The lipid composition of the primary cilia membrane is emerging as a critical regulator of cilia formation, maintenance and function. Here, we show that conditional deletion of the phosphoinositide ...5'-phosphatase gene Inpp5e, mutation of which is causative of Joubert syndrome, in terminally developed mouse olfactory sensory neurons (OSNs), leads to a dramatic remodeling of ciliary phospholipids that is accompanied by marked elongation of cilia. Phosphatidylinositol (4,5)-bisphosphate PI(4,5)P2, which is normally restricted to the proximal segment redistributed to the entire length of cilia in Inpp5e knockout mice with a reduction in phosphatidylinositol (3,4)-bisphosphate PI(3,4)P2 and elevation of phosphatidylinositol (3,4,5)-trisphosphate PI(3,4,5)P3 in the dendritic knob. The redistribution of phosphoinositides impaired odor adaptation, resulting in less efficient recovery and altered inactivation kinetics of the odor-evoked electrical response and the odor-induced elevation of cytoplasmic Ca2+. Gene replacement of Inpp5e through adenoviral expression restored the ciliary localization of PI(4,5)P2 and odor response kinetics in OSNs. Our findings support the role of phosphoinositides as a modulator of the odor response and in ciliary biology of native multi-ciliated OSNs.
Abstract
The tumor microenvironment (TME) is composed of highly heterogeneous structures and cell types that dynamically influence and communicate with each other. The constant interaction between a ...tumor and its microenvironment plays a critical role in how the cancer develops, progresses, and responds to therapies. Traditionally, Hematoxylin and Eosin (H&E) staining has been used to annotate and characterize tissues and associated pathologies. Recent single analyte approaches spatially interrogate targeted or transcriptome-wide expression of RNA in tissue sections, while others capture phenotypes using a limited number of protein markers. However, for a more comprehensive understanding of the unique characteristics of cell types, cell states, and cell-cell interactions within the TME, multiple layers of information are needed and must be studied together.
Here we demonstrate a novel, streamlined multiomic spatial assay that integrates histological staining and imaging with simultaneous transcriptome-wide gene expression and highly multiplexed protein expression profiling from the same formalin-fixed paraffin embedded (FFPE) tissue section. In short, tissue sections from archived FFPE samples were placed on slides containing arrayed capture oligos with unique positional barcodes. The H&E or immunofluorescence stained tissues were then imaged, followed by incubation with transcriptome-wide probes and a high-plex DNA-barcoded antibody panel containing intra- and extracellular markers. Transcriptome probes and antibody-barcodes were then spatially captured on the slide and converted into sequencing-ready libraries. Our data analysis and interactive visualization software enable interrogation of all data layers (H&E/immunofluorescence, RNA, protein) from the same tissue section.
We apply this method to simultaneously measure gene and protein expression within the TME of human breast cancer and melanoma FFPE samples using whole transcriptome probes and an immune-oncology antibody panel. The data enables comparison and correlation of multiple analytes and their patterns within the same sample section. In addition, this simultaneous detection enables marker-guided regional selection and differential gene expression analysis on the defined regions. Taken together, our data demonstrates that a spatially resolved, multiomic approach provides a more comprehensive understanding of cellular behavior in and around tumors, yielding new insights into disease progression, predictive biomarkers, drug response and resistance, and therapeutic development.
Citation Format: Cedric Uytingco, Jennifer Chew, Naishitha Anaparthy, Jun D. Chiang, Christina Galonska, Karthik Ganapathy, Ryo Hatori, Alexander Hermes, Layla Katiraee, Anna-Maria Katsori, William Nitsch, Patrick Roelli, Joe Shuga, Rapolas Spalinskas, Mesruh Turkekul, Benton Veire, Dan Walker, Neil Weisenfeld, Stephen R. Williams, Zachary Bent, Marlon Stoeckius. Multiomic characterization of the tumor microenvironment in FFPE tissue by simultaneous protein and gene expression profiling abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3814.
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