Molecular Biomarkers of Cancer Stem/Progenitor Cells Associated with Progression, Metastases, and Treatment Resistance of Aggressive Cancers

Abstract

The validation of novel diagnostic, prognostic, and predictive biomarkers and therapeutic targets in tumor cells is of critical importance for optimizing the choice and efficacy of personalized therapies. Importantly, recent advances have led to the identification of gene-expression signatures in cancer cells, including cancer stem/progenitor cells, in the primary tumors, exosomes, circulating tumor cells (CTC), and disseminated cancer cells at distant metastatic sites. The gene-expression signatures may help to improve the accuracy of diagnosis and predict the therapeutic responses and overall survival of patients with cancer. Potential biomarkers in cancer cells include stem cell–like markers [CD133, aldehyde dehydrogenase (ALDH), CD44, and CD24], growth factors, and their cognate receptors [epidermal growth factor receptor (EGFR), EGFRvIII, and HER2], molecules associated with epithelial–mesenchymal transition (EMT; vimentin, N-cadherin, snail, twist, and Zeb1), regulators of altered metabolism (phosphatidylinositol-3′ kinase/Akt/mTOR), and drug resistance (multidrug transporters and macrophage inhibitory cytokine-1). Moreover, different pluripotency-associated transcription factors (Oct3/4, Nanog, Sox2, and Myc) and microRNAs that are involved in the epigenetic reprogramming and acquisition of stem cell–like properties by cancer cells during cancer progression may also be exploited as molecular biomarkers to predict the risk of metastases, systemic treatment resistance, and disease relapse of patients with cancer.

Introduction

Significant advancement in basic and clinical oncology during the last few years has led to earlier diagnosis and more effective therapeutic management of patients with leukemias and organ-confined tumors in the clinics (1-3). Although the surgical tumor resection may result in some cases to a complete remission, the rapid cancer progression of aggressive cancers to locally invasive and metastatic stages is generally associated with the development of resistance mechanisms by cancer cells to current antihormonal, radiation, and/or chemotherapeutic treatments and disease relapse (1-3). At the present time, the metastatic cancers remain the leading cause of the death of patients with cancer. Therefore, many research efforts have been made to identify and validate novel molecular biomarkers and therapeutic targets in cancer cells at primary and secondary tumors to prevent cancer progression and metastases and optimize the genetic- and proteomic-based individualized treatments of patients with cancer (Fig. 1; refs. 4-28).

Figure 1.

Schematic representation of functions of cancer stem/progenitor cells during cancer progression and metastasis and characterization of their biomarkers. The scheme shows cancer stem/progenitor cells endowed with stem cell–like properties and which can generate the total cancer cell population at the primary and secondary tumors. Moreover, the exosomes released by cancer cells, which may contribute to the malignant transformation of other cancer cells via the transfer of oncogenic products and drug resistance–associated molecules such as EGFRvIII and P-glycoprotein, are also illustrated. The possibility to perform the characterization of molecular gene signature and biomarkers of cancer cells, exosomes, and CTCs, including cancer stem/progenitor cells expressing stem cell–like markers, is also indicated.

Importantly, accumulating lines of evidence have revealed that the shedding of cancer cells from the primary tumors into the lymphatic vessels and peripheral circulation can occur very early during the cancer development and be dependent of cellular origin, genetic alterations, and aggressiveness of cancer subtypes (16, 29-41). Hence, some patients who undergo a complete surgical tumor resection with negative margins may show the presence of circulating tumor cells (CTC) in the peripheral blood and disseminated tumor cells at the regional lymph nodes and distant tissues and organs (Fig. 1; refs. 16, 29-41). Consequently, CTCs that are able to survive in the bloodstream and spread at distant sites can persist and contribute to metastases and disease relapse even after an effective and apparently curative surgical resection of the primary tumor. In this regard, a growing body of experimental evidence has also revealed that cancer stem/progenitor cells endowed with stem cell–like properties, also designated as cancer-, tumor-, and metastasis-initiating cells, can provide critical functions for tumor growth, metastases at near and distant tissues and organs, treatment resistance, and disease relapse. In fact, it has been shown that the most cancers may originate from the malignant transformation of immature tissue-resident stem/progenitor cells or their early differentiated progenies endowed with a high self-renewal ability and aberrant differentiation potential (2, 42-44). The cancer stem/progenitor cells expressing specific stem cell–like markers such as CD133, CD44^high, nestin, aldehyde dehydrogenase (ALDH^high), and high levels of ATP-binding cassette (ABC) multidrug transporters have also been identified and isolated from primary and secondary neoplasms, including leukemias, melanomas, brain tumors, and the most epithelial cancers and cancer cell lines (9,17, 24, 44-76). It has been shown that cancer stem/progenitor cells were able to give rise to the total tumor cell mass, including differentiated cancer cells that reconstituted the histological architecture and molecular characteristics of primary and secondary tumors closely resembling to original patient’s tumors in vivo (9, 17, 45-57, 59-66, 68, 69, 71, 77). Moreover, the data from recent studies have indicated that cancer stem/progenitor cells may be more resistant than their differentiated progenies to current antihormonal, radiation and chemotherapeutic treatments, and targeted therapies (17, 22-25, 44, 52, 53, 56-64, 68, 70, 72, 77-94).

We review here recent advances on the characterization of gene products often altered in cancer stem/progenitor cells and their differentiated progenies during primary cancer progression and dissemination through the peripheral circulation and metastases. The emphasis is on molecular gene signatures, epithelial–mesenchymal transition (EMT)-like and stem cell–like biomarkers detected in cancer cells and exosomes associated with cancer progression, treatment resistance, disease relapse, and poor outcome of patients with cancer.

Heterogeneity of Cancers

The most cancers are heterogeneous diseases and include some biological subtypes characterized by specific genetic and/or epigenetic alterations occurring in cancer stem/progenitor cells and their differentiated progenies during cancer initiation and progression to locally invasive and metastatic stages (4, 6, 7, 44, 60, 78, 80, 95-109). Although the molecular events that govern the spread of cancer stem/progenitor cells and their differentiated progenies at preferential metastatic sites remain not precisely defined, some lines of evidence suggest that particular gene signatures of cancer cells at primary cancers may be associated with their propensity at metastasizing at specific tissues and organs (4, 6, 7, 95-102). More particularly, the alterations in the gene products involved in the acquisition of aggressive and migratory properties by highly tumorigenic cancer stem/progenitor cells during the EMT program at the primary tumor may result in their invasion to near lymph nodes and tissues, dissemination through the peripheral circulation, and metastases at distant sites (Fig. 1; refs. 30, 42, 90, 91, 96, 97, 110-126). Moreover, different gene products have also been identified in exosomes released by tumor cells and CTCs with stem cell–like features that may be used as diagnostic, prognostic, and/or predictive biomarkers. In this regard, we review the gene products frequently altered in cancer cells, during cancer progression to locally advanced and metastatic states, exosomes and CTCs that have been associated with treatment resistance, disease relapse, and poor overall survival of patients with cancer.

Molecular gene signatures and stem cell–like biomarkers associated with aggressive and metastatic cancers

Some tissue-specific and whole genome approaches for molecular profiling of complex gene signatures of tumors and cancer cell lines have been developed in the last few years and led to the discovery of specific gene sets related to particular cancer types and which may predict the risk of metastatic dissemination at specific tissues and organs and overall outcome of patients with cancer (Fig. 1; refs. 4-15). Moreover, the in vitro and in vivo characterization of stem cell–like markers and functional features of cancer stem/progenitor cells in tumor tissue specimens from patients with cancer and cancer cell lines has also revealed their major implications in tumor growth, metastases, treatment resistance, and disease relapse (Fig. 1; refs. 15-27). We are reporting here the gene signatures and stem cell–like biomarkers of cancer cells associated with particular subtypes of aggressive cancers, including hormone-independent prostate and breast cancers.

Prostate cancer

Prostate cancer is among the most commonly diagnosed malignancies affecting men (127-129). Although an earlier diagnosis of patients with prostate cancer has led to an improvement of their effective treatment by tumor resection surgery and/or radiation therapy, the locally advanced and metastatic castration-resistant prostate cancers (CRPC) still represent the second leading cause of cancer-related death (127-129). In fact, antihormonal treatments and first-line docetaxel-based chemotherapies of metastatic CRPCs are only palliative and result in the death of most patients after about 12 to 19 months (127-129). Therefore, this inefficacy of current treatments against CRPCs underlines the importance to identify novel biomarkers to predict the risk of propagation to CRPCs. Of clinical relevance, a 11-gene signature related to the activation of polycomb-group protein BMI-1 pathway, which provides critical functions for the self-renewal of diverse normal and cancer stem cells, has notably been identified through a comparative genomic approach using primary and metastatic tumor specimens from transgenic adenocarcinoma of the mouse prostate model and patients with prostate cancer (4). The 11-gene signature includes 8 upregulated genes (Ki67, cyclin B1, gastrulation brain homeobox 2, budding uninhibited by benzimidazoles 1 “BUB1” that acting as serine/threonine-protein kinase, kinetochore associated 2, ubiquitin-specific protease 22, host cell factor 1, and ring finger protein 2) and 3 downregulated genes (ankyrin 3, fibroblast growth factor receptor 2, and carboxylesterase 1; ref. 4). This 11-gene signature, which is associated with an upregulation of stem cell–resembling expression profile of the BMI-1–regulated pathway, has been shown to be a powerful predictor of patients with prostate cancer progression to locally invasive and metastatic stages (4). The analyses of this 11-gene signature of BMI-1–driven pathway in 1,153 clinical tumor specimens from patients with cancer diagnosed with 11 different types of cancer have also indicated that the expression of these gene products was associated with a short interval to disease recurrence, distant metastases, and death after therapy of patients with cancer (4). These cancer types comprise acute myeloid leukemia, lymphoma, mesothelioma, medulloblastoma, glioma, and prostate, breast, lung, ovarian, and bladder epithelial cancers (4). Importantly, high expression levels and/or activities of stem cell–like markers such as CD133, CD44, integrin-α₂, nestin, ALDH1, and CD49f in primary prostatic adenocarcinomas and bone metastases from patients and prostate cancer cell lines have also been associated with their aggressive and invasive phenotypes and treatment resistance (17-26, 28). Particularly, the data from immunohistochemical analyses of integrin-α₂, integrin-α₆, and hepatocyte growth factor receptor (MET) in prostate cancer cells have revealed that an increase of expression levels of these markers in prostatic adenocarcinoma specimens from patients with prostate cancer was associated with an enhanced risk of local invasion and bone metastasis (130). It has also been reported that several ALDH isoforms were expressed in specimens of primary prostate tumors with matched bone metastases and prostate cancer cell lines, and ALDH^high PC3M-Pro4 cells rapidly formed primary prostatic tumor and metastasized at distant sites in vivo as compared with ALDH^low PC3M-Pro4 cells (18, 19). Of therapeutic interest, the knockdown of ALDH7A1 expression in PC3M-Pro4 cells has also been observed to result in a decrease of the integrin-α₂^high/integrin-α_v^high/CD44⁺ PC3M-Pro4 stem/progenitor cell subpopulation and gene products involved in the EMT process and invasion, including snail, snail2, twist, and extracellular molecule, osteopontin (19). Moreover, the ALDH7A1 silencing in PC3M-Pro4 cells significantly inhibited their clonogenic and migratory abilities in vitro as well as their capacity to form bone metastases in vivo (19). In the same way, it has also been observed that prostate-specific antigen (PSA^−/lo) prostate cancer cells from LNCaP, LAPC4, or LAPC9 cell lines or tumor specimens from patients with prostate cancer expressing stem cell–like markers such as ALDH, CD44, integrin-α₂β₁, and with a long-term tumor-propagating ability were able to give rise to the total mass of PSA⁺ prostate cancer cells in vivo (17). It has also been noted that PSA^−/lo LAPC9 cells were resistant to androgen deprivation in castrated hosts, and high grade and recurrent prostatic adenocacinoma specimens of patients were enriched in PSA^−/lo prostate cancer cells (17). These observations suggest that PSA^−/lo prostate cancer cells may play critical functions for the tumor regrowth after antihormonal treatment initiation.

In addition, we have observed that sonic hedgehog (SHH/GLI-1), epidermal growth factor receptor (EGFR), pAkt, nuclear factor-κB (NF-κB), and macrophage inhibitory cytokine-1 (MIC-1) are frequently overexpressed in prostate cancer cells, including CD133⁺ prostate cancer stem/progenitor cells, detected in prostatic adenocarcinoma and bone metastasis specimens from patients with prostate cancer (23, 24). The high expression levels and/or activities of these oncogenic products were also detected in the side population from tumorigenic and invasive WPE1-NB26 cells and metastatic PC3M-LN4 endowed with a high prostasphere-forming ability (23-25). Moreover, the targeting of hedgehog, EGFR, pAkt, NF-κB, or MIC-1 by using cyclopamine, gefitinib, Akt inhibitor III, partenolide, or anti-MIC-1 antibody was effective at inducing the apoptotic effects on side population and non-side population cells from WPE1-NB26 and PC3M-LN4 cell lines and improving cytotoxic effects induced by docetaxel on these side population and non-side population cells (23, 24). Recent studies have also revealed that the changes in the expression levels and activities of hypoxia-inducible factors (HIF) and microRNAs (miRNA), which are short noncoding RNA molecules of about 22 nucleotides that are involved in the stringent control of normal stem cell behavior often occur in cancer cells including prostate cancer cells (27, 28, 131, 132). HIFs and miRNAs may be involved in the epigenetic reprogramming and acquisition of stem cell–like features by cancer cells (27, 28, 131, 132). More specifically, the upregulation of HIF-1α and HIF-2α has been associated with the increased expression of pluripotency-associated transcription factors (Nanog, Oct4, and Sox2) and EMT program-associated molecules in prostate cancer cells under normoxic and hypoxic conditions during prostate cancer progression to locally invasive and metastatic stages (27, 28). The downregulation of some tumor-suppressor miRNAs such as mi-R34a, let-7b, miR-106a, and miR-141 combined with an upregulation of miR-301 and miR-452 has also been observed to regulate the proliferation and tumorigenicity of CD133/CD44/integrin-α₂β₁ prostate cancer stem/progenitor cell population and side population cells in vitro and in vivo (131, 132).

Breast cancer

Breast cancer is among the most common cancers in women (127, 133, 134). Although a high survival rate is observed for early-stage patients with breast cancer treated by tumor resection surgery and adjuvant treatments, the propagation to locally invasive and metastatic disease state is generally associated with the development of resistance mechanisms by cancer cells to current antihormonal treatments, targeted therapies, radiation, and chemotherapies (127, 133, 134). Typically, breast cancer is a complex and highly heterogeneous disease that encompasses distinct cancer subtypes characterized by the malignant transformation of the basal and/or luminal breast epithelial cells in the mammary gland (135, 136). In this regard, the data from analyses of the expression levels of gene products in breast cancer cells, including breast cancer stem cells (BCSCs) with stem cell–like features, have indicated that breast cancer subtypes may exhibit cellular and functional heterogeneities (5-14, 137, 138). It has been observed that CD44^high/CD24^−/low BCSCs are frequently detected in basal-like subtypes of breast cancer whereas these immature cancer cells are very low in erbB2/HER2⁺ breast tumors (8). Moreover, a transcriptional analysis of basal-like breast cancer cell lines has led to the identification of 2 major cancer subclasses in which basal B seemed mesenchymal-like, whereas basal A may have either a luminal-like or basal-like morphology. It has been observed that a CD44^high/CD24^low epithelial basal A cell subpopulation isolated from triple-negative HMT3909S13 breast cancer cell line exhibited cancer stem cell features such as mammosphere formation and resistance to the standard chemotherapy as well as tumor-initiating capacity in vivo (6). In contrast, CD44^high/CD24⁻ mesenchymal-like basal B cell fraction does not exhibit these functional features (6). The data from comparative quantitative proteomic and gene array analyses of CD44^high/CD24^low BCSCs with an epithelial-like morphology have also indicated that these immature cancer cells expressed lipolysis-stimulated lipoprotein receptor, RAB25, S100A14, and mucin 1 (MUC1; ref. 6). These CD44^high/CD24^low BCSCs also expressed a novel 31-gene signature that was related at the presence of distant metastases in estrogen receptor negative (ER⁻)-human breast cancer specimens (6). Conversely, the results from another investigation have revealed that CD44^high/CD24^low BCSCs that were detected in patient’s primary tumors exhibited a gene signature as the claudin-low molecular subtype, which is characterized by the expression of several EMT program-associated molecules (10). It has been noted that the CD44^high/CD24^low BCSCs expressing mesenchymal markers were enriched in patient’s primary tumors after either endocrine letrozole-based therapy or docetaxel-based chemotherapy (10). These findings suggest that breast cancer cells endowed with an epithelial or mesenchymal phenotype can exhibit cancer stem cell properties dependent of breast cancer subtypes and be involved in the metastasis formation and treatment resistance.

On the other hand, the data from analyses of global gene expression profiles have also indicated that a common subset of 81 genes was differentially expressed between ER⁻ and ER⁺ breast tumor specimens and breast cancer cell lines as well as in androgen-independent versus androgen-sensitive prostate cancer cell lines (11). It has been noted that some signaling elements of EGFR pathway were enriched in ER⁻ breast and androgen-independent prostate cancer cell lines, suggesting that the sustained activation of this tumorigenic cascade may contribute to the growth and survival of these hormone-independent cancer cells (11). Interestingly, it has been reported that an 8-gene signature (EGFR, integrin-α₃, myosin light chain kinase, retinoic acid–induced protein 14, AHNAK nucleoprotein, glutaminase, interleukin-32, and nicotinamide N-methyltransferase) was associated with the invasion and sensitivity of a panel of 78 tumor cell lines to chemotherapeutic drugs, including paclitaxel, docetaxel, erlotinib, everolimus, and dasatinib (14). This set of 8 genes also predicted the relapse-free survival for patients with breast cancer (14).

Other cancer types

Recent investigations have also led to the discovery of specific gene signatures and potential biomarkers in cancer tissue specimens for improving the personnel management of patients with other aggressive cancers such as brain, melanoma, esophageal squamous cell carcinoma (ESCC) and lung, pancreatic, colorectal, esophageal, and ovarian cancers (Table 1; refs. 3, 14, 70, 139-149). For instance, the real-time PCR data from gene expression profiles of brain tumor stem-like cells (BTSCs) from 5 human glioblastoma multiforme (GBM) and matched GBM tissue specimens have indicated that SHH and GLI1 hedgehog signaling elements were coexpressed only at high levels in BTSCs 1 to 3 and not in BTSCs 4 and 5 (141). It has also been noted that the SHH/GLI1 cascade-induced BTSC proliferation via phosphatidylinositol-3′ kinase (PI3K)/Akt/mTOR, was inhibited by small hairpin RNA (shRNA) against human GLI1 or rapamycin in vitro and in an intracranial tumor model in athymic mice (141). Of clinical interest, higher expression levels of SHH, patched receptor 1 (PTCH1), and GLI1 have also been detected in phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-expressing brain tumors than in PTEN-deficient brain tumors and associated with a poorer survival time of GBM patients (141). Furthermore, it has been shown that melanoma cells from patient with melanoma specimens expressing high levels of ALDH1A1 and ALDH1A3 isoforms displayed a high self-renewal ability, and were more resistant to chemotherapeutic agents and more tumorigenic than ALDH⁻ melanoma cells in non-obese diabetic (NOD)/severe combined immonodeficient (SCID) mice (139). It has also been noticed that the silencing of the ALDH1A gene by shRNA led to an arrest in cell cycle and apoptosis of ALDH1A⁺ melanoma cells, inhibition of tumor growth in vivo, and sensitized the ALDH1A⁺ melanoma cells to cytotoxic effects of chemotherapeutic drugs (139). These findings implicate that ALDH1A and ALDH3A isozymes are putative biomarkers for melanoma stem cells and constitute attractive therapeutic targets for treating melanoma patients. In the same way, human pancreatic ductal adenorcinoma (PDAC) specimens have also been observed to contain a side population exhibiting the gene expression signatures that are associated with pancreatic cancer stem cell–like characteristics and resistance to chemotherapeutic drugs (144). Among these genes, expression levels of ABCB1 multidrug transporter and CXC chemokine receptor 4 (CXCR4) were correlated with a worse survival of patients with pancreatic cancer (144).

Table 1.

Characterization of molecular biomarkers detected in patient tissue specimens and their clinical significances in diverse aggressive and metastatic cancers

Results and clinical significance
Cancer type	Biomarker detection method	Number of patients/ samples	Biomarkers and their diagnostic, prognostic, and predictive potentials	Ref.
11 different cancer  types including  hematologic, brain,  and epithelial cancers	Microarray and  qRT-PCR analyses	1,153 clinical tumor  specimens from patients  with cancer diagnosed  with acute myeloid  leukemia, lymphoma,  mesothelioma,  medulloblastoma,  glioma, and prostate,  breast, lung, ovarian, or  bladder epithelial  cancers	An 11-gene signature that was  associated with an upregulation of  stem cell–resembling expression  profile of the BMI-1–regulated  pathway has been shown to be a  powerful predictor of prostate cancer  progression and metastases. The  expression of this 11-gene signature  has also been associated with a short  interval to disease recurrence, distant  metastases and death after therapy of  patients with 11 different cancer  types.	(4)
Prostate cancer	Immunhistochemical  staining	Tissue microarray (TMA)  containing primary  prostate tumor sections  (n = 30) as well as 10  primary prostate tumors  and their 10 matched  bone metastases from  patients with prostate  cancer.	ALDH7A1 isoform was highly  expressed in 83% clinical specimens  of primary prostate tumors (TMA) as  well as in 80% and 90% of prostate  cancers and their matched bone  metastases, respectively. These data  suggest that ALDH7A1 could  constitute a potential biomarker for  the stratification of patients with  prostate cancer at risk of developing  metastatic disease.	(18)
Prostate cancer	Immunhistochemical  staining of PSA+ and  PSA−/lo cells in  prostate cancer  sections	Primary tumor specimens  from untreated patients  with Gleason score 7 (n =  10) and 9 or 10 (n = 10),  and treatment-failed  patients (n = 23)	High grade and recurrent prostatic  adenocacinoma specimens of  patients were enriched in PSA−/lo  prostate cancer cells suggesting that  PSA−/lo prostate cancer cells with  stem cell–like properties may play  critical functions in the tumor regrowth  after treatment initiation.	(17)
Prostate cancer	Immunhistochemical  staining	76 primary tumor  specimens from patients  with prostate cancer with  Gleason scores 6–10  plus 30 bone metastasis  specimens from patients  with prostate cancer.	The expression levels of EGFR,  Ser473-pAkt, NF-κB p65, and MIC-1  proteins were significantly enhanced  in the same subset of 76 cases of  prostate cancer specimens during the  disease progression and detected in a  small subpopulation of CD133+  prostate cancer cells and the bulk  tumor mass of CD133− prostate  cancer cells. All of these biomarkers  were also overexpressed in 80% to  100% of 30 prostate cancer  metastasis bone tissue specimens  indicating their potential use as  diagnostic and prognostic  biomarkers.	(24)
Breast cancer	Analysis of a gene  expression signature  from CD44+/CD24−/low  cells and  mammosphere (MS)-  forming cancer cells in  breast cancer  specimens	The gene expression  signature was analyzed in  36 tumors (18 luminal  A/B, 13 basal-like, and  5 ErbB2-enriched). For  therapeutic significance  of signature, 18 patients  with breast cancer pairs  before and after therapy  with letrozole or 12  patient pairs before and  after docetaxel therapy  were used.	A gene expression signature common  to both CD44+/CD24−/low BCSCs  and MS-forming breast cancer cells  was mainly detected in patient’s  primary tumors as the claudin-low  molecular subtype expressing  several EMT program-associated  molecules. The CD44+/CD24−/low-  MS and claudin-low signatures were  also enriched in patient’s primary  tumors after either endocrine  letrozole-based therapy or  docetaxel-based chemotherapy.  These results support the  potential application of the CD44+/  CD24−/low-MS signature to predict  the treatment resistance and  relapse.	(10)
Breast and prostate  cancers	Comparative analyses  of profiles of hormone-  sensitive and  -independent breast  cancers and prostate  cancer cell lines with  gene expression data  sets from patient’s  breast cancers.	Breast ER-status signature  was established from  ER− (hormone-  independent) and ER+  (hormone-dependent)  invasive breast cancer  (n = 295) profile datasets  and 18 different breast  cancer cell lines (10 of  them ER−). The gene  signature data sets from  8 androgen-sensitive and  androgen-independent  prostate cancer cell lines  was also used.	A common subset of 81 genes was  differentially expressed between ER−  and ER+ breast tumor specimens and  breast cancer cell lines as well as in  androgen-independent versus  androgen-sensitive prostate cancer  cell lines. EGFR signaling elements  were also enriched in ER− breast and  androgen-independent prostate  cancer cell lines suggesting that the  EGFR tumorigenic cascade may  contribute to hormone-independent  phenotypes of the breast cancer and  prostate cancer cells.	(11)
Breast and lung cancers	Comparative analyses  of invasion assay and  Affymetrix gene  expression data on a  panel of cancer cell  lines with chemo-  sensitivity data.	Invasion-associated genes  established from 60  human cancer cell lines  developed by the  National Cancer Institute  were compared with  chemo-sensitivity data of  99 anticancer drugs and  clinical data from  adjuvant chemotherapy  cohorts in 508 breast and  71 patients with lung  cancer and a cohort of  untreated patients  (controls).	An 8-gene signature (EGFR, integrin-α3,  myosin light chain kinase, retinoic  acid induced protein 14, AHNAK  nucleoprotein, glutaminase,  interleukin-32, and nicotinamide  N-methyltransferase) was  associated with the invasion and  sensitivity of a panel of 60 tumor cell  lines to paclitaxel, docetaxel,  erlotinib, everolimus, and dasatinib.  The 8-gene signature also  predicted the relapse-free survival  for patients with breast and lung  cancer.	(14)
Non–small cell lung  cancer (NSCLC)	Immunohistochemical  analysis	Various histological tissue  specimens of lung  squamous cell  carcinoma (SCC) and  lung adenocarcinomas  in 2 independent large  TMA sets (n1 = 287 and  n2 = 511) were used.	A marked nuclear expression of SOX2  embryonic stem cell transcriptional  factor was detected in all normal  bronchial epithelia, alveolar  bronchiolization structures, and  premalignant lesions in SCC  development (hyperplasia, dysplasia,  and carcinoma in situ). In contrast,  SOX2 protein expression was not  seen in all normal alveoli and atypical  adenomatous hyperplasias.  Moreover, SOX2 expression was  greatly higher in lung SCCs compared  with lung adenocarcinomas.	(143)
Pancreatic cancer	Whole-genome  expression analyses of  side population cells  and main non-side  (non-side population)  cell fraction isolated  from human pancreatic  cancer resections  specimens by FACS	Side population cells were  obtained from human  PDAC specimens (n = 32)  and the prognostic value  of the side population  gene signature was  validated in a large  independent series of  PDAC patients (n = 78).	32 PDAC specimens contained a side  population cell subpopulation exhibiting  an upregulation of genes associated  with markers of putative pancreatic  cancer stem and therapy resistance.  The validation of side population  cell–derived gene signatures of 32 or  10 up- or downregulated genes in a  different set of 78 PDAC samples has  also indicated that the expression levels  of ABCB1 multidrug transporter and  CXCR4 were correlated with worse  patient survival suggesting their  potential used as prognostic biomarkers  and therapeutic targets.	(144)
Brain cancer	The mRNA expression  levels of PTEN, SHH,  PTCH1, and GLI1 were  determined by real-  time PCR assays.	55 GBM tissue specimens  of patients (age range =  29–75) who were  diagnosed with de novo  glioblastoma and treated  with external beam  radiation therapy to 60 Gy  and chemo-therapy were  used. The GBM samples  were separated into a  PTEN-expressing group  and a PTEN-deficient  group on the basisof their  PTEN expression.	The expression levels of SHH and GLI1  were significantly higher in PTEN-  expressing GBM specimens than in  PTEN-deficient GBMs. The higher  expression levels of SHH, PTCH1,  and GLI1 in PTEN-coexpressing GBM  specimens were also associated with  reduced survival of patients. These  data indicate that hedgehog signaling  elements may constitute potential  prognostic biomarkers and molecular  targets for individualized treatment of  GBM patients overexpressing SHH/  GLI1.	(141)
Melanoma	Analyses of ALDH activity  by Aldefluor assay and  gene expression by  microarray analyses,  qRT-PCR and  immunohischemistry	3 primary and 6 metastatic  tumors from melanoma  patients	8 patient’s tumors harbored small  ALDH+ subpopulations, ranging from  0.08% to 1.15%. The gene expression  signatures of melanoma cells  expressing high levels of ALDH1A and  ALDH3A from patient’s melanoma  specimens were characterized by  expression of some genes associated  with stem cell functions.	(139)

Altogether, these studies have led to the identification and validation of gene expression signatures and potential diagnostic, prognostic, and predictive biomarkers related to stem cell–like cancer cells, EMT program, treatment resistance, and relapse of different aggressive cancers. Future investigations are however necessary to validate the expression of these gene signatures and their implications in the treatment resistance on larger cohorts of tumor tissue specimens from patients with cancer. In this regard, recent studies have also revealed the possibility to analyze the phenotypic features of exosomes secreted by cancer cells and CTCs, including circulating cancer stem/progenitor cells, to assess the tumor stage and risk of cancer progression, metastases, and disease relapse of patients with cancer.

Molecular biomarkers of exosomes

Accumulating lines of evidence have indicated that exosomes that are secreted by normal and cancer cells can act as key mediators in signal transduction mechanisms that are involved in short- and long-distance cell-to-cell communication (150-157). In fact, the exosomes are small microvesicles (sized about 50–100 nm) derived from cellular endocytosis that may contain specific lipids, proteins, mRNAs, and miRNAs encapsulated in a cholesterol-rich phospholipid membrane (150-157). More particularly, the exosomes that are released by cancer cells in their surrounding microenvironment and peripheral circulation may adhere via their antigenic surface molecules or transfer their cytoplasmic contents through membrane fusion to their targeted host cells, including other cancer cells, stromal cells, endothelial cells, and immune cells (Fig. 1; refs. 150-157). Hence, the shedding of exosomes by cancer cells under normoxic and hypoxic conditions may modulate immune system and promote the malignant behavior of recipient cells in their tumor microenvironment and angiogenesis as well as the formation of premetastatic niches, metastases at distant tissues and organs, and treatment resistance (Fig. 1 and Table 2; refs. 150-160). Importantly, recent advances in the development of isolation techniques of exosomes from some types of body fluids or cancer cell culture supernatants using centrifugation, ultrafiltration, immunoprecipitation, magnetic activated cell sorting(MACS), and immunoaffinity capture methods have led to an improvement of the purity of exosomal preparations (150-159, 161-164). Hence, several studies carried with these isolation methods have indicated the presence of exosomes in blood, plasma, serum, lymph, urine, cerebrospinal fluid, or ascites from patients with melanoma and brain, ovarian, breast, pancreatic, colon, bladder, prostate, and lung cancers as well as conditioned medium of different cancer cell types (Fig. 1 and Table 2; refs. 150-159, 161-164). It has also been observed by proteomic profiling that different exosomal preparations of different cancer types frequently expressed a set of biomarkers. The common biomarkers of most exosomes include tubulin, actin, tetraspanins (CD9, CD63, CD81, and CD82), Alix, TSG101, clathrin, small GTPases (Rab proteins), annexins, heat-shock proteins (Hsp70 and Hsp90), 14-3-3 proteins, and MHC class I molecules that are involved in the biosynthesis, cytoskeletal structure, and trafficking of exosomes (159, 162-164). Furthermore, tumor cell–derived exosomes also harbored specific RNAs, miRNAs, and proteins, including stem cell–like markers (CD133, CD44, and/or CD105) and oncogenic products (EGFR, EGFRvIII mutant, erbB2/HER2/Neu, MET, claudins, and/or FASN) that are related to cancer progression, metastases, and treatment resistance (150-159, 161-166). For instance, the data from proteomic analyses of highly purified exosomes derived from 2 ovarian cancer cell lines, OVCAR-3 and IGROV1, have indicated that several proteins of tumorigenic pathways that are overexpressed in ovarian cancer tissues and associated with migration, invasion, immune modulation, angiogenesis, and metastasis were also detected in exosomes (162). These oncogenic products comprise EGFR, epithelial cell surface antigen (EpCAM), proliferation cell nuclear antigen (PCNA), tubulin β-3 chain (TUBB3), apolipoprotein E (APOE), and claudin 3 (CLDN3; ref. 162). Moreover, the exosomes expressing T-cell receptor (TCR), CD20, HLA-DR, B7-2, HER2, CA125, and histone H2A have been detected in 85.4% (35/41) of ascites samples from patients with ovarian cancer (167). It has also been shown that the exosomes induced the apoptosis of precursor and mature dendritic cells and peripheral blood mononuclear cells (PBMC) and impaired the cytotoxic activity of PBMCs in the presence of dendritic cells suggesting their implication in the modulation of antitumor immunity (167).

Table 2.

Potential applications of molecular biomarkers of circulating exosomes for the diagnosis, prognosis, and prediction of treatment responses of diverse aggressive and metastatic cancers

Potential application of exosomes	Cancer type	Exosome source and purification and detection methods	Results and their significances	Ref.
Diagnostic  biomarkers	Ovarian cancer	Tumor-derived exosomes  were isolated from  peripheral blood of 50  women diagnosed with  serous papillary  adenocarcinoma of the  ovary (stages I–IV) using a  MACS procedure with  anti-EpCAM coupled to  magnetic microbeads.	An 8 diagnostic miRNA signature (miR-21,  miR-141, miR-200a, miR-200c, miR-200b,  miR-203, miR-205, and miR-214) was detected  in ovarian tumor-derived EpCAM-exosomes  and ovarian tumor specimens isolated from  same patients with cancer. In contrast, these  exosomal miRNAs were not be detected in  normal controls. Moreover, the levels of  circulating exosomes detected in women  increased with the stages I–IV of ovaian cancer.	(166)
Diagnostic  biomarkers	Prostate cancer	Exosomes were prepared  by differential  centrifugation from urine  samples of 11 patients  with prostate cancer.	The expression of prostate cancer biomarkers,  including prostate cancer gene-3 (PCA-3) and  TMPRSS2; ERG gene fusion, was detected in  urinary exosomes suggesting their potential  use for the diagnosis and monitoring of patients  with prostate cancer.	(161)
Diagnostic  biomarker	Brain tumor	Exosomes were purified by  differential centrifugation  from serum samples from  25 GBM patients and 30  healthy individuals.	Tumor-specific and constitutively active EGFRvIII  mutant was detected by RT-PCR in serum  exosomes from 7 of 25 (28%) GBM patients  whereas EGFRvIII mRNA was not detected in  serum exosomes from 30 normal individuals  used as controls.	(152)
Diagnostic and  prognostic  biomarker	Esophageal  squamous cell  cancer (ESCC)	Exosomes were extracted  from the serum samples  of patients with ESCC  and benign diseases  without systemic  inflammation.	The exosomal miR-21 expression was  significantly upregulated in serum samples  from patients with ESCC versus benign  diseases without systemic inflammation.  Exosomal miR-21 positively correlated with  tumor progression and aggressiveness, lymph  node status, and the presence of metastasis  with inflammation and clinical stage without  inflammation.	(165)
Diagnostic and  prognostic  biomarkers	Melanoma	Exosomes were purified by  centrifugation from  peripheral blood samples  from stage I–IV  melanoma subjects.	An exosome-specific melanoma signature  comprised of MET, tyrosinase-related protein-  2, very late antigen-4, HSP70, and HSP90 has  been associated with stage and outcome of  patients with melanoma.	(169).
Predictive biomarker	Breast cancer	Exosomes were isolated  from HER2-  overexpressing breast  carcinoma cell lines  SKBR3 and BT474 or  breast cancer patient’s  serum samples.	Exosomes from HER2+ SKBR3 and BT474 tumor  cell-conditioned supernatants or breast cancer  patients’ serum expressing HER-2 have been  observed to interact with humanized antibody  trastuzumab. Moreover, the data from  functional assays have also revealed that  exosomes suppressed the inhibitory activity of  trastuzumab on the SKBR3 cell proliferation  whereas lapatinib had no effects. These  findings suggest that HER2+ exosomes can  modulate the sensitivity of breast cancer cells to  trastuzumab, and consequently HER2-driven  tumor aggressiveness.	(172)
Diagnostic and  predictive  biomarker	Prostate cancer	Exosomes were purified by  ultracentrifugation from  plasma and serum  samples from 39 patients  with prostate cancer, 20  benign prostatic  hyperplasia (BPH)  patients, 8 recurrent  prostate cancers and 16  healthy controls.	Survivin level was significantly upregulated in the  tumor-derived exosomes compared with those  from BPH and controls. Exosomal survivin level  was also higher in patients that had relapsed  after chemotherapeutic treatment compared  with controls.	(171)
Diagnostic,  prognostic and  predictive  biomarkers	Brain cancer	Profiling of circulating  exosomes labeled with  target-specific magnetic  nanoparticles was  directly performed in  blood samples of 24  GBM patients and 8  healthy volunteers using  microfluidic chip.  Exosomes were detected  with miniaturized nuclear  magnetic resonance  system	Data of protein profiling of circulating exosomes  have indicated that the expression of a  combination of 4 markers (EGFR, EGFRvIII  mutant, podoplanin, and cytosolic isocitrate  dehydrogenase 1) considerably improved the  detection accuracy (>90%) as compared with a  single marker (<76). Circulating exosome  profiling also predicted treatment outcomes of  GBM patients and differentiated between  responders and nonresponders to a treatment  with alkylating agent, temozolomide plus  radiation.	(170)

In addition, the tumor-specific and constitutively active form of EGFR designated as EGFRvIII mutant has also been detected in serum exosomes from 7 of 25 patients with glioblastoma (152). It has been noted that EGFRvIII protein expressed by glioma cells may be transferred via exosomes to other glioma cells lacking EGFRvIII, and thereby promoted their oncogenic transformation via the activation of PI3K/Akt and mitogen-activated protein kinase (MAPK) pathways (Figs. 1 and 2A and Table 2; ref. 153). These molecular events induced the changes in the expression of EGFRvIII-regulated genes such as vascular growth factor “VEGF,” Bcl-xL, and p27 in host glioma cells and increased their anchorage-independent growth capacity (153). It has also been observed that exosomes released from glioblastoma tumor cells also contained mRNA, miRNA, and angiogenic proteins that could be transferred to host cells, including normal brain microvascular endothelial cells, and thereby stimulate the tubule formation (153). In the same way, cancer stem cells expressing the mesenchymal stem cell marker CD105 from human renal cell carcinoma also released exosomes that contained proangiogenic mRNAs and miRNA (168). It has also been noted that the exosomal mRNAs and miRNA in turn activated the growth of normal endothelial cells and induced the vessel formation and angiogenesis after implantation in SCID mice (168). Moreover, the treatment of SCID mice with exosomes from CD105⁺ cells was effective at promoting the formation of lung metastases induced by intravenous injection of renal carcinoma cells (168).

Figure 2.

Schematic representation of functions of exosomes released by cancer cells in cancer progression, metastasis, angiogenesis, and treatment resistance and their potential therapeutic applications. A, the oncogenic molecules such as EGFR or EGFRvIII, which can be shed from cancer cells via exosome secretion and transferred to other adjacent cancer cells and/or endothelial cells. Hence, the activated EGFR or EGFRvIII may stimulate MAPK and Akt pathways and expression of their targeted genes, including VEGF, in host cancer cells and endothelial cells in their surrounding tumor microenvironment. VEGF released by endothelial cells in turn can activate in an autocrine manner the VEGFR2 cascade and tumor angiogenesis. Of therapeutic interest, the inhibition of exosome secretion from cancer cells carried by shRNA silencing of Rab27a/b, using an inhibitor of sphingomyelinase (GW4869) or annexin/diannexin and which represent potential strategies to prevent the promoting effects of exosomes on tumor progression, angiogenesis, and metastasis is indicated. B, the elimination of tumor cell–derived exosomes from the entire circulatory system, which can be performed by extracorporeal hemofiltration using novel medical devices such as an affinity plasmapheresis platform designated as Aethlon ADAPT system is illustrated. C, the systemic delivery of therapeutic agents incorporated in cellular or synthetic exosomes and which are able to inhibit the functions of tumor-specific mRNAs, miRNAs such as spherical nucleic acid constructs, and/or oncogenic products in target cancer cells is also illustrated.

Importantly, several lines of experimental evidence have also indicated that the exosomes may be involved in the formation of premetastatic niches and transfer of more aggressive and drug-resistance phenotypes to cancer cells and modulation of treatment responses of patients with cancer (Table 2; refs. 150, 169-175). For instance, CD133-expressing exosomes purified from FEMX-I cell line, which was derived from a lymph node metastasis of a patient with malignant melanoma, have been observed to express several prometastatic proteins, including CD44, MAPK4K, GTP-binding proteins, ADAM10, and annexin A2 and high levels of miRNAs with cancer-related functions (173). It has been shown that primary melanoma-derived exosomes can induce the recruitment and growth of melanoma cells to sentinel lymph nodes in vivo by inducing extracellular matrix deposition and vascular proliferation in the lymph nodes (174). Moreover, the exosomes from highly metastatic melanomas also contributed to the premetastatic niche formation through the upregulation of the MET oncoprotein in CD45⁻/KIT^low/+/tyrosine kinase with immunoglobulin-like and EGF-like domains-2 (TIE2⁺) bone marrow-derived progenitor cells (Fig. 1; ref. 169). The enhanced expression of MET by bone marrow progenitor cells resulted in their reprogramming toward provasculogenic and prometastatic phenotypes (169). The gene signature of exosomes released by melanoma, including MET, tyrosinase-related protein-2, very late antigen-4, HSP70, and HSP90, has also been associated with a poor prognosis of patients with melanoma (Table 2; ref. 169). Moreover, it has been reported that the exosomes isolated from serum samples of patients with prostate cancer promoted the proliferation and invasion of prostate cancer cell lines whereas the exosomes from age-matched healthy controls have not significant effects (150). The exposure of DU145 and 22Rv1 prostate cancer cells to exosomes from serum samples of patients with prostate cancer treated with docetaxel was also effective at enhancing their docetaxel resistance as compared with exosomes from untreated prostate cancer patient’s sera (150). It has also been noticed that docetaxel-resistant DU145RD and 22Rv1RD cell lines secreted the exosomes expressing P-glycoprotein, which could be transferred and conferred docetaxel resistance to parental DU145 and 22Rv1 cell lines (Fig. 1; ref. 150). The PCR-based analyses of urine samples from patients with prostate cancer have also indicated that the transcripts of prostate cancer gene-3 (PCA-3) and TMPRSS2;ERG gene fusion were detected in urinary exosomes suggesting their potential use as a noninvasive method for the diagnosis and follow-up of patients with prostate cancer (161). Moreover, it has also been shown that the exosomes released by SKBR3 breast cancer cells overexpressing HER2 oncoprotein interfered with the antitumoral activity of the anti-HER2 antibody, trastuzumab (Herceptin; ref. 172).

Therapeutic applications of exosomes

The molecular targeting of tumor cell–derived exosomes by interfering with their biosynthesis, secretion, and/or interactions with cell antigens or receptors also represents a promising approach to prevent the cancer progression, premetastatic niche formation, and metastases at distant sites (Fig. 2; refs. 160, 169, 176-179). For example, the targeting of neutral sphingomyelinase 2, which is involved in the ceramide biosynthesis and upregulation of exosome secretion, with chemical drug GW4869 has been shown to inhibit the lung metastases in lung cancer cell-bearing mice in vivo (176). Moreover, the interference with molecular pathways of exosome secretion by shRNA silencing of small GTPase Rab27a or Rab27b or 2 Rab27 effectors, known as Slp4 and Slac2b, also inhibited the exosome secretion by cervical adenocarcinoma HeLa cells, 4T1 and TS/A mammary carcinoma cells, and melanoma cells (169, 177, 178). It has also been noted that Rab27a blockade resulted in decreased primary tumor growth and lung dissemination of a metastatic carcinoma (4T1), but not of a nonmetastatic carcinoma (TS/A; ref. 177). In A431 human tumor xenografts in mice, angiogenic endothelial cells stained positively for human EGFR and phospho-EGFR, whereas treatment with annexin homodimer, diannexin which cloak phosphatidylserine residues on the surfaces of exosomes led to a reduction of tumor growth rate and microvascular density (Fig. 2A; ref. 160). Of particular clinical interest, a novel medical device using an affinity plasmapheresis platform termed as Aethlon ADAPT system was also effective to perform extracorporeal hemofiltration and elimination of tumor cell–derived exosomes from the entire circulatory system (Fig. 2B; ref. 179).

Recent investigations have also revealed that exosomes could be used as natural cell–derived membrane vehicles for systemic delivery of therapeutic agents interfering with the functions of tumor-specific mRNAs, miRNAs, and oncogenic products in target cells as well as immune regulatory tools in cancer immunotherapy (Fig. 2C; refs. 180-183). As a matter of fact, exosomes engineered to deliver spherical nucleic acid constructs into PC3 prostate cancer cells downregulated miR-21 target by approximately 50% (182). Furthermore, it has been shown exosomes that are released by tumor cells and dendritic cells may be used to elicit-specific immune responses against established tumors (180, 181). For instance, the data from a phase I clinical trial have indicated that a combination of autologous ascites-derived exosomes with granulocyte macrophage colony-stimulating factor (GM-CSF) was safe and well tolerated and induced tumor antigen-specific cytotoxic T-cell responses without side effects in patients with advanced colorectal cancer (180).

Together these data suggest important roles of exosomes secreted by cancer cells in their surrounding microenvironment in the acquisition of a more malignant behavior by host cancer cells and promoting immune escape and tumor neovascularization during cancer progression. Moreover, the exosomes can play critical functions in the establishment of premetastatic niches, recruitment, and homing of cancer cells at distant tissues and organs, metastases, and treatment resistance. Hence, these results support the interest to use the exosome-specific biomarkers as new diagnostic, prognostic, and predictive indicators and to develop new exosome-based-targeted therapies for treating aggressive and metastatic cancers.

Molecular biomarkers of CTCs

The enumeration of CTCs isolated from whole blood samples of patients with metastatic cancers before or posttreatment initiation and characterization of their molecular biomarkers may constitute a noninvasive and real-time method for improving accuracy of diagnosis and prognosis and choice of their therapeutic management, including use of appropriate targeted therapy (16, 31-35, 184). Several strategies, including CellSearch platform, microfluidic devices, and cell size–based separation methods, have been developed to detect, enrich, and isolate rare CTCs from peripheral blood samples (Fig. 1 and Table 3; refs. 16, 31-34). These detection techniques involve the CTC immunocapture from peripheral blood samples using different specific antibodies or size-based filters (Fig. 1 and Table 3; refs. 16, 31-34). More particularly, the CellSearch technology, which consists to use immunomagnetic beads coated with an antibody directed against EpCAM molecules, can allow researchers to isolate epithelial cancer cells expressing EpCAM surface marker (16, 31, 33-35, 184, 185). In a second step, the analyses of different biomarkers of the interest expressed by captured CTCs can be performed by quantitative real-time reverse transcription PCR (qRT-PCR) using DNA extracted from CTCs or immunofluorescence staining of CTCs (Fig. 1; refs. 16, 31, 34, 184). The biomarkers of CTCs include stem cell–like markers (CD133, CD44, ALDH, and ABC multidrug transporters), cytokeratins, EGFR, EGFRvIII, HER2, and K-Ras as well as the absence of the leukocyte-specific marker CD45 (Table 3; refs. 16, 31, 34, 184). Hence, it has been observed that the number and phenotypic features of CTCs captured using CellSearch assay, microfluidic CTCships, and other detection techniques given significant information on the treatment benefit and helped to predict the overall outcome of patients with locally advanced and metastatic breast, prostate, ovarian, pancreatic, lung, colorectal, and pancreatic cancers (Table 3; refs. 16, 31-35, 37, 184, 186-189). For instance, the characterization of CTCs from patients with metastatic CRPC has indicated that the majority of CTCs coexpressed epithelial proteins (EpCAM, cytokeratins, and E-cadherin), mesenchymal markers (vimentin, N-cadherin, and O-cadherin), and the stem cell–like marker CD133 (34).

Table 3.

Characterization of molecular biomarkers of CTCs and their clinical significances in diverse aggressive and metastatic cancers

Results and clinical significance
Cancer type	CTC detection method	Number of CTCs detected by patients/samples	Biomarkers and their diagnostic, prognostic, and predictive potentials	Ref.
Breast cancer	AdnaTest Breast Cancer  Select for detection of  EpCAM, MUC1, HER2  and β-actin transcripts	CTCs were detected by  AdnaTest in 19% of 502  blood samples from  patients with breast  cancer. Blood samples  from 502 primary patients  with breast cancer versus  30 healthy donor  samples	29% samples positive for CTCs were  characterized by RT-PCR by the  expression of at least one EMT marker  (twist, Akt2, and PI3Kα) whereas 97% of  30 healthy donor samples investigated  were negative. Moreover, 14% samples  positive for CTCs expressed ALDH1  stem cell–like marker whereas 95% of 30  healthy donor samples were negative.	(38)
Breast cancer	AdnaTest Breast Cancer  Select for detection of  EpCAM, MUC1, and  HER2 transcripts	226 blood samples from 39  patients with metastatic  breast cancer obtained  during their follow-up of  chemotherapy,  hormonal, or  trastuzumab antibody-  based treatment	CTCs were detected by AdnaTest in 31%  of 226 blood samples. The samples  positive for CTCs were characterized by  RT-PCR by the expression of at least one  EMT marker (twist, Akt2, and PI3Kα) and  ALDH1 in 62% and 69% cases as  compared with 7% and 14% in blood  samples negative for CTCs, respectively.  The EMT and ALDH1 expression levels in  blood samples from patients unresponsive  to therapy were of 62% and 44% relative  to only 10% and 5% in responders.	(39)
Breast cancer	CellSearch EpCaM-based  immunocapture method	Blood samples from 16  women with metastatic  breast cancer with  disease progression	Data from analyses performing by  CellSearch system and immunostaining  have revealed that over 75% of CTCs  detected in blood samples from patients  with metastatic cancers breast cancer  coexpressed epithelial markers (EpCAM  and E-cadherin) and mesenchymal  proteins (vimentin and N-cadherin).	(34)
Breast cancer	Microfluidic capture of  CTCs with epithelial- and  tumor-specific  antibodies directed  against EpCAM, EGFR,  and HER2	Blood samples from 41  patients with metastatic  breast cancer at various  stages of treatment.	CTCs were detected in 41% of blood  samples from patients with breast  cancer. CTCs expressed epithelial  (cytokeratins, EpCAM, and cadherin 1)  and/or mesenchymal markers  (fibronectin, cadherin 2, and serpin  peptidase inhibitor, clade E). The  enrichment of mesenchymal CTCs was  associated with disease progression  and expression of TGF-β signaling  pathway components.	(37)
Prostate cancer	CellSearch EpCaM-based  immunocapture method	Blood samples from 41 men  with metastatic  castration-resistant  prostate cancer with  disease progression	Data from analyses performing by Cell  Search System and immunostaining  have indicated that over 80% of CTCs  detected in blood samples from  metastatic CRPC patients coexpressed  epithelial markers (EpCAM, cytokeratin,  and E-cadherin), mesenchymal proteins  (vimentin, N-cadherin, and O-cadherin),  and CD133 stem cell–like marker.	(34)
Ovarian cancer	The monocyte blood  fraction containing CTCs  was enriched by 2-layer  density gradient and RNA  extracted from the  enriched cell fraction	Blood samples from 216  patients with epithelial  ovarian cancer obtained  before primary treatment  and 6 months after  adjuvant platinium-  based chemotherapy  and 39 cases of healthy  women	CTCs were detected in 24.5% of the  baseline and 20.4% of the follow-up  samples. The detection of CTCs  correlated with the presence of ascites,  suboptimal debulking and elevated CA-  125, and human epididymis protein 4  (HE-4) levels. Moreover, the CTC  detection was more frequent in  platinium resistant patients than in the  group sensitive to platinium treatment  and indicative of poor outcome of  patients.	(187)
Pancreatic cancer	CellSearch system	Blood samples from 26  patients with pancreatic  cancer	CTCs expressing cytokeratins were  detected in 42% of blood samples and  patients with pancreatic cancer with  CTCs exhibited significantly shorter  overall survival.	(188)
Pancreatic cancer	CellSearch system or  isolation by size of  epithelial tumor cells  (ISET)	Blood samples from 54  patients with pancreatic  cancer	CTCs were detected by CellSearch  EpCaM-based immunocapture  method and ISET in 40% versus 93% of  patients with pancreatic cancer,  respectively. The immunostaining  analyses have also indicated that CTCs  expressed variable levels of EpCaM,  pan-cytokeratin, E-cadherin, vimentin,  and cytokeratin 7.	(186)
Colorectal cancer	Pre- and postoperative  EpCAM based-flow  cytometry analyses	Blood samples from 76  patients with colorectal  cancer undergoing  surgical resection	CTCs were detected in 71% patients  preoperatively, and all metastatic  patients showed high levels of CTCs.  Colorectal tumor surgical resection was  associated with a significant decrease  in CTCs. A high postoperative level of  CTCs was also related to cancer  relapse. The progression-free survival  rate of patients with colorectal  cancer without CTCs was increased  from 16% to 86%, with a reduction in  the risk of disease relapse greater than  90%.	(185)
Lung cancer	Centrifugal force-based  separation technique  termed dean flow  fractionation (DFF)	Blood samples from 20  patients with metastatic  lung cancer at different  stages of treatment  versus 20 blood samples  from healthy individuals	5 to 88 CTCs were detected by DFF in all  blood samples analyzed from patients  with metastatic lung cancer whereas  healthy subjects had a negligible  number of CTCs. Moreover, the data  from immunostaining have revealed  that CTCs detected in blood samples  from patients with advanced lung  cancer coexpressed cytokeratin and  CD133 stem cell–like marker.	(40)
Non–small cell lung  cancer (NSCLC)	CellSearch system or  mutation detection in  circulating tumor DNA	Blood samples from 41  patients with relapsed or  refractory NSCLC  enrolled in a single-arm  phase II multicenter trial  with erlotinib and  pertuzumab	CTCs were detected using CellSearch  system in 78% of patients with NSCLC  and a greater baseline of CTC counts  was associated with a response to  treatment by response evaluation  criteria in solid tumor (RECIST). A  decrease of CTC counts upon  treatment of patients was also related  with FDG-PET and RECIST response  and longer progression-free survival.  Data from RT-PCR analyses have also  indicated that the detection of mutation  in EGFR in ctDNA and DNA isolated  from CTCs was associated with a most  substantial decrease in CTC counts  over the course of treatment of NSCLC  patients with erlotinib and pertuzumab.	(184)
Epithelial  malignancies	Multimarker quad-  μ-nuclear magnetic  resonance (μ-NMR)	Biopsies of 58 patients with  epithelial malignancies  including breast (n = 4),  gastrointestinal (n = 17),  genitourinary (n = 4),  gynecologic (n = 7),  pancreatic (n = 10), lung  (n = 9), and poorly  differentiated  adenocarcinoma (n = 7)  and 6 patients with  benign diagnosis	Data from quad μ-NMR expression  profiles of the biopsies obtained by the  fine needle aspiration of 58 patients  with different epithelial malignancies  have revealed that the combined  analyses of EpCAM, HER2, EGFR, and  MUC1 markers may be used to achieve  the diagnosis of patients with cancer in  99.2% of samples.	(189)
Melanoma	RNA isolation from whole  blood samples	Blood sample from 230  melanoma patients,  including 154 patients at  early stages I–II and 76   patients at late stages  III–IV versus 152 cases of  healthy individuals	Data from quantitative RT-PCR analyses  have indicated that 92% of melanoma  patients expressed MLANA, TGF-β2,  PAX3d, MCAM, and ABCG5 multidrug  transporter. Importantly, the  expression of MLANA and ABCG5  could predict disease recurrence  whereas MLANA expression was  associated with a poor outcome of  patients with melanoma after  treatment.	(41)

In addition, the results of qRT-PCR analyses of CTCs from patients with breast cancer have also indicated a direct relationship between the expression of stem cell–like markers such as Nanog, Oct3/4, Sox-2, nestin, and CD34 on CTCs and the stage of the disease progression (36). Moreover, the analyses of blood samples from 502 patients with breast cancer with the AdnaTest BreastCancer Select for the detection of EpCAM, MUC1, HER2, and β-actin transcripts have revealed that CTCs were detected in 19% of samples, and 29% samples expressed at least one of EMT markers (twist1, Akt2, and PI3Kα) whereas ALDH1 stem cell–like marker was detected in 14% of the samples (38). In another study, CTCs were also detected in 31% of 226 blood samples from 39 patients with metastatic breast cancer and 62% were positive for at least one of EMT markers and 69% for ALDH1 (39). It has also been noticed that the expression of EMT markers and ALDH1 was detected in 62% and 44% of patients with breast cancer that were nonresponders to the therapy whereas only 10% and 5% of the responders expressed these markers, respectively suggesting their potential use as predictive biomarkers (39).

More recently, single CTCs and multicellular clusters in a viable state have also been isolated from bloodstream of patients with metastatic lung cancers by dean flow fractionation (40). This size-based separation method may overcome the problems associated with the immunocapture with antibodies that is dependent of antigens expressed by CTCs (40). The number of CTCs detected by dean flow fractionation in whole blood samples from 20 patients with metastatic lung cancer was 39.1 ± 24.8 CTCs/mL whereas CTCs in blood samples from 20 healthy individuals was negligible (0.79 ± 0.42 CTCs/mL; ref. 40). It has also been observed that the CTC population detected in blood samples from patients with advanced lung cancer contained cells coexpressing cytokeratin and CD133 stem cell–like marker (40). Moreover, the qRT-PCR analyses of peripheral blood samples from patients with primary melanoma (early stages 0–II, n = 154) and metastatic melanoma (late stages III–IV, n = 76) have indicated that all samples contained CTCs (41). It has also been noted that melan-A known as melanoma antigen and stem cell–like marker, ABCB5 multidrug transporter were expressed in 45% and 49% of patients showing melanoma recurrence after treatment initiation, respectively (41).

The use of specific biomarkers of CTCs, including stem cell–like markers, represents a promising strategy for improving the diagnosis, prognosis, and therapeutic management of patients with aggressive and metastatic cancers.

Conclusions and Perspectives

The characterization of gene products expressed in tumor tissue specimens and CTCs from patients with cancer and exosomes released by cancer cells has led to the identification and validation of some gene signatures and potential stem cell–like markers. These altered gene products could be used as noninvasive diagnostic, prognostic, and predictive tools in conjunction for monitoring the risk of tumor progression and metastases and determining the choice of therapeutic treatments of patients with cancer. Furthermore, the molecular targeting of specific markers of cancer stem/progenitor cells, CTCs, and exosomes also represents a potential strategy could prevent cancer progression and metastases and overcome the treatment resistance and disease relapse of patients with cancer.

Future investigations are however required to optimize the sensitivity and reproducibility of immunocapture methods used for the enrichment and enumeration of CTCs and circulating exosomes from whole blood samples to consider their low number, potential heterogeneity, and specific biomarkers. Indeed, the detection and purification of rare CTCs, including circulating cancer stem cells, and circulating exosomes may be obscured by the nonspecific background of other hematopoietic cell types and overwhelming material in whole blood samples. Therefore, the optimization of in vivo detection platforms suitable for a continuous monitoring of whole blood volume could markedly improve the probability to detect rare CTCs, including circulating cancer cells with stem cell–like features. Moreover, the use of additional antibodies directed against epithelial, mesenchymal, and/or stem cell–like surface markers of CTCs could be included in immunoaffinity capture methods, such as CellSearch platform and microfluidic ships, to detect the EpCAM⁻ CTCs or exosomes. It will also be of interest to further establish the relationship between the expression levels of EMT process- and stem cell–like markers detected on captured CTCs and exosomes and the stage of tumor progression, treatment response, and survival rate using a large cohort of patients with cancer. Hence, the combined use of these new biomarkers detected in primary and metastatic tumor tissues, CTCs, and exosomes should lead to the development of multiplex biomarker approaches for improving the accuracy of current diagnostic and prognostic tests and efficacy of individualized treatments for patients with cancer. Of clinical relevance, the development of new nanotheranostic platforms for the combined in vivo detection by imaging and nanobubble-based targeted therapies of CTCs and circulating exosomes also may constitute a promising approach for real-time diagnosis and treatment of patients with aggressive and metastatic cancers.