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Are Cancer Stem Cells a Prime Target for Therapy?

by Emilie T.E. Gross, PhD, November 22, 2018 at 01:00 PM | Tags

Cancer stem cell hypothesis compared with normal stem cell differentiation

Cancer stem cell hypothesis compared with normal stem cell differentiationCancer stem cells (CSC) represent one of the most challenging targets for cancer therapy today. Here we review the background to this phenomenon and explore the various experimental models that are used to investigate anticancer therapies targeting CSCs.

What are Cancer Stem Cells?

Cancer heterogeneity is a fact. However, over the past two decades, cancer hierarchy has emerged as a novel paradigm, transforming our vision of tumor heterogeneity and therapeutic management of cancer. Similarly to normal tissues, cancers contain a minor stem cell-like cell compartment fueling the mass generation of differentiated daughter cancer cells.

In normal cellular physiology, stem cells are defined by their ability to divide asymmetrically. Depending on their molecular and cellular environmental context, stem cells can either:

  • Self-renew and generate a copy of the original stem cell, or
  • Undergo differentiation and produce the differentiated cellular lineages of a given tissue.

Similarly, cancer stem cells (CSC) can also self-renew, as well as generate a progeny of differentiated cancer cells.

Most studies investigating CSC however, have studied tumor-initiating cells. These are cells which:

  • Initiate tumor formation in animal models (with this capacity being confined to CSC according to the CSC paradigm)
  • As well as giving rise to non-CSC by differentiation (cancer cells which lack the ability to self-renew and differentiate).

How do Cancer Stem Cells Arise?

The existence of CSC has been the subject of a highly controversial scientific debate ever since their first identification in acute myeloid leukemia (AML) back in 1997.

CSC emergence has been attributed to:

  • The oncogenic transformation of normal stem cells.
  • The dedifferentiation of mature tumor cells driven by the activation of morphogenous pathways during cancer progression.

Oncogenic Transformation of Normal Stem Cells

The best evidence for CSC originating from normal stem cells was provided by three back-to-back articles published in 2012. These studies tracked brain, colon, and skin stem cells during carcinogenesis using genetic markers. In the tissues investigated, the transformed stem cells were the cellular precursors of cancer, as well as the key cellular targets to achieve durable tumor regression.

Somatic Reprogramming

With the advent of the induced pluripotent stem cell (iPSC) era, reprogramming differentiated cells to a pluripotent stage is now a far less complicated process than previously thought. This pluripotent reprogramming is driven by the sole expression of just four transcription factors: Nanog, Sox2, Oct-4, and Klf4, as well as the oncogenic protein c-myc.

By extension, a number of stimuli from the microenvironment can also induce activation of stem cell molecular programs in cancer cells. These include inflammation, hypoxia, TGF-beta, and chemotherapeutic pressure. The most studied stem cell pathway in cancer is perhaps the Wnt/beta-catenin pathway.

Current CSC Research and Targeting

Two decades of extensive research in this field have identified tumor-initiating cells in virtually all cancers, including melanoma, breast, colon, lung, brain, liver, AML, chronic myeloid leukemia (CML), and more.

On top of their regenerative properties, CSC are also responsible for metastasis, cancer resistance to conventional therapies (chemotherapies and radiotherapies), and cancer recurrence post-treatment.

As a result, cancer stem cells represent the ultimate, and probably most challenging, target for cancer therapies.

How to Preclinically Define Agent Efficacy on CSC

While the CSC paradigm is firmly established in hematopoietic cancers such as AML and CML, the existence of CSC in solid tumors is more of a controversial issue. Across a variety of indications, CSC are defined by specific extracellular markers which allow the identification of both CSC and non-CSC compartments. Breast and colon cancers are the best-documented indications regarding the existence of solid tumor CSC.

Cancer Stem Cell Markers

Tumor type CSC phenotype Reference
AML CD34+/CD38+ Bonnet and Dick, 1997
Brain CD133+ Sinh et al., 2004
Breast CD44+/CD24-/low Al-Hajj et al., 2003
Colon CD133+ O'Brien et al., 2004
Melanoma ABCG5+, CD20+ Shatton et al., 2008
Multiple myeloma CD138-, CD20+ Matsui et al., 2008
Prostate CD133+/CD44+/α1 β2+ Collins et al., 2005

Alternatively, with a lack of specific markers, a variety of functional tests can be used to define “stemness” among cancer samples. These include side population identification, ALDH assay, and the tumorsphere culture enabling anchorage-independent 3D growth.

The CSC percentage in a given sample is dependent on a number of variables. These include whether the sample was obtained after 2D or 3D in vitro growth, or in vivo growth in animal models. In addition, the degree of host immunodeficiency is also another factor which impacts the frequency of CSC in tumors. The various murine models can be:

  • immunocompetent (wildtype): syngeneic (relying on the engraftment of a murine cancer cell line) and primary models developed in a WT background (for which carcinogenesis is either spontaneous or induced chemically or genetically). These immunocompetent models allow you to test cancer immunotherapies.

  • immunodeficient (e.g. NOD-SCID, RAG-/- which lack adaptive immunity): mostly used for patient-derived xenografts (PDX) or traditional cell line derived xenograft models

  • severely immunodeficient (e.g. RAG-/- x γc-/-, NSGTM which lack adaptive immunity and some components of the innate immune system): again used for cell line or patient-derived xenograft models

Immunocompetent Models

Syngeneic transplants, as well oncogene and chemically-induced models, offer the most complete picture when it comes to understanding the microenvironment’s influence on cancer stemness. These models also allow the investigation of CSC targeting by immunotherapies.

One great advantage of syngeneic and cell line-derived xenograft models is the amount of published evidence regarding their hierarchy as well as their CSC phenotype. Nevertheless, syngeneic tumor models pose the problem of overextended in vitro culture that can potentially alter the heterogeneity population dynamics and the cell’s tumor initiating capacities.

CSC Populations in Common Syngeneic Models

Cancer Type Cell Line Mouse Strain CSC phenotype Reference
Adenocarcinoma JC Balb/c N/A  
Bladder MBT-2 C3H/He Oct-3/4 Chang et al., 2008
Breast 4T1 Balb/c Side population Kruger et al., 2006
  EMT-6 Balb/c N/A  
Colon CT-26.WT Balb/c CD44 (not exclusive) Dotse and Bian, 2016
  MC38 C57BL/6 CD44+ ALDH1+ Jinushi et al., 2001
Fibrosarcoma WEHI-164 Balb/c N/A  
Kidney Renca Balb/c Side population Nishizawa et al., 2012
      Encapsulation of tumor colony, Sca1, Oct4, CD44 Smith et al., 2011
Lung LL/2 (LLC1) C57BL/6 Sphere, CD133, CD34, Sox2, Oct4 Zhang et al., 2018
      Side population Zhang et al., 2012
  KLN205 DBA/2 N/A  
Lymphoma A20 Balb/c N/A  
Melanoma B16-F10 C57BL/6 CD133+ CD44+ Zhao et al., 2017
        Dou et al., 2007
Pancreatic Pan02 C57BL/6 ALDH+ Zhang et al., 2016
Prostate RM-1 C57BL/6 N/A  

CSC Populations in Common Xenograft Models

Cancer Type Model Mouse Strain CSC Phenotype Reference
Breast MCF-7 NOD-SCID CD44+ CD24low Vazquez-Santillan et al., 2016
Afzali et al., 2016
Side population Han and Crowe, 2009
Colon carcinoma COLO205 Balb/c nude CD133+ CD44+ Li et al., 2012
CD133+ Zangiacomi et al., 2014
HCT-116 Balb/c nude CD44+ Yeung et al., 2010
CD133+ Botchkina et al., 2009
Lung A549 Balb/c nude CD44+ CD24-/low Balla et al., 2015
Lymphoblast Raji SCID/Beige ALDH+, Sox2, Nanog Ryu et al., 2017
Ovarian TOV-21G Athymic Nude ALDH+ Wang et al., 2012
TOV112D NCR nu/nu NA  
Prostate 22Rv1 Balb/c nude ALDH+ Nishida et al., 2012
CD133+ Kanwal et al., 2018
DU145 Balb/c nude CD44+ integrinα2β1+ CD133+  Chen and Wang, 2012
CD133+ CD44+ Wei et al., 2007
Wang et al., 2013
PC-3 Balb/c nude CD133+ CD44+ Wang et al., 2013

In comparison to transplant models, murine primary models have multiple advantages in reflecting cancer progression, and microenvironmental changes during the various phases of the oncogenic process. On the other hand, oncogene-driven models may fail to recapitulate tumor heterogeneity and hierarchy, leaving chemically-induced or spontaneous murine tumor models as probably the best primary model option.

These chemically-induced or spontaneous murine tumor models, however, have the tendency to develop cancers which do not completely model their human cancer counterpart (e.g. methylchlorantherene (MCA)-induced sarcoma, New Zealand black mice for Chronic lymphocytic leukemia).

Finally, when considering syngeneic and primary models for cancer stem cells studies, species specificity needs to be considered. A number of cancer stem markers, such as Stem cell antigen 1 (Sca-1), are specifically confined to murine stem cell biology and therefore cannot be transposed to human cancers. This means also that their associated signaling pathways also cannot serve as therapeutic targets for human diseases.

Patient-Derived Xenografts

Patient-derived xenografts (PDX) potentially offer the most relevant options when looking for CSC therapeutics, excluding immuno-oncology drugs. PDX are never passaged in vitro and their CSC percentage, phenotype, and molecular signatures should be therefore comparable to their original parental tumor. PDX, unlike traditional xenografts, also lack any in vitro-induced or genetically engineered immortalization which might alter their CSC phenotype.

The CSC proportion in PDX samples is likely to be influenced by whether the patients were subjected to prior therapies or if the sample comes from a metastatic site. Immunotherapies also have their part to be played in PDX CSC targeting strategies with the use of humanized mice.

Conclusions

The CSC paradigm has revolutionized our understanding of tumor biology and created opportunities for novel therapeutic strategies targeting CSC. Exploring the efficacy of novel cancer therapeutics requires a range of models and tracking techniques to fully assess the potential of therapies to target CSC and achieve durable and complete eradication of tumors.


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