Anti-cancer effect of melittin-derived peptide TB511 in organoid model of NSCLC

다국어 초록 (Multilingual Abstract)

Anti-cancer effect of melittin-derived peptide TB511 in organoid model of NSCLC Juyeon Jo Department of Korean Medicine Graduate School, Kyung Hee University, Seoul, Korea. Directed by Prof. Hyunsu Bae K.M.D., Ph.D. Lung cancer has the highest mortality rate and is divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). The main subtypes of NSCLC are adenocarcinoma, squamous cell carcinoma, and large cell carcinoma, accounting for approximately 80–85% of all lung cancers. NSCLC is commonly treated with chemotherapy in more than 50% of patients; however, immunotherapy use has steadily increased in recent years. The tumor microenvironment (TME) shapes disease progression and influences therapeutic response. Most aggressive solid tumors have high levels of myeloid cell infiltration, namely tumor-associated macrophages (TAM). Recapitulation of the interaction between the different cellular players of the TME, along with the extracellular matrix (ECM), is critical for understanding the mechanisms underlying disease progression. This particularly holds true for the prediction of therapeutic response(s) to standard therapies and interrogation of efficacy of TME-targeting agents. Typically, tumor-associated macrophages (TAMs), an abundant population of leukocytes in lung cancer, are affected by tumor microenvironment (TME) and shift towards either a pro-tumor (M2-like) or an anti-tumor phenotype (M1-like). M2-polarized macrophages are one of the primary tumor-infiltrating immune cells and were reported to be associated with the promotion of cancer cell growth, invasion, metastasis, and angiogenesis. TAMs are considered a potential target for adjuvant anti-cancer therapies, and recent therapeutic approaches targeting the M2 polarization of TAMs have shown encouraging results. Peptide drug conjugates are a newly designed therapeutic drug. It is designed to kill only the desired target by linking a peptide that delivers the drug to the target and a cytotoxic agent. TB511 was designed to link a melittin-modified peptide known to target M2-like macrophages with KLA(KLAKLAK)2, which is known to disrupt the mitochondrial membrane as a cytotoxic agent. In this study, to determine whether TB511 induces apoptosis and exhibits anti-cancer effects through target protein identified in M2 macrophages, a tumor microenvironment was established using the 3D-3 multicellular spheroid method. Spheroids were established from non-small cell lung cancer cells (A549) or carboplatin-resistant lung cancer cells (carboplatin-resistant A549), Cancer-associated Fibroblasts (CAF), and Tumor-associated Macrophages (TAM), grown in 3D culture using a Matrigel matrix. To confirm the efficacy of treating non-small cell lung cancer spheroids with TB511, changes in spheroid size due to THP-1 apoptosis and immunofluorescence staining were performed for Ki-67, a tumor growth factor and EMT-related markers. A549 or carboplatin-resistant A549, CAF, and THP-1 cells were co- cultured using the Matrigel matrix, and the formation of the spheroids was confirmed through the expression of type Ⅰ collagen. When the tumor spheroids were treated with TB511, THP-1 cells decreased in a concentration-dependent manner, suggesting that TB511 induces apoptosis through a target protein identified in M2 macrophages. The size of the spheroids significantly decreased as the number of THP-1 cells decreased in a concentration-dependent manner by TB511. As the size of the lung cancer tumor spheroids decreased, the expression level of ki-67 and vimentin also decreased. Taken together, TB511 exhibits anti-cancer effects by specifically targeting M2 macrophages in the non-small cell lung cancer tumor microenvironment formed by 3D-3 organoids, causing apoptosis and suppressing tumor proliferation. Keywords: NSCLC ( non-small cell lung cancer), TAM (tumor associated macrophages), peptide-drug conjugates, TB511, TME (tumor microenvironment), spheroid, 3D-3 organoid, CAF (cancer-associated fibroblast)

목차 (Table of Contents)

• Abstract - i
• Contents - iv
• List of Figures v
• Ⅰ. Introduction 1
• Ⅱ. Materials and methods 6
• Ⅲ. Results 9
• Ⅳ. Discussion 32
• V. References 40