Topological-Proteomics of Breast Cancer Intra-Tumor Heterogeneity Reveals Diversity within Single Tumors

Introduction.

One of the major obstacles in breast cancer treatment is its high degree of heterogeneity and ambiguous classification. The molecular and histological subtypes are routinely defined in the clinic to determine treatment modalities; however, these distinct subtypes co-exist in single tumors and therefore lead to therapy resistance. Here we aim to delineate the proteomic landscapes of breast cancer intratumoral diversity based on clinical-pathological parameters using mass spectrometry-based microproteomics.

Materials and methods.

Using histopathological analysis of immunostained formalin-fixed paraffin-embedded (FFPE) tissues, we assembled more than 300 tumor regions comprising of different histopathological and molecular subtypes that originate from 35 patients. Cancer regions were isolated using laser-capture microdissection (LCM). Proteins were solubilized and digested following the SP3 protocol. Peptides were labeled with TMT 10-plex and analyzed on the Q-Exactive HF mass spectrometer.

Results and discussion.

MaxQuant analysis identified a total of more than 8300 proteins. Topological proteomic analysis revealed proteomic portraits that are associated with receptor expression levels and histological characteristics. The main parameter for the segregation of tumor regions was based on the level of heterogeneity. Tumor regions within a homogeneous environment demonstrate elevated immune activity in receptor-negative (TN) and ER-positive/PR-positive tumors. These proteins were previously shown to be associated with immune response, and suggest potential relevance to immunotherapy in breast cancer. Interestingly, tumor regions within a heterogeneous environment show involvement of metabolic reprogramming with elevated TCA cycle, oxidative phosphorylation, and fatty acid metabolism. For instance, TN regions within receptor-positive environment displayed shared features of basal-like and hormone-positive tumors.

Conclusion.

These findings suggest that integrated topo-proteomic landscape of inter- and intratumoral heterogeneity can serve as a platform for deciphering the mechanisms underlying tumorigenesis, distinct profiles of cancer cell subpopulations, cancer evolution, and therapy resistance.