Abstract Rationale Lymphangioleiomyomatosis (LAM) is a rare multisystem neoplastic disease characterized by diffuse cystic lung destruction. Previous research observed lymphangiogenesis in LAM cell hotspot regions, supporting the lymphatic cleft theory, which suggests that LAM cell clusters closely interact with lymphatic endothelial cells (LECs). However, a systematic and quantitative analysis of lymphatic (and vascular) networks across different lung regions is lacking, limiting further development of lymphangiogenesis-targeted therapies. Methods We implemented multiplex immunohistochemistry (mIHC) analyses, comparing lung tissues from 10 LAM-group patients (5 from transplant surgery, 5 from surgery for treating pneumothorax or diagnosis) and 10 control-group patients. We co-stained for LAM cells (SMA, PMEL), LECs (LYVE1, PROX1), and vascular endothelial cells (CD34, ERG). Subsequently, whole slide imaging and quantitative analysis with QuPath were performed to calculate indicators including the proportion (%), the density (cells/mm2 tissue), and the area ratio (%) of each cell phenotype. Regions of interest (ROIs) in areas of alveolar septa, visceral pleura, and LAM nodules were further analyzed. Results Compared to controls, LAM lungs exhibited a significant and widespread increase of lymphangiogenesis within the whole range of lung tissue slides. Generally, the overall density of LYVE1+ LECs indicating lymphatics of all sizes was higher in LAM lung tissues (median 363 vs. 159 cells/mm2 tissue, p0.05, one-sided test). Moreover, this increase was also significant in ROIs of alveolar septa (median 421 vs. 129 cells/mm2 tissue, p0.05, one-sided test) and visceral pleura (median 184 vs. 59 cells/mm2 tissue, p0.05, one-sided test) without apparent LAM cell infiltration. For LYVE1+/PROX1+ LECs indicating larger lymphatics, the density is higher in LAM nodules compared to alveolar septal regions (p0.05, two-sided test), indicating spatial co-localization of larger lymphatic structures with tumors. In contrast, the density of CD34+/ERG+ vascular endothelial cells lacks significant differences between LAM and control lungs. Similar comparison results are observed with the other two indicators, the proportion and the area ratio. Conclusions Our study provides quantitative evidence for the existence of pervasive lymphangiogenesis, but not angiogenesis, in LAM lungs, which even widely affects areas of alveolar septa and visceral pleura without signs of LAM cell invasion. Those results indicate that secretory factors related to lymphangiogenesis, namely VEGF-D, may drive global lymphatic abnormalities in whole lung tissues, extending the theoretical basis for developing lymphangiogenesis-targeted therapies. Future research may enlarge the sample size of research and focus on combination strategies targeting both lymphangiogenesis and mTOR to further improve long-term outcomes in LAM patients. This abstract is funded by: The National Key R&D Program of China (2022YFC2703901) and the National Natural Science Foundation of China (U20A20341)
Zhao et al. (Fri,) studied this question.