Methods for analysis of the cancer microenvironment and their potential for disease prediction, monitoring and personalized treatments

Abstract

A tumor does not consist of a homogenous population of cancer cells. Therefore, to understand cancer, the tumor microenvironment and the interplay between the different cell types present in the tumor has to be taken into account, and how this regulates the growth and survival of the cancer cells. To achieve a full picture of this complex interplay, analysis of tumor tissue should ideally be performed with cellular resolution, providing activity status of individual cells in this heterogeneous population of different cell-types. In addition, in situ analysis provides information on the architecture of the tissue wherein the cancer cells thrive, providing information of the identity of neighboring cells that can be used to understand cell-cell communication. Herein we describe how padlock probes and in situ PLA can be used for visualization of nucleic acids and protein activity, respectively, directly in tissue sections, and their potential future role in personalized medicine.

Figure 1

Detection of nucleic acids with padlock probes. a A single stranded stretch of DNA containing the target site is generated by enzymatic digestion of DNA or reverse transcription of RNA, producing a free 3'-end. b A perfect hybridization of a padlock probe will bring the 5'-and 3'- end of the padlock probe together (arrow), c as the gap is sealed by ligation a circular DNA molecule is created. d The free 3'-end of the target sequence will act as a primer for RCA to produce a concatameric RCA-product, complementary to the padlock probe, to which fluorescence-labeled detection oligonucleotides can hybridize, for visualization. e Detection of HER2 (green dots) and β-actin (red dots) transcripts using padlock probes in fresh frozen tissue sections from breast cancer [38]. Nuclei are counter-stained with DAPI (blue), scale bar equals 50 μm.

Figure 2

Detection of protein interactions with in situ PLA. a Proximal binding of PLA-probes to interacting proteins will guide the b hybridization of circularization probes, c allowing them to be connected by ligation (arrows) and thereby creating a circular reporter molecule of the protein interaction. d The oligonucleotide on one of the PLA-probes will then act as primer for RCA to generate an RCA product that will be an elongation of the PLA-probe. Fluorescence-labeled detection oligonucleotides are then used to visualize the RCA-product. e Detection of Mucin2 glycosylation (Sialyl-Tn) (red dots) in formalin-fixed paraffin-embedded tissue sections from intestinal metaplasia [49]. Nuclei are counterstained with Hoechst 33342 (blue), using the autofluorescence of the tissue (green) to visualize the histology, scale bar equals 50 μm.