Key result
Co-culturing lung fibroblasts with breast cancer cells and monocytes increases α-SMA expression ~2.3-fold for screening.
Why the study?
Targeting the activation of cancer-associated fibroblasts (CAFs) has been proposed to hinder metastatic spread, but an in vitro phenotypic screening assay to assess this activation was lacking.
A novel 96-well phenotypic screening assay was successfully developed to measure the activation of cancer-associated fibroblasts, enabling medium-to-high-throughput screening of potential anti-metastatic compounds.
May enable high-throughput CAF inhibitor screening; leaves open clinical translation to anti-metastatic therapies.
Background In cancer metastasis, tumor cells condition distant tissues to create a supportive environment, or metastatic niche, by driving the activation of cancer-associated fibroblasts (CAFs). These CAFs remodel the extracellular matrix, creating a microenvironment that supports tumor growth and compromises immune cell function, enabling cancer cells to evade immune detection. Consequently, targeting the activation of CAFs has been proposed as a therapeutic strategy to hinder metastatic spread. Our objective was to develop the first in vitro phenotypic screening assay capable of assessing this activation process. Methods Human primary lung fibroblasts were co-cultured with highly invasive breast cancer cells (MDA-MB-231) to identify changes in the expression of selected genes using RT-qPCR. An In-Cell ELISA (ICE)-based assay using human lung fibroblasts, MDA-MB-231 cells and human monocytes (THP-1 cells) was developed to measure the activation of CAFs. Another ELISA assay was used to measure released osteopontin. Results When lung fibroblast were co-cultured with MDA-MB-231 cells, among the 10 selected genes, the genes for osteopontin (SPP1), insulin like growth factor 1 (IGF1), periostin (POSTN) and α-smooth muscle actin (α-SMA, ACTA2) elicited the greatest fold change (55-, 37-, 8- and 5-fold respectively). Since osteopontin, IGF-1 and periostin are secreted proteins and α-SMA is an intracellular cytoskeleton protein, α-SMA was chosen to be the readout biomarker for the ICE assay. When fibroblasts were co-cultured with MDA-MB-231 cells and monocytes in the 96 well ICE assay, α-SMA expression was increased 2.3-fold yielding a robust Z′ of 0.56. A secondary, low throughput assay was developed by measuring the release of osteopontin which showed a 6-fold increase when fibroblasts were co-cultured with MDA-MB-231 cells and monocytes. Discussion This phenotypic assay is the first to measure the activation of CAFs in a 96-well format, making it suitable for medium-to high-throughput screening of potential therapeutic compounds. By focusing on observable cellular phenotypic changes rather than targeting specific molecular pathways, this assay allows for a broader and unbiased identification of compounds capable of modulating CAF activation.
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Ilg et al. (2025) studied Cancer-associated fibroblast activation. Co-culture with MDA-MB-231 breast cancer cells and THP-1 monocytes vs. Fibroblasts cultured alone was evaluated on α-SMA expression fold change (p=<0.05). Co-culture of primary human lung fibroblasts with breast cancer cells and monocytes increased α-SMA expression 2.3-fold, establishing a phenotypic assay with a robust Z' of 0.56 for screening.
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