Cancer stem cells: A roadblock in disease treatment

Dear Editor,

Cancer stem cells (CSCs) represent a rare subpopulation found within tumors, characterized by the ability to undergo self-renewal and continuous proliferation. Rather than being a static and rigidly organized subset, CSCs exhibit a highly dynamic state within tumors. This intrinsic plasticity enables CSCs to not just transition between quiescent and proliferative states but also adopt epithelial or mesenchymal phenotypes in response to various stresses, in turn making their eradication through conventional therapeutic approaches very difficult.

CSCs were successfully isolated for the first time in 1997 from acute myeloid leukemia and identified as cells having extensive proliferative potential along with CD34⁺CD38⁻ phenotype.^[1] Thereafter, the concept of CSCs was expanded to solid tumors, and CSC populations have been well-characterized for breast, colorectal, lung, pancreatic cancers, etc.^[2]

CSCs exhibit a number of similarities with the normal stem cells (NSCs), particularly in terms of their self-renewal and differentiation capabilities. In fact, CSCs commonly take over the molecular mechanisms associated with self-renewal and differentiation of NSCs to accelerate tumor progression. CSC behavior is governed by an intricate signaling network mainly comprised of Notch, Sonic Hedgehog, wingless-related integration site (WNT)/β-catenin, transforming growth factor-beta (TGF-β), Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3), phosphatidylinositol 3-kinase (PI3K)/Akt pathways. Several key stemness factors participate in the transcriptional control of CSCs across various cancer types, namely, octamer-binding transcription factor 4 (Oct4), SRY (sex-determining region Y)-box 2 (Sox2), c-Myc, kruppel-like factor 4 (KLF4), and NANOG.^[3]

Surface markers play a key role in the identification and characterization of CSCs. Some of the commonly expressed stemness-related markers by CSCs have been listed in Supplementary Table 1. Nonetheless, these markers may differ in expression levels and/or patterns across cancer types. Western blotting remains the gold standard for CSC identification based on these specific marker/s’ expression. The evolution of cell sorting technologies, such as fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and microfluidics, has progressed from exploiting physical properties of CSCs (e.g., these specific cell surface markers) to using functional characteristics such as dye efflux, Ca²⁺ concentration, and pH. Recent advancements in single-cell sequencing and spatial transcriptomics have further revealed the underlying heterogeneity among CSC populations. Next-Generation Sequencing (NGS) data analysis approaches such as Ribo-Seq, RIP-Seq, CLIP-Seq, and ribonucleic acid (RNA) velocity provide valuable insights into CSC heterogeneity, lineage dynamics, and stemness/differentiation traits.^[4]

The tumor microenvironment (TME) significantly influences the pathological role of CSCs. Various components of TME, including extracellular matrix and non-tumor cells such as immune cells, cancer-associated fibroblasts (CAF), and endothelial cells, are well-known for their ability to modulate CSC activity through paracrine signaling.^[5] Hypoxic TME allows CSCs to remain in a relatively dormant and undifferentiated state. Epithelial to mesenchymal transition (EMT), a hallmark of cancers, has been extensively linked to increased invasiveness and resistance to apoptosis. Tumor-associated macrophages promote EMT and maintain stemness features through TGF-β signaling in CSCs. Interestingly, hypoxia and EMT are closely correlated since hypoxia-inducible factor-1 (HIF-1) activates numerous transcription factors involved in EMT, including twist and snail.^[6] EMT also prompts CSC-like phenotype, since EMT transcription factors activate pluripotency regulators (Oct4, Sox2, and NANOG) and sustained signaling through WNT/β-catenin, Notch, and Hedgehog pathways.^[7]

CSCs exhibit metabolic adaptability, which enables them to switch between different energy sources depending on the diverse environmental conditions. This metabolic plasticity is intimately regulated by Notch, Hedgehog, and PI3K signaling pathways in CSCs, resulting in the development of distinct signaling-metabolism feedback loops, which ensure that CSC characteristics are maintained.^[2] The role of CSCs in disease progression and relapse has been depicted in Figure 1.

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Figure 1: Tumor microenvironment (TME) acts as a niche for cancer stem cells (CSCs) and plays an essential role in the promotion and maintenance of CSCs. Cells within TME, including tumor-associated macrophages, cancer-associated fibroblasts, tumor endothelial cells, and extracellular matrix components, govern CSC behavior. CSCs play a crucial role in tumor initiation, progression, maintenance, metastasis, therapeutic resistance, and relapse.

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Due to their active involvement in metastasis as well as therapy resistance, CSCs are being considered as key targets for improving cancer treatment outcomes. CSCs resist therapy through a wide range of intrinsic and extrinsic mechanisms, which include metabolic reprogramming, drug efflux transporters, aldehyde dehydrogenase (ALDH) activity, and quiescence. CSCs actively interact with adjacent stromal cells within the TME, causing impaired responses to immunotherapy.^[8] Several therapeutic approaches aimed at specifically targeting these mechanisms are currently in preclinical and clinical development. For example, targeting proliferative CSCs having high telomerase activity using cyclin-dependent kinase 4/6 (CDK 4/6) inhibitors^[9] or promoting CSC differentiation.^[10,11] Various novel compounds and distinct epigenetic modulators are also currently under investigation for their potential to selectively target CSC phenotypes. Furthermore, there is a rising interest in chimeric antigen receptor T-cells (CAR T-cells) for effective targeting of antigens enriched on the surface of CSCs.^[12] Other cutting-edge technologies, such as clustered regularly interspaced short palindromic repeats (CRISPR)-based functional screening and multi-omics-based profiling, have significantly contributed towards the characterization of CSC vulnerabilities.

A substantial limitation that is hindering the clinical translation of several CSC-targeted therapies is CSC’s adaptive plasticity, which enables them to bypass most of the single-target therapeutic interventions. Therefore, designing multi-target interventions so that there is no compensatory shift in the energy utilization of CSCs is essential.^[1]

Combinatorial regimens have been proposed that target both the rapidly proliferating tumor cells and quiescent CSCs along with their immunosuppressive niche.^[6]

CSC research should also prioritize establishing early detection strategies since CSCs are believed to be closely associated with the disease pathology. This involves inclusion of exosomal RNA signatures and liquid biopsy approaches for the identification of CSCs. Similarly, extracellular vesicles are potential non-invasive biomarkers for monitoring the activity of CSCs.^[2]

Altogether, the ultimate challenge lies in translating fundamental biological insights into clinically viable diagnostic and therapeutic strategies. Incorporation of advanced technologies, such as single-cell monitoring in association with spatial profiling, into clinical trials for efficient tracking of CSC dynamics in real time is warranted.^[12] As we progress toward the clinical application of CSC-targeted therapies, a multidisciplinary approach that integrates various concepts of immunotherapy, systems biology, synthetic biology, and bioinformatics-guided precision medicine is essential to disrupt CSC-driven tumor progression and thereby improve long-term patient outcomes.^[13]