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. Multi-modal 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 spatiallydefined 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 multi-modal analysis using a modified multiplex immunofluorescence (MxIF) protocol. Importantly, we have formulated Poloxamer 407 to polymerize and crosslink 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.3 Introduction:

Spatial host-microbiome sequencing

“…Slides were then placed in a hybridization chamber, and 75µl of preheated FISH solution (0.9M NaCl, 20mM Tris pH 7 (both Thermo Fisher Scientific, USA), 0.1% SDS (Sigma-Aldrich, USA)), a FISH oligonucleotide detection probe (0.06ug/ul)) were added to each well and incubated for two hours at 25°C. Oligonucleotide detection FISH probes (IDT, USA) were used depending on the target of interest: probe EUB338 (5'-/Cy5/GCTGCCTCCCGTAGGAGT-3') for all bacteria, probe non338 (5'-/Cy5/ACTCCTACGGGAGGCAGC-3') as a negative control, probe Lab158 (5'-/Cy5/GGTATTAGCAYCTGTTTCCA-3') [81][82][83] to target ASF360, probe Lac435 (5'-/Cy5/TCTTCCCTGCTGATAGA-3') [84,85] to target ASF502 and probe Bac303 (5'-/Cy5/CCAATGTGGGGGACCTT-3') [8,83] to target ASF519. After the two hours incubation, FISH solution was removed and wells were washed with 100µl 1X PBS, before the hybridization chamber was removed and slides were dipped 12 times in 50ml 1x PBS before air dried.…”

“…probe EUB338 (5'-/Cy5/GCTGCCTCCCGTAGGAGT-3') for all bacteria, probe non338 (5'-/Cy5/ACTCCTACGGGAGGCAGC-3') as a negative control, probe Lab158 (5'-/Cy5/GGTATTAGCAYCTGTTTCCA-3') [81][82][83] to target ASF360, probe Lac435 (5'-/Cy5/TCTTCCCTGCTGATAGA-3') [84,85] to target ASF502 and probe Bac303 (5'-/Cy5/CCAATGTGGGGGACCTT-3') [8,83] to target ASF519. After the two hours incubation, FISH solution was removed and wells were washed with 100µl 1X PBS, before the hybridization chamber was removed and slides were dipped 12 times in 50ml 1x PBS before air dried.…”