Integrating AI-Powered Digital Pathology and Imaging Mass Cytometry Identifies Key Classifiers of Tumor Cells, Stroma, and Immune Cells in Non-Small Cell Lung Cancer.

Artificial intelligence (AI)-powered approaches are becoming increasingly used as histopathologic tools to extract subvisual features and improve diagnostic workflows. On the other hand, hi-plex approaches are widely adopted to analyze the immune ecosystem in tumor specimens. Here, we aimed at combining AI-aided histopathology and imaging mass cytometry (IMC) to analyze the ecosystem of non-small cell lung cancer (NSCLC). An AI-based approach was used on hematoxylin and eosin (H&E) sections from 158 NSCLC specimens to accurately identify tumor cells, both adenocarcinoma and squamous carcinoma cells, and to generate a classifier of tumor cell spatial clustering. Consecutive tissue sections were stained with metal-labeled antibodies and processed through the IMC workflow, allowing quantitative detection of 24 markers related to tumor cells, tissue architecture, CD45+ myeloid and lymphoid cells, and immune activation. IMC identified 11 macrophage clusters that mainly localized in the stroma, except for S100A8+ cells, which infiltrated tumor nests. T cells were preferentially localized in peritumor areas or in tumor nests, the latter being associated with better prognosis, and they were more abundant in highly clustered tumors. Integrated tumor and immune classifiers were validated as prognostic on whole slides. In conclusion, integration of AI-powered H&E and multiparametric IMC allows investigation of spatial patterns and reveals tissue relevant features with clinical relevance. SIGNIFICANCE: Leveraging artificial intelligence-powered H&E analysis integrated with hi-plex imaging mass cytometry provides insights into the tumor ecosystem and can translate tumor features into classifiers to predict prognosis, genotype, and therapy response.

Two pediatric observations of spinal extradural arteriovenous fistulas presenting with epidural hemorrhages and cord compression.

We report two cases of acute spinal cord compression in children with low-flow spinal epidural arteriovenous fistulas (SEAVFs) and discuss the clinical presentation and management of these vascular anomalies. While most low-flow SEAVFs without radiculomedullary drainage are benign lesions typically diagnosed incidentally, we suggest that asymptomatic lesions may warrant aggressive management in specific circumstances, including lesions diagnosed at an early age or in patients under anticoagulation therapy. Our observations also emphasize that patients with a "spontaneous" epidural hemorrhage should undergo dedicated preoperative or postoperative vascular imaging to identify a possible underlying vascular anomaly.

Monocyte differentiation within tissues: a renewed outlook.

When recruited to mammalian tissues, monocytes differentiate into macrophages or dendritic cells (DCs). In the past few years, the existence of monocyte-derived DCs (moDCs) was questioned by the discovery of new DC populations with overlapping phenotypes. Here, we critically review the evidence for monocyte differentiation into DCs in tissues and highlight their specific functions. Recent studies have shown that monocyte-derived macrophages (moMacs) with distinct life cycles coexist in tissues, both at steady state and upon inflammation. Integrating studies in mice and humans, we highlight specific features of moMacs during inflammation and tissue repair. We also discuss the notion of monocyte differentiation occurring via a binary fate decision. Deciphering monocyte-derived cell properties is essential for understanding their role in nonresolving inflammation and how they might be targeted for therapies.

Distinct responses of newly identified monocyte subsets to advanced gastrointestinal cancer and COVID-19.

Monocytes are critical cells of the immune system but their role as effectors is relatively poorly understood, as they have long been considered only as precursors of tissue macrophages or dendritic cells. Moreover, it is known that this cell type is heterogeneous, but our understanding of this aspect is limited to the broad classification in classical/intermediate/non-classical monocytes, commonly based on their expression of only two markers, i.e. CD14 and CD16. We deeply dissected the heterogeneity of human circulating monocytes in healthy donors by transcriptomic analysis at single-cell level and identified 9 distinct monocyte populations characterized each by a profile suggestive of specialized functions. The classical monocyte subset in fact included five distinct populations, each enriched for transcriptomic gene sets related to either inflammatory, neutrophil-like, interferon-related, and platelet-related pathways. Non-classical monocytes included two distinct populations, one of which marked specifically by elevated expression levels of complement components. Intermediate monocytes were not further divided in our analysis and were characterized by high levels of human leukocyte antigen (HLA) genes. Finally, we identified one cluster included in both classical and non-classical monocytes, characterized by a strong cytotoxic signature. These findings provided the rationale to exploit the relevance of newly identified monocyte populations in disease evolution. A machine learning approach was developed and applied to two single-cell transcriptome public datasets, from gastrointestinal cancer and Coronavirus disease 2019 (COVID-19) patients. The dissection of these datasets through our classification revealed that patients with advanced cancers showed a selective increase in monocytes enriched in platelet-related pathways. Of note, the signature associated with this population correlated with worse prognosis in gastric cancer patients. Conversely, after immunotherapy, the most activated population was composed of interferon-related monocytes, consistent with an upregulation in interferon-related genes in responder patients compared to non-responders. In COVID-19 patients we confirmed a global activated phenotype of the entire monocyte compartment, but our classification revealed that only cytotoxic monocytes are expanded during the disease progression. Collectively, this study unravels an unexpected complexity among human circulating monocytes and highlights the existence of specialized populations differently engaged depending on the pathological context.

Elevated Intrarenal Resistive Index Predicted Faster Renal Function Decline and Long-Term Mortality in Non-Proteinuric Chronic Kidney Disease.

Background. Intrarenal resistive index (RI) ≥ 0.80 predicts renal outcomes in proteinuric chronic kidney disease (CKD). However, this evidence in non-proteinuric patients with CKD of unknown etiology is lacking. In this study, we assessed the effect of intrarenal RI on renal function and all-cause mortality in non-proteinuric patients with CKD of unknown etiology despite an extensive diagnostic work-up. Methods. Non-proteinuric CKD patients were evaluated in a retrospective longitudinal study. Progression of renal disease was investigated by checking serum creatinine levels at 1, 3, and 5 years and defined by a creatinine level increase of at least 0.5 mg/dL. The discrimination performance of intrarenal RI in predicting the 5-year progression of renal disease was assessed by calculating the area under the receiver operating characteristic curve (AUROC). Results. One-hundred-thirty-one patients (76 ± 9 years, 56% males) were included. The median follow-up was 7.5 years (interquartile range 4.3−10.5) with a cumulative mortality of 53%, and 5-year renal disease progression occurred in 25%. Patients with intrarenal RI ≥ 0.80 had a faster increase of serum creatinine levels compared to those with RI < 0.80 (+0.06 mg/dL each year, 95% CI 0.02−0.10, p < 0.010). Each 0.1-unit increment of intrarenal RI was an independent determinant of 5-year renal disease progression (odds ratio 4.13, 95% CI 1.45−12.9, p = 0.010) and predictor of mortality (hazards ratio 1.80, 95% CI 1.05−3.09, p = 0.034). AUROCs of intrarenal RI for predicting 5-year renal disease progression and mortality were 0.66 (95% CI 0.57−0.76) and 0.67 (95% CI 0.58−0.74), respectively. Conclusions. In non-proteinuric patients with CKD of unknown etiology, increased intrarenal RI predicted both a faster decline in renal function and higher long-term mortality, but as a single marker, it showed poor discrimination performance.

Histopathological and Immune Prognostic Factors in Colo-Rectal Liver Metastases.

Prognostic studies are increasingly providing new tools to stratify colo-rectal liver metastasis patients into clinical subgroups, with remarkable implications in terms of clinical management and therapeutic choice. Here, the strengths and hurdles of current prognostic tools in colo-rectal liver metastasis are discussed. Alongside more classic histopathological parameters, which capture features related to the tumor component, such as tumor invasion, tumor growth pattern and regression score, we will discuss immune mediators, which are starting to be considered important features. Their objective quantification has shown significant results in prognostication studies, with most of the work focused on adaptive immune cells, namely T cells. As for macrophages, they are only starting to be appreciated and we will present recent advances in evaluation of macrophage morphological features. Deeper knowledge acquired by multiparametric analyses is rapidly uncovering the variety of immune players that should be assessed. The future projection is to implement deep-learning histopathological tools and to integrate histopathological and immune metrics in multiparametric scores, with the ultimate objective to achieve a deeper resolution of the tumor features and their relevance for colo-rectal liver metastasis.

Manipulation of Glucose Availability to Boost Cancer Immunotherapies.

The orchestration of T cell responses is intimately linked to the execution of metabolic processes, both in homeostasis and disease. In cancer tissues, metabolic alterations that characterize malignant transformation profoundly affect the composition of the immune microenvironment and the accomplishment of an effective anti-tumor response. The growing understanding of the metabolic regulation of immune cell function has shed light on the possibility to manipulate metabolic pathways as a strategy to improve T cell function in cancer. Among others, glucose metabolism through the glycolytic pathway is central in shaping T cell responses and emerges as an ideal target to improve cancer immunotherapy. However, metabolic manipulation requires a deep level of control over side-effects and development of biomarkers of response. Here, we summarize the metabolic control of T cell function and focus on the implications of metabolic manipulation for the design of immunotherapeutic strategies. Integrating our understanding of T cell function and metabolism will hopefully foster the forthcoming development of more effective immunotherapeutic strategies.

The neuro-immune axis in cancer: Relevance of the peripheral nervous system to the disease.

The peripheral nervous system and the immune system are critically involved in the surveillance of our body, having the ability to sense the environment, recognize danger signals and orchestrate an appropriate response. Despite the fact that these two systems have been historically considered autonomous entities, a large body of evidence has shown how they interact in many homeostatic responses and how these interactions are critically involved in pathologic contexts too, including inflammation, infection and autoimmunity. In the context of cancer, where it is already known that inflammation plays a key role, the cross-regulation of immune cells and neural components is still somewhat unexplored. Detailed characterization of the mediators and pathways involved in neuro-immune interactions in cancer is expected to provide insights into the pathogenesis of disease and open new possibilities related to therapeutic strategies.

Macrophage morphology correlates with single-cell diversity and prognosis in colorectal liver metastasis.

It has long been known that in vitro polarized macrophages differ in morphology. Stemming from a conventional immunohistology observation, we set out to test the hypothesis that morphology of tumor-associated macrophages (TAMs) in colorectal liver metastasis (CLM) represents a correlate of functional diversity with prognostic significance. Density and morphological metrics of TAMs were measured and correlated with clinicopathological variables. While density of TAMs did not correlate with survival of CLM patients, the cell area identified small (S-TAM) and large (L-TAM) macrophages that were associated with 5-yr disease-free survival rates of 27.8% and 0.2%, respectively (P < 0.0001). RNA sequencing of morphologically distinct macrophages identified LXR/RXR as the most enriched pathway in large macrophages, with up-regulation of genes involved in cholesterol metabolism, scavenger receptors, MERTK, and complement. In single-cell analysis of mononuclear phagocytes from CLM tissues, S-TAM and L-TAM signatures were differentially enriched in individual clusters. These results suggest that morphometric characterization can serve as a simple readout of TAM diversity with strong prognostic significance.

Multiplexed Optical Sensors in Arrayed Islands of Cells for multimodal recordings of cellular physiology.

Cells typically respond to chemical or physical perturbations via complex signaling cascades which can simultaneously affect multiple physiological parameters, such as membrane voltage, calcium, pH, and redox potential. Protein-based fluorescent sensors can report many of these parameters, but spectral overlap prevents more than ~4 modalities from being recorded in parallel. Here we introduce the technique, MOSAIC, Multiplexed Optical Sensors in Arrayed Islands of Cells, where patterning of fluorescent sensor-encoding lentiviral vectors with a microarray printer enables parallel recording of multiple modalities. We demonstrate simultaneous recordings from 20 sensors in parallel in human embryonic kidney (HEK293) cells and in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and we describe responses to metabolic and pharmacological perturbations. Together, these results show that MOSAIC can provide rich multi-modal data on complex physiological responses in multiple cell types.

Dysregulation of NIPBL leads to impaired RUNX1 expression and haematopoietic defects.

The transcription factor RUNX1, a pivotal regulator of HSCs and haematopoiesis, is a frequent target of chromosomal translocations, point mutations or altered gene/protein dosage. These modifications lead or contribute to the development of myelodysplasia, leukaemia or platelet disorders. A better understanding of how regulatory elements contribute to fine-tune the RUNX1 expression in haematopoietic tissues could improve our knowledge of the mechanisms responsible for normal haematopoiesis and malignancy insurgence. The cohesin RAD21 was reported to be a regulator of RUNX1 expression in the human myeloid HL60 cell line and during primitive haematopoiesis in zebrafish. In our study, we demonstrate that another cohesin, NIPBL, exerts positive regulation of RUNX1 in three different contexts in which RUNX1 displays important functions: in megakaryocytes derived from healthy donors, in bone marrow samples obtained from adult patients with acute myeloid leukaemia and during zebrafish haematopoiesis. In this model, we demonstrate that alterations in the zebrafish orthologue nipblb reduce runx1 expression with consequent defects in its erythroid and myeloid targets such as gata1a and spi1b in an opposite way to rad21. Thus, also in the absence of RUNX1 translocation or mutations, additional factors such as defects in the expression of NIPBL might induce haematological diseases.

Metabolome of Pancreatic Juice Delineates Distinct Clinical Profiles of Pancreatic Cancer and Reveals a Link between Glucose Metabolism and PD-1+ Cells.

Better understanding of pancreatic diseases, including pancreatic ductal adenocarcinoma (PDAC), is an urgent medical need, with little advances in preoperative differential diagnosis, preventing rational selection of therapeutic strategies. The clinical management of pancreatic cancer patients would benefit from the identification of variables distinctively associated with the multiplicity of pancreatic disorders. We investigated, by 1H nuclear magnetic resonance, the metabolomic fingerprint of pancreatic juice (the biofluid that collects pancreatic products) in 40 patients with different pancreatic diseases. Metabolic variables discriminated PDAC from other less aggressive pancreatic diseases and identified metabolic clusters of patients with distinct clinical behaviors. PDAC specimens were overtly glycolytic, with significant accumulation of lactate, which was probed as a disease-specific variable in pancreatic juice from a larger cohort of 106 patients. In human PDAC sections, high expression of the glucose transporter GLUT-1 correlated with tumor grade and a higher density of PD-1+ T cells, suggesting their accumulation in glycolytic tumors. In a preclinical model, PD-1+ CD8 tumor-infiltrating lymphocytes differentially infiltrated PDAC tumors obtained from cell lines with different metabolic consumption, and tumors metabolically rewired by knocking down the phosphofructokinase (Pfkm) gene displayed a decrease in PD-1+ cell infiltration. Collectively, we introduced pancreatic juice as a valuable source of metabolic variables that could contribute to differential diagnosis. The correlation of metabolic markers with immune infiltration suggests that upfront evaluation of the metabolic profile of PDAC patients could foster the introduction of immunotherapeutic approaches for pancreatic cancer.

Macrophages at the crossroads of anticancer strategies.

Macrophages are essential elements in the tumor microenvironment, where they can promote tumor growth but also influence the efficacy of anticancer strategies. In conventional therapies, chemotherapy and radiotherapy, TAMs play a dichotomous role, contributing to antitumor activity or hindering the efficacy of cytoreductive therapies. Macrophages express checkpoint ligands and are therefore targets of immunotherapy approaches based on checkpoint inhibitors. Targeted therapies with monoclonal antibodies elicit TAMs to engage in antitumor functions such as antibody-dependent phagocytosis through the activation of Fc receptors. New approaches to exploit macrophage effector functions induced by therapeutic antibodies are under investigation. Finally, strategies aimed at targeting TAM recruitment, survival and functional polarization are advancing towards the clinic. Collectively, TAM-centered strategies will hopefully complement conventional and unconventional anticancer therapies to achieve improved therapeutic benefit.

Health Care Expenditures of Medicare Beneficiaries with Normal Pressure Hydrocephalus.

BACKGROUND: Normal pressure hydrocephalus (NPH) is an underdiagnosed and undertreated condition affecting the elderly population and with costs associated with its surgical management reported to be less than those associated with conservative management. OBJECTIVE: To determine if the rate of diagnosis of NPH has improved over the last decade, the rate of treatment has increased, and if surgical treatment costs and socioeconomic factors related to receipt of treatment have changed over time compared with conservative therapy. METHODS: A retrospective study based on data from a nationally representative random sample of 2,378,637 Medicare beneficiaries (2006-2010) was performed. Shunt surgery, shunt revision, replacement, and removal were analyzed as independent variables. RESULTS: A total of 2321 patients with NPH were included, with 580 (24.99%) receiving a first shunt procedure. The adjusted effect of the procedure is that total 5-year expenditures are $11,676 more per patient (P < 0.001) than expenditures associated with nonsurgical management. Shunt revision ($22,715, P < 0.01) and/or replacement ($46,607, P < 0.001) add significantly to 5-year expenditures. Socioeconomic factors including African American race (P = 0.006); age 75-79 years (P = 0.024), 80-84 years (P < 0.001), and ≥85 years (P < 0.001); and Medicaid (P < 0.001) have significant negative associations with shunt surgery. CONCLUSIONS: There was a 1.66-fold increase in the rate of diagnosis of NPH, from 0.12% in 1999 to 0.2% in 2008. The total costs per surgical patient rose by approximately 145% to 160% comparing 2001 and 2010. This increase was mainly due to hospital (by 167% to 168%) and home health costs (by 118% to 148%). Providing appropriate care across the socioeconomic spectrum warrants further study and requires identifying the factors that limit access to care.

Large-Scale Production of Mature Neurons from Human Pluripotent Stem Cells in a Three-Dimensional Suspension Culture System.

Human pluripotent stem cells (hPSCs) offer a renewable source of cells that can be expanded indefinitely and differentiated into virtually any type of cell in the human body, including neurons. This opens up unprecedented possibilities to study neuronal cell and developmental biology and cellular pathology of the nervous system, provides a platform for the screening of chemical libraries that affect these processes, and offers a potential source of transplantable cells for regenerative approaches to neurological disease. However, defining protocols that permit a large number and high yield of neurons has proved difficult. We present differentiation protocols for the generation of distinct subtypes of neurons in a highly reproducible manner, with minimal experiment-to-experiment variation. These neurons form synapses with neighboring cells, exhibit spontaneous electrical activity, and respond appropriately to depolarization. hPSC-derived neurons exhibit a high degree of maturation and survive in culture for up to 4-5 months, even without astrocyte feeder layers.

Rapid cellular turnover in adipose tissue.

It was recently shown that cellular turnover occurs within the human adipocyte population. Through three independent experimental approaches--dilution of an inducible histone 2B-green fluorescent protein (H2BGFP), labeling with the cell cycle marker Ki67 and incorporation of BrdU--we characterized the degree of cellular turnover in murine adipose tissue. We observed rapid turnover of the adipocyte population, finding that 4.8% of preadipocytes are replicating at any time and that between 1-5% of adipocytes are replaced each day. In light of these findings, we suggest that adipose tissue turnover represents a possible new avenue of therapeutic intervention against obesity.

A high-efficiency system for the generation and study of human induced pluripotent stem cells.

Direct reprogramming of human fibroblasts to a pluripotent state has been achieved through ectopic expression of the transcription factors OCT4, SOX2, and either cMYC and KLF4 or NANOG and LIN28. Little is known, however, about the mechanisms by which reprogramming occurs, which is in part limited by the low efficiency of conversion. To this end, we sought to create a doxycycline-inducible lentiviral system to convert primary human fibroblasts and keratinocytes into human induced pluripotent stem cells (hiPSCs). hiPSCs generated with this system were molecularly and functionally similar to human embryonic stem cells (hESCs), demonstrated by gene expression profiles, DNA methylation status, and differentiation potential. While expression of the viral transgenes was required for several weeks in fibroblasts, we found that 10 days was sufficient for the reprogramming of keratinocytes. Using our inducible system, we developed a strategy to induce hiPSC formation at high frequency. Upon addition of doxycycline to hiPSC-derived differentiated cells, we obtained "secondary" hiPSCs at a frequency at least 100-fold greater than the initial conversion. The ability to reprogram cells at high efficiency provides a unique platform to dissect the underlying molecular and biochemical processes that accompany nuclear reprogramming.