Browsing by Author "Jäntti, Joona"

Ovarian cancer is the most lethal gynaecological malignancy and is among the leading causes of cancer-related deaths among women. Chemotherapy resistance is a major obstacle in treating high-grade serous carcinoma (HGSC), leading to a low 5-year survival rate (35-40%). This resistance often develops even after the initial response to treatment. Cellular signalling changes are known to play a role in this process and transcription factors convey these signals of various pathways to transcriptional outputs. Understanding which pathways become active during treatment is crucial for developing new therapies and understanding how chemotherapy resistance develops. This thesis presents a novel method to record transcriptional pathway activation at single-cell level. It utilizes two plasmid constructs: one expressing doxycycline (DOX)-inducible Cas12a enzyme from Acidaminococcus (AsCas12a) and another containing single guide RNAs (sgRNA) under a stress-responsive promoter. The AsCas12a enzyme cleaves DNA at specific sites upon encountering the sgRNAs, leaving a permanent record of pathway activation. In the stress recording workflow that we developed the cells are first lentivirally transduced with both plasmid constructs. Then, treatment with DOX and a specific stress inducer (e.g., TNF-α for NF-κB pathway activation) jointly triggers targeted AsCas12a activity and genomic level records are produced. Our findings demonstrate tight control by both DOX and the stress inducer. No editing occurred without both plasmids and both inducers, demonstrating the system's timed activation. The method opens the door to studying the activation of different transcriptional pathways in HGSC. By simply modifying the response promoter sequence in the design, the system can be adapted to record various pathways. Ultimately, recording the activation of various pathways at the single-cell level holds promise for identifying key vulnerabilities of the adaptive mechanism that can be targeted for improved therapies in HGSC.