According to the Canadian Cancer Society, nearly 1 in 2 Canadians will be diagnosed with cancer. Despite advances in diagnosis and treatment, cancer continues to be a death sentence for many patients due to relapse, metastasis at distant sites, drug resistance and the toxicities associated with select therapeutic treatment approaches [1, 2]. Studies in the past two decades have identified a subpopulation of cancer cells called cancer stem cells (CSCs) or tumour-initiating cells (TICs) as being solely responsible for tumour initiation, progression, relapse, metastasis and drug resistance [3-6]. Consequently, selective targeting of CSCs within the tumour cell population was initially thought to be a very promising therapeutic strategy to treating cancer [7, 8]. This essay will, therefore, elucidate on the characteristics of CSCs, the challenges and potentials of the proposed CSC therapy, and offer suggestions on the strategies to wholly targeting cancer to prevent relapse, metastasis and drug resistance.
Cancer Stem cells
Two models currently exist to account for tumour growth and the heterogeneity within tumours. The clonal evolution model argues that all cells within a tumour have the capacity to propagate all the various cell types in a tumour mass, and subclonal differences resulting from both genetic and epigenetic changes acquired during development are responsible for the intercellular variations . Conversely, the CSC model suggests that all of the inherent characteristics of tumour, including initiation, heterogeneity, recurrence and metastasis, are being driven by a subpopulation of cells termed cancer stem cells [10, 11]. See Figure 1 below for an illustration of the two models. Although the first evidence of stem-cell like cancer cells was reported as early as 1937 , it was not until 1997 that Bonnet and Dick  provided indisputable evidence of the existence of a subpopulation of CD34+ CD38– cells in acute myeloid leukemia (AML) patients that possessed the ability to self-renew, proliferate and differentiate into other cancer cells. CSCs were subsequently identified in a broad spectrum of human solid tumours, including breast (CD44+CD24–/low cells) , brain (CD133+ cells) , prostate (CD44+CD24– cells) [16, 17], among others. As one could imagine, identification of these CSCs was made possible due to specific cell surface markers they were expressing. It is, however, paramount to state that certain markers are not restricted to a particular tumour, and that cancers not expressing the associated markers above have been found to be also tumorigenic. To put this into perspective, CD133– cells in brain tumours have been found to possess high tumorigenic activity . Apart from identifying CSCs using their cell surface markers by fluorescence activated cell sorting (FACS), other methods, including spheroid formation assay , side population assay  as well as a method based on the enzymatic activity of aldehyde dehydrogenase (ALDH)  have been used for the isolation and subsequent evaluation of CSCs.There are considerable evidence that these CSCs have different cells of origin [22, 23], and that, depending on the cancer types, CSCs may originate from somatic stem cells, partially differentiated progenitor cells or differentiated cells that have acquired stemness through a number of dysregulated mechanisms [24, 25]. Certain signalling pathways have been attributed to the stemness phenotypes of CSCs, including, but not limited to, Wnt, JAK/STAT, Hedgehog, Notch and FAK signalling pathways [26, 27]. Figure 2 depicts a simple schematic representation of these different pathways and some of the targeting strategies that have been proposed to block these signalling pathways. The identification of these stemness propagating pathways, the characterisation of the properties of the CSCs themselves (promoting relapse and drug resistance, for example) as well as the isolation of specific cell surface markers as highlighted above have fuelled interests in designing targeted therapies solely against CSCs. The next section will focus mainly on the limitations and some successes that have characterised this ambitious goal.
CSC targeted therapies – a reality or an illusion?
Compelling evidence exists to suggest that targeted therapy has been somewhat successful in the clinic in the eradication of tumours. For example, olaparib, a PARP inhibitor, is used clinically to treat patients with metastatic HER2-negative breast cancers that also have mutations in the BRCA1 or BRCA2 gene . But, as was previously proposed, would designing drugs targeting certain features that drive CSC phenotypes hold the cure to cancer? To answer this question, this section of the essay will discuss some of the limitations of targeting CSCs. First, although it may be possible to design drugs that target certain features of CSCs, it has been suggested that multiple CSC subsets, consisting of undifferentiated cells with different origins, may exist within a tumour. For example, studies suggest the existence of both CD133+ and CD133– CSC subpopulations with different origins [18, 29]. While targeted therapy against CD133+ CSCs may eliminate them, the CD133– subpopulation will be resistant ; thus, allowing the CD133– subpopulation to grow, divide and replenish the CSC population. Consequently, some monoclonal antibodies, including Lintuzumab against CD33, have been discontinued, despite showing some modest benefit [7, 30]. Another limitation to targeting CSC is that they share the same cell surface markers (e.g., CD133+) as normal stem cells . Therefore, designing drugs against these markers will not be specific in targeting the CSC population within the tumour. For example, the selective CSC inhibitor, salinomycin, shows increased toxicity to normal CD4+ T cells at concentrations effective against CD4+ T cell leukemia cells .
Additionally, CSCs within certain tumours are known to possess a lower proliferative rate, display an elevated level of quiescence , and have efficient DNA repair mechanisms  compared to other cells within the same tumour. Consequently, these heterogeneous features of CSCs suggest that targeting them with drugs that damage DNA will not have a profound cytostatic effect on them. Compounding this further is the fact that CSCs also possess increased expression of anti-apoptotic proteins , ABC transporters (involved in increased efflux of chemotherapeutic agents)  and increased level of ALDH and oxidant scavengers (both function to metabolise chemotherapeutic agents and reactive oxygen species ) that ultimately contribute to their resistance to chemo- and radiotherapy. Another hurdle in the CSC therapy is that eliminating CSC may not change disease outcome. This is because new CSCs are likely to be generated via the spontaneous dedifferentiation of non-CSCs due to cellular plasticity . It would, therefore, be beneficial targeting both the differentiated cancer cells and CSCs with combinatorial therapies. Another factor that has limited the use of CSC targeted therapy is similarity in the many genes and signalling pathways that regulate both the stemness pathway and normal stem cells . For example, proper regulation and homeostasis of intestinal stem cells are mediated via Wnt signalling [38, 39]. Hence, there have been concerns about toxicity and off-target effects of targeted therapies against dysregulated proteins in the signalling pathways. Finally, the efficacy of these targeted CSC therapies is not guaranteed due to redundancy in the signalling pathways . To overcome this challenge, recent clinical trials are using high-throughput screening techniques to specifically target CSCs. The afore-mentioned challenges and limitations, therefore, weaken the claim that cancer can be cured by solely targeting CSCs.
Despite these hurdles or challenges, the subtle surface marker differences and the dysregulation of the signalling pathways in CSCs have been exploited as potential therapeutic targets . Currently, some of these targeted therapies against CSCs have been investigated in different phases of randomised clinical trials (see table 1 below). For example, the Sonic Hedgehog pathway inhibitor, vismodegib, is currently being used to treat basal cell carcinoma  and has been evaluated to treat patients with Hedgehog pathway mutations that lead to medulloblastoma . Majority of these studies are, however, combining with standard-of-care chemotherapy, and none have demonstrated clinical efficacy as single agent inhibitor [7, 27]. The preceding point illustrates the potential of using combination therapy to target both the CSCs and the differentiated tumour , thereby overcoming cancer cell heterogeneity and plasticity. Other therapeutic strategies that are currently being exploited in combating CSCs are depicted in Figure 3 below. While efforts have been made to specifically target CSCs with the aim to completely eradicate the tumour mass, as shown by the many targeted monotherapies, there have been no clinically approved single agent therapies against CSCs , suggesting that targeting CSCs might not improve clinical outcomes.
Conclusions and future perspectives
Understanding tumour initiation, progression, and how to combat it has been the focus of cancer research for many decades. While the identification of CSCs and the elucidation of their signalling pathways have paved the way to targeting them, there are considerable challenges yet to be overcome as highlighted above. The idea of solely targeting CSCs is unrealistic given that they (CSCs) share so many characteristics with normal stem cells, together with the high intratumoural heterogeneity within CSCs. While studies evaluating monotherapies against CSCs have shown promise in early phase studies, combining anti-CSC therapies with other traditional or targeted therapies against not just the CSC subpopulation, but the bulk of the tumour represents one approach to eradicating cancers. In addition, there is the need to develop efficacious techniques to selectively target CSCs, while sparing normal stem cells. Finally, overcoming the challenge of CSC therapy resistance using innovative techniques, such as immunotherapy, represents another avenue yet to be investigated.
- Chen X, Lowe M, Herliczek T, Hall MJ, Danes C, Lawrence DA, and Keyomarsi K. Protection of normal proliferating cells against chemotherapy by staurosporine-mediated, selective, and reversible G(1) arrest. J Natl Cancer Inst, 2000. 92(24): p. 1999-2008.
- Theobald DE. Cancer pain, fatigue, distress, and insomnia in cancer patients. Clin Cornerstone, 2004. 6 Suppl 1D: p. S15-21.
- Adorno-Cruz V, Kibria G, Liu X, Doherty M, Junk DJ, Guan D, Hubert C, Venere M, Mulkearns-Hubert E, Sinyuk M, Alvarado A, Caplan AI, Rich J, Gerson SL, Lathia J, and Liu H. Cancer stem cells: targeting the roots of cancer, seeds of metastasis, and sources of therapy resistance. Cancer research, 2015. 75(6): p. 924-929.
- Diehn M, Cho RW, Lobo NA, Kalisky T, Dorie MJ, Kulp AN, Qian D, Lam JS, Ailles LE, Wong M, Joshua B, Kaplan MJ, Wapnir I, Dirbas FM, Somlo G, Garberoglio C, Paz B, Shen J, Lau SK, Quake SR, Brown JM, Weissman IL, and Clarke MF. Association of reactive oxygen species levels and radioresistance in cancer stem cells. Nature, 2009. 458(7239): p. 780-3.
- Li X, Lewis MT, Huang J, Gutierrez C, Osborne CK, Wu MF, Hilsenbeck SG, Pavlick A, Zhang X, Chamness GC, Wong H, Rosen J, and Chang JC. Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J Natl Cancer Inst, 2008. 100(9): p. 672-9.
- Liu H, Patel MR, Prescher JA, Patsialou A, Qian D, Lin J, Wen S, Chang YF, Bachmann MH, Shimono Y, Dalerba P, Adorno M, Lobo N, Bueno J, Dirbas FM, Goswami S, Somlo G, Condeelis J, Contag CH, Gambhir SS, and Clarke MF. Cancer stem cells from human breast tumors are involved in spontaneous metastases in orthotopic mouse models. Proc Natl Acad Sci U S A, 2010. 107(42): p. 18115-20.
- Yakisich JS. Challenges and limitations of targeting cancer stem cells and/or the tumour microenvironment. Drugs and Therapy Studies, 2012. 2: p. 48-55.
- Vinogradov S and Wei X. Cancer stem cells and drug resistance: the potential of nanomedicine. Nanomedicine (Lond), 2012. 7(4): p. 597-615.
- Campbell LL and Polyak K. Breast tumor heterogeneity: cancer stem cells or clonal evolution? Cell Cycle, 2007. 6(19): p. 2332-8.
- Tang DG. Understanding cancer stem cell heterogeneity and plasticity. Cell Res, 2012. 22(3): p. 457-72.
- Ajani JA, Song S, Hochster HS, and Steinberg IB. Cancer stem cells: the promise and the potential. Semin Oncol, 2015. 42 Suppl 1: p. S3-17.
- Furth J, Kahn MC, and Breedis C. The Transmission of Leukemia of Mice with a Single Cell. The American Journal of Cancer, 1937. 31(2): p. 276-282.
- Bonnet D and Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med, 1997. 3(7): p. 730-7.
- Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, and Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A, 2003. 100(7): p. 3983-8.
- Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, Henkelman RM, Cusimano MD, and Dirks PB. Identification of human brain tumour initiating cells. Nature, 2004. 432(7015): p. 396-401.
- Patrawala L, Calhoun T, Schneider-Broussard R, Li H, Bhatia B, Tang S, Reilly JG, Chandra D, Zhou J, Claypool K, Coghlan L, and Tang DG. Highly purified CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells. Oncogene, 2006. 25(12): p. 1696-708.
- Collins AT, Berry PA, Hyde C, Stower MJ, and Maitland NJ. Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res, 2005. 65(23): p. 10946-51.
- Beier D, Hau P, Proescholdt M, Lohmeier A, Wischhusen J, Oefner PJ, Aigner L, Brawanski A, Bogdahn U, and Beier CP. CD133(+) and CD133(-) glioblastoma-derived cancer stem cells show differential growth characteristics and molecular profiles. Cancer Res, 2007. 67(9): p. 4010-5.
- Somervaille TC and Cleary ML. Identification and characterization of leukemia stem cells in murine MLL-AF9 acute myeloid leukemia. Cancer Cell, 2006. 10(4): p. 257-68.
- Hirschmann-Jax C, Foster AE, Wulf GG, Nuchtern JG, Jax TW, Gobel U, Goodell MA, and Brenner MK. A distinct “side population” of cells with high drug efflux capacity in human tumor cells. Proc Natl Acad Sci U S A, 2004. 101(39): p. 14228-33.
- Cheung AM, Wan TS, Leung JC, Chan LY, Huang H, Kwong YL, Liang R, and Leung AY. Aldehyde dehydrogenase activity in leukemic blasts defines a subgroup of acute myeloid leukemia with adverse prognosis and superior NOD/SCID engrafting potential. Leukemia, 2007. 21(7): p. 1423-30.
- Driessens G, Beck B, Caauwe A, Simons BD, and Blanpain C. Defining the mode of tumour growth by clonal analysis. Nature, 2012. 488(7412): p. 527-530.
- Visvader JE. Cells of origin in cancer. Nature, 2011. 469(7330): p. 314-322.
- Jamieson CH, Ailles LE, Dylla SJ, Muijtjens M, Jones C, Zehnder JL, Gotlib J, Li K, Manz MG, Keating A, Sawyers CL, and Weissman IL. Granulocyte-macrophage progenitors as candidate leukemic stem cells in blast-crisis CML. N Engl J Med, 2004. 351(7): p. 657-67.
- Lim E, Vaillant F, Wu D, Forrest NC, Pal B, Hart AH, Asselin-Labat ML, Gyorki DE, Ward T, Partanen A, Feleppa F, Huschtscha LI, Thorne HJ, Fox SB, Yan M, French JD, Brown MA, Smyth GK, Visvader JE, and Lindeman GJ. Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers. Nat Med, 2009. 15(8): p. 907-13.
- Pattabiraman DR and Weinberg RA. Tackling the cancer stem cells – what challenges do they pose? Nature reviews. Drug discovery, 2014. 13(7): p. 497-512.
- Ramos EK, Hoffmann AD, Gerson SL, and Liu H. New Opportunities and Challenges to Defeat Cancer Stem Cells. Trends Cancer, 2017. 3(11): p. 780-796.
- Caulfield SE, Davis CC, and Byers KF. Olaparib: A Novel Therapy for Metastatic Breast Cancer in Patients With a BRCA1/2 Mutation. Journal of the advanced practitioner in oncology, 2019. 10(2): p. 167-174.
- Lottaz C, Beier D, Meyer K, Kumar P, Hermann A, Schwarz J, Junker M, Oefner PJ, Bogdahn U, Wischhusen J, Spang R, Storch A, and Beier CP. Transcriptional profiles of CD133+ and CD133- glioblastoma-derived cancer stem cell lines suggest different cells of origin. Cancer Res, 2010. 70(5): p. 2030-40.
- Jurcic JG. What Happened to Anti-CD33 Therapy for Acute Myeloid Leukemia? Current Hematologic Malignancy Reports, 2012. 7(1): p. 65-73.
- Karsten U and Goletz S. What makes cancer stem cell markers different? SpringerPlus, 2013. 2(1): p. 301-301.
- Boehmerle W and Endres M. Salinomycin induces calpain and cytochrome c-mediated neuronal cell death. Cell Death Dis, 2011. 2: p. e168.
- Roesch A, Fukunaga-Kalabis M, Schmidt EC, Zabierowski SE, Brafford PA, Vultur A, Basu D, Gimotty P, Vogt T, and Herlyn M. A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth. Cell, 2010. 141(4): p. 583-94.
- Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, Dewhirst MW, Bigner DD, and Rich JN. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature, 2006. 444(7120): p. 756-60.
- Feuerhake F, Sigg W, Hofter EA, Dimpfl T, and Welsch U. Immunohistochemical analysis of Bcl-2 and Bax expression in relation to cell turnover and epithelial differentiation markers in the non-lactating human mammary gland epithelium. Cell Tissue Res, 2000. 299(1): p. 47-58.
- Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, Morris JJ, Lagutina I, Grosveld GC, Osawa M, Nakauchi H, and Sorrentino BP. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med, 2001. 7(9): p. 1028-34.
- Chaffer CL, Brueckmann I, Scheel C, Kaestli AJ, Wiggins PA, Rodrigues LO, Brooks M, Reinhardt F, Su Y, Polyak K, Arendt LM, Kuperwasser C, Bierie B, and Weinberg RA. Normal and neoplastic nonstem cells can spontaneously convert to a stem-like state. Proc Natl Acad Sci U S A, 2011. 108(19): p. 7950-5.
- Pinto D, Gregorieff A, Begthel H, and Clevers H. Canonical Wnt signals are essential for homeostasis of the intestinal epithelium. Genes Dev, 2003. 17(14): p. 1709-13.
- Booth C, Brady G, and Potten CS. Crowd control in the crypt. Nat Med, 2002. 8(12): p. 1360-1.
- Chen K, Huang YH, and Chen JL. Understanding and targeting cancer stem cells: therapeutic implications and challenges. Acta Pharmacol Sin, 2013. 34(6): p. 732-40.
- Von Hoff DD, LoRusso PM, Rudin CM, Reddy JC, Yauch RL, Tibes R, Weiss GJ, Borad MJ, Hann CL, Brahmer JR, Mackey HM, Lum BL, Darbonne WC, Marsters JC, de Sauvage FJ, and Low JA. Inhibition of the Hedgehog Pathway in Advanced Basal-Cell Carcinoma. N Engl J Med, 2009. 361(12): p. 1164-1172.
- Robinson GW, Orr BA, Wu G, Gururangan S, Lin T, Qaddoumi I, Packer RJ, Goldman S, Prados MD, Desjardins A, Chintagumpala M, Takebe N, Kaste SC, Rusch M, Allen SJ, Onar-Thomas A, Stewart CF, Fouladi M, Boyett JM, Gilbertson RJ, Curran T, Ellison DW, and Gajjar A. Vismodegib Exerts Targeted Efficacy Against Recurrent Sonic Hedgehog-Subgroup Medulloblastoma: Results From Phase II Pediatric Brain Tumor Consortium Studies PBTC-025B and PBTC-032. J Clin Oncol, 2015. 33(24): p. 2646-54.
- Dubrovska A, Elliott J, Salamone RJ, Kim S, Aimone LJ, Walker JR, Watson J, Sauveur-Michel M, Garcia-Echeverria C, Cho CY, Reddy VA, and Schultz PG. Combination therapy targeting both tumor-initiating and differentiated cell populations in prostate carcinoma. Clin Cancer Res, 2010. 16(23): p. 5692-702.
- Carnero A and Lleonart M. The hypoxic microenvironment: A determinant of cancer stem cell evolution. Bioessays, 2016. 38 Suppl 1: p. S65-74.