Optimization of tumor xenograft dissociation for the profiling of cell surface markers and nutrient transporters

Abstract

Metabolic adaptations and changes in the expression of nutrient transporters are known to accompany tumorigenic processes. Nevertheless, in the context of solid tumors, studies of metabolism are hindered by a paucity of tools allowing the identification of cell surface transporters on individual cells. Here, we developed a method for the dissociation of human breast cancer tumor xenografts combined with quantification of cell surface markers, including metabolite transporters. The expression profiles of four relevant nutrient transporters for cancer cells' metabolism, Glut1, ASCT2, PiT1 and PiT2 (participating to glucose, glutamine and inorganic phosphate, respectively), as detected by new retroviral envelope glycoprotein-derived ligands, were distinctive of each tumor, unveiling underlying differences in metabolic pathways. Our tumor dissociation procedure and nutrient transporter profiling technology provides opportunities for future basic research, clinical diagnosis, prognosis and evaluation of therapeutic responses, as well as for drug discovery and development. Markers of tumor cell metabolism are of valuable importance for basic and clinical cancer research. The identification of metabolic alterations that accompany cancer processes is becoming critical for refining the clinical evaluation and treatment of patients, 1,2 as well as for further drug discovery and development. While metabolomics relies mainly on the quantification of anabolic and catabolic end products, 3 the bidirectional fluxes of nutrients and metabolites, as well as the efflux of toxins and pharmaceutical compounds, are ensured by nutrient transporters. 4 However, the monitoring of metabolite and nutrient transporters at the cell surface has been hampered by the paucity, if not total lack, of exofacial antibodies to such transporters. 5,6 Cell entry of gamma-and deltaretroviruses occurs via recognition of membrane metabolite transporters by retroviral envelope glycoproteins (Env). 7,8 Viral entry occurs following the specific binding of the amino-terminal receptor binding domain (RBD) of an individual Env with its cognate receptor. We designed RBD probes, derived from gamma-and deltaretroviral Env, as specific ligands that bind to these cell surface transporters. Binding of defined ligands allows the quantification of distinct sets of metabolite transporters at the cell surface. 9-13 Furthermore, while cell surface labeling is readily achieved on cultured cell lines and circulating hematopoietic cells, singlecell labeling of solid tumors has remained problematic. Indeed, such analyses require dissociation methods that yield

Human breast cancer and lymph node metastases express Gb3 and can be targeted by STxB-vectorized chemotherapeutic compounds

“…Single-cell suspensions were prepared from excised tumors as previously described [ 23 ]. Briefly, tumors were removed from sacrificed mice, minced and incubated twice in non-enzymatic dissociation buffer (Invitrogen) followed by mild enzyme digestion step including collagenase III (200 U/ml; Sigma-Aldrich, St Louis, MO, USA), DNase I (200 U/ml; Sigma-Aldrich), each incubation for 30 min at 37°C.…”

Ionizing radiation sensitizes tumors to PD-L1 immune checkpoint blockade in orthotopic murine head and neck squamous cell carcinoma

“…For flow cytometric analysis of tumor tissue, tumors were digested into single-cell suspension as previously reported. 22 Briefly, tumors were finely cut and placed in HBSS solution containing 200U of Collagenase III (Worthington) for 60 minutes with gentle shaking every 15 minutes. After the incubation period, tumor pieces were passed through a 100um nylon mesh.…”

XPR1 mutations are a rare cause of primary familial brain calcification

“…Cell surface expression of phosphate transporters was monitored with soluble ligands derived from the receptorbinding domain (RBD) of different retroviral envelope glycoproteins. Production of and binding with RBD from X-MLV (XRBD), and koala retrovirus (KoRBD), or with a soluble ligand derived from the surface unit (SU) of amphotropic-MLV (ASU), used to detect XPR1, PiT1, and PiT2, respectively were performed as previously described [10,15]. Briefly, 5 9 10 5 cells were resuspended in PBA (PBS with 2 % FBS) containing the adequate RBD and incubated for 30 min at 37°C, followed by two washes with PBA and incubation with an Alexa Fluor 488-conjugated anti-mouse IgG1 antibody (Life Technologies; 1:5000) for 20 min at 4°C.…”