Highly multiplexed imaging of tissues with subcellular resolution by imaging mass cytometry

Cancer is a tissue disease. Heterogeneous cancer cells and normal stromal and immune cells form a dynamic ecosystem that evolves to support tumor expansion and ultimately tumor spread. The complexity of this dynamic system is the main obstacle in our attempts to treat and heal the disease. The study of the tumor ecosystem and its cell-to-cell communications is thus essential to enable an understanding of tumor biology, to define new biomarkers to improve patient care, and ultimately to identify new therapeutic routs and targets.

To study and understand the workings of the tumor ecosystem, highly multiplexed image information of tumor tissues is essential. Such multiplexed images will reveal which cell types are present in a tumor, their functional state, and which cell-cell interactions are present. To enable multiplexed tissue imaging, we developed imaging mass cytometry (IMC). IMC is a novel imaging modality that uses metal isotopes of defined mass as reporters and currently allows to visualize over 50 antibodies and DNA probes simultaneously on tissues with subcellular resolution. In the near future, we expect that over 100 markers can be visualized. We applied IMC for the analysis of breast cancer samples in a quantitative manner. To extract biological meaningful data and potential biomarkers from this dataset, we developed a novel computational pipeline called histoCAT geared for the interactive and automated analysis of large scale, highly multiplexed tissues image datasets. Our analysis reveals a surprising level of inter and intra-tumor heterogeneity and identify new diversity within known human breast cancer subtypes as well as a variety of stromal cell types that interact with them.

In summary, our results show that IMC provides targeted, high-dimensional analysis of cell type, cell state and cell-to-cell interactions within the TME at subcellular resolution. Spatial relationships of complex cell states of cellular assemblies can be inferred and potentially used as biomarkers. We envision that IMC will enable a systems biology approach to understand and diagnose disease and to guide treatment.