Véronique Serre-Beinier

Selective targeting of cancer cells is a major challenge for cancer therapy. Many cancer cells overexpress the cystine/glutamate antiporter xCT/CD98, an l-cystine transport system that strengthens antioxidant defences, thereby promoting tumour survival and progression. Here, we show that the d-enantiomer of cysteine (d-Cys) is selectively imported into xCT/CD98-overexpressing cancer cell lines and impairs their proliferation, particularly under high oxygen concentrations. Intracellular d-Cys specifically inhibits the mitochondrial cysteine desulfurase NFS1, a key enzyme of cellular iron–sulfur protein biogenesis, by blocking sulfur mobilization due to steric constraints. NFS1 inhibition by d-Cys affects all cellular iron–sulfur cluster-dependent functions, including mitochondrial respiration, nucleotide metabolism and maintenance of genome integrity, leading to decreased oxygen consumption, DNA damage and cell cycle arrest. d-Cys administration diminishes tumour growth of human triple-negative breast cancer cells implanted orthotopically into the mouse mammary gland. Hence, d-Cys could represent a simple therapy to selectively target those forms of cancer characterized by overexpression of xCT/CD98.

Lung cancer is the leading cause of cancer mortality worldwide, with a median survival rate at 5 years of less than 20%. While molecular mapping aids in selecting appropriate therapies, it cannot predict personalized treatment response and long-term efficacy. For addressing these challenges, there is a great need for functional tests. Within this context, we developed patient-derived spheroids (PDS) from tumor and adjacent normal tissue to biomimic the respective tissue for assessing the personalized drug treatment response in vitro. Surgically resected lung specimens were used to generate spheroids using a two-step culture procedure. Flow cytometry and immune staining enabled the characterization of different cell populations resulting from the lung samples. PDS phenotype, cell proliferation and apoptosis were evaluated. Differential gene expression between tumor and adjacent normal tissue was analyzed via RT-qPCR. PDS drug sensitivity was assessed using a cell metabolic assay in response to two chemotherapeutic drug combinations. Cellular and molecular analysis revealed the proportion of epithelial cells, fibroblasts, and immune cells in the patients’ tissue samples. Subsequently, PDS models from tumor and normal lung were successfully established using the expanded epithelial cells. As a proof of concept, an analysis of the drug treatment using PDS of lung adenoid cystic carcinoma exhibited a dose-dependent effect in response to cisplatin/etoposide and cisplatin/paclitaxel. Our spheroid model of both tumor and non-tumor lung cells holds great promise for enhancing the treatment efficacy in the cancer patients.

Selective targeting of cancer cells is a major challenge for cancer therapy. Many cancer cells overexpress the cystine/glutamate antiporter xCT/CD98 to increase the import of L-cystine for antioxidant defense, thus supporting tumour cell survival and progression^_1,2. We have exploited this feature of cancer cells by transforming xCT/CD98 overexpression into a selective vulnerability. We found that the D-enantiomer of cysteine (Cys), but not other D-amino acids, impaired proliferation of numerous xCT/CD98-expressing cancer cell lines, whereas xCT/CD98-non-overexpressing (cancer) cells were unaffected. D-Cys acquired by xCT/CD98 specifically inhibited the cysteine desulfurase NFS1, the sulfur donor of cellular iron-sulfur protein biogenesis³. NFS1 was able to form the D-Cys-ketimine intermediate, but, due to steric incompatibility, failed to transfer the D-Cys sulfur to its active-site Cys381 residue, thereby blocking enzymatic activity. This led to a general defect of all cellular processes relying on iron-sulfur proteins, including mitochondrial respiration, nucleotide metabolism, and nuclear genome maintenance, resulting in reduced oxygen consumption, accumulation of DNA damage, and cell cycle arrest. In vivo D-Cys administration diminished tumour growth of human triple-negative breast cancer cells implanted orthotopically into the mouse mammary gland. Hence, D-Cys could represent a simple therapy to selectively target those forms of cancer characterized by overexpression of xCT/CD98.

Defining the ontogeny of tumor-associated macrophages (TAM) is important to develop therapeutic targets for mesothelioma. We identified two distinct macrophage populations in mouse peritoneal and pleural cavities, the monocyte-derived, small peritoneal/pleural macrophages (SPM), and the tissue-resident large peritoneal/pleural macrophages (LPM). SPM rapidly increased in tumor microenvironment after tumor challenge and contributed to the vast majority of M2-like TAM. The selective depletion of M2-like TAM by conditional deletion of the Dicer1 gene in myeloid cells (D ^−/− ) promoted tumor rejection. Sorted SPM M2-like TAM initiated tumorigenesis in vivo and in vitro, confirming their capacity to support tumor development. The transcriptomic and single-cell RNA sequencing analysis demonstrated that both SPM and LPM contributed to the tumor microenvironment by promoting the IL-2-STAT5 signaling pathway, inflammation, and epithelial–mesenchymal transition. However, while SPM preferentially activated the KRAS and TNF-α/NFkB signaling pathways, LPM activated the IFN-γ response. The importance of LPM in the immune response was confirmed by depleting LPM with intrapleural clodronate liposomes, which abrogated the antitumoral memory immunity. SPM gene signature could be identified in pleural effusion and tumor from patients with untreated mesothelioma. Five genes, TREM2, STAB1, LAIR1, GPNMB , and MARCO , could potentially be specific therapeutic targets. Accordingly, Trem2 gene deletion led to reduced SPM M2-like TAM with compensatory increase in LPM and slower tumor growth. Overall, these experiments demonstrate that SPM M2-like TAM play a key role in mesothelioma development, while LPM more specifically contribute to the immune response. Therefore, selective targeting of monocyte-derived TAM may enhance antitumor immunity through compensatory expansion of tissue-resident TAM.

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9–18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [¹⁸F]-2-fluoro-2-deoxy-D-glucose ([¹⁸F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [¹⁸F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced. This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

Malignant pleural mesothelioma (MPM) is a thoracic aggressive cancer caused by asbestos exposure, which is difficult to diagnose and treat. Here, we characterized an in vivo orthotopic xenograft model consisting of human mesothelioma cells (designed as H2052/484) derived from a pleural NCI-H2052 tumor injected in partially immunodeficient athymic mice. We assessed tumor formation and tumor-dependent patterns of inflammation. H2052/484 cells conserved their mesothelioma phenotype and most characteristics from the parental NCI-H2052 cells. After intra-thoracic injection of H2052/484 cells, thoracic tumors developed in nearly all mice (86%) within 14 days, faster than from parental NCI-H2052 cells. When the mice were euthanized, the pleural lavage fluid was examined for immune cell profiles. The pleural immune cell population increased with tumor development. Interestingly, the proportion of myeloid-derived suppressor cell and macrophage (especially CD206⁺ M2 macrophages) populations increased in the pleural fluid of mice with large mesothelioma development, as previously observed in immunocompetent mice. This reliable orthotopic model recapitulates human mesothelioma and may be used for the study of new treatment strategies.

Summary: Chronic exposure to intraperitoneal asbestos triggered a marked response in the mesothelium well before tumor development. Macrophages, mesothelial precursor cells, cytokines and growth factors accumulated in the peritoneal lavage. Transcriptome profiling revealed YAP/TAZ activation in inflamed mesothelium with further activation in tumors, paralleled by increased levels of cells with nuclear YAP/TAZ. Arg1 was one of the highest upregulated genes in inflamed tissue and tumor. Inflamed tissue showed increased levels of single nucleotide variations, with an RNA-editing signature, which were even higher in the tumor samples. Subcutaneous injection of asbestos-treated, but tumor-free mice with syngeneic mesothelioma tumor cells resulted in a significantly higher incidence of tumor growth when compared to naïve mice supporting the role of the environment in tumor progression. Highlights: Asbestos increases levels of cytokines and growth factors in mesothelium environment Recruitment of macrophages and mesothelial precursor cells prior to tumor development YAP/TAZ signaling upregulated in pre-neoplastic tissues and cancer Increased RNA-editing and somatic mutations as early steps in tumor development

Https://doi.org/10.18632/oncotarget.28134 Dylan A.J. Gendre, Edis Ameti, Wolfram Karenovics, Nadja Perriraz-Mayer, Frédéric Triponez, Véronique Serre-Beinier

Role of MIF/CD74 signaling pathway in the development of pleural mesothelioma