Co-localization of a miRNA and its Putative Target using Multispectral Imaging Peter J. Dwyer, Ph.D.,1 Gerard Nuovo, M.D.,2 Dhanrajan Tiruchinapalli, Ph.D.,1 Carlo Croce, M.D.2 Seeing life in a new light. 1Cambridge Research and Instrumentation, Inc, Woburn, Massachusetts, USA 2The Ohio State University, Columbus, Ohio, USA

Background Molecular Imaging: a Part of the Puzzle (miRNAs) are potent post-transcriptional regulators of coding . Patterns of misexpression What? Where ? Who ? Why ? of miRNAs during cancer suggest a key function of Identify miRNA, mRNA, &/or Develop theranostic tumorigenesis. miR221 and miR222 are encoded by a Develop multi-parameter in- Correlate signatures with clinical protein signatures for the medical capabilities cluster on the X . They share similar seed situ molecular methods outcome and compounds sequence and appear to have identical target which is disease/disorder p27(kip1)/CDKN1B mRNA. p27 mRNA is a member of the Cip/Kip family of cyclin/cycling dependent kinase inhibitors involved in proliferation and cell cycle regulation. Studies have shown that p27 mRNA and protein are down-regulated during tumor invasiveness. In addition, miRNA microarray analysis has shown an increase in miR221/miR222 levels in Dx + Rx hepatocellular carcinoma with increased aggressiveness of the tumor, this in turn was associated with decreased TIMPS, p27 and PTEN levels1.

Hepatocellular carcinoma (HCC) and precancerous cervical cancer are some of the most common malignant tumors in the world. Recently high-throughput miRNA microarray and LNA-based real-time PCR profiling of tumor vs normal samples revealed a cluster of miRNA associated genes which are deregulated contributing to tumor progression and metastasis2. Furthermore, the mTOR signaling pathway NBT Blue-miR222 in Liver Cancer Panel A: RGB image of fresh P27, pAKT, pS6 and PTEN are unregulated during Original color appearance PFFE human Liver caner liver cancer cell proliferation leading to HCC development. An tissue stained for miR222. interesting observation in precancerous cervical tissue Panel B: Nuance spectral samples is an increase in the expression of muscle specific library used to unmix into miR206. However, studies only reveal the individual component images level of specific microRNAs in tissue extracts, that consist of potentially heterogeneous population of cells. NBT Blue-miR222 (blue), B DAB-PTEN (green) and Nova Microscopy-based multiple probe molecular imaging revealed Red-P27 (Red). Panels C, D, the differential localization of microRNAs 206 and 221/222 and E represent the with p27 and PTEN proteins in distinct epithelial and liver spectrally unmixed individual HCC tissue samples, respectively. Multispectral imaging signals present in the sample. (MSI) techniques, based on CRI’s microscope-based Nuance Panel F: composite image. system have been shown to simultaneously image and Panel G: Pseudo quantify the expression of microRNA as well as proteins in A F fluorescence image of miR 3 RGB 20X Unmixed Composite tissue sections , even in the presence of spatial and spectral UNMIX 222 (Purple) and p27 (Red) overlap. MSI methodologies can spectrally characterize and computationally eliminate auto fluorescence, thus revealing Spectrally unmixed component images undetectable molecular targets.

CRi’s NuanceTM system is capable of detecting low abundant miRNAs and proteins using Fluorescence and Chromogenic (brightfield) labeling techniques. There was an inverse correlation between microRNAs 221/222 and P27/PTEN levels. In cervical precancerous tissue a co-expression of miR206 and p27 protein in epithelial cells was determined.

NBT Blue-miR222 C DAB-PTEN D Nova Red-p27 E Pseudo FL Composite G

Spectral imaging instrumentation NBT Blue-miR206 in Cervical Precancer Bright field color appearance Panel A: RGB image of a Pseudo Color Image Display formalin-fixed, paraffin- embedded (FFPE) cervical precancer stained for miR 206. Panel B: Nuance spectral

Example of positive cell library used to unmix into individual component images B NBT Blue-miR206 (blue) and Nova Red-P27 (red). Panels C: Pseudo brightfield image showing the co-expression of miR 206 with p27. Panels D: Brightfield image with yellow UNMIX Yellow Cells are co-labeled with pixels showing miR206 co- RGB 20X A miR-206 and p27 C expression with p27 in cervical precancer. Panel E: Nuance Percentage positivity of miR206 with p27 statistical analysis showing the percentage co-expression. Panel F: shows mir221/222 seed sequence in P27 mRNA 3’UTR. Spectral Data Acquisition • Take images at different wavelengths with a CCD-equipped 5 Quantum dot vials Nuance. E • Assemble the data into a “cube” in memory. Autofluorescence-I F • This creates a spectrum at every Yellow Pixels miR 206 + p27 D pixel (x,y) of the image. λ Summary – Microscopy-based multiple probe molecular imaging revealed differential localization of microRNAs 221/222 with p27 and 650 nm PTEN proteins in distinct epithelial cells of liver HCC tissue samples respectively. – 625 There was an inverse correlation between miR 221/222 expression and p27/PTEN protein levels. nm x – In cervical precancerous tissue samples, we observed positive co-expression of miR206 with p27 protein in epithelial cells. 600 – nm We observed an increase in miR 221/222 levels in hepatocellular carcinoma correlate with increased aggressiveness of the tumor. This in turn was associated with decreased TIMP3, p27 and PTEN levels. 575 TM nm – CRi Nuance system is capable for detecting low abundant miRNAs and proteins using Fluorescence and Chromogenic (brightfield) labeling techniques. 550 nm y References 1. miR-221 overexpression contributes to liver tumorigenesis. Pineau et al., Proc. Natl. Acad. Sci. 107: 264-9, 2009. Poster at Cambridge Healthtech Institute, microRNA in Human 2. miRNAs and cancer. Visone R. and Croce C.M. Am J Pathol. 174: 1131-8, 2009. 3. In-situ miRNA expression Profiling Using Multispectral imaging to elucidate pathways involved in neurogenerative disease. Lynch, D.T. and Levenson, R. Biotechniques 44: 5, 2008. Disease and Development, March 22-24, 2010, Boston, Mass. www.cri-inc.com