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 6

LAMFs represent an activated lung fibroblast phenotype compared with HLFs.

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LAMFs represent an activated lung fibroblast phenotype compared with HLF...
(A) Relative expression of SMαA comparing HLFs and LAMFs from 3 independent donors with a representative Western blot, with quantification normalized to β-actin. (B and C) Representative SMαA (green, B) and phase contrast (C) images of spheroids generated from HLFs and LAMFs; scale bars = 100 μm. (D and E) Quantification of changes in the compactness (D) and solidity (E) of spheroids over 7 days comparing LAMFs and HLFs. Each dot indicates a spheroid and a minimum of 11 (range 11–78) spheroids were evaluated. N = 3 biological replicates per cell type. (F) Kinase array for LAMFs representing expression in LAMFs relative to the average signal intensity across all proteins evaluated. (G) Heatmap of canonical pathways comparing the integrated spatial transcriptomics data with the kinase array data showing kinase-related pathway data shown have a cutoff z-score > 1 and log10P value > 1.5; orange is higher pathway activation and blue is pathway inhibition. (H) quantitative reverse transcription PCR comparing gene expression of PDGFRB and TGFB1I1 in HLFs and LAMFs. Data represent mean ± SEM. Panels A and H are analyzed with an unpaired Student’s t test and panels D and E with a Mann-Whitney U 2-tailed test with significance represented by *P < 0.05, **P < 0.01, ****P < 0.0001.