Abstract 4597: TIMP1 is a major contributor of the angiogenic priming of tumor associated fibroblasts in lung adenocarcinoma
Authors: Valdivia Natalia Isabel Diaz , Duch Paula , Ikemori Rafael , Monso Eduard , Reguart Noemi , Alcaraz Jordi 
Published: April 2023

Abstract
Background: Lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) are the most common histologic subtypes in lung cancer. Intriguingly, ADC and SCC exhibit distinct responses to the multi-tyrosine kinase inhibitor nintedanib and other antiangiogenic drugs, suggesting that angiogenesis may depend on the histologic subtype in lung cancer. In addition, tumor-associated fibroblasts (TAFs) are known regulators of angiogenesis, and we have recently reported that TAFs exhibit enhanced fibrosis and response to nintedanib in ADC compared to SCC, owing to the stronger epigenetic repression of SMAD3 in SCC-TAFs caused by excessive exposure to cigarette smoke particles. However, how the histotype-dependent fibrotic phenotype in TAFs impacts their pro-angiogenic functions remains unknown.
Methods: A panel of angiogenesis markers was assessed in patient samples using publicly available databases. The pro-angiogenic function of the conditioned medium of TAFs from ADC and SCC patients pre-activated with TGF-β1 was analyzed in vitro using migration and network formation assays of endothelial cells. The secretion of pro-angiogenic factors in TGF-β1-activated TAFs was analyzed using an angiogenesis antibody blot array. Selected factors were functionally validated in vitro and in vivo using genetic models.
Results: All angiogenesis markers were consistently upregulated in ADC compared to SCC concomitantly with a lower necrosis in ADC, unveiling a larger angiogenesis in ADC. We also observed that the conditioned medium of TGF-β1-activated TAFs elicited a larger endothelial cell network formation and migration in ADC than SCC, revealing that ADC-TAFs exhibit enhanced angiogenesis. Our immunoblot array analysis identified a subset of pro-angiogenic factors that were selectively overexpressed in ADC-TAFs compared to SCC-TAFs, including TIMP-1. Notably, TIMP-1 overexpression in ADC-TAFs was SMAD3-dependent and knocking down TIMP-1 by siRNA in ADC-TAFs impaired angiogenesis in vitro and in tumor xenografts in vivo.
Conclusions: Our results reveal a larger angiogenesis in ADC compared to SCC, and implicate the TGF-β1/SMAD3/TIMP-1 pathway in the enhanced angiogenesis of ADC-TAFs. Our findings identify a biological process underlying the poor response of SCC to antiangiogenic therapies like nintedanib, and provide a rationale for the earlier metastasis of ADC patients compared to SCC patients based on the enhanced pro-angiogenic role of ADC-TAFs.
Citation Format: Natalia Isabel Diaz Valdivia, Paula Duch, Rafael Ikemori, Eduard Monso, Noemi Reguart, Jordi Alcaraz. TIMP1 is a major contributor of the angiogenic priming of tumor associated fibroblasts in lung adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4597.