Targeting fibroblast–endothelial cell interactions in LAM pathogenesis using 3D spheroid models and spatial transcriptomics
Sinem Koc-Gunel, Emily C. Liu, Lalit K. Gautam, Ben A. Calvert, Shubha Murthy, Noa C. Harriott, Janna C. Nawroth, Beiyun Zhou, Vera P. Krymskaya, Amy L. Ryan
Sinem Koc-Gunel, Emily C. Liu, Lalit K. Gautam, Ben A. Calvert, Shubha Murthy, Noa C. Harriott, Janna C. Nawroth, Beiyun Zhou, Vera P. Krymskaya, Amy L. Ryan
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Research Article Cell biology Pulmonology

Targeting fibroblast–endothelial cell interactions in LAM pathogenesis using 3D spheroid models and spatial transcriptomics

Abstract

Lymphangioleiomyomatosis (LAM) is a progressive lung disease with limited treatments, largely because of an incomplete understanding of its pathogenesis. Lymphatic endothelial cells (LECs) invade LAM cell clusters, which include human melanoma black-45–positive epithelioid cells and smooth muscle α-actin–expressing LAM-associated fibroblasts (LAMFs). Recent evidence shows that LAMFs resemble cancer-associated fibroblasts, with LAMF-LEC interactions contributing to disease progression. To explore these mechanisms, we used spatial transcriptomics on LAM lung tissues and identified a gene cluster enriched in kinase signaling pathways linked to myofibroblasts and coexpressed with LEC markers. Kinase arrays revealed elevated PDGFR and FGFR in LAMFs. Using a 3D coculture spheroid model of primary LAMFs and LECs, we observed increased invasion in LAMF-LEC spheroids compared with non-LAM fibroblasts. Treatment with sorafenib, a multikinase inhibitor, significantly reduced invasion, outperforming rapamycin. We also verified tuberous sclerosis complex 2–deficient renal angiomyolipoma (TSC2-null AML) cells as key VEGF-A secretors; VEGF-A was suppressed by sorafenib in both TSC2-null AML cells and LAMFs. These findings highlight VEGF-A and basic FGF as potential therapeutic targets and suggest multikinase inhibition as a promising strategy for LAM.

Authors

Sinem Koc-Gunel, Emily C. Liu, Lalit K. Gautam, Ben A. Calvert, Shubha Murthy, Noa C. Harriott, Janna C. Nawroth, Beiyun Zhou, Vera P. Krymskaya, Amy L. Ryan

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Figure 7

LAMF-LEC organoids have increased invasion into the ECM.

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LAMF-LEC organoids have increased invasion into the ECM. (A) Spatial hea...
(A) Spatial heatmap of localization of LEC genes in LAM_D1 tissue (SOX18, PDPN, LYVE1, and VEGFR3). Scale bars represent 2 mm. (B) Colocalization of core LEC gene signature and LAM-core signature genes in LAM lung tissue LAM_D1. Scale bars represent 2 mm. (C) Violin plots showing highest expression of both LEC signature genes and LAM-core signature genes in blue cluster 4, which spatially maps to histological regions of the lung tissue representing LAM nodules. The blue arrow is highlighting the cluster that is represented by the blue dots on the image above (original magnification, ×10). (D) Representative immunofluorescence (IF) images of LAMF-LEC spheroids with CellTracker Red–labeled LECs and CellTracker Green–labeled LAMFs 24 hours after seeding in 3D culture conditions. Scale bars represent 100 μm. (E) Quantification of changes in the compactness, perimeter, and solidity of the cocultured spheroids over 3 days comparing LAMFs and HLFs. Each dot indicates a spheroid and a minimum of 11 (range 11–78) spheroids were evaluated. (F) Representative images of LAMF-LEC spheroids embedded in ECM after 7 days. Scale bars represent 100 μm. (G) Quantification of changes in the compactness, perimeter, and solidity of the cocultured spheroids over 7 days comparing LAMFs and HLFs. Each dot indicates a spheroid and a minimum of 11 (range 11–78) spheroids were evaluated. (H) Representative phase contrast images of HLFs and LAMFs after 7 days of 3D culture. (Original magnification, ×10.) In all experiments N = 3, n = 9 experimental repeats. Data shown represent mean ± SEM. Panels E and G are analyzed using a Mann-Whitney U 2-tailed test significance represented by *P < 0.05, **P < 0.01, and ***P < 0.001.