysms treated initially with single versus multiple overlapping PEDs nor are there significant differences in procedure‐related complications. Single PED flow diversion may be preferred whenever possible, with multiple PED constructs reserved for extenuating clinical circumstances as may be encountered with giant aneurysms

Comparison between transradial and transfemoral mechanical thrombectomy for ICA and M1 occlusions: insights from the Stroke Thrombectomy and Aneurysm Registry (STAR)

BackgroundThe role for the transradial approach for mechanical thrombectomy is controversial. We sought to compare transradial and transfemoral mechanical thrombectomy in a large multicenter database of acute ischemic stroke.MethodsThe prospectively maintained Stroke Thrombectomy and Aneurysm Registry (STAR) was reviewed for patients who underwent mechanical thrombectomy for an internal carotid artery (ICA) or middle cerebral artery M1 occlusion. Multivariate regression analyses were performed to assess outcomes including reperfusion time, symptomatic intracerebral hemorrhage (ICH), distal embolization, and functional outcomes.ResultsA total of 2258 cases, 1976 via the transfemoral approach and 282 via the transradial approach, were included. Radial access was associated with shorter reperfusion time (34.1 min vs 43.6 min, P=0.001) with similar rates of Thrombolysis in Cerebral Infarction (TICI) 2B or greater reperfusion (87.9% vs 88.1%, P=0.246). Patients treated via a transradial approach were more likely to achieve at least TICI 2C (59.6% vs 54.7%, P=0.001) and TICI 3 reperfusion (50.0% vs 46.2%, P=0.001), and had shorter lengths of stay (mean 9.2 days vs 10.2, P<0.001). Patients treated transradially had a lower rate of symptomatic ICH (8.0% vs 9.4%, P=0.047) but a higher rate of distal embolization (23.0% vs 7.1%, P<0.001). There were no significant differences in functional outcome at 90 days between the two groups.ConclusionsRadial and femoral thrombectomy resulted in similar clinical outcomes. In multivariate analysis, the radial approach had improved revascularization rates, fewer cases of symptomatic ICH, and faster reperfusion times, but higher rates of distal emboli. Further studies on the optimal approach are necessary based on patient and disease characteristics