Application of Stem Cells in Targeted Therapy of Breast Cancer : A Systematic Review

Breast cancer remains the most common malignancy among women worldwide, with an increase in incidence from 10.9 to approximately 20 million new cases per year by the year 2020, and a growing annual mortality from 6.6 to more than 10 million (Parkin et al., 2001; Roukos et al., 2007; Lehmann et al., 2011). In spite of the clonal origin of tumors, increasing evidence in hematopoetic malignancies (Clarke et al., 2006) and many solid cancers suggest that the tumor cell populations are heterogeneous in terms of proliferation and differentiation (Massard et al., 2006). This feature could be well clarified by “cancer stem cell” hypothesis, and may answer to the ever increasing questions such as cancer progression and drug resistance. Cancer Stem Cells (CSCs) or cancer initiating cells (CICs) are a small population of cancer cells within tumors, which poses stem cell features like self-renewal, capability to develop multiple lineages, and capacity of proliferation (Heppner, 1984; Reya et al, 2001; Clarke et al., 2006; Dwyer et al., 2007; Bohl et al., 2011). Although the definite origin of CSCs is not completely identified yet, various possible origins for CSCs have been proposed including adult stem cells existing in many tissues, from a population of


Introduction
Breast cancer remains the most common malignancy among women worldwide, with an increase in incidence from 10.9 to approximately 20 million new cases per year by the year 2020, and a growing annual mortality from 6.6 to more than 10 million (Parkin et al., 2001;Roukos et al., 2007;Lehmann et al., 2011).
In spite of the clonal origin of tumors, increasing evidence in hematopoetic malignancies (Clarke et al., 2006) and many solid cancers suggest that the tumor cell populations are heterogeneous in terms of proliferation and differentiation (Massard et al., 2006).This feature could be well clarified by "cancer stem cell" hypothesis, and may answer to the ever increasing questions such as cancer progression and drug resistance.Cancer Stem Cells (CSCs) or cancer initiating cells (CICs) are a small population of cancer cells within tumors, which poses stem cell features like self-renewal, capability to develop multiple lineages, and capacity of proliferation (Heppner, 1984;Reya et al, 2001;Clarke et al., 2006;Dwyer et al., 2007;Bohl et al., 2011).Although the definite origin of CSCs is not completely identified yet, various possible origins for CSCs have been proposed including adult stem cells existing in many tissues, from a population of
Despite advances in detection and treatment of metastatic cancers, applying radiotherapy, chemotherapy, immunotherapy, drug combination, and gene therapy with some vehicles such as viral vectors (Behbod and Rosen, 2005), mortality from cancer remains high (Schultz and Weber, 1999;Stockler et al., 2000;Al-Hajj et al., 2003).The importance of CSCs relies on the potential role of these cells in re-initiation and maintenance of tumor growth, which is the main cause of recurrence and relapse of tumors (Bohl et al., 2011).
It is believed that cancer targeted therapies especially stem cell targeted therapy are superior to current treatments such as traditional chemotherapy or radiotherapy to overcome recurrence, metastasis and chemo-resistance.Commonly used anti-cancer therapies can shrink primary and metastatic tumors, nevertheless such effects are usually transient and relapse of most metastatic cancers frequently occur (Reya et al., 2001), which are attributed to CSCs.There is sufficient evidence that CSCs are relatively chemo, radio and endocrine resistant, indicating that novel CSC-targeted therapies are required to achieves a true cure and elimination of cancer (Ablett et al., 2012;Reya et al., 2001).Standard therapies in combination with CSC-targeted therapies could provide effective treatment strategy by de-bulking the tumour mass and preventing recurrence (Chaffer et al., 2011;Gupta et al., 2011).There are several potential ways of targeting CSC including inhibition of self-renewal signaling pathways thus inducing differentiation or apoptosis, targeting resistance mechanisms or targeting the CSC niche that supports them.Monoclonal antibodies raised against specific components of signaling pathways or cell surface antigens on CSCs have been used to target these cells specifically (Ablett et al., 2012).Another important therapeutic option could be development of specific anti-CSC drugs targeting specific markers and pathways (Orian-Rousseau, 2010).
Although considerable progress has been made to identify CSCs, there is still the need to fully characterize the CSCs in terms of cell surface markers.No universal cell surface antigen, or combination of antigens, for the purification of breast CSCs by antibody techniques yet have been identified (Ablett et al., 2012).Many putative CSC markers are not merely restricted to CSCs, therefore the main goal in targeted therapy is specific destruction of CSCs while protecting normal cells (Deonarain et al., 2009).
In addition to surface markers, CSCs share some key signaling pathways with normal stem cells which can be mutated in CSCs and be considered as attractive targets for cancer therapies (Soltanian and Matin, 2011).A distinguished understanding of signaling pathways between normal and CSCs is required to prevent destroying normal stem cells, as this is the key point to perfect accomplishment of anti-CSC therapies (Deonarain et al., 2009).
Signaling pathways including Wnt, Hedgehog (Hh) and Notch play important roles in cell proliferation regulations and contribute to the self-renewal of stem cells and/or progenitor cells in a variety of organs, including the haematopoietic and nervous systems (Austin and Kimble, 1987;Henrique et al., 1997;Chan et al., 1999;Gailani and Bale, 1999;Wechsler-Reya and Scott, 1999;Zhu and Watt, 1999;Polakis, 2000;Zhang and Kalderon, 2001;Al-Hajj et al., 2003).
One of the novel targeted therapy modalities could be therefore signaling pathways.Blocking an abnormally active Hh pathway using an Hh antagonist in non-smallcell lung cancer (NSCLC) resulted in significant decrease in cell viability and malignancy (Yuan et al., 2007).Also, Notch ligand protein blocking antibody (ADLL4) was used to inhibit the Notch pathway inhuman breast cancer xenografts, leading to a significant reduction of tumor growth and a strong decrease of CD44+breast CSCs (Hoey et al., 2009).
There are two different hypotheses for interaction of stem cells and cancer leading to various applications of stem cells in targeted therapy of breast cancer.The first one is targeting CSC markers or pathways involved in CSCs using monoclonal antibodies as a novel strategy to improve the outcome of cancer therapy (Deonarain et al., 2009).The second one applies stem cells particularly Mesenchymal Stem Cells (MSCs) as promising platform for cell and gene therapy of incurable cancers (Ozawa et al., 2008).The high tropism of MSCs to cancers, as well as their ability to engraft, survive, and proliferate in the tumor without any immunogenicity and toxicity to the host, makes them ideal vehicle for tumor-selective drug delivery.MSCs migrate to sites of tumorigenesis and are utilized as efficient cellular vehicle for the targeted delivery anti-neoplastic therapy to both primary tumors and their metastases (El-Haibi and Karnoub, 2010).Several preclinical studies support the basis for genetically modified MSC to deliver therapeutics to tumor sites; include glioma, melanoma, Kaposi's sarcoma, Ewing sarcoma, as well as carcinomas of the colon, ovary, breast (Studeny et al., 2004;Nakamizo et al., 2005;Khakoo et al., 2006;Komarova et al., 2006;Karnoub et al., 2007;Menon et al., 2007;Coffelt et al., 2009;Duan et al., 2009;El-Haibi and Karnoub, 2010).Despite these approaches, the basic mechanisms involved in the homing of MSCs to sites of malignant growth are still only partially defined.
This systematic review explores the recent burgeoning evidence focusing on these two separate concepts based on selected key words to investigate the application of cancer stem cells as specific targeting modalities in breast cancer and also describes the use of stem cells as cellular

Search strategy
Specific key words were agreed to be searched within Medline (Pubmed), ISI web of knowledge, Gateway, Ovid and Embase for original research articles published between January 2007 and May2012.Included keywords were "breast cancer", "breast neoplasm", "stem cell" combined with "targeted therapy" or "targeted", "therapy" or "therapeutics".

Study inclusion criteria
Published papers were included if the following criteria were met: clinical trials at any phase, either discusses about the application of cancer stem cells as specific targeting modalities in breast cancer or describes the use of stem cells as cellular vehicles for breast tumor targeted delivery therapy, published within the recent five years, English, and predetermined key words existed.Meanwhile, review papers, any type of articles other than original research, exclusive animal experimental research (without additional human experiments), duplicate or sliced research articles, and research protocols were excluded in this systematic review.Review papers, commentaries, editorials, letters, and books were also excluded from the study.
Reference lists of identified papers were reviewed and the Cochrane Libraries was searched for any systematic review in this field or similar subjects.We limited the search to humans, cancer, title/abstract in English papers published within 5 years until May 2012.Abstracts were reviewed by three independent researchers (ZM, EG and EE), then relevant papers were identified and full papers were obtained for scrutiny regarding the methodology and main findings by all authors.Specific parts of the included papers were then entered in a standard table.
Search strategy is schemed in Figure 1.

Search results
Nine out of 32 studies, with heterogeneous study design, met the predefined inclusion criteria and all were reviewed Of these, six studies were phase I (Dwyer et al., 2007;Woodward et al., 2007;Vlashi et al., 2009;Grisendi et al., 2010;Dwyer et al., 2011;Milane et al., 2011)and three were phase II clinical trials (Table 1) (Ueno et al., 2009;Resetkova et al., 2010;Viens et al., 2010).There was no published paper regarding phase III clinical trial on stem cell targeted therapy of breast cancer within the period of this study.Although our search investigated all papers over the period of 5 years, the papers possessed our inclusion criteria ranged from Jan 2007 to May 2011.As mentioned earlier, to ease distinguishing both concepts of application of CSCs in breast cancer, the results are presented here separately.

The use of stem cells in combination with therapeutic agents
The role of tumor-secreted monocyte chemotactic protein-1 (MCP-1) in stimulating Mesenchymal Stem Cells (MSC) migration has been studied in a phase I clinical trial by Dwyer et al. (2007) which supports the capability of MSCs as tumor targeted delivery vehicles for therapeutic agents.MSC have been also used as cellular vehicles for tumor targeted delivery of therapeutic agents (Dwyer et al., 2011).Adipose Derived Mesenchymal stromal/stem cells (AD-MSC) armed with TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) has been offered as proficient tools for cell-based gene therapy for incurable cancers (Grisendi et al., 2010).
Autologous stem cell transplantation (HSCT) was applied in combination with 166-Holmium (Ho)-DOTMP to avoid the anticipated myelosuppression confirming that this combination shows an acceptable toxicity profile in bone metastatic breast cancer patients (Ueno et al., 2009).
A randomized clinical trial (RCT) conducted by PEGASE Group evaluated the value of high dose chemotherapy (HDC) in combination with hematopoietic stem cell transplantation (HSCT) and the value of targeted therapies in non-metastatic breast cancer.This study revealed that this combitaion only improve pathological complete response (pCR), while it could not significantly improve overall survival (Viens et al., 2010).The clinical trials in which either stem cell used as gene therapy or HSCT used in combination with chemo/radiotherapy have been summarized in Table 2.

Targeting cancer stem cells
The 26S proteasome as the main regulator of many processes within proliferating cells has been recently introduced for targeting of CSCs.Therefore reduced 26S proteasome activity couldbe applied for identification, tracking, and targeting of this subpopulation (Vlashi et al., 2009).
Wnt/β-catenin signaling pathway in stem/progenitor cells, which is responsible for radioresistency, has been Multi-drug resistant (MDR) cancer, known as cancer cell with stem cell properties, was targeted with a nanocarrier system by binding to the EGFR receptor and subsequently delivered drug solutions, paclitaxel (PTX) (Milane et al., 2011) and lonidamine (LON) to the site of a tumor.These EGFR targeted combination nanoparticles decreased tumor volume and also expression of hypoxic and MDR associated proteins in the orthotopic breast cancer model.This nanocarrier system could be used as a model for the design of other MDR cancer therapies (Milane et al., 2011).
Another study conducted by Resetkova et al to investigate the relevance of ALDH1 as a putative cancer stem cell marker in breast cancer, did not demonstrate any correlation between ALDH1 expression with response to neoadjuvant therapy or overall survival after chemotherapy, in breast cancer patients (Resetkova et al., 2010).The clinical trials (phase I and II) in which CSCs were targeted with different mechanisms, have been summarized in Table 3.

Discussion
There are two major concepts regarding interaction of stem cells and cancer.This discrepancy was the main cause of heterogeneity in study designs that we were encountered in this systematic review.In the present study, we identified nine recent clinical trials based on predetermined criteria, in which either stem cells applied in combination with therapeutic agents as cellular vehicles or CSCs targeted for breast cancer therapy.Therefore we discuss different concepts of "cancer" and "stem cells" under various sub headings: The use of stem cells in combination with therapeutic agents, in the first series of studies, MSCs were applied as vehicle to transfer therapeutic agent in incurable breast cancers.Dwyer et al. (2007) investigated the role of tumor-secreted MCP-1 in stimulating MSC migration in a phase I clinical trial, indicating the important role of MSCs as a vehicle for in vivo tumor-targeted therapy because of specific migration to tumors.They determined systemic levels of the chemokine in a cohort of breast cancer patients and age-matched controls concluding that MCP-1levels were significantly higher in postmenopausal breast cancer patients than the age-matched control group, supporting the capability of MSCs as tumor-targeted delivery vehicles for therapeutic agents.Although this data supports a potential role for MSCs as attractive delivery agents in tumor-targeted therapy, further studies are needed to clarify the factors that facilitate MSC migration and engraftment to provide the clinical application of this novel approach.
Moreover, in a recent study Dwyer et al. (2011) showed that MSC-mediated expression of the sodium iodide symporter (NIS) is potentially is appropriate for imaging, tracking and therapy of breast cancer.By injection of MSC-NIS, human NIS (hNIS) gene expression in various tumor sites occurred and a significant reduction of tumor growth was observed.The major advantage of this strategy was the ability to track MSCs migration noninvasively before therapy, supporting the application of MSCs as a vehicle in novel therapy of breast cancer.The persistence of MSCs after treatment and their role in the tumor microenvironment is still unclear.Further improvement  DOI:http://dx.doi.org/10.7314/APJCP.2013.14.5.2789Application of Stem Cells in Targeted Therapy of Breast Cancer might be achieved using repeated dose of MSC-NIS and radioiodide, as other studies have demonstrated that tumor irradiation stimulates increased MSC engraftment (Klopp et al., 2007;Spaeth et al., 2008;Zielske et al., 2009;Kim et al., 2010).This approach may cause greater stimulation of MSC engraftment in remaining disease and could be applied as an effective treatment in metastatic cancer.
In another study AD-MSC has been used as cellular vectors to deliver proapoptotic molecules TRAIL for breast cancer treatment (Grisendi et al., 2010).Although antitumor activity of recombinant human TRAIL has been confirmed in several studies (Grisendi et al., 2010), its application in vivo is limited by a short half-time in plasma, due to a rapid renal clearance.To overcome this limitation, stably transduced AD-MSC used as a constant source of TRAIL production targeting a variety of tumor cell lines including breast cancer.AD-MSC TRAIL is localized into tumors and mediated apoptosis without extensive toxicities to normal tissues after injection into mice.In spite of liver toxicity of recombinant TRAIL (Jo et al., 2000), the functional liver enzyme were normal in TRAIL-treated mice.This study indicated that cell therapy with AD-MSC TRAIL alone or in combination with sensitizing agents (Bortezomib) successfully targets TRAIL-resistant cancers, which is a new potential strategy in cancer therapy (Grisendi et al., 2010).
These studies confirm the notable experimental possibilities of MSC-based antineoplastic cellular therapy, and underline their potential application in breast cancer treatment.Some of these approaches are already in various phases of clinical trials; however, their efficacy and clinical safety in breast cancer patients remain to be determined.
Based on our search strategy we found a series of clinical trial phase II in which stem cells applied in combination with radiotherapy or chemotherapy to enhance their efficacy.For example Ueno et al determined the safety and efficacy of radiopharmaceutical agent, named 166-Holmium (Ho)-DOTMP, for irradiating malignant cells and adjacent marrow in bone metastatic breast cancer women (Ueno et al., 2009).This finding confirms that 166-Ho-DOTMP in combination with autologous stem cell transplantation had an acceptable toxicity profile when used in bone-metastatic breast cancer.Two out of 6 patients remained progression free without evidence of disease for more than 6 years, achieving long term remission (Ueno et al., 2009).Evaluation of other side effects and the probability of secondary hematologic malignancies were limited in this study due to small number of cases.
In a more recent study, a collection of RCTs by PEGASE Group (Viens et al., 2010) have been conducted in France to examine the value of high dose chemotherapy (HDC) with hematopoetic stem cell transplantation (HSCT) and the vlaue of targeted therapies in non metastatic inflammatory breast cancer (IBC), which revealed an appropriate pathological complete response rate by HDC.The other parts of these ongoing clinical trials recently have been published which was not in the time frame of this study (Viens et al., 2010).
HDC combined with autologous HSCT has been applied in several solid tumors to overcome tumor chemoresistance, indicating that this combination may improve tumor response rates or relapse-free survival (RFS), especially in selected subsets of patients (Banna et al., 2007).However, Banna et al. (2007) reviewed solid tumor trials concluding that there was no overall benefit for the use of this combination.
In a more recent systematic review Berry et al concluded that combined use of HDC with HSCT prolonged RFS and overall survival (OS)in high-risk primary breast cancer compared with control, whereas a statistically significant benefit was not observed in OS (Berry et al., 2011).
Targeting cancer stem cells, cancer stem cells are of particular interest in the literature, for their ability in initiation and maintenance of tumor growth and their potential role in early relapses and resistance to current therapies (Reya et al., 2001;Heppner, 1984;Al-Hajj et al., 2003;Clarke et al., 2006;Massard et al., 2006;Dwyer et al., 2007;Aboody et al., 2008;Nakshatri, 2010;Bohl et al., 2011;Lehmann et al., 2011).Despite nearly a decade after the introduction of tumorogenicity of CSCs in breast cancer (Al-Hajj et al., 2003), only a few clinical trials have been performed to confirm this hypothesis.In the second group of studies, cancer stem cells were targeted via their characteristics such as markers or signaling pathways.In a study by Vlashi et al. (2009) the 26S proteasome as the main regulator of many processes within a proliferating cell has been applied for imaging, tracking and targeting of CSCs.Reduced proteasome activity may occur simultaneously with the expression of stem cell markers and lack of differentiation markers (Vlashi et al., 2009).CSCs may be either immunologically silent or express antigens leading to failure in current targeted immunotherapy approaches.This system enables screening of novel compounds thatmight alter 26S proteasome function specifically in CSCs leding to novel targeted therapies against this subpopulation.Therefore reduced 26S proteasome activity could be assumed as a general feature of CSCs and could be easily used to identify, track and target this subpopulation in vitro and in vivo (Vlashi et al., 2009).Although CSCs can be selectively targeted via a proteasome-based dependent suicide gene, this population with low protease activity may be a heterogenous population that needs further identification.To obtain a pure CSC population, ZsGreenpositive cells are required to be further purified.
The Wnt, Notch and Hedgehog (Hh) pathways are developmental pathways that are commonly activated in different cancer types.The mutation of these pathways have been frequently occurred in many types of cancers particularly within subpopulation of CSCs (Dickinson and McMahon, 1992;Kintner, 1992;Ruiz i Altaba, 1999;Weissman, 2000;Reya et al., 2001;Barker and Clevers, 2006;Visvader and Lindeman, 2008;Shackleton et al., 2009;Curtin and Lorenzi, 2010;Li and Clevers, 2010;Snippert et al., 2010).This finding provides an opportunity for specifically targeting CSCs which are responsible for tumor initiation, progression, recurrence and metastasis (Curtin and Lorenzi, 2010).Significant progress has been made in developing therapeutics targeting Notch and Hh (Luistro et al., 2009;Robarge et al., 2009), whereas the Wnt pathway has been more challenging for targeted therapy (Curtin and Lorenzi, 2010).
Wnt/β-catenin signaling pathway has been suggested to be an attractive target for directed anti-stem cell therapy (Woodward et al., 2007).The subpopulation of stem/progenitor cells which remain after breast cancer radiotherapy, may lead to recurrent disease.It was hypothesized that radio resistance of this subpopulation is partially mediated by Wnt/β-catenin signaling pathway, which is involved in stem cell survival.Thus, radioresistance of CSCs was investigated by treating primary BALB/c mouse mammary epithelial cells with clinically relevant doses of radiation which resulted in enrichment of normal progenitor cells (Woodward et al., 2007).This data confirm that progenitor cells in the mammary gland are more resistant to radiation compared to non-progenitor cells, indicating that overexpression of the Wnt/β-catenin pathway may enhance the radioresistance of stem/progenitor cells.Therefore, targeting Wnt/βcatenin pathway that is responsible for self-renewal can be a potential therapeutics strategy (Woodward et al., 2007).Although mutation of β-catenin may not commonly occurred in human breast cancers, various studies showed the role of Wnt signaling pathway in pathogenesis of breast cancer (Lin et al., 2000;Jain et al., 2002;Wong et al., 2002;Klopocki et al., 2004) suggesting a link between Wnt signaling and DNA damage response in epithelial cells (Ayyanan et al., 2006).
As a result of its role in different cancers, the Wnt signaling pathway is a major target for therapeutic intervention.Wnt inhibition could be used in combination with classic chemotherapeutic agents ;i.e if the CSCs were targeted accompanied with a Wnt pathway inhibitor, more effective response would be expected (Curtin and Lorenzi, 2010).Some triple negative breast cancer cell lines have been shown to express ligands and markers of abnormal Wnt/β-catenin signaling without common mutations in the pathway.Wnt signaling in these cells can be inhibited by overexpression of endogenous inhibitors (Bafico et al., 2004).Moreover, the Wnt pathway regulates epithelialmesenchymal transition (EMT), an important component of metastasis.Therefore it can be hypothesized that the Wnt pathway in breast CSCs may offer an exceptional opportunity to target metastasis as a main cause of morbidity in many types of cancers (Kim et al., 2002;Muller et al., 2002;Liebner et al., 2004;Thiery et al., 2009;Curtin and Lorenzi, 2010).Another clinical challenge in conventional therapy of cancer is the development of multi-drug resistant (MDR) cancer that often slow down the treatment as it results in metastatic disease (Harris and Hochhauser, 1992;Yague et al., 2007).Because MDR is resistant to many current therapies, there is a demand for new drug combinations with less toxicity for treating MDR.
Two models of cancer stem cells have been proposed:cancer initiating stem cells, which originate as stem cells, but alter into cancer causing cells; and cancer derived stem cells, which are a population of cancer cells with stem-like properties, which known as MDR cells (Milane et al., 2011).In line with the second model, various studies have shown that cell stressors such as hypoxia, which are capable in inducing cancer aggression and MDR phenotypes, also increase stem-like properties (Harris, 2002;Semenza, 2003;Cosse and Michiels, 2008;Han et al., 2008;Semenza, 2008).In addition, many MDR cells over-express epidermal growth factor receptor (EGFR) (Franovic et al., 2007).Milane et al utilized this expression through development of EGFRtargeted, polymer blend nano-carriers to combat MDR cancer using paclitaxel (PTX) (Milane et al., 2011) and lonidamine (LON) (Del Bufalo et al., 1996;Ravagnan et al., 1999;Li et al., 2002),where a novel orthotopic model of MDR human breast cancer in nude mice was developed to evaluate the safety and efficacy of nano-particle (NP) treatment.They observed that treatment with the EGFRtargeted LON/PTX nano-particles decreased tumor density and altered the MDR phenotype of the tumor xenografts, which means that combination of LON/PTX therapy using EGFR-targeted NPs could be used as a new approach for the treatment of MDR cancer.Although this approach provides a solution to chemotherapy related toxicity through the use of a nano-carrier system, more in vivo experiments are needed to approve this combination (Milane et al., 2011).
To evaluate the prognostic impact and relevance of ALDH1 as a putative cancer stem cell marker in breast cancer (Resetkova et al., 2010) four cohorts series including an adjuvantly treated series of 245 invasive cancers, a neoadjuvantly treated series of 34 cases, and two series of 58 and 40 triple negative cases were studied by immunohistochemistry (IHC).In spite of prevoius studies that stated the role of CSCs in resistance to chemotherapies, this study showed that unexpected expression of ALDH1 in breast tumor cell did not correlate with response to neoadjuvant therapy, diseasefree or overall survival following adjuvant or neoadjuvant chemotherapy.Therefore,based on Resetkova's study ALDH1 can not be suggested as a useful marker for targeted therapy of breast cancer (Resetkova et al., 2010).
Currently, two methods used to measure breast CSC activity include FACS using antibodies to cell surface markers or intracellular enzymes such as ALDH, using the ALDEFLUOR assay, and mammosphere assay.A previous immunohistochemical (IHC) study performed by Ginestier et al in a large series of breast cancer patients, demonstrated a correlation between ALDH1 expression and poor prognosis (Ginestier et al., 2007).Whether this is because of increased number or activity of breast CSCs remains unknown.Therefore, prior to application of IHC markers as alternative measures of breast CSC expression in clinical trials, they first need to be correlated with functional assays of CSC activity (Ablett et al., 2012 ).The clinical effectiveness of putative breast CSC markers such as ALDH1, CD44 and CD24 to identify and monitor breast CSCs using IHC is complicated due to heterogeneous nature of disease and the existence of different populations of breast CSCs within a single tumour (Park et al., 2010).
In contrast, one may critique that the present method for evaluating CSCs by injecting cells into mice may introduce a selection bias for human cells capable of DOI:http://dx.doi.org/10.7314/APJCP.2013.14.5.2789 Application of Stem Cells in Targeted Therapy of Breast Cancer surviving and proliferating in the mouse microenvironment with foreign growth factors and cytokines (Kelly et al., 2007;Quintana et al., 2008;Ablett et al., 2012).
Recently the merged model of the clonal evolution model and the CSC hypothesis has been proposed in order to clarify tumour maintenance and progression, which predicts that the frequency of CSCs varies significantly in each patient and appears to be dependent on the type of breast cancer and the dominant mutations, gene amplifications and deletions (Ablett et al., 2012).
In order to assess breast CSCs in clinical trials, it is important to use reliable methods for identification and isolation of breast CSCs, as well as developing novel therapeutic endpoints which reflect their expression and/ or function.Therefore, novel clinical settings should be able to measure CSC frequency as well as tumour volume.Moreover, a clinical test must have a high sensitivity and specificity, be acceptable to the patient, logistically reasonable, quick to perform and cost-effective (Ablett et al., 2012).
Tumour formation and serial transplantation assays which have been used the 'gold standard' in vivo methods for measuring breast CSC activity, are technically challenging, expensive, possessing ethical consequences, and would be impractical to apply in clinical settings.However, alternative in vitro methods such as colonyforming assays and identification of cell surface markers have been employed in pre-surgical window trials to assess the efficacy of treatments on the breast CSCs before and after treatment (Yu et al., 2007;Li et al., 2008).Though, the technical expertise, time and expense for implementing these assays limits their application in large scale clinical settings (Ablett et al., 2012).
Study publication bias, publication bias, as a matter of limitation in any systematic review, should be considered in our study, where research with positive and desirable results is potentially more attractive to be published.Considering predetermined inclusion and exclusion criteria, likewise other systematic reviews, restricted the occurrence of selection bias.
In conclusion, this systematic reviewinvestigated two different models concerning stem cells and cancer based on specific key words demonstrating an ever increasing experimental data on various applications of stem cells in targeted therapy of breast cancer.
In the absence of large prospective clinical trials so farto investigate mid and long term outcomes, a few conclusions canbe drawn from this systematic review.Applying MSCs as potenial vehicles to transfer therapeutic agents could be a contemporary therapeutic approach for breast cancer.However, this approach remains an experimental treatment and needs to be confirmed in large scale randomized clinical trials.In addition, combination of hematopoietic stem cell transplantation (HSCT) with high-dose chemotherapy (HDCT) in breast cancer patients could not significantly improve overall survival.
Novel CSC-targeted therapies in breast cancer are very challenging due to heterogenecity of disease.The major challenge is to determine which of CSC properties could be targeted or which CSC biomarkers are appropriate to measure the efficacy of the novel CSC therapies.
Although utilizing biomarkers had been proposed to identify and assess CSC activity in clinical trials, there are no universal molecular marker to isolate breast CSCs properly and suitable for use in clinical trials, but they will be achievable in near future.It could be concluded that there are clear uncertainties over the clinical application of stem cells in targeted therapy of breast cancer, yet warrants confirmation in appropriately designed controlled trials.

Figure 1 .
Figure 1.Flow Diagram of the Procedure used to Select Relevant Articles DOI:http://dx.doi.org/10.7314/APJCP.2013doi.org/10.7314/APJCP..14.5.2789Application of Stem Cells in Targeted Therapy of Breast Cancer vehicles for breast tumor targeted delivery therapy in the most recent clinical trials.