Citation: Saar, M.; Jaal, J.; Meltsov, A.; Laasfeld, T.; Lust, H.; Kasvandik, S.; Lavogina, D. Exploring the Molecular Players behind the Potentiation of Chemotherapy Effects by Durvalumab in Lung Adenocarcinoma Cell Lines. Pharmaceutics 2023, 15, 1485. https://doi.org/10.3390/ pharmaceutics15051485 Academic Editor: Xiaoyong Wang Received: 11 March 2023 Revised: 5 May 2023 Accepted: 9 May 2023 Published: 12 May 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). pharmaceutics Article Exploring the Molecular Players behind the Potentiation of Chemotherapy Effects by Durvalumab in Lung Adenocarcinoma Cell Lines Marika Saar 1,2,3 , Jana Jaal 1,4 , Alvin Meltsov 5,6 ,Tõnis Laasfeld 7,8 , Helen Lust 1 , Sergo Kasvandik 9 and Darja Lavogina 1,5,7, * 1 Institute of Clinical Medicine, Faculty of Medicine, University of Tartu, 50406 Tartu, Estonia 2 Instituteof Pharmacy, University of Tartu, 50411 Tartu, Estonia 3 Pharmacy, Tartu University Hospital,50406 Tartu, Estonia 4 Haematology and Oncology Clinic, Tartu University Hospital, 50406 Tartu, Estonia 5 Competence Centre on Health Technologies, 50411 Tartu, Estonia 6 Department of Genetics and Cell Biology, GROW School for Oncology and Developmental Biology, Maastricht University, 6200 MD Maastricht, The Netherlands 7 Instituteof Chemistry, University of Tartu, 50411 Tartu, Estonia 8 Department of Computer Science, University of Tartu, 51009 Tartu, Estonia 9 Proteomics Core Facility, Institute of Technology, University of Tartu, 50411 Tartu, Estonia * Correspondence: darja.lavogina@ut.ee; Tel.: +372-737-5296 Abstract: Immune checkpoint inhibitors are increasingly used in combination with chemotherapy for the treatment of non-small cell lung cancer, yet the success of combination therapies is relatively limited. Thus, more detailed insight regarding the tumor molecular markers that may affect the responsiveness of patients to therapy is required. Here, we set out to explore the proteome of two lung adenocarcinoma cell lines (HCC-44 and A549) treated with cisplatin, pemetrexed, durvalumab, and the corresponding mixtures to establish the differences in post-treatment protein expression that can serve as markers of chemosensitivity or resistance. The mass spectrometry study showed that the addition of durvalumab to the treatment mixture resulted in cell line- and chemotherapeutic agent- dependent responses and confirmed the previously reported involvement of DNA repair machinery in the potentiation of the chemotherapy effect. Further validation using immunofluorescence also indicated that the potentiating effect of durvalumab in the case of cisplatin treatment was dependent on the tumor suppressor RB-1 in the PD-L1 weakly positive cells. In addition, we identified aldehyde dehydrogenase ALDH1A3 as the general putative resistance marker. Further studies in patient biopsy samples will be required to confirm the clinical significance of these findings. Keywords: lung adenocarcinoma; cisplatin; pemetrexed; durvalumab; proteomics; chemosensitiv- ity; resistance 1. Introduction Non-small cell lung cancer (NSCLC) is one of the leading and deadliest types of cancer, causing annually 1.8 million deaths worldwide [1]. The treatment of NSCLC depends on the stage, histology, and tumor cell features, including genetic driver alter- ations and the expression of markers that predict the efficacy of immunotherapy (e.g., programmed death-ligand 1, PD-L1) [2]. Despite the emergence of targeted therapy strate- gies, chemotherapy remains the backbone of neoadjuvant and adjuvant treatment before and after surgery in early-stage NSCLC as well as the optimal first-line treatment option in metastatic NSCLC [3,4]. In recent decades, however, the choice of therapies has been complemented by PD-1/PD-L1 axis-targeting immune checkpoint inhibitors (ICIs), which are frequently used in combination with chemotherapy. Pharmaceutics 2023, 15, 1485. https://doi.org/10.3390/pharmaceutics15051485 https://www.mdpi.com/journal/pharmaceutics