# Effect of Bleomycin Hydrolase Expression in Tumor Tissue on the Therapeutic Effectiveness of Electrochemotherapy

## TL;DR

This study shows that higher levels of an enzyme called bleomycin hydrolase in tumors reduce the effectiveness of electrochemotherapy, suggesting it could be used to predict treatment outcomes.

## Contribution

The study identifies bleomycin hydrolase as a potential predictive biomarker for electrochemotherapy effectiveness in tumors.

## Key findings

- Tumors with higher BLMH content responded poorly to electrochemotherapy.
- BLMH mRNA levels strongly correlated with bleomycin sensitivity in vitro.
- In vivo, BLMH expression moderately correlated with complete tumor response rates.

## Abstract

Electrochemotherapy (ECT) is a cancer treatment that combines application of short electrical pulses with chemotherapy drugs to improve their delivery into tumor cells. Bleomycin is one of the most effective drugs used in ECT, but its success can vary depending on the tumor type. One possible reason is the presence of bleomycin hydrolase (BLMH), an enzyme that inactivates bleomycin. In this study, we measured BLMH levels in different mouse tumor cell lines and tumors grown in animals. Thereafter we compared the levels of BLMH with tumor response to ECT and found a correlation with BLMH content, i.e., tumors with higher BLMH content responded poorly. These findings suggest that levels of BLMH could serve as a predictive marker for ECT effectiveness. Testing for BLMH before treatment may help personalize therapy and identify patients most likely to benefit from ECT.

Background: Electrochemotherapy (ECT) enhances the intracellular delivery of chemotherapeutic agents, most notably bleomycin, through electroporation. Despite high response rates, tumor sensitivity to ECT varies, highlighting the need for predictive biomarkers. Bleomycin hydrolase (BLMH), an enzyme that metabolically inactivates bleomycin, may influence treatment outcomes. This study investigated the relationship between BLMH expression and bleomycin-based ECT effectiveness. Methods: BLMH expression was evaluated at the mRNA and protein levels in six murine tumor cell lines and their corresponding syngeneic tumors using qPCR, immunofluorescence, and immunohistochemistry. Correlations between BLMH expression and tumor response to ECT were assessed both in vitro and in vivo. Results: BLMH expression varied significantly among tumor models, without consistent patterns across cancer types. In vitro, BLMH mRNA levels strongly correlated with IC30 values for bleomycin (R = 0.74), while the correlation weakened at IC50 doses, suggesting enzyme saturation. In vivo, BLMH expression levels moderately correlated with complete tumor response rates following ECT (R = 0.50). Differences between in vitro and in vivo expression highlighted the role of the tumor microenvironment. Conclusions: High BLMH expression reduces tumor sensitivity to bleomycin-based ECT, supporting its role as a predictive biomarker. Measuring BLMH levels may help stratify patients and personalize ECT application; however, it is not the sole factor for response prediction. Future studies in clinical tumor samples are warranted to evaluate its predictive value and to develop integrated biomarker models.