Rapid phenotypic screening for anti-fibrotic activity

Our innovative assay (1) uses In-Cell ELISA to monitor the inhibition of metastatic activity by your test compound.

Metastasis is responsible for approximately 90% of all cancer-related deaths. For tumour cells to successfully metastasise, they must develop a metastatic niche at the distant secondary site. To do this, cancer cells activate resident fibroblasts at the secondary site, recruiting them as cancer-associated fibroblasts (CAFs). CAFs remodel the extracellular matrix, creating a microenvironment which supports tumour growth and helps cancer cells evade detection by immune cells.

Our phenotypic screening method can be used to quantify the ability of any small molecule compound or biological agent to inhibit cancer cell-induced CAF activation, a pivotal step in the metastatic cascade.

Our assay utilises cancer cell lines (an example is highly invasive MDA-MB-231 breast cancer cell line), and healthy patient-derived lung fibroblasts, as the lung is a very common metastatic site for breast cancer. In co-culture, MDA-MB-231 cells activate primary lung fibroblasts into tumour-promoting CAFs. This change in cell phenotype in the fibroblasts is monitored by measuring expression of phenotypic markers using In-Cell ELISA. Inhibition of fibroblast activation by a test compound is evidence of anti-metastatic activity.

This novel, rapid and robust assay can be used to screen a large library of potential drug candidates at low cost. The assay can also be adapted to use other cancer cell lines.

Figure 1: Outline of our phenotypic assay. Patient-derived lung fibroblasts are seeded into 96-well plates. After 24 hours, lung fibroblasts are primed with MDA-MB-231 cells, plus test compounds. After 6 days of co-incubation, CAF activation can be quantified by measuring the expression of α-SMA.

Figure 2: Concentration response curves (CRCs) of two compounds tested in our In-cell ELISA assay.
A) In-cell ELISA image output: negative control wells are outlined in grey (wells containing fibroblasts only), positive control wells are outlined in blue (fibroblasts in co-culture with cancer cells). The CRC wells contain fibroblasts and cancer cells in co-culture and are outlined in yellow (compound 1) and pink (compound 2), with concentrations ranging between 10 µM and 1 pM. Each compound concentration was tested in triplicate. Green stain denotes expression of α-SMA (CAF marker), red stain denotes DRAQ5 nuclear stain.
CRCs of compound 1 (B) and 2 (C): circles denote percentage of cancer cell-induced CAF activation (ratio of α-SMA/DRAQ5 staining), and crosses denote percentage of total cell viability (DRAQ5 staining only). Data presented as mean ± SD (n=3).

We have already screened 1,953 FDA-approved drugs using this assay and have identified hit compounds which can prevent metastatic niche formation.

Screening assay service

Our screening assay is available as a service.

Assays are conducted on 96-well plates. Any compound showing a positive effect can be further investigated by constructing concentration-response curves. Promising candidate compounds can then be taken forward to have their anti-metastatic activity confirmed in functional phenotypic assays, such as total ECM production, and migration/invasion assays.

Get in touch

For more information, contact Dr Marc Kuiper at [email protected] and/or Dr Kirsty McKenzie at [email protected]

References

1. Ilg, M. M., Lapthorn, A. R., Harding, S. L., Minhas, T., Koduri, G., Bustin, S. A., and Cellek, S. (2025) ‘Development of a phenotypic screening assay to measure activation of cancer-associated fibroblasts’, Front. Pharmacol., 16, 1526495. Available at: https://doi.org/10.3389/fphar.2025.1526495