Visualization of tumor-resident bacteria in colorectal cancer metastases tissue section using Fluorescent In Situ Hybridization

Deneft, Joséphine;et.al.
(2025) 12th Edition of the IREC PhD Day (2025) — Location: Woluwe Saint-Lambert, Maisin Auditorium, UCLouvain (10.October.2025)

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The intra-tumoral microbiome is a growing area of research in advanced and metastatic colorectal cancer (mCRC). We previously investigated the presence of tumour-resident bacteria in surgical frozen tissue samples from CRC primary tumours (PT) and metastases (MT) from different sites, both with paired-normal adjacent tissue (NAT) using 16S rRNA gene sequencingbased microbiome profiling. We analysed 615 frozen samples (147 patients, 4 distinct cohorts), allowing us to validate the existence of bacteria in CRC PT and MT and to identify a distinctive bacterial signature in mCRC samples including Bacteroides, Fusobacterium, Clostridia class, and Prevotella. We further developed a validation approach to confidently detect the presence of bacterial DNA in CRC MT samples by Fluorescent In Situ Hybridization (FISH) with microscopy. We selected Fusobacterium and Bacteroides as most suitable genera for visualization through imaging based on their CRC specificity, sharing between PT and MT, high “Predominance Score”, and biological relevance. We selected eight highly colonized samples based on 16S rRNA amplicon sequencing. As Fusobacterium was more specific to CRC MT, metastatic samples were exclusively chosen for this genus, while for Bacteroides, we selected CRC MT samples from different sites and one CRC PT sample as a positive control, as this genus was most predominant in CRC PT. FISH was performed on tissue section from fresh frozen CRC samples embedded in Optimal Cutting Temperature (OCT) compound. Tissue sections were fixed in PFA, dehydrated in ethanol gradient, and hybridized with three sets of probes given the high abundance of host cells and DNA in MT samples and the risk of unspecific binding: a genus-specific probes for Bacteroides (Bfra602) or Fusobacterium (Fuso227), the universal bacterial probe EUB338 as positive-control, and the nonsense probe NON-EUB338 as negative control. To minimize autofluorescence in CRC MT samples, probes were labelled with infrared fluorophores (Alexa 660, 594 and 750), while in CRC PT EUB338 and Bacteroides probes were labelled with Alexa 488 and 561 respectively. After overnight hybridization at 60 °C, tissue sections were washed, counterstained with DAPI, mounted with ProLong™ Diamond Antifade Mountant, and imaged using a Leica Thunder microscope, with standardized post-acquisition adjustments. A genus was considered positively identified when hybridization occurred with both the genus-specific and EUB338 probes but not with the NON-EUB338 probe. Using this approach, we microscopically validated bacterial presence in all our mCRC samples. We successfully detected both Fusobacterium and Bacteroides in di􀆯erent metastatic organs. Validating our previous sequencing results, bacterial signals were confirmed in all selected samples, in varying proportions, and were primarily organized into microcolonies in certain regions of the tumor tissue.
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Deneft, J., & et al. (2025). Visualization of tumor-resident bacteria in colorectal cancer metastases tissue section using Fluorescent In Situ Hybridization. 12th Edition of the IREC PhD Day (2025), Woluwe Saint-Lambert, Maisin Auditorium, UCLouvain. https://hdl.handle.net/2078.5/272075