Abstract 1099: Resistance to anti-EGFR therapy is transferred horizontally between lung cancer cells through caveolin-associated intercellular trafficking
Authors: Neto Susana Junqueira , Oliveira Ana Rita , Marques Joana Fernandes , Fernandes Gabriela , Hespanhol Venceslau , Moura Conceição Souto , Melo Sónia Almeida , Costa José Luís , Machado José Carlos 
Published: June 2022

Abstract
Despite initial benefit from anti-EGFR therapy, patients with EGFR-mutated lung cancer (LC) inevitably relapse. Knowing that the majority of the relapsing cancer cells do not carry the resistance mutation, we hypothesized that resistance to anti-EGFR therapy can be transferred horizontally between LC cells, through intercellular communication. Using a cell culture system that allows communication of secreted molecules and extracellular vesicles, we performed indirect co-culture of Erlotinib-sensitive LC HCC827 cells (S cells) and Erlotinib-resistant LC H1975 cells (R cells, carrying the EGFR T790M resistance mutation), where S cells were treated with conditioned medium from R cells under selective pressure of Erlotinib (EGFR inhibitor). In vivo, immunodeficient mice (Rag2-/-Il2rg-/-) were engrafted with S cells in a single flank (n=12), or with S and R cells in opposite flanks (n=20) and treated with Erlotinib three times a week. Our results showed that resistance to anti-EGFR therapy can be transferred in vitro from R to S cells. Through digital PCR we provide evidence that transfer of resistance is neither due to cell contamination, nor transfer of DNA molecules with the T790M mutation. We also showed that resistance to anti-EGFR therapy can be transferred in vivo from R to S cells, originating “de novo” resistant tumors (SR tumors) and thereby decreasing progression-free survival of the animals. Using DNA fingerprinting we demonstrated that SR tumors do not contain R cells, ruling out cell migration as an explanation for the growth of the SR tumors. Moreover, the acquired resistance phenotype is stable over several in vivo passages of the tumor. We have analyzed the SR tumors with NGS and did not detect any known resistance mutations to anti-EGFR therapy. Through RNA-Seq and pathway analysis we found that SR tumors show significant changes in genes involved in intracellular trafficking, as evidenced by increased expression of CAV1 and CAV2. This increment of caveolins expression was validated in the SR tumors by real-time PCR and immunohistochemistry. CAV1 and CAV2 expression analysis was also performed in human LC biopsies, showing that in a subset of EGFR-mutated LC there is an increase in caveolins expression from the untreated primary to the anti-EGFR-treated relapsing tumor. Our results show that the anti-EGFR resistance phenotype can be transferred from R to S cells both in vivo and in vitro, and that it results in reprogramming of the expression profile of SR cells. Our findings point out a key role for caveolin-associated intercellular trafficking in the acquisition of resistance to targeted therapy in cancer. This challenges the current paradigm of cell division and selection as the sole mechanism underlying transmission of mutation-driven therapy resistance, and reveals an unforeseen level of plasticity in cancer. PhD Scholarship SFRH/BD/115099/2016
Citation Format: Susana Junqueira Neto, Ana Rita Oliveira, Joana Fernandes Marques, Gabriela Fernandes, Venceslau Hespanhol, Conceição Souto Moura, Sónia Almeida Melo, José Luís Costa, José Carlos Machado. Resistance to anti-EGFR therapy is transferred horizontally between lung cancer cells through caveolin-associated intercellular trafficking [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1099.