VistoSeg: a MATLAB pipeline to process, analyze and visualize high resolution histology images for Visium spatial transcriptomics data

Motivation: Recent advances in spatially-resolved transcriptomics technologies such as the 10x Genomics Visium platform have enabled the generation of transcriptome-wide spatial expression maps within intact tissue. However, steps for processing the high-resolution histology images, extracting relevant features from the images, and integrating them with the gene expression data remain unresolved. Results: We describe VistoSeg, a MATLAB pipeline to process, analyze, and interactively visualize the high-resolution images from the 10x Genomics Visium platform. The output from VistoSeg can then be integrated with the spatial-molecular information in downstream analyses using any programming language, such as R or Python. Availability: VistoSeg is available at https://github.com/LieberInstitute/VistoSeg with a tutorial at http://research.libd.org/VistoSeg

Ex vivo intact tissue analysis reveals alternative calcium-sensing behaviors in parathyroid adenomas

Context The biochemical basis for clinical variability in primary hyperparathyroidism (PHPT) is poorly understood. Objectives To define parathyroid tumor biochemical properties associated with calcium sensing failure in PHPT patients, and to relate differences in these profiles to variations in clinical presentation. Design Pre-operative clinical data were evaluated for correlation to parathyroid tumor biochemical behavior. Setting An endocrine surgery referral center at a large, public university hospital. Patients and Other Participants A sequential series of 39 patients undergoing surgery for PHPT. Main Outcome Measures An intact tissue, ex vivo interrogative assay was employed to evaluate the calcium-sensing capacity of parathyroid adenomas relative to normal donor glands. Tumors were functionally classified based on calcium dose-response curve profiles, and clinical parameters were compared among the respective classes. Changes in the relative expression of CASR, RGS5, and RCAN1, three key components in the calcium/PTH signaling axis were evaluated as potential mechanisms for calcium-sensing failure. Results Parathyroid adenomas grouped into three distinct functional classes. Tumors with diminished calcium sensitivity were the most common (18 of 39) and were strongly associated with reduced bone mineral density (p=0.0009). Tumors with no calcium sensing deficit (11 of 39) were associated with higher pre-operative PTH (p = 0.036). A third group (6/39) displayed a non-sigmoid calcium/PTH response curve; four of these six tumors expressed elevated RCAN1. Conclusions Calcium-sensing capacity varies among parathyroid tumors but down-regulation of the calcium sensing receptor (CASR) is not an obligate underlying mechanism. Differences in tumor calcium responsiveness may contribute to variations in PHPT clinical presentation.

Quantitative imaging of intracellular density with ratiometric stimulated Raman scattering microscopy

Cell size and density impact a wide range of physiological functions, including tissue homeostasis, growth regulation, and osmoregulation. Both are tightly regulated in mammalian cells. In comparison, density variation of a given cell type is much smaller than cell size, indicating that maintenance of cell type-specific density is important for cell function. Despite this importance, little is known about how cell density affects cell function and how it is controlled. Current tools for intracellular cell density measurements are limited either to suspended cells or cells growing on 2D substrates, neither of which recapitulate the physiology of single cells in intact tissue. While optical measurements have the potential to measure cell density in situ and noninvasively, light scattering in multicellular systems prevents direct quantification. Here, we introduce an intracellular density imaging technique based on ratiometric stimulated Raman scattering microscopy (rSRS). It quantifies intracellular drymass density through vibrational imaging of macromolecules. Moreover, water is used as an internal standard to correct for aberration and light scattering. We demonstrate real-time measurement of intracellular density quantification and show that density is tightly regulated across different cell types and can be used to differentiate cell types as well as cell states. We further demonstrate dynamic imaging of density change in response to osmotic challenge as well as intracellular density imaging of a 3D tumor spheroid. Our technique has the potential for imaging intracellular density in intact tissue and understanding density regulation and its role in tissue homeostasis.

spatialLIBD: an R/Bioconductor package to visualize spatially-resolved transcriptomics data

Motivation: Spatially-resolved transcriptomics has now enabled the quantification of high-throughput and transcriptome-wide gene expression in intact tissue while also retaining the spatial coordinates. Incorporating the precise spatial mapping of gene activity advances our understanding of intact tissue-specific biological processes. In order to interpret these novel spatial data types, interactive visualization tools are necessary. Results: We describe spatialLIBD, an R/Bioconductor package to interactively explore spatially-resolved transcriptomics data generated with the 10x Genomics Visium platform. The package contains functions to interactively access, visualize, and inspect the observed spatial gene expression data and data-driven clusters identified with supervised or unsupervised analyses, either on the user's computer or through a web application. Availability: spatialLIBD is available at http://bioconductor.org/packages/spatialLIBD.

EVALUATION OF THE PROGNOSTIC SIGNIFICANCE OF SOME BIOLOGICAL FACTORS IN LOCAL AND GENERALIZED CLEAR CELL RENAL CANCER

Purpose of the study. To evaluate the prognostic significance of biological factors VEGF-A, sVEGF-R1, VEGF-D, FGF, EGF, EGFR, IGF-1, IGF-2, IGFBP-1, IGFBP-2, somatotropin-releasing factor (GHRH) in kidney tissues (tumour tissue, tissue of the perifocal zone and conditionally intact tissue) in local and generalized clear cell renal cancer using ROC analysis.Materials and methods. Two groups of patients were included in the study. Group 1 comprised 50 patients with local kidney cancer (T1–3N0M0), while group 2 comprised 50 patients with metastatic kidney cancer (T1–4N0M1). 10% cytosolic fractions of the kidney tumour tissue were examined. The content of growth factors — somatotropinreleasing factor (GHRH), somatotropin-releasing factor (GHRH) — was determined by the ELISA assay using standard test systems. An assessment of prognostically unfavourable factors that significantly affect the generalization of the tumour process was carried out using binary logistic regression and ROC analysis.Results. The performed ROC analysis revealed diagnostically significant progression biomarkers and their critical values for clear cell renal cancer (for conditionally intact tissue, these values were: VEGF-A ≤ 9107.9 pg/g of tissue; VEGF-R1 ≤ 122.8 ng/g of tissue; FGF ≤ 364.7 pg/g of tissue; IGF-2 ≤ 148 ng/g of tissue; for perifocal tissue, VEGF-A ≤ 5839.6 pg/g of tissue; for tumour tissue, VEGF-A > 9622.5 pg/g of tissue, FGF ≤ 435.1 pg/g of tissue, somatotropin-releasing factor (GHRH) ≤ 158.6 ng/g of tissue). The obtained data contribute to optimization of the disease prognosis.Conclusion. It is established that the most prognostically significant markers of clear cell renal cancer progression include VEGF-A, FGF, somatotropin-releasing factor (GHRH), which can serve as an additional criterion for the differential diagnosis of progression and monitoring of clear cell renal cancer.

Application of microRNA-34a for diagnosis of pleomorphic adenomas of salivary glands

Objective. Determination of expression of microRNA-34a in tissues of pleomorphic adenomas of large salivary glands, adjacent to tumor salivary gland tissue, intact tissue of a salivary gland, which was not connected with the tumor, and in a venous blood as well. Materials and methods. The investigation was conducted in 20 patients, suffering benign tumors of large salivary glands (pleomorphic adenomas). Expression was estimated, using adverse transcription and quantitative polymerase chain reaction in regime of a real time. Results. Analysis of the expression level for microRNA-34a was conducted in the tumoral tissue, adjacent to the tumor salivary gland tissue, and in intact tissue of salivary gland, which lacked a link with the tumor, the venous blood in patients, suffering pleomorphic adenomas of large salivary glands, and there was revealed, that it have appeared the highest in the salivary gland adjacent to the tumor - (1052.02 ± 367.20, comparing with the same index in the intact gland - 47.72 ± 28.93). Conclusion. Level of expression of microRNA-34a in the tissues of pleomorphic adenomas of salivary glands, which is in 11 times higher, than in norm (in intact tissue of salivary gland), may be applied as a genetic marker for verification of these tumors.