Understanding internal tumor heterogeneity by topological proteomic profiling

Solid tumors form heterogeneous cellular communities, composed of diverse cancer cells and multiple stromal cell types that affect tumor growth, metastatic dissemination and drug response. The cancer cell diversify through rapid accumulation of mutations; and the stroma is often reprogrammed by the cancer cells to support cell growth. This tumor evolution leads to high diversity between patients, but also between cells and regions within the same tumors. Analysis of the various levels of heterogeneity is associated with the technological challenge of analyzing minute sample amounts, and at the same time analyzing multiple samples that would represent the inter-patient heterogeneity. Our study examined more than 200 macrodissected breast cancer patient samples. Unsupervised analysis of this cohort unraveled a novel breast cancer subtype, which was then validated in independent proteomic datasets, but not in RNA-based analysis. Tackling internal tumor heterogeneity, we laser-capture microdissected more than 350 breast cancer regions according to their histopathological characteristics. LC-MS/MS analysis of all of these tumor regions revealed the large extent of heterogeneity within breast tumors, and could identify features that are shared between distinct regions that are indistinguishable using clinical histopathological parameters. Last, moving towards single cell analyses, we are able to separate whole tumors into single cells, and quantify more than 1500 proteins in single cells, using TMT-based multiplexing. Ultimately, these studies will unravel the intricate relationships within the tumor mass, to unravel novel mechanisms of tumor progression.