Cancer Stem Cells: From Bench to Bedside

Abstract

Objective clinical responses to anticancer treatments often do not translate into substantial improvements in overall survival. Recent data suggesting many cancers arise from rare self-renewing cells (cancer stem cells) that are biologically distinct from their more numerous differentiated progeny, may explain this paradox. Current anticancer therapies have been developed to target the bulk of the tumor mass (i.e., the differentiated cancer cells). Although treatments directed against the bulk of the cancer may produce dramatic responses, they are unlikely to result in long-term remissions if the rare cancer stem cells are also not targeted. Better understanding the biology of cancer stem cells as well reexamining both our preclinical and clinical drug development paradigms to include the cancer stem cell concept, have the potential to revolutionize the treatment of many cancers.

Historical Perspective

It has been clear since at least the 1970s, that only a minority of cells from most hematologic malignancies and solid tumors are clonogenic in vitro and in vivo.^1;2 Investigators called these rare clonogenic cells “tumor stem cells.”^1;2 However, this low clonogenic potential could represent proliferative capacity exclusively restricted to a small subset of cancer cells, or alternatively all the cells within a cancer retaining the capacity to proliferate but only at a low rate. Insufficient tools available at the time precluded investigators from distinguishing which of these two possibilities explained the low clonogenicity of most cancers.

Fialkow and his colleagues first suggested that chronic myeloid leukemia (CML) arose from a transformed hematopoietic stem cell nearly 40 years ago, when they showed that both granulocytes and red blood cells from CML patients had a common cell of origin.³ The stem cell origin of CML was confirmed nearly 15 years ago when several groups, using characteristics known to define normal HSC, identified and isolated CML stem cells capable of expansion ex vivo.⁴ Dick and colleagues extended these observations more than ten years ago, showing that primitive hematopoietic stem cells purified from patients with both AML⁵ and CML⁶ would generate leukemia in vivo when injected into NOD/SCID mice. More recently, cancer stem cells that are biologically distinct from the differentiated cells that make up the bulk of the tumor have also been found in myelodysplastic syndromes,⁷ breast cancer,⁸ multiple myeloma,⁹ brain cancer,^10;11 and lung cancer.¹² However, the biologic and clinical relevance of cancer stem cells remains unclear.

The Paradox of Response and Survival

A cardinal principle of cancer therapeutics has been that evidence of a clinical response will translate into improved survival. The major advantage of using clinical response as the primary endpoint of therapeutic trials is that it is measurable over weeks to months, allowing the stepwise process of drug development to occur more rapidly and efficiently. In contrast, demonstrating a survival benefit adds significant complexity to clinical trial design, usually requiring the accrual of large patient numbers and long follow-up to provide statistical significance.

Although clinical responses can clearly decrease side-effects and improve quality of life, there is surprisingly little evidence that disease response is an appropriate surrogate for survival.¹³ In fact, there are numerous examples in which response does not predict for an improved survival. Indolent lymphoma patients who achieved a complete remission with conventional-dose therapies in the pre-rituximab era did not experience a survival advantage over similar patients treated with a “watch and wait” approach.¹⁴ In multiple myeloma, neither the magnitude nor the kinetics of clinical response had an impact on survival.¹⁵ Even the most intensive therapy for myeloma, blood or marrow transplantation (BMT),^16;17 provided no overall survival advantage in the recently published national intergroup trial¹⁸ or a recent meta-analysis.¹⁹ Similarly, significant clinical responses in pancreatic cancer²⁰ and prostate cancer²¹ have not translated into survival benefits. Further, despite new treatments that now produce complete remissions in the majority of women with ovarian carcinoma, cures are rare.²²

Cancer Stem Cells and Cancer Therapeutics: The Dandelion Phenomenon

Emerging data with one of the most successful new anticancer agents has helped shed light on this paradox that response and survival are not always linked. Imatinib has replaced interferon-alpha (IFN) as the standard-of-care for newly diagnosed CML patients, based on an interim analysis of a multicenter, randomized trial showing higher response rates for imatinib.²³ With up to five years of follow-up, most of the complete cytogenetic remissions to imatinib remain durable.²⁴ However, it now appears that imatinib may not be able to completely eradicate CML. CML patients who achieve the best responses to imatinib (reverse-transcriptase polymerase chain reaction negativity for BCR-ABL transcripts) can relapse quickly when the drug is discontinued,^25–27 or even progress while remaining on the drug.²⁸

BCR-ABL gene amplification or mutations prevent productive imatinib binding,²⁹ and secondary genetic mutations capable of driving BCR-ABL independent leukemic growth may also be present, even at initial diagnosis.³⁰ However, these genetic mechanisms of resistance are probably not responsible for the persistent CML in most patients treated with imatinib. Several investigators have now provided evidence that imatinib has differential effects on CML cells depending on their state of differentiation: while imatinib is toxic to differentiated CML progenitors, CML stem cells may be relatively or even completely resistant to the drug.^31–33 The basis for the differential activity of imatinib toward CML stem cells and their differentiated progeny is likely multifactorial.³³ CML stem cells share many biologic properties with their normal counterparts,⁴ that likely limit the effectiveness of therapeutic strategies targeting BCR-ABL signaling. Hematopoietic stem cells are largely quiescent and normally express high levels of ATP-binding cassette (ABC) transporters, such as the multidrug resistance-1 gene³⁴ and ABCG2.³⁵ Both of these factors may limit the cellular uptake of imatinib, which is a substrate for the ABC transporters.^36;37 Maybe most important, BCR-ABL appears to have different effects on CML stem cells and their differentiated progeny.³³ The cellular expansion in CML occurs primarily in the differentiated progenitors, rather than the in stem cell pool.^4;38 Moreover, BCR-ABL expression appears to be required for the survival of CML progenitors, but the same does not appear to be true for CML stem cells where the BCR-ABL gene can be silent.^4;39 These data suggest that BCR-ABL may produce only subtle effects in CML stem cells, and thus its inhibition may similarly have only minor consequences for these cells.³³ Therefore, based on the longevity (possibly greater than 10 years) of their normal counterparts, CML stem cells likely survive for years even if BCR-ABL activity is completely inhibited;⁴ eventually, because of intrinsic genomic instability, CML stem cells and their progeny may develop genetic resistance to imatinib.

The rapid responses induced in CML patients by imatinib²³ are likely a consequence of its impressive activity against differentiated CML progenitors that make up the bulk of the leukemia. An inability to cure CML^25–28 in the face of such potent initial activity is consistent with CML stem cell resistance to imatinib. This pattern of activity is analogous to cutting a dandelion off at ground level; although this will eliminate the visible portion of the weed, the unseen root also needs to be eliminated to prevent regrowth of the weed (Figure 1).^13;33;40 Conversely, the slow, but occasionally durable, responses seen in IFN-treated patients⁴¹ is consistent with reports showing that IFN’s activity is directed principally at the rare CML stem cells.^33;42 This treatment effect mimics attacking just the root of the dandelion; although this has no immediately discernible effect on the weed, over time the weed will eventually wither and die if its root has been eliminated (Figure 1).^13;33;40

Figure 1. The dandelion phenomenon.

Most current anticancer therapies have been developed to target bulk tumor cell populations. The anticancer effects of such treatments are analogous to mowing dandelions; although this will eliminate the visible portion of the weeds (differentiated cancer cells responsible for the bulk of the tumor), the unseen roots (cancer stem cells) also need to be eliminated to prevent regrowth of the weeds. Equally problematic for drug development are therapies directed principally at the rare cancer stem cells. This treatment effect mimics attacking just the roots of the dandelions; since no immediately discernible effect on the weeds (differentiated cancer cells) will be seen, such potentially curative therapies could be prematurely abandoned.

The “dandelion phenomenon” appears to apply to other cancers as well. Although multiple myeloma is characterized by neoplastic plasma cells, these cells appear to be terminally differentiated like their normal counterparts. The myeloma plasma cells that form the bulk of the tumor actually arise from a minute population of less differentiated cancer stem cells that resemble memory B cells and have the ability to self-renew, differentiate, and maintain the disease.⁹ It appears that most cancer stem cells arise from normal counterparts with stem cell features; although not stem cells in the classic sense, memory B cells could be considered “honorary” stem cells - i.e., they are long-lived, self-renew, and differentiate into plasma cells in order to maintain long-term immune memory. We found that the novel anti-myeloma agents, bortezomib and lenalidomide, have little activity against myeloma stem cells in vitro, despite being quite active against the plasma cells.⁴³ Conversely, rituximab and alemtuzumab eliminated myeloma stem cells in vitro, but had no activity against myeloma plasma cells that lack the relevant target antigens (CD20 and CD52, respectively). Although rituximab’s activity in multiple myeloma has been disappointing,⁴⁴ parameters typically used to follow clinical response in myeloma (i.e., monoclonal immunoglobulin level and percentage plasma cells in the marrow) primarily measure the effect of therapies on the terminally differentiated plasma cells. The long survival of the myeloma plasma cells could have obscured activity against the myeloma stem cells responsible for maintaining the disease. Perhaps a longer duration of rituximab treatment could ultimately have demonstrated clinical responses using traditional criteria, by inhibiting new myeloma cell production for a sufficient period of time to allow terminally differentiated myeloma plasma cells to undergo spontaneous apoptosis (Figure 1).

Gemtuzumab, anti-CD33 antibody conjugated to calicheamycin, has been approved to treat AML. Although the AML blasts usually express CD33, the AML stem cells more closely resemble normal primitive hematopoietic and may not express markers of more differentiated cells,⁵ including CD33.⁴⁵ Clinical studies are also looking at monoclonal antibody conjugates directed at B cell markers, such as CD19, in acute lymphocytic leukemia (ALL).^46;47 CD19 is a marker of B cell differentiation and not seen on many ALL stem cells.^48;49 Thus, targeting markers such as CD33 in AML and CD19 in ALL is unlikely to improve the curability of leukemia patients whose leukemia stem cells do not also express these antigens.

Targeting Cancer Stem Cells

Currently, the search for novel anticancer therapies is primarily focused on oncogenes that are specific for (e.g., BCR-ABL in CML), or over-expressed in (e.g., c-myc or bcl-2), selected cancers. However, attacking cancer-specific targets has met with variable success, and many of the most effective anticancer therapies, such as rituximab in lymphomas, high-dose cytotoxic therapy, or the allogeneic graft-versus-tumor effect, show limited or even no tumor selectivity. Targeting a cancer-specific pathway could fail for several reasons. It is likely that many cancers have already acquired multiple oncogenic mutations, even at initial diagnosis, capable of driving tumor growth; in such cases, targeting only one oncogene might be expected to generate limited activity. Further, as already discussed, even when the initiating oncogenic event is targeted as with imatinib in CML, inherent properties of stem cells may make the target inaccessible or no longer relevant.^33;40

Properties shared with normal stem cells not only appear to be responsible for cancer stem cell resistance to many anticancer agents, they may also lead to the development of novel therapies active across many malignancies. In fact, prospective targets shared with normal stem cells may have particularly strong anticancer potential since their conserved expression suggests a critical function retained by the cancer stem cells. Several signaling pathways that are important for the generation and maintenance of normal stem cells during embryonic development [e.g, Notch, Wnt, and Hedgehog (Hh)]⁵⁰ and/or postnatally (e.g., telomerase⁵¹ and growth factors) also appear to be important for the growth of many cancers. Preliminary data suggest that inhibition of these pathways, even when they are not mutated or over-expressed, may produce potent antitumor activity across a range of malignancies, possibly because of the key roles these pathways play in stem cell maintenance and growth.

While toxicity from lack of tumor-specificity is an obvious concern for shared stem cell targets, there are several potential differences between normal stem cells and cancer stem cell that may provide a therapeutic ratio for shared targets. Normal stem cells have normal cell cycle checkpoints that are likely to protect them from cellular damage or crisis. The stage of differentiation at which cancers arise may also provide a therapeutic ratio for approaches targeting cancer. Although many cancers may arise from tissue stem cells, they may not be the most primitive tissue stem cells as exemplified by CML.⁴ Accordingly, if a therapy equally eliminated both CML stem cells and their normal counterparts, the existence of more primitive normal stem cells should replenish the normal progenitor pool.³³

Another example of a shared stem cell target potentially providing tumor selectivity is telomerase, where differences between cancer stem cells and their normal counterparts in the interplay of telomere length and telomerase should provide a therapeutic ratio. Normal stem cells require telomerase to prevent telomere shortening leading to replicative senescence. However, even in the absence of telomerase, normal stem cells can maintain replicative capacity for some period of time because of their relatively long telomeres. Accordingly, telomerase knockout mice show deficits only after 4–6 generations.⁵² In addition, the major cause of death in dyskeratosis congenita, a congenital disease that results from loss of function mutations in telomerase components, is bone marrow failure but this usually does not manifest until the second or third decade of life.⁵³ In contrast, uninterrupted telomerase activity may be absolutely required for the maintenance and growth of most malignancies, in order to stabilize the short telomeres that characterize cancer cells. Accordingly, crossing telomerase knock-out mice with INK4a^{−/− 54} or APC^{min 55} mice predisposed to cancer, significantly lowered the development of cancers in these mice. Thus, the differential in telomere length between normal (long) and cancer (short) stem cells, should provide telomerase inhibition differential sensitivity.

Because of the difficulty assessing the effects of therapies on the rare cancer stem cells responsible for relapse, the development of such approaches requires new clinical paradigms and methodologies.¹³ We believe these new paradigms should rely heavily on preclinical modeling, eliminate traditional measures of clinical response as trial endpoints, and utilize novel preclinical assays to evaluate the fate of cancer stem cells . Preclinical studies should assess the effects of therapies on both cancer stem cell and differentiated cancer cell populations. Using the correct preclinical models, it may be possible to develop a detailed understanding of the mechanisms of action of new treatments, as well as strategies for optimizing activity; this could potentially allow a fully developed new approach to be taken directly from the “bench to the bedside.” However, effective preclinical models for cancer stem cells may ease the task of clinical trial development, but will not eliminate the need for new clinical paradigms. Evaluating the efficacy of treatments against cancer stem cells should be possible by utilizing these treatments after debulking the differentiated cells that constitute the majority of the tumor. In cancers where clinical debulking is successful (i.e., complete remissions are common but transient), studying therapies after induction of remission should permit using duration of remission as a measure of activity against the cancer stem cells. The fate of cancer stem cells could also be assessed as secondary laboratory endpoints using newly developed preclinical assays.

Conclusions and Future Directions

Traditional response criteria measure tumor bulk and may not reflect changes populations of rare cancer stem cells.¹³ Therapies directed against the bulk of the cancer may produce dramatic responses, but are unlikely to result in long-term remissions if the rare cancer stem cells responsible for maintaining the disease are also not targeted (Figure 1). Since many currently active treatments have been developed to target the differentiated cancer cells, they may have little activity against the biologically distinct cancer stem cells. Standard response parameters may not only potentially overestimate the effect of therapy on the minute population of stem cells, but may also underestimate it. As with IFN for CML and rituximab in myeloma, therapy selectively directed at cancer stem cells will not immediately eliminate the differentiated tumor cells; such therapy therefore might be prematurely abandoned if clinical activity is judged solely by standard response criteria that reflect the effects of treatment on the bulk of the cancer (Figure 1).

Thus, improving the results of cancer therapy requires identification and better understanding the biology of cancer stem cells, as well as reexamining both our preclinical and clinical drug development paradigms to include the cancer stem cell concept. Studies into identifying and characterizing cancer stem cells from hematologic malignancies have been greatly facilitated by a comprehensive understanding of cell surface antigen expression throughout lymphohematopoietic differentiation. In contrast, little is known about the cell surface phenotype associated with the growth and development of many non-lymphohematopoietic tissues. However, shared stem cell properties may not only be important causes of drug resistance and important targets for novel anticancer therapies, they may also aid in identification of cancer stem cells. Active cellular efflux of the DNA binding dye Hoechst 33342 by ABC membrane transporters, especially ABCG2/BCRP, is at least partly responsible for the Hoechst “side population” (SP) that is characteristic of stem cells from many tissues.⁵⁶ Aldehyde dehydrogenase (ALDH), specifically the ALDH1 family, mediates the synthesis of intracellular all-trans-retinoic acid that is required for the growth of normal stem cells in the hematopoietic system and other tissues.⁵⁷ The role of ALDH is not limited to retinoic acid metabolism, as it is also involved in the detoxification of a variety of compounds such as ethanol and the cytotoxic alkylator cyclophosphamide.⁵⁷ Similar to Hoechst SP, high expression of ALDH appears to be a marker for stem cells from many tissues,^58;59 and methodology to isolate viable cells by ALDH activity using a fluorescent labeled aldehyde substrate (Aldefluor) is now available.⁵⁸ Data from several groups suggest that SP and ALDH may identify cancer stem cells from a variety of malignancies,^43;60 and may be particularly useful in those malignancies where little is known about the phenotypes associated with the differentiation program of the tissues of origin.