Uncovering tumor−stroma inter-relationships using MALDI Mass spectrometry imaging

Abstract

Tumorigenesis involves a complex interplay between genetically modified cancer cells and their adjacent normal tissue, the stroma. We used an established a breast cancer mouse model to investigate this interrelationship. Conditional activation of Rho-associated protein kinase (ROCK) in a model of mammary tumorigenesis enhances tumor growth and progression by educating the stroma and enhancing the production and remodeling of the extracellular matrix. We used peptide matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) to quantify the proteomic changes occurring within tu-mors and their stroma in their regular spatial context. Peptides were ranked according to their ability to discriminate between the two groups, using a receiver operating characteristic (ROC) tool. Peptides were identified by LC-MS/MS and protein expression was validated by quantitative immunofluorescence using an independent set of tumor samples. We have identified and validated four key proteins upregulated in ROCK-activated mammary tumors relative to those expressing kinase-dead ROCK, namely collagen I, α-SMA, Rab14 and tubulin-β4. Rab14 and tubulin-β4 are expressed within tumor cells, whereas collagen I is localized within the stroma. α-SMA is predominantly localized within the stroma but is also expressed at higher levels in the epithelia of ROCK-activated tumors. High expression of COL1A, the gene encoding the pro-α 1 chain of collagen, corre-lates with cancer progression in two human breast cancer genomic datasets, and high expression of COL1A and ACTA2, (the gene encoding α-SMA) are associated with a low survival probability (COLIA p=0.00013, ACTA2 p=0.0076) in estrogen receptor negative breast cancer patients. To investigate whether ROCK-activated tumor cells cause stromal cancer-associated fibroblasts (CAFs) to upregulate expression of collagen I and α-SMA, we treated cancer-associated fibroblasts with medium conditioned by primary mammary tumor cells in which ROCK had been activated. This led to abundant production of both proteins in CAFs, clearly highlighting the inter-relationship between tumor cells and CAFs and identifying CAFs as the potential source of high levels of collagen 1 and α-SMA and associated enhancement of tissue stiffness. Our research emphasizes the capacity of MALDI MSI to quantitatively assess tumor-stroma inter-relationships and to identify potential prognostic factors for cancer progression in human patients, using sophisticated mouse cancer models.