Drug Screening in 3D Microtumors Reveals DDR1/2-MAPK12-GLI1 as a Vulnerability in Cancer-Associated Fibroblasts

Interactions between cancer cells and surrounding stromal cells are critical for tumor biology and treatment response. We compare drug screening results from conventional 2D cancer cell lines with 3D tumor tissues and find that, on average, three times more drugs are effective in 3D microtumors. We confirmed the effectiveness of doramapimod, a compound that reduces 3D microtumor viability and suppresses tumor growth in mouse models, has no effect on cancer cell growth in monolayers. Mechanistically, doramapimod targets DDR1/2 and MAPK12 kinases in cancer-associated fibroblasts (CAFs), decreasing extracellular matrix (ECM) production and enhancing interferon signaling. These kinases regulate ECM through GLI1 activity in CAFs, independently of canonical hedgehog signaling. Inhibiting the DDR1/2-MAPK12-GLI axis enhances the effectiveness of chemotherapy and immunotherapy in patient tumor slices and preclinical models. These findings highlight the importance of DDR1/2-MAPK12-GLI axis in CAF function and demonstrate the utility of 3D tissue models in identifying microenvironment-specific therapeutic targets. Table S4. KPC and E0771 tumor-bearing mice were treated with doramapimod or vehicle control. Treatment was administered according to standard protocols, and tumors were harvested at the experimental endpoint. Tumor tissues were processed for total RNA extraction and downstream transcriptomic analysis. Table S5. Human breast cancer-associated fibroblasts (CAFs) were treated with doramapimod or vehicle control under standard culture conditions. At the end of the treatment period, cells were harvested and total RNA was extracted for downstream transcriptomic analysis. Table S7. Human breast cancer-associated fibroblasts (CAFs) were transfected with siRNA targeting DDR1, DDR2, or MAPK12, or with a non-targeting control. Transfections were performed using standard protocols, and cells were maintained under standard culture conditions for 48–72 hours. At the endpoint, cells were harvested and total RNA was extracted for downstream analysis. Table S8. Human breast cancer-associated fibroblasts (CAFs) were treated with GANT61 or vehicle control under standard culture conditions. Following treatment, cells were harvested and total RNA was extracted for downstream transcriptomic analysis.