2023年 · 第103卷第02期

Lymphangioleiomyomatosis (LAM) is a rare lung disease characterized by cystic lung destruction, progressive lung function decline, and lung failure. The pathology of LAM is characterized by the hyperproliferation of abnormal smooth muscle-like LAM cells in the lung, and the abnormal expression of multiple proteases [mainly matrix metalloproteinase-2 (MMP-2), MMP-9 and cathepsin K] in LAM cells, which may directly lead to lung structural destruction and cyst formation. Rapamycin is the primary treatment for patients with LAM, but the disease continues to progress after discontinuation. At present, pulmonary function tests [such as forced expiratory volume in the first second (FEV1)] are the standard method for monitoring the burden of disease and treatment response in patients with LAM. However, FEV1 levels are prone to technical variations and the response to treatment is slow, so quantitative, accurate and rapid-response biomarkers are urgently needed to monitor disease progression and treatment prognosis in patients with LAM. Activity-based nanosensors are emerging biosensors that detect dysregulated proteases in the body and release a reporter to provide urinary readings of disease. Due to the widespread dysregulation of protease regulation in LAM, this study developed a nanosensor panel containing 14 peptide substrates, which was tracheally instilled into the lungs of LAM model mice, and urine was collected for mass spectrometry to determine the nanosensor cleavage products, monitoring the response of LAM mice to rapamycin treatment, and distinguishing healthy from LAM mice, treated from untreated mice through machine learning. It was found that the multiactivity-based nanosensor PP03 (cleaved by MMP, aspartic acid and cysteine proteases) (P<0.001) and PP10 (cleaved by serine, aspartate and cysteine proteases) (PThe cleavage signal of =0.017) is significantly different between LAM mice and normal mice, and the machine learning model can be strongly classified. Within 2 d after rapamycin treatment, the cleavage signals of PP03 and PP10 returned to normal in LAM mice, and the machine learning model enabled accurate classification of treatment response [area under the curve (AUC) =0.94 in the untreated group].

Lymphangioleiomyomatosis is a multisystem disease that causes cystic changes in the lungs and respiratory failure. The loss of 1/2 gene function in tuberous sclerosis (TSC) leads to dysregulation of mammalian rapamycin target protein (mTOR) activity and hyperproliferation of abnormal smooth muscle cells (i.e., LAM cells). LAM cells interact with fibroblasts, lymphoendothelial cells, and various inflammatory cells to form LAM nodules similar to "cancer nests" in the lungs. In addition, mast cell aggregation was also found in LAM nodules, but its function and mechanism are still unclear. Mast cells are granular hematopoietic cells that reside in tissues and play a key role in the pathological processes of allergy, asthma, fibrosis and arthritis. Trypsins are serine proteases, which are one of the most secreted substances by mast cells and act as fibroblast mitogens in lung diseases. This study analyzed the interaction between LAM cells and LAM-associated fibroblasts (LAF), evaluated the relationship between LAM cells and mast cell migration through 3D spheroid co-culture, and constructed LAM mouse allogeneic transplantation tumor model to explore the effect of mast cells and their trypsin secreted on LAM. It was found that LAM cells/LAF co-culture induced LAF to secrete multiple CXC chemokines, and the number of trypsin-like mast cells expressing CXC chemokine receptors (CXCRs) in LAM lungs was significantly increased (P<0.05）。 LAM spheroids attract mast cells, a process inhibited by pharmacology of CXCR1 and CXCR2 and CRISPR/cas9 inhibition. LAM spheroids caused mast cell trypsin-like release, induced fibroblast proliferation, and increased LAM spheroid size (P=0.002）； Trypsin-like inhibitors APC366 and sodium tryptonate (SCG) inhibit mast cell-induced spheroid growth. In vivo, SCG reduced mast cell activation and lung tumor burden in LAM mice (P=0.004）。