Multimodal Imaging of Hypoxia in Breast Cancer

Abstract

Knowing “what” and “where” brings us closer to “how”. In this project, we have used mass spectrometers to know “what” is present inside breast cancer cells and “where” these molecules are located in human breast tumor xenograft models. Understanding the molecular composition of breast tumors is a crucial step towards the development of new anti-cancer treatment strategies. In the present research project we performed experiments in collaboration with a state-of-the-art proteomics facility located in the Netherlands Proteomic Centre in Utrecht, in which we identified and quantified more than 6, 000 different proteins from breast cancer cells. We focused on hypoxia-induced proteome changes and identified hundreds of up- and down-regulated proteins in hypoxic breast cancer cells. The up-regulated proteins belong to cellular pathways responsible for cell survival, energy metabolism, apoptosis, heme metabolism and immune system modulation. Next, we employed the mass spectrometry imaging facility at the FOM-Institute AMOLF in Amsterdam and the ICMIC molecular imaging facility at The Johns Hopkins University School of Medicine, where we combined bright field/fluorescence microscopy, mass spectrometry imaging and histological tissue staining to visualize and explore the molecular composition of breast tumor xenograft tissue. Microscopy provided information about the localization of hypoxic as well as necrotic tumor regions, while mass spectrometry imaged biomolecular distributions in these heterogeneously distributed tumor regions. In order to co-register the images obtained by different imaging modalities we developed a novel fiducial marker system. Fiducial markers such as cresyl violet, Ponceau S, and bromophenol blue possess a combination of optical and molecular properties that resulted in a clear optical and mass spectrometric signature. This system allowed us to precisely overlay multiple 2D images by providing reference points. It also proved to be a powerful tool for 3D volume reconstruction of different breast tumor microenvironments imaged in parallel by bright field, fluorescence, and mass spectrometric imaging. With our 3D MSI technique we were able to visualise biomolecular distributions in different tumor microenvironments, in all three dimensions and with high molecular specificity. One of the major goals of the project was to localize and investigate effects of hypoxia in breast tumors. A genetically modified breast cancer cell line expressing tdTomato red fluorescent protein under hypoxic conditions was used to generate breast tumor xenograft models. Traditionally, fluorescent proteins are imaged by fluorescence microscopy. Here, for the first time we employed mass spectrometry imaging to visualize the distribution of the tdTomato red fluorescent protein directly from breast tumor tissue. While microscopic detection utilizes this protein’s intrinsic feature of fluorescence, MSI detects this protein’s unique amino acid sequence. Multimodal imaging visualized intact phospholipids in viable tumor tissue while a higher concentration of lysolipids was detected in necrotic regions. Hypoxic tumor regions contained different metabolites, lipids and proteins than well oxygenated, normoxic tumor regions. Biomolecules such as acylcarnitines, a number of different phosphatidylcholines and sphingomyelins were mostly detected in hypoxic tumor regions. Multimodal imaging led us to thus far unexplored scientific territories and shed light on processes occurring in different microenvironments inside breast tumors.