Cancer stem cells: landscape, challenges and emerging therapeutic innovations

Abstract

Cancer stem cells (CSCs) constitute a highly plastic and therapy-resistant cell subpopulation within tumors that drives tumor initiation, progression, metastasis, and relapse. Their ability to evade conventional treatments, adapt to metabolic stress, and interact with the tumor microenvironment makes them critical targets for innovative therapeutic strategies. Recent advances in single-cell sequencing, spatial transcriptomics, and multiomics integration have significantly improved our understanding of CSC heterogeneity and metabolic adaptability. Metabolic plasticity allows CSCs to switch between glycolysis, oxidative phosphorylation, and alternative fuel sources such as glutamine and fatty acids, enabling them to survive under diverse environmental conditions. Moreover, interactions with stromal cells, immune components, and vascular endothelial cells facilitate metabolic symbiosis, further promoting CSC survival and drug resistance. Despite substantial progress, major hurdles remain, including the lack of universally reliable CSC biomarkers and the challenge of targeting CSCs without affecting normal stem cells. The development of 3D organoid models, CRISPR-based functional screens, and AI-driven multiomics analysis is paving the way for precision-targeted CSC therapies. Emerging strategies such as dual metabolic inhibition, synthetic biology-based interventions, and immune-based approaches hold promise for overcoming CSC-mediated therapy resistance. Moving forward, an integrative approach combining metabolic reprogramming, immunomodulation, and targeted inhibition of CSC vulnerabilities is essential for developing effective CSC-directed therapies. This review discusses the latest advancements in CSC biology, highlights key challenges, and explores future perspectives on translating these findings into clinical applications.

Fig. 1

Historical evolution of CSC research. The concept of CSCs has evolved through distinct scientific milestones across centuries. (19th century—Early Theory of Tumor Origin): In 1858, “Omnis cellula e cellula” indicated that tumors arise from pathological alterations in normal cells. In 1867, the embryonal rest hypothesis was proposed, suggesting that tumors originate from dormant embryonic cells. (20th century—Experimental Evidence & Identification): Early experimental studies demonstrated that single-cell transplantation could initiate leukemia (1937) and that teratocarcinoma cells were capable of tumor initiation (1941). Further evidence has shown that undifferentiated germinal cells are the origin of tumor development (1960). In the 1990s, CSCs were first identified in leukemia (1994–1995), laying the foundation for CSC theory. (21st century - Expanding CSC Concept Across Cancer Types): In 2003, CSCs were first identified in solid tumors such as breast cancer and glioblastoma, followed by lung cancer (2005) and other malignancies, including colon cancer, head and neck squamous cell carcinoma (HNSCC), pancreatic cancer (2007), and melanoma (2008). (21^st century – Technological and therapeutic innovations in CSC Research): Since the early 2010s, single-cell sequencing technologies have enabled high-resolution analysis of CSC heterogeneity (2011–2012). In 2015, a preclinical study demonstrated the feasibility of targeting CSCs via CAR-T-cell therapy. In 2018, machine learning was used to develop stemness indices on the basis of transcriptomic and epigenetic data, providing a pan-cancer framework for CSC quantification and therapeutic target discovery. Created with BioRender.com

Fig. 2

Functional roles and characteristics of CSCs. CSCs play pivotal roles in tumor initiation, progression, metastasis, recurrence, and therapeutic resistance. Similarities to NSCs enable CSCs to exhibit self-renewal and differentiation properties, contributing to tumor heterogeneity. Tumor initiation is driven by a subset of CSCs known as TICs, which possess the capacity to form tumors upon transplantation into a mouse model. CSCs also promote tumor growth through HIF1α-induced VEGF signaling, enhancing angiogenesis. Tumor progression is further supported by genetic alterations, such as mutations in KRAS and TP53, which contribute to the acquisition of more malignant characteristics. Metastasis occurs through EMT, where CSCs downregulate E-cadherin and upregulate N-cadherin, a process known as the cadherin switch, to increase motility and facilitate intravasation into the bloodstream as circulating tumor cells (CTCs). Recurrence is linked to CSC quiescence in the G0 phase, resistance to therapy-induced oxidative stress via ROS detoxification mechanisms (SOD, CAT, GPX, and GSH), and the capacity for tumor regrowth following treatment. The regulatory network involving the Wnt/β-catenin, Notch, and Hedgehog signaling pathways further supports CSC maintenance and therapy resistance, underscoring their role as key drivers of cancer persistence. Created with BioRender.com

Fig. 3

Tumor microenvironmental factors influencing CSC formation. The tumor microenvironment plays a crucial role in CSC induction and maintenance by modulating key factors such as hypoxia, proinflammatory signals, and stromal interactions. Hypoxia stabilizes hypoxia-inducible factors (HIF-1α and HIF-2α), which promote VEGF-mediated angiogenesis and upregulate self-renewal transcription factors (OCT4, NANOG, and SOX2), thereby driving CSC-like properties in cancer cells. Proinflammatory signals further contribute to CSC formation, as cytokines such as TNF-α, IL-6, and IL-8, which are secreted by TAMs and CAFs, activate key pathways (NF-κB, JAK/STAT, and COX-2) that increase CSC survival and promote the conversion of non-CSCs (differentiated cancer cells) into CSC-like cells. Additionally, secretion factors such as TGF-β and HGF in the tumor microenvironment promote EMT, which facilitates CSC emergence. Endothelial cells contribute by releasing Jagged-1, activating Notch signaling, and further enhancing CSC self-renewal and survival. Finally, cellular plasticity permits the dedifferentiation of differentiated cancer cells into CSCs through intrinsic factors, such as genetic and epigenetic alterations, and extrinsic cues from the tumor microenvironment, thereby contributing to tumor heterogeneity and therapy resistance. Created with BioRender.com

Fig. 4

Biomarkers and their regulatory roles in CSC maintenance and regulation. CSCs are characterized by a range of membrane-integrated and intracellular biomarkers that regulate key signaling pathways involved in stemness, therapy resistance, and cellular plasticity. Membrane-associated markers, such as CD44, EpCAM, LGR5, CD133, EGFR, CXCR4, and CD24, contribute to CSC properties by modulating pathways, including the Wnt/β-catenin, PI3K/AKT, JAK/STAT, Notch, Hedgehog, and mTOR pathways. These markers facilitate CSC survival, EMT, angiogenesis, and oncogenic signaling stabilization. Additionally, intracellular CSC markers, including OCT4, SOX2, and NANOG, play essential roles in self-renewal, pluripotency, and therapy resistance as transcription factors. ALDH1, through the RA signaling pathway, further enhances CSC properties by influencing cellular plasticity and metabolic adaptation. RAR-mediated RA signaling contributes to CSC maintenance and drug resistance. Created with BioRender.com

Fig. 5

Signaling pathways and metabolic adaptation in CSCs. a Core signaling pathways involved in CSC maintenance. The Notch, Hedgehog, and PI3K/AKT/mTOR pathways regulate CSC self-renewal, quiescence, therapy resistance, and survival. Notch signaling, which is activated by ligand binding to NOTCH1-4, promotes transcriptional changes via the NICD-CSL-RBPJ complex in the canonical pathway, whereas non-canonical Notch signaling interacts with SMAD and NF-κB to modulate CSC plasticity. Hedgehog signaling is activated by ligands such as IHH, DHH, and SHH, leading to GLI transcription factor activation, which supports tumorigenesis. The PI3K/AKT/mTOR pathway enhances CSC maintenance through downstream activation of mTORC1 and mTORC2. mTORC2 is stimulated by PI3K signaling and phosphorylates AKT at Ser473, which in turn activates mTORC1. This axis upregulates stemness-associated transcription factors such as OCT4, SOX2 and NANOG, contributing to quiescence, therapy evasion, and enhanced DNA repair capacity. b Metabolic regulation of CSCs and their interplay with signaling pathways. CSCs exhibit metabolic plasticity, shifting between glycolysis, OXPHOS, and lipid metabolism on the basis of microenvironmental conditions. HIFs upregulate GLUT1/3 to increase glucose uptake, fueling glycolysis and the TCA cycle. PI3K/AKT signaling inhibits TSC2, leading to mTORC1 activation, which in turn promotes SREBP1-mediated de novo lipogenesis, supporting CSC growth through membrane synthesis and ribosomal biogenesis. FASN-mediated lipid synthesis further sustains CSC survival, whereas oxidative metabolism generates ROS, influencing epigenetic modifications. These interconnected pathways highlight the adaptability of CSC metabolism and its critical role in therapy resistance. Created with BioRender.com

Fig. 6

Metabolic plasticity of CSCs and tumor-specific metabolic adaptations. a Cancer stem cells adapt their metabolism on the basis of nutrient and oxygen availability: basal-like breast CSCs predominantly rely on glycolysis (nutrient-rich, hypoxic conditions), colorectal CSCs utilize a hybrid glycolytic/OXPHOS phenotype, and glioma or luminal breast CSCs primarily employ OXPHOS (nutrient-poor, oxygen-rich environments). This metabolic flexibility supports CSC survival and therapy resistance. b Glutamine metabolism supports CSC proliferation and redox homeostasis. Pancreatic CSCs are strongly dependent on glutamine metabolism and utilize glutamate and aspartate as key intermediates to fuel the TCA cycle and sustain nucleotide biosynthesis under nutrient-limited conditions. Additionally, glutamine metabolism contributes to redox balance, as the conversion of glutamine-derived α-KG to OAA supports NADPH generation via malate metabolism, which maintains the GSH/GSSG cycle to mitigate oxidative stress. Created with BioRender.com

Fig. 7

Tumor microenvironmental factors shaping CSC dynamics. The TME provides essential cues that regulate CSC survival, plasticity, and resistance to therapy. At the core, hypoxia induces CSC maintenance by stabilizing HIFs, which activate key signaling pathways such as the Notch, Hedgehog, and Wnt/β-catenin pathways, promoting self-renewal and metabolic adaptation. CSCs exploit immune evasion mechanisms, including the upregulation of PD-1/PD-L1 and the secretion of immunosuppressive cytokines (TGF-β, IL-10, and VEGF), to suppress cytotoxic T-cell (T_C) responses and escape immune surveillance. The ECM also plays a pivotal role, where stiffening due to increased collagen, laminin, and fibronectin deposition reinforces CSC survival and plasticity through YAP/TAZ activation, whereas MMP-mediated ECM degradation facilitates tumor invasion and metastasis. Furthermore, stromal cells, including MSCs and CAFs, support CSC maintenance by releasing EVs containing growth factors, metabolic substrates, and miRNAs. Additionally, cysteine metabolism in stromal cells contributes to CSC GSH production, enhancing stemness, proliferation, and EMT. These TME components collectively create a supportive niche, reinforcing CSC-driven tumor progression and therapy resistance. Created with BioRender.com

Fig. 8

Metabolic symbiosis between CSCs and the tumor microenvironment. CSCs establish metabolic symbiosis with stromal cells to sustain energy production and resist therapy-induced stress. Glucose metabolism: CAFs undergo aerobic glycolysis, producing lactate as a metabolic byproduct. CSCs take up lactate via MCT1/MCT4 and utilize it for oxidative metabolism, reducing glucose dependency and conferring resistance to glycolysis-targeting therapies. Amino acid metabolism: Tumor-associated stromal cells supply CSCs with glutamine, which is converted into glutamate via GLS and further fuels the TCA cycle or contributes to GSH synthesis, protecting CSCs from oxidative stress. Lipid metabolism: Adipocytes and TAMs release free FAs, which CSCs take up via the CD36 and FATP2 transporters for FAO. This provides CSCs with a stable energy source and enhances resistance to chemotherapy-induced stress. Additionally, TAMs secrete immunosuppressive cytokines, contributing to a protumor immune microenvironment. These interconnected metabolic exchanges support the maintenance, survival, and therapeutic resistance of CSCs, making them critical therapeutic targets. Created with BioRender.com

Fig. 9

Mechanisms of therapy resistance in CSCs. CSCs employ multiple resistance mechanisms that limit the effectiveness of current therapeutic strategies. One major mechanism involves elevated expression of antiapoptotic factors such as BCL2, BCL-XL, and Survivin, which inhibit apoptosis pathways. Additionally, enhanced DNA damage repair mechanisms activated in CSCs effectively counteract therapies designed to induce lethal DNA damage. CSCs frequently exist in a quiescent state (in the G₀ phase) and are maintained by cell cycle inhibitors such as p21 and p27, thereby evading therapies that target proliferative cells. Moreover, CSCs exhibit robust drug efflux capacity mediated by ABC transporters (e.g., ABCB1 and ABCG2), which actively export chemotherapeutic drugs, reducing intracellular drug accumulation and effectiveness. Increased ALDH activity in CSCs facilitates the detoxification of intracellular aldehydes to less toxic carboxylic acids, resulting in decreased ROS levels. This enhanced detoxification activity contributes to CSC survival and resistance to oxidative stress-inducing therapies. Collectively, these diverse resistance mechanisms underscore the necessity for combinational therapeutic strategies targeting the multifaceted vulnerabilities of CSCs. Representative therapeutic strategies that counteract these resistance mechanisms, such as venetoclax (BCL2 inhibitor), PARP inhibitors (targeting DNA repair), CDK4/6 inhibitors (disrupting quiescence), and disulfiram (ALDH inhibitor), are indicated in red. Created with BioRender.com