ADK-VR2, a cell line derived from a treatment-naïve patient with SDC4-ROS1 fusion-positive primarily crizotinib-resistant NSCLC: a novel preclinical model for new drug development of ROS1-rearranged NSCLC

Abstract

Background: ROS1 fusions are driver molecular alterations in 1-2% of non-small cell lung cancers (NSCLCs). Several tyrosine kinase inhibitors (TKIs) have shown high efficacy in patients whose tumors harbour a ROS1 fusion. However, the limited availability of preclinical models of ROS1-positive NSCLC hinders the discovery of new drugs and the understanding of the mechanisms underlying drug resistance and strategies to overcome it. Methods:The ADK-VR2 cell line was derived from the pleural effusion of a treatment-naïve NSCLC patient bearing SDC4-ROS1 gene fusion. The sensitivity of ADK-VR2 and its crizotinib-resistant clone ADK-VR2 AG143 (selected in 3D culture in the presence of crizotinib) to different TKIs was tested in vitro, in both 2D and 3D conditions. Tumorigenic and metastatic ability was assessed in highly immunodeficient mice. In addition, crizotinib efficacy on ADK-VR2 was evaluated in vivo.Results: 2D-growth of ADK-VR2 cells was partially inhibited by crizotinib. On the contrary, the treatment with other TKIs, such as lorlatinib, entrectinib and DS-6051b, did not result in cell growth inhibition. TKIs showed dramatically different efficacy on ADK-VR2 cells, depending on the cell culture conditions. In 3D culture, ADK-VR2 growth was indeed almost totally inhibited by lorlatinib and DS-6051b. The clone ADK-VR2 AG143 showed higher resistance to crizotinib treatment in vitro, compared to its parental cell line, in both 2D and 3D cultures. Similarly to ADK-VR2, ADK-VR2 AG143 growth was strongly inhibited by ^ ORCID: 0000-0001-6105-4895. lorlatinib in 3D conditions. Nevertheless, ADK-VR2 AG143 sphere formation was less affected by TKIs treatment, compared to the parental cell line. In vivo experiments highlighted the high tumorigenic and metastatic ability of ADK-VR2 cell line, which, once injected in immunodeficient mice, gave rise to both spontaneous and experimental lung metastases while the crizotinib-resistant clone ADK-VR2 AG143 showed a slower growth in vivo. In addition, ADK-VR2 tumor growth was significantly reduced but not eradicated by crizotinib treatment. Conclusions:The ADK-VR2 cell line is a promising NSCLC preclinical model for the study of novel targeted therapies against ROS1 fusions and the mechanisms of resistance to TKI therapies.

Pre-clinical modelling of ROS1+ non-small cell lung cancer

“…The tumorigenic and metastatic abilities were studied in immunocompetent mice after being intravenously injected with ADK-VR2 cells. Metastatic lesions were detected but they were not significantly reduced upon crizotinib administration [96]. Thus, this novel cell line constitutes a great asset to explore the biology of ROS1 fusions.…”

Virus-like Particle (VLP) Vaccines for Cancer Immunotherapy

“…The tumor mutational burden (TMB) that leads to neoantigen formation is a major problem in several cancers (e.g., melanoma, lung adenocarcinoma, stomach adenocarcinoma, colorectal carcinoma, and sarcomas), mainly due to the consequent acquired resistance to therapies, especially immune checkpoint inhibitors [ 95 , 96 , 97 ]. On the contrary, it represents an advantage for immunotherapy because neoantigen-specific T cells are less likely to be eliminated during tumor immune evasion.…”

“…The growth of established B16-pIR-HH tumors was significantly inhibited by immunization using VLP-pulsed DCs, resulting in a higher survival rate of vaccinated mice [175]. A further multiepitope VLP vaccine was designed by Ding et al by loading HBc VLP of four HBx-dominant CTL epitopes (HBx (115)(116)(117)(118)(119)(120)(121)(122)(123) , HBx (92)(93)(94)(95)(96)(97)(98)(99)(100) , HBx (140)(141)(142)(143)(144)(145)(146)(147)(148) , or HBx (52)(53)(54)(55)(56)(57)(58)(59)(60) ). VLP-pulsed dendritic cells in both HLA-A*0201 transgenic (Tg) mice and peripheral blood lymphocytes from HLA-A2(+)/HBx(+) HBVinfected hepatocellular carcinoma (HCC) patients showed CTL responses against epitopeloaded VLPs.…”