Abstract B17: Xenospheres: a comprehensive patient-derived in vitro model to study response and resistance to targeted therapies in metastatic colorectal cancer
Authors: Luraghi Paolo , Bigatto Viola , Reato Gigliola , Cipriano Elia , Sassi Francesco , Isella Claudio , Bertotti Andrea , Trusolino Livio , Comoglio Paolo Maria , Boccaccio Carla 
Published: March 2017

Abstract
Although EGFR targeted therapy has improved the survival outcome of patients with metastatic colorectal cancer, lack of response or emergence of secondary resistance frequently occurs as result of genetic alterations that hyperactivate the RAS pathway or by activation of compensatory pathways by signals from the tumor microenvironmet that can also induce a stem-like phenotype. From patient-derived xenografts (“xenopatients”) of colorectal cancer liver metastases, we generated primary sphere cultures (“xenospheres”) that display the properties of cancer stem-like cells. Indeed, these cells can long-term self-propagate in vitro, and form phenocopies of original patient tumors in vivo (“spheropatients”), maintaining the same response to anti-EGFR therapy.
We obtained a panel of xenospheres derived from more than 40 different xenopatients, all genetically characterized for the presence of mutations in genes that are predictive for response to EGFR targeted therapy. The panel included xenospheres harboring mutations in RAS genes (Rasmut), or recently identified rare predictors of resistance: ERBB2 amplification/mutation, MET amplification, EGFR mutation in the extracellular domain, and IGF2 overexpression.
Moreover, we conducted an extensive exome-sequencing analysis of 10 xenospheres, for which the exome data of the matched xenopatients were available, thus allowing a complete and robust comparison. Xenospheres displayed a good genetic stability and a remarkable correspondence with xenopatients, in both the mutational and gene expression profiles.
While RASmut xenospheres displayed self-sustained proliferative ability, those lacking mutations affecting the RAS pathway (RASwt) were strongly dependent of exogenous growth factors. We thus assessed the response of all RASwt xenospheres to ligands of the EGF family (TGFA, EREG, AREG, HB-EGF, NRG1), or bFGF, or HGF, testing both proliferation and response to EGFR inhibition (cetuximab). We found that TGFA and HB-EGF induced proliferation and resistance to cetuximab with almost the same potency of EGF, while EREG and AREG had a weak mitogenic activity and did not protect against cetuximab treatment, accordingly with observations in patients and xenopatients. The different proliferative activity (and protection against cetuximab) can be at least in part explained by the effect on receptor stabilization and exposure to the cell membrane exerted by different ligands. Interestingly, we found that, among all EGF family ligands, NRG1 (binding ERBB3/4) had the most potent mitogenic activity and protection against cetuximab treatment.
NRG1 was able to replace EGF in sustaining xenosphere propagation, without altering the global gene expression profile, implying that it could sustain cancer-stem like cell properties similarly to EGF. To study the role of NRG1 in vivo we generated both an autocrine and a paracrine mode, by inducing its expression infecting either RASwt xenospheres or a cell line of murine fibroblasts. NRG1-expressing RASwt xenospheres became growth factor independent, resistant to cetuximab but sensitive to lapatinib (a dual EGFR/ERBB2 small molecule). Similar results were obtained using the conditioned medium of NRG1-expressing murine fibroblasts. By injecting parental and modified RASwt xenospheres into immunocompromised mice, we found that NRG1-expressing xenospheres had a strongly increased tumor onset, and that these tumors were resistant to cetuximab but sensitive to lapatinib treatment, suggesting its possible use in the clinical practice of RASwt patients.
We conclude that xenospheres are a robust patient-derived in vitro model of colorectal cancer-stem like cells, amenable to study molecular mechanisms of response or resistance to targeted therapies.
Citation Format: Paolo Luraghi, Viola Bigatto, Gigliola Reato, Elia Cipriano, Francesco Sassi, Claudio Isella, Andrea Bertotti, Livio Trusolino, Paolo Maria Comoglio, Carla Boccaccio. Xenospheres: a comprehensive patient-derived in vitro model to study response and resistance to targeted therapies in metastatic colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B17.