Targeting lung cancer-associated fibroblasts as a therapeutic approach to suppress mediastinal lymph node metastasis in non-small cell lung cancer

Abstract

Cancer-associated fibroblasts (CAFs) are pivotal in the tumour microenvironment of non-small cell lung cancer (NSCLC), but their precise role in lymphangiogenesis and mediastinal lymph node metastases is unknown. This study sought to bridge this gap by examining the lymphangiogenic function of lung CAFs and elucidating the mechanism through which they promote lymphangiogenesis. Furthermore, we aimed to investigate potential therapeutic approaches utilizing a novel orthotopic xenograft model enriched with lung CAFs. Lung CAFs were generated by co-culturing normal human lung fibroblasts (MRC-5) with NSCLC cell lines (H358 and HCC827), or through treatment with TGFβ1. Orthotopic xenograft models were established by implanting NSCLC cells into mouse lung, with or without lung CAFs. The efficacy of CAF inhibitors, ABT- 199, was assessed for their ability to inhibit lymphangiogenesis and prevent mediastinal lymph node metastasis utilizing H358 (with or without H358-CAFs) orthotopic xenograft model. Tumour growth was monitored using micro-computed tomography (micro-CT), with response evaluated according to RECIST 1.1 criteria. Confirmation of mediastinal lymph node metastasis was conducted through histological methods, including H&E staining and immunohistochemistry. Western blot and immunofluorescence were used to evaluate Nicotinamide N- methyltransferase (NNMT) and collagen expression in lung CAFs. NNMT inhibition (NNMTi) or siRNA targeting NNMT assessed potential reduction of collagen synthesis. The efficacy of NNMTi, in vivo, was evaluated for its capability in mitigating desmoplasia and lymphangiogenesis utilizing HCC827 (with or without HCC827-CAFs) orthotopic xenograft model. Activation of lung CAFs was successfully achieved through co-culture with NSCLC cell lines. Co-administration of cancer cells with lung CAFs resulted in tumours displaying heightened peritumoral lymphangiogenesis and increased incidence of mediastinal lymph node metastasis compared to tumours lacking CAFs. Conversely, ABT-199 treatment significantly reduced lung CAFs, effectively inhibiting tumour growth, lymphangiogenesis, and mediastinal lymph node metastasis in the H358-CAFs-rich orthotopic xenograft model. NNMT overexpression in NSCLC cells and lung CAFs was unveiled through Western blot and immunofluorescence analyses. TGFβ1 treatment induced normal lung fibroblast (MRC-5) conversion into α-SMA+ CAFs, elevating collagen levels and upregulating NNMT and SMAD2/3 expression. siRNA targeting NNMT led to decreased α-SMA and Col 1A1 expression in TGFβ1-activated myofibroblasts. NNMTi led to decreased α-SMA, Col 1A1, Col 3A1 and SMAD2/3 expression in TGFβ1-activated myofibroblasts. NNMTi administration significantly reduced

lung CAFs, suppressing tumour burden, proliferation, lymphangiogenesis, and collagen deposition in the HCC827-CAFs-rich orthotopic xenograft model. In summary, lung CAFs promote tumour-associated lymphangiogenesis and mediastinal lymph node metastases in NSCLC. NNMT serves as a regulatory factor in orchestrating the conversion of normal lung fibroblasts into myofibroblasts and governing collagen synthesis via the TGFβ-SMAD2/3 signalling pathway. NNMTi and ABT-199 targeting lung CAFs show promise in reducing desmoplasia and restraining lymphangiogenesis in CAFs-rich NSCLC. Targeting lung CAFs presents an encouraging therapeutic approach to limit lung cancer spread through the lymphatic system.