Single-cell spatial analysis of tumor immune architecture in diffuse large B-cell lymphoma.

Multiplexed immune cell profiling of the tumor microenvironment (TME) in cancer has improved our understanding of cancer immunology, but complex spatial analyses of tumor-immune interactions in lymphoma are lacking. Here, we used imaging mass cytometry (IMC) on 33 cases of diffuse large B-cell lymphoma (DLBCL) to characterize tumor and immune cell architecture and correlate it to clinicopathological features such as cell of origin, gene mutations, and responsiveness to chemotherapy. To understand the poor response of DLBCL to immune checkpoint inhibitors (ICI), we compared our results to IMC data from Hodgkin lymphoma, a cancer highly responsive to ICI, and observed differences in the expression of PD-L1, PD-1, and TIM-3. We created a spatial classification of tumor cells and identified tumor-centric subregions of immune activation, immune suppression, and immune exclusion within the topology of DLBCL. Finally, the spatial analysis allowed us to identify markers such as CXCR3, which are associated with penetration of immune cells into immune desert regions, with important implications for engineered cellular therapies. This is the first study to integrate tumor mutational profiling, cell of origin classification, and multiplexed immuno-phenotyping of the TME into a spatial analysis of DLBCL at the single-cell level. We demonstrate that, far from being histopathologically monotonous, DLBCL has a complex tumor architecture, and that changes in tumor topology can be correlated with clinically relevant features. This analysis identifies candidate biomarkers and therapeutic targets such as TIM-3, CCR4, and CXCR3 that are relevant for combination treatment strategies in immuno-oncology and cellular therapies.

Severe intraventricular hemorrhage causes long-lasting structural damage in a preterm rabbit pup model.

BACKGROUND: Intraventricular hemorrhage causes significant lifelong mortality and morbidity, especially in preterm born infants. Progress in finding an effective therapy is stymied by a lack of preterm animal models with long-term follow-up. This study addresses this unmet need, using an established model of preterm rabbit IVH and analyzing outcomes out to 1 month of age. METHODS: Rabbit pups were delivered preterm and administered intraperitoneal injection of glycerol at 3 h of life and approximately 58% developed IVH. Neurobehavioral assessment was performed at 1 month of age followed by immunohistochemical labeling of epitopes for neurons, synapses, myelination, and interneurons, analyzed by means of digital quantitation and assessed via two-way ANOVA or Student's t test. RESULTS: IVH pups had globally reduced myelin content, an aberrant cortical myelination microstructure, and thinner upper cortical layers (I-III). We also observed a lower number of parvalbumin (PV)-positive interneurons in deeper cortical layers (IV-VI) in IVH animals and reduced numbers of neurons, synapses, and microglia. However, there were no discernable changes in behaviors. CONCLUSIONS: We have established in this preterm pup model that long-term changes after IVH include significant wide-ranging alterations to cortical organization and microstructure. Further work to improve the sensitivity of neurocognitive testing in this species at this age may be required. IMPACT: This study uses an established animal model of preterm birth, in which the rabbit pups are truly born preterm, with reduced organ maturation and deprivation of maternally supplied trophic factors. This is the first study in preterm rabbits that explores the impacts of severe intraventricular hemorrhage beyond 14 days, out to 1 month of age. Our finding of persisting but subtle global changes including brain white and gray matter will have impact on our understanding of the best path for therapy design and interventions.

Large Extracellular Vesicle Characterization and Association with Circulating Tumor Cells in Metastatic Castrate Resistant Prostate Cancer.

Liquid biopsies hold potential as minimally invasive sources of tumor biomarkers for diagnosis, prognosis, therapy prediction or disease monitoring. We present an approach for parallel single-object identification of circulating tumor cells (CTCs) and tumor-derived large extracellular vesicles (LEVs) based on automated high-resolution immunofluorescence followed by downstream multiplexed protein profiling. Identification of LEVs >6 µm in size and CTC enumeration was highly correlated, with LEVs being 1.9 times as frequent as CTCs, and additional LEVs were identified in 73% of CTC-negative liquid biopsy samples from metastatic castrate resistant prostate cancer. Imaging mass cytometry (IMC) revealed that 49% of cytokeratin (CK)-positive LEVs and CTCs were EpCAM-negative, while frequently carrying prostate cancer tumor markers including AR, PSA, and PSMA. HSPD1 was shown to be a specific biomarker for tumor derived circulating cells and LEVs. CTCs and LEVs could be discriminated based on size, morphology, DNA load and protein score but not by protein signatures. Protein profiles were overall heterogeneous, and clusters could be identified across object classes. Parallel analysis of CTCs and LEVs confers increased sensitivity for liquid biopsies and expanded specificity with downstream characterization. Combined, it raises the possibility of a more comprehensive assessment of the disease state for precise diagnosis and monitoring.

Long-term neurological effects of neonatal caffeine treatment in a rabbit model of preterm birth.

BACKGROUND: Neonatal caffeine treatment might affect brain development. Long-term studies show conflicting results on brain-related outcomes. Herein we aimed to investigate the long-term effects of neonatal caffeine administration in a rabbit model of preterm birth. METHODS: Preterm (born day 29) and term (day 32) pups were raised by wet nurses and allocated to treatment with saline or caffeine for 7 or 17 days. At pre-puberty, neurobehavioral tests were performed and brains were harvested for immunostaining of neurons, synapses, myelin, and astrocytes. RESULTS: Survival was lower in preterm saline pups than in controls, whereas caffeine-treated preterm pups did not differ from term control pups. Preterm saline pups covered less distance compared to controls and were more likely to stay in the peripheral zone of the open field. Corresponding differences were not seen in preterm caffeine pups. Preterm animals had lower neuron density compared to controls, which was not influenced by caffeine treatment. Synaptic density, astrocytes, and myelin were not different between groups. CONCLUSION: Caffeine appeared to be safe. All preterm rabbits had lower neuron density but anxious behavior seen in preterm saline rabbits was not seen in caffeine-treated preterm pups.

Multiplex protein detection on circulating tumor cells from liquid biopsies using imaging mass cytometry.

Molecular analysis of circulating and disseminated tumor cells (CTCs/DTCs) has great potential as a means for continuous evaluation of prognosis and treatment efficacy in near-real time through minimally invasive liquid biopsies. To realize this potential, however, methods for molecular analysis of these rare cells must be developed and validated. Here, we describe the integration of imaging mass cytometry (IMC) using metal-labeled antibodies as implemented on the Fluidigm Hyperion Imaging System into the workflow of the previously established High Definition Single Cell Analysis (HD-SCA) assay for liquid biopsies, along with methods for image analysis and signal normalization. Using liquid biopsies from a metastatic prostate cancer case, we demonstrate that IMC can extend the reach of CTC characterization to include dozens of protein biomarkers, with the potential to understand a range of biological properties that could affect therapeutic response, metastasis and immune surveillance when coupled with simultaneous phenotyping of thousands of leukocytes.