Heterogeneous expression of EPCAM in human circulating tumour cells from patient-derived xenografts

Chiara Agnoletto,Marco Galasso,Stefano Cairo,Laura Brulle-Soumare,Fabio Corrà,Lorenzo Pasquali,Jean-Gabriel Judde,Federica Baldassari,Linda Minotti,Stefano Volinia

doi:10.1186/s40364-018-0145-8

Chiara Agnoletto, Marco Galasso + Show 8 more

Open Access

https://doi.org/10.1186/s40364-018-0145-8

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Abstract

BackgroundWe aim to characterize the heterogeneous circulating tumour cells (CTCs) in peripheral blood, independently of physical or immunological purification, by using patient-derived xenografts (PDXs) models. CTC studies from blood generally rely on enrichment or purification. Conversely, we devised a method for the inclusive study of human cells from blood of PDX models, without pre-selection or enrichment.MethodsA qRT-PCR assay was developed to detect human and cancer-related transcripts from CTCs in PDXs. We quantified the EPCAM and keratins CTC markers, in a PDX cohort of breast cancer. The murine beta actin gene was used for normalization. Spearman’s rho coefficients were calculated for correlation.ResultsWe demonstrated, for the first time, that we can quantify the content of CTCs and the expression of human CTC markers in PDX blood using human-specific qRT-PCR. Our method holds strong potential for the study of CTC heterogeneity and for the identification of novel CTC markers.ConclusionsThe identification and the relative quantification of the diverse spectrum of CTCs in patients, irrespective of EPCAM or other currently used markers, will have a great impact on personalized medicine: unrestricted CTCs characterization will allow the early detection of metastases in cancer patients and the assessment of personalized therapies.

Highlights

We aim to characterize the heterogeneous circulating tumour cells (CTCs) in peripheral blood, independently of physical or immunological purification, by using patient-derived xenografts (PDXs) models
RNA was extracted from the whole blood cell pellet, reverse-transcribed and tested for expression of the human Epithelial cell adhesion molecule [Homo sapiens] (EPCAM) and Keratin 19 [Homo sapiens] (KRT19) CTC markers
It is in our plans to extend the number of human CTC markers in the assay, the cohort of PDX mice, to incorporate CTC counting by genomic DNA and to perform in parallel single-cell RNA sequencing

Summary

Introduction

We aim to characterize the heterogeneous circulating tumour cells (CTCs) in peripheral blood, independently of physical or immunological purification, by using patient-derived xenografts (PDXs) models. CTC studies from blood generally rely on enrichment or purification. We devised a method for the inclusive study of human cells from blood of PDX models, without pre-selection or enrichment. Metastases from primary tumours account for the great majority of cancer-related deaths [1]. This process is thought to involve a series of sequential steps, including the release of circulating tumour cells (CTCs) into blood stream [1, 2]. CTCs have been detected in the peripheral blood of patients with advanced cancers of different origin [3].

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