title: Ex-vivo characterization of circulating colon cancer cells distinguished in stem and differentiated subset provides useful biomarker for personalized metastatic risk assessment.
abstract: Circulating tumor cells (CTCs) represent one of the most interesting target in improving diagnosis, prognosis and treatment. Herein we evaluate the possibility of using an emo-cytometric approach on the evaluation of the heterogeneous population of CTCs to improve personalized metastatic risk assessment. We benchmarked ex vivo behavior of distinct subsets of circulating colon tumor cells with correspondent clinical behavior of patients from which we isolated CTCs.
  
  
  
  Isolation and CTC expansion were performed by a gradient protocol. In vitro characterization was determined by flow cytometry, immunofluorescence, western blotting and proteomic profiling. Cell sorter was performed with immunomagnetic beads. Confocal microscopy was used to evaluate tissue sections. Kaplan Mayer curves was cared for through Medcalc program.
  
  
  
  We collected heterogeneous CTCs, derived from the whole blood of seven patients affected by colon cancer, expressing CD133(pos)CD45(neg) (5 ± 1) and (2 ± 1) and CK20(pos)CD45(neg) of (29 ± 3) (11 ± 1) cells/ml in Dukes D and A stage respectively. Proliferation rate of 57 ± 16 %, expression for CXCR4(pos) of 18 ± 7 % and detectable levels of IL-6, IL-8 and SDF-1 cytokines in conditioned culture medium characterized short-time expanded-CTCs (eCTCs). ECTCs organized in tumor sphere were CD45(neg)CD133(pos) while in adhesion were CXCR4(pos)CK20(pos). These two subsets were separately injected in mice. The first group of xenografts developed superficial lesions within 2 weeks. In the second group, in absence of growing tumour, the survival of injected eCTCs was monitored through SDF-1 serum levels detection. The detection of human cancer cells expressing CK20, in mice tissues sections, suggested a different biological behaviour of injected eCTC-subsets: tumorigenic for the first and disseminating for the second. The benchmarking of the experimental data with the clinical course highlights that patients with prevalence of circulating cancer stem cells (CD45(neg)CD133(pos)) have a lower overall survival. Conversely, patients with prevalence of circulating differentiated cells (CXCR4(pos)CK20(pos)) have a low disease-free survival.
  
  
  
  On the basis of the heterogeneous composition and despite the low number of CTCs, it was possible to distinguish two subgroups of CTCs, suggesting a different clinical outcome. CTC-subsets detailing is useful to better define the metastatic-risk personalized score thus improving disease management and reducing patient care cost.
authors.affiliation: IRC-FSH Laboratories, Department of Health Science, University "Magna Graecia", Viale Europa, Località Germaneto, 88100, Catanzaro, Italy. nataliamalara@unicz.it.
authors.forename: Natalia
authors.initials: N
authors.lastname: Malara
authors.affiliation: Cellular Toxicological Laboratory, Department of Health Science, Salvatore Venuta Campus, University "Magna Graecia", 88100, Catanzaro, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Trunzo
authors.affiliation: Medical Oncologic Department of Experimental and Clinical Medicine, Salvatore Venuta Campus and Cancer Centre of Excellence, "Magna Graecia", 88100, Catanzaro, Italy.
authors.forename: Umberto
authors.initials: U
authors.lastname: Foresta
authors.affiliation: Medical Oncologic Department of Experimental and Clinical Medicine, Salvatore Venuta Campus and Cancer Centre of Excellence, "Magna Graecia", 88100, Catanzaro, Italy.
authors.forename: Nicola
authors.initials: N
authors.lastname: Amodio
authors.affiliation: Bionem Laboratories, Department of Experimental and Clinical Medicine, Salvatore Venuta Campus, University "Magna Graecia", 88100, Catanzaro, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: De Vitis
authors.affiliation: Oncologic Surgery Unit, Cancer Centre of Excellence, "Magna Graecia", 88100, Catanzaro, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Roveda
authors.affiliation: Oncologic Surgery Unit, Cancer Centre of Excellence, "Magna Graecia", 88100, Catanzaro, Italy.
authors.forename: Mariagiovanna
authors.initials: M
authors.lastname: Fava
authors.affiliation: Bionem Laboratories, Department of Experimental and Clinical Medicine, Salvatore Venuta Campus, University "Magna Graecia", 88100, Catanzaro, Italy.
authors.forename: MariaLaura
authors.initials: M
authors.lastname: Coluccio
authors.affiliation: IRC-FSH Laboratories, Department of Health Science, University "Magna Graecia", Viale Europa, Località Germaneto, 88100, Catanzaro, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Macrì
authors.affiliation: Department of Experimental and Clinical Medicine, Salvatore Venuta Campus, University "Magna Graecia", 88100, Catanzaro, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Di Vito
authors.affiliation: Cellular Toxicological Laboratory, Department of Health Science, Salvatore Venuta Campus, University "Magna Graecia", 88100, Catanzaro, Italy.
authors.forename: Nicola
authors.initials: N
authors.lastname: Costa
authors.affiliation: Department of Health Science, Salvatore Venuta Campus, University "Magna Graecia", 88100, Catanzaro, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Mignogna
authors.affiliation: Department of Health Science, Salvatore Venuta Campus, University "Magna Graecia", 88100, Catanzaro, Italy.
authors.forename: Domenico
authors.initials: D
authors.lastname: Britti
authors.affiliation: IRC-FSH Laboratories, Department of Health Science, University "Magna Graecia", Viale Europa, Località Germaneto, 88100, Catanzaro, Italy.|Cellular Toxicological Laboratory, Department of Health Science, Salvatore Venuta Campus, University "Magna Graecia", 88100, Catanzaro, Italy.
authors.forename: Ernesto
authors.initials: E
authors.lastname: Palma
authors.affiliation: IRC-FSH Laboratories, Department of Health Science, University "Magna Graecia", Viale Europa, Località Germaneto, 88100, Catanzaro, Italy. mollace@unicz.it.|Cellular Toxicological Laboratory, Department of Health Science, Salvatore Venuta Campus, University "Magna Graecia", 88100, Catanzaro, Italy. mollace@unicz.it.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Mollace
mesh_terms: D000328:Adult; D000368:Aged; D000818:Animals; D014408:Biomarkers, Tumor / Q000378:metabolism*; D002448:Cell Adhesion; D002453:Cell Cycle; D002454:Cell Differentiation; D049109:Cell Proliferation; D048430:Cell Shape; D002470:Cell Survival; D003110:Colonic Neoplasms / Q000473:pathology*; D016207:Cytokines / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D016513:Mice, SCID; D008875:Middle Aged; D009362:Neoplasm Metastasis; D009360:Neoplastic Cells, Circulating / Q000473:pathology*; D014411:Neoplastic Stem Cells / Q000473:pathology*; D057285:Precision Medicine; D018570:Risk Assessment; D023041:Xenograft Model Antitumor Assays
chemical_list: D014408:Biomarkers, Tumor; D016207:Cytokines
keywords: Circulating colon tumor differentiated/stem cells; Metastatic-risk stratification