There is currently a great interest in understanding direct interactions between human-associated microbial populations and the tumor microenvironment. In this study, we aimed to develop a chromogenic in situ hybridization (CISH) assay for universal detection of bacteria in formalin fixed paraffin embedded (FFPE) cancer tissues. This was accomplished using the RNAscope assay (Advanced Cell Diagnostics) with a CISH probe set designed to target the most highly conserved region in the bacterial 16S ribosomal RNA (rRNA) gene. Optimization of the 16S rRNA CISH assay was conducted using FFPE mouse colon, Mycobacterium tuberculosis infected rabbit lung, and Propionibacterium acnes infected mouse prostate tissues. Positive control (peptidylprolyl isomerase B- PPIB) and negative control (maize –Zm) stains were also conducted on the tissues. We additionally developed immunohistochemistry (IHC) assays with antibodies against lipopolysaccharide (LPS) and lipoteichoic acid (LTA) to visualize gram negative and gram positive bacteria, respectively, as a means to verify the results of the CISH assay. As our laboratory has a specific interest in the interactions between prostate infections and prostate cancer, we used this technique to interrogate a series of 10 radical prostatectomy specimens that contained a very high degree of acute and chronic inflammation, and were suspicious for the presence of an infectious organism. Both CISH and IHC were conducted in FFPE radical prostatectomy samples.

The 16S rRNA CISH assay detected bacteria in all of the control tissues. Interestingly, 3 of the 10 human radical prostatectomy samples that we examined showed bacterial 16S rRNA signal concentrated in areas with acute inflammation where the glandular lumens were filled with neutrophils. Two of the radical prostatectomy samples that showed positive bacterial 16S rRNA CISH signal also showed positive LPS staining indicative of the presence of gram negative bacteria. The remaining positive sample was suspicious for a gram positive intracellular organism. This study shows preliminary data supporting robust detection of bacteria in a subset of human prostate samples. We expect the frequency of bacterial infections of this nature present in radical prostatectomy specimens would be low, and that we were only able to identify these cases because we screened for samples with very high levels of inflammation. Detection of both bacterial RNA and protein in the tissues corroborates the strength and reliability of the 16S rRNA CISH. The use of these two methods concurrently can provide valuable information on bacterial presence in a range of clinical samples. In the future, we plan to use these methods to probe for bacterial presence and in understanding the spatial dynamics of the urinary microbiome. We expect that this method may also be of broader use in the study of many infection-associated cancers.

Citation Format: Eva Shrestha, Karen S. Sfanos. Development of an RNA hybridization technique for the in situ visualization of bacteria in cancer tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5136.