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The reduced effectiveness of chemotherapy in many patients undergoing treatment highlights the need for novel drug combinations that target drug resistance mechanisms contributing to tumor survival. Dynamic conditions within the tumor microenvironment influence the response to anti-cancer drugs. Accordingly, identifying effective drug concentrations and interactions (additive, synergistic, or antagonistic) in relevant tumor tissue models will inform new treatment combinations. To address this need for combinatorial chemotherapeutic (CTx) screening assays, we have developed a new assay called CombiCTx, which uses a device with three reservoirs containing gels loaded with anti-cancer drugs. The drug-loaded device is inverted and placed in a standard culture dish above cancer cells, and both are then enclosed in gel. Drugs diffuse from the reservoirs and expose cancer cells to overlapping dynamic drug gradients. We imaged diffusion of the anti-cancer drug doxorubicin in the assay using time-lapse microscopy, and established an imaging protocol for quantifying MDA-MB-231 breast cancer cell survival responses along drug gradients. Finally, evaluating combination effects of navitoclax and gemcitabine with CombiCTx revealed localized effects of navitoclax, attributed to limited diffusion, while gemcitabine seemed to diffuse readily throughout the assay and revealed a mild synergy in navitoclax affected regions. These data demonstrate the capacity of CombiCTx to evaluate the cytotoxic effects of anti-cancer drug combinations while accounting for drug diffusion differences, which is relevant in the context of the 3D tumor environment and may thereby help inform clinical treatment strategies.

Hepatocellular carcinoma (HCC) remains a major therapeutic challenge with limited systemic treatment options and suboptimal response rates. Anthracyclines such as doxorubicin (DOX) and idarubicin (IDA) are frequently used in clinical settings, including transarterial chemoembolization (TACE), yet their efficacy and adverse effects in HCC remain poorly defined. In this long-term in vivo study, we performed a head-to-head comparison of DOX and IDA in a chemically induced mouse model of HCC, using repeated dosing (twice weekly for three weeks) to mimic clinical exposure similar to TACE. Both treatments induced significant weight loss and spleen enlargement, with IDA exhibiting a more pronounced systemic impact. While neither compound significantly altered tumor burden, both agents unexpectedly reduced hepatic collagen deposition and fibrosis. Mechanistically, DOX decreased hepatic stellate cell (HSC) activation without major changes in fibrotic markers, whereas IDA paradoxically increased HSC activation and upregulated TGF-β and CTGF expression. Both DOX and IDA activated endoplasmic reticulum (ER) stress pathways in non-tumorous liver tissue, particularly through the PERK axis. IDA treatment was associated with a strong upregulation of ATF4 in hepatocytes and enhanced macrophage recruitment, suggesting an impact on the hepatic microenvironment. Despite comparable tumor control, the divergent stromal and inflammatory responses may help explain differences in toxicity and long-term outcomes observed clinically. Our findings emphasize the need to consider microenvironmental and stress-related pathways when selecting and optimizing anthracycline regimens for TACE.