Multiomic Analyses of Polycellular and Polymicrobial Assemblages Within Human Biofluids

Objectives: While diverse in origin and composition, human biofluids have enormous promise as minimally invasive diagnostic samples for precision medicine. Methods for single-cell and spatial multiomics are rapidly progressing for tissues; however, more work is needed to capture, profile, and analyze human and microbial cells in biofluids. Here, we describe a comprehensive strategy for multiplexed imaging and analysis of polycellular and polymicrobial components. Our aim was to explore the dynamic 2D spatial relationships between organisms that exist in both healthy and diseased patients, and investigate how changes in the assemblages’ structure and compositions can be used as indicators of changing health status.
Methods: Nasal swabs, endotracheal tubes, and saliva samples were simultaneously collected from adult patients (UNC-IRB-17-2677) and slowly frozen to -80C using a cryopreservation solution for storage and transport. Once thawed, RNA sequencing was performed on all matched samples to reveal differences between biofluids within and between subjects, and identify microbial targets. Additionally, cells were fixed and adhered to poly-L-lysine-coated glass slides using centrifugation for imaging. Highly-multiplexed immunohistochemistry, RNA-in situ hybridization, histological staining, multi-length scale/super-resolution FISH and computational imaging were employed iteratively to profile host and known microbiome species.
Results: Sampling from multiple sites revealed diverse populations of adaptive (CD3+; CD8+; CD20+) and innate (CD14+; CD68+) immune cells, as well as diverse epithelial (KRT14+; KRT19+; KRT4+; KRT10+; differentiated pan-KRT+) cells across all sample types. Profiling of microbial populations showed unique microbial niches across the inhalation interface. Additionally, host cells and microbes were found to co-exist in multi-cellular assemblages, enriched on epithelial populations. These assemblages varied in size, shape, and microbial composition.
Conclusions: Inhalation interface biofluids are distinct in cell types, with multiple kingdoms represented. Improvements in spatial profiling methods shown here allow for the novel evaluation of human biofluid samples to discover how epithelial cells may serve as substrates for bacterial assemblages.