Uncovering Tumor–Stroma Inter-relationships Using MALDI Mass Spectrometry Imaging

Tumorigenesis involves a complex
interplay between genetically
modified cancer cells and their adjacent normal tissue, the stroma.
We used an established breast cancer mouse model to investigate this
inter-relationship. 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 tumors 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 tool. Peptides were identified by liquid
chromatography tandem mass spectrometry, 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,
correlates with cancer progression in two human breast cancer genomic
data sets, 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 CAFs 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.