Abstract B142: Dissociation of preclinical primary human cancer xenografts for cell surface transportome profiling.
Authors: Petit Vincent , Massonnet Gerald , Maciorowski Zofia , Touhami Jawida , Fontaine Jean-Jacques , Thuleau Aurelie , Laval Julie , Chateau-Joubert Sophie , Battini Jean-Luc , Sitbon Marc , Decaudin Didier 
Published: June 2012

Background: Tumor cell metabolism is of growing interest in both basic and clinical cancer research. A better understanding of underlying molecular mechanisms involving metabolite transport in normal and tumor cells should help drug discovery and development. Specific exofacial ligands to metabolite transporters derived from the receptor binding domain (RBD) of retrovirus envelope proteins were developed and used to quantify cell surface metabolite transporters. While cell surface labelling can be readily performed on cultured cell lines, analysis of single cells from solid tumors is more challenging. In this study, we developed a robust method for the disaggregation of tumor cells from human breast cancers grown as xenografts in mice. This procedure was then used to analyse the expression profiles of 4 cell membrane metabolite transporters involved in cell proliferation and tumorigenesis: Glut1, ASCT2, PiT1, and PiT2.

Materials and methods: Eight primary human breast cancer xenografts were used for ex vivo experiments (Marangoni et al 2007). We developed an optimized disaggregation protocol to obtain maximum viable cell recovery from the xenografts. The protocol was validated for presence of CD44+ tumor cells and for cell viability using caspase 3 and DAPI exclusion and subsequently, applied to the xenografts for flow cytometry analyses, immunohistochemistry and ex vivo cell culture. Expression profiles of 4 metabolite transporters were assessed in 5 different human breast cancer xenografts.

Results: The optimal dissociation protocol developed for these tumors combined mild non-enzymatic (non-enzymatic dissociation buffer) and enzymatic (collagenase III/DNase I) steps, followed by cell purification on a dual density Ficoll gradient. Less than 10% of resulting DAPI negative tumor cells were caspase 3 positive. Dissociated cells showed sustained viability in in vitro cultures for at least 12 days. The numbers of CD44+ cells determined by flow cytometry corresponded to those observed by IHC. The expression profiles of Glut1, ASCT2, PiT1, and PiT2 were distinct for each of the five human breast cancers, and metabolite transporter profiles were highly conserved for xenografts derived from the same tumor.

Conclusions: Mouse xenograft implants of human breast cancer tumors were used to optimize and validate a dissociation method for the production of viable single cells. Cell suspensions were then assessed for cell surface metabolite transporters expression by flow cytometry. The expression patterns of four metabolite transporters, Glut1, ASCT2, PiT1, and PiT2 showed distinctive signature profiles for each group of xenografts, indicative of specific metabolic adaptations that can be tracked with our ligands for each tumor.

Reference:

1. Marangoni E et al, CCR 2007;13:3989–3998.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B142.