Hillberg, Frank

Abstract The two most common lung cancer subtypes are adenocarcinoma (ADC) and squamous cell carcinoma (SCC). Even though both subtypes are epithelial in origin, it is now clear that tumor associated fibroblasts (TAFs) are key regulators of tumor progression and response to therapies. Nintedanib is an antifibrotic drug that targets the tumor stroma and has been clinically approved to treat lung ADC patients owing to the therapeutic benefits exhibited by this drug selectively in ADC (but not in SCC) in the LUME-1 cinical trial. We have implicated recently both ADC-TAFs and ADC cancer cells in the selective effects of nintedanib in ADC, since this drug reduced the growth and invasion induction elicited by the secretome of TGF-β-activated ADC-TAFs on a panel of ADC cells, whereas such reduction was not observed in SCC-TAFs. However, the key molecules involved in the aberrant TAF-carcinoma crosstalk in ADC remain unknown. TIMP-1 is a multifunctional protein that has been associated with poor prognosis in lung cancer and is downregulated by nintedanib in a bleomycin model of pulmonary fibrosis. Our preliminary results revealed that the TIMP-1 receptor CD63 is overexpressed in ADC patients compared to SCC. Therefore, our working hypothesis was that nintedanib reduces ADC cells growth and invasion by abrogating the TAF-carcinoma crosstalk driven by TIMP-1 and CD63.To test this hypothesis, we used primary TAFs obtained with the patient informed consent, and using protocols approved by the Ethics Committee of the Hospital Clinic. TGF-β1-activated ADC-TAFs and SCC-TAFs were treated with nintedanib, and their secreted TIMP-1 was determined by ELISA. Two high-CD63 cell lines (H1437 and H23) were used in some experiments, and siRNA was used to knock-down either TIMP-1 in TAFs or CD63 in cancer cellsOur in vitro results showed that the secretion of TIMP-1 was significantly larger in ADC-TAFs compared to SCC-TAFs. Likewise, nintedanib elicited a higher TIMP-1 downregulation in ADC-TAFs compared SCC-TAFs. Of note, TIMP-1 and CD63 were both implicated in the pro-tumorigenic crosstalk, since knocking-down TIMP-1 in ADC-TAFs or CD63 in ADC cells was sufficient to abrogate the growth and invasion enhancement elicited by the secretome of TAFs. Moreover, CD63 was necessary to enhance the invasion of ADC cells upon stimulation with recombinant TIMP-1. In addition, we found that knocking-down TIMP-1 in ADC-TAFs was sufficient to compromise the inhibitory effects of nintedanib on the growth and invasion enhancement elicited by the secretome of TAFs on ADC cells. Collectively, our results support a novel TAF-carcinoma crosstalk driven by TIMP-1 and CD63 in lung ADC, and support that such heterotypic crosstalk may underlie the aberrant tumor-promoting effects of ADC-TAFs that are selectively downregulated by nintedanib. Citation Format: Paula Duch, Natalia Díaz-Valdivia, Marta Gabasa, Rafael Ikemori, Marselina Arshakyan, Frank Hillberg, Noemí Reguart, Derek Radisky, Jordi Alcaraz. Stromal TIMP-1 drives tumor progression in lung adenocarcinoma through CD63 interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3167.

Abstract Tumor associated fibroblasts (TAFs) are important regulators of tumor growth and resistance to therapies. We have recently shown that lung TAFs in vitro respond to the antifibrotic drug nintedanib in adenocarcinoma (ADC) but not squamous cell carcinoma (SCC). We also showed that the tumor-promoting effects of TAFs are driven by different mechanisms in ADC and SCC, which remain to be elucidated. Tissue inhibitor of metalloproteinases 1 (TIMP-1) has been associated with poor prognosis in lung cancer, its expression is downregulated by nintedanib, and our preliminary results reveal that its putative receptor, CD63, is overexpressed in ADC patients compared to SCC, supporting a selective crosstalk between TAFs and cancer cells in ADC through TIMP-1 and CD63. The aim of this study was to test this hypothesis using in vitro preclinical models. Primary fibroblasts were obtained with the patient informed consent, and using protocols approved by the Ethics Committee of the Hospital Clinic. ADC-TAFs and SCC-TAFs were stimulated with TGF-β1 in the presence or absence of nintedanib, and the TIMP-1 content in their conditioned medium was determined by ELISA. TIMP-1 was knocked-down in ADC-TAFs by siRNA, and the corresponding conditioned medium was used to stimulate growth and invasion of the high-CD63 ADC cell line, H1437. Likewise, CD63 expression in H1437 cells was reduced by siRNA. Our in vitro results showed that TIMP-1 secretion induced by TGF-β1 is significantly larger in ADC-TAFs compared to SCC-TAFs. Likewise, nintedanib elicited a higher downregulation of secreted TIMP-1 in ADC-TAFs compared to SCC-TAFs. Of note, TIMP-1 from ADC-TAFs was necessary to induce growth and invasion of H1437 cells. Likewise, knocking-down CD63 in H1437 ADC cells was sufficient to reduce the growth and invasion elicited by the conditioned medium of TGF-β1 activated ADC-TAFs. Collectively, our results unveil a novel stroma-carcinoma crosstalk driven by TIMP-1 and CD63 selectively in lung ADC, and support that such heterotypic crosstalk may underlie the aberrant tumor-promoting effects of ADC-TAFs that are selectively downregulated by nintedanib. Citation Format: Paula Duch, Marta Gabasa, Rafael Ikemori, Marselina Arshakyan, Frank Hillberg, Noemí Reguart, Derek Radisky, Jordi Alcaraz. TIMP-1 in tumor-associated fibroblasts drives tumor progression in lung adenocarcinoma through CD63 interaction [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5091.