Clinically adaptable polymer enables simultaneous spatial analysis of colonic tissues and biofilms

Abstract

Microbial influences on host cells depend upon the identities of the microbes, their spatial localization, and the responses they invoke on specific host cell populations. Multimodal analyses of both microbes and host cells in a spatially resolved fashion would enable studies into these complex interactions in native tissue environments, potentially in clinical specimens. While techniques to preserve each of the microbial and host cell compartments have been used to examine tissues and microbes separately, we endeavored to develop approaches to simultaneously analyze both compartments. Herein, we established an original method for mucus preservation using Poloxamer 407 (also known as Pluronic F-127), a thermoreversible polymer with mucus-adhesive characteristics. We demonstrate that this approach can preserve spatially-defined compartments of the mucus bi-layer in the colon and the bacterial communities within, compared with their marked absence when tissues were processed with traditional formalin-fixed paraffin-embedded (FFPE) pipelines. Additionally, antigens for antibody staining of host cells were preserved and signal intensity for 16S rRNA fluorescence in situ hybridization (FISH) was enhanced in poloxamer-fixed samples. This in turn enabled us to integrate multimodal analysis using a modified multiplex immunofluorescence (MxIF) protocol. Importantly, we have formulated Poloxamer 407 to polymerize and cross-link at room temperature for use in clinical workflows. These results suggest that the fixative formulation of Poloxamer 407 can be integrated into biospecimen collection pipelines for simultaneous analysis of microbes and host cells.

Spatial host–microbiome sequencing reveals niches in the mouse gut

“…Slides were then placed in a hybridization chamber, and 75 μl of preheated FISH solution (0.9 M NaCl and 20 mM Tris, pH 7 (both Thermo Fisher Scientific), 0.1% SDS (Sigma-Aldrich) and a FISH oligonucleotide detection probe (0.06 μg ul −1 )) was added to each well and incubated for 2 h at 25 °C. Oligonucleotide detection FISH probes (IDT) were used depending on the target of interest: probe EUB338 (5′-/Cy5/ GCTGCCTCCCGTAGGAGT-3′) for all bacteria; probe non-338 (5′-/Cy5/ ACTCCTACGGGAGGCAGC-3′) as a negative control; probe Lab158 (5′-/Cy5/GGTATTAGCAYCTGTTTCCA-3′) [65][66][67] to target ASF360; probe Lac435 (5′-/Cy5/TCTTCCCTGCTGATAGA-3′) 68,69 to target ASF502; and probe Bac303 (5′-/Cy5/CCAATGTGGGGGACCTT-3′) 8,67 to target ASF519. After the 2-h incubation, FISH solution was removed, and wells were washed with 100 μl of 1× PBS before the hybridization chamber was removed and slides were dipped 12 times in 50 ml of 1× PBS before Article https://doi.org/10.1038/s41587-023-01988-1 being air dried.…”

The Fast Track for Intestinal Tumor Cell Differentiation and In Vitro Intestinal Models by Inorganic Topographic Surfaces

“…The cells were co-stained with DAPI for the nucleus and FITC-UEA. UEA binds to MUC2-positive mucus released by goblet cells [23] and to fucose, found on M cells in Peyer's patches in mouse intestine [24][25][26] and on intestinal mucus [27]. Fucose plays an important role in intestinal health and cross-talk with the microbiota [28].…”

“…WGA binds to collagen type 1 of the extracellular matrix and in fibrotic tissue [33] or to glycans on the mucus layer in the intestine [27]. It was also used for the detection of sialic acid and acetylglucosamine present on M cells in a Caco2/Raji B cells [34,35].…”

Mucus, commensals, and the immune system

“…A recently described approach utilizing a specialized fixative formulation was demonstrated to overcome this concern. 40 …”