Malignant pleural mesothelioma (MPM) is a fatal cancer, and one of the primary risk factors remains asbestos exposure. Developing robust, safe, and efficient therapeutic targets and models that accurately recapitulate the disease is critical for improving MPM therapeutic strategies. This study aims to modulate nucleolin (NCL), a protein ubiquitously expressed in the cell nucleus and specifically present in the cytoplasmic membrane of some cancer malignancies, and develop a robust in vivo orthotopic model to explore additional mechanistic insights on tumor drug targeting as was successfully performed by our group previously (doi: 10.1016/j.nantod.2021.101095).

Murine malignant mesothelioma AB1, AB12, and AB22 cell lines were used to generate NCL-overexpressing models via lentiviral transduction of the pFUGW plasmids encoding NCL tagged to HA and membrane-bound NCL-overexpressing models via plasmid transfection using the pDisplay™ Mammalian Expression Vector. Inducible downregulated NCL models were generated upon DNA transfection of constructs carrying a reporter gene and an shRNA targeting the coding sequence of human NCL gene under a tetracycline-responsive promoter (TRE) and a puromycin resistance cassette under the human CMV promoter. Individual clones were generated via limiting dilution technique and were characterized by FACS, Western Blotting, and q-RT-PCR. Cells were visualized using the Axio Observer Z1 system while controlling temperature (37ºC) and humidity. Balb/c mice were implanted intrapleurally with increasing concentrations of luciferase-expressing MPM cells (kindly provided by Dr. Marco Bianchi, San Raffaele Hospital, Milan, Italy), and tumor growth was evaluated by bioluminescence. Tumors and adjacent tissue were collected at the endpoint and included in OCT.

All MPM cell lines showed positive expression of NCL in the cell membrane in over 50% of the cells. NCL overexpressing MPM cells display stable NCL expression over time, as detected by q-RT-PCR and western blotting. Specific membrane-bound NCL expression is currently being evaluated. Inducible downregulation of NCL was detected in pool transfected cells in the presence of doxycycline over time. Intrapleural administration of 0.5x106 AB1 cells generated tumors that recapitulated the course of disease, up to 13 days. No metastases were observed outside the pleural compartment.

These findings highlight the importance of versatile in vitro models that can be effectively manipulated to develop tailored and effective cancer therapies. New treatment strategies can be further evaluated by using this robust in vivo orthotopic immunocompetent mouse model, an invaluable platform in the context of MPM.