Defining the Tumor Microenvironment by Integration of Immunohistochemistry and Extracellular Matrix Targeted Imaging Mass Spectrometry

SIMPLE SUMMARY: Extracellular matrix within the tumor microenvironment influences signaling, controls molecular diffusion of nutrients and growth factors, alters immunogenicity, and contributes to disease progression and therapeutic response. ECM is secreted by multiple cell types, including tumor cells, fibroblasts, and immune cells, yet there are limited approaches that link the cell type to the ECM proteins within the heterogeneous tumor microenvironment. Here, we show that integrating immunohistochemistry (IHC) with extracellular matrix (ECM) imaging mass spectrometry allows ECM proteomic profiling based on patterns of diverse cell types and proteins in tissue. The developed approach is demonstrated using phosphatase and tensin homolog (PTEN) staining and ECM imaging/proteomics on the same tissue sections in normal breast and in a tissue microarray of breast tumor and normal adjacent tissue. The data suggests that PTEN expression in tumor and in normal adjacent tissue may be associated with different collagen types and regulation by post-translational sites of modification. ABSTRACT: Breast stroma plays a significant role in breast cancer risk and progression yet remains poorly understood. In breast stroma, collagen is the most abundantly expressed protein and its increased deposition and alignment contributes to progression and poor prognosis. Collagen post-translation modifications such as hydroxylated-proline (HYP) control deposition and stromal organization. The clinical relevance of collagen HYP site modifications in cancer processes remains undefined due to technical issues accessing collagen from formalin-fixed, paraffin-embedded (FFPE) tissues. We previously developed a targeted approach for investigating collagen and other extracellular matrix proteins from FFPE tissue. Here, we hypothesized that immunohistochemistry staining for fibroblastic markers would not interfere with targeted detection of collagen stroma peptides and could reveal peptide regulation influenced by specific cell types. Our initial work demonstrated that stromal peptide peak intensities when using MALD-IMS following IHC staining (αSMA, FAP, P4HA3 and PTEN) were comparable to serial sections of nonstained tissue. Analysis of histology-directed IMS using PTEN on breast tissues and TMAs revealed heterogeneous PTEN staining patterns and suggestive roles in stromal protein regulation. This study sets the foundation for investigations of target cell types and their unique contribution to collagen regulation within extracellular matrix niches.