Advanced drug delivery platforms target cancer stem cells.

Human head and neck squamous cell carcinoma (HNSCC) is a highly malignant cancer associated with major morbidity and mortality. In this study, we determined that human HNSCC-derived HSC-3 cells contain a subpopulation of cancer stem cells (CSCs) characterized by high levels of CD44v3 and aldehyde dehydrogenase-1 (ALDH1) expression. These tumor cells also express several stem cell markers (the transcription factors Oct4, Sox2, and Nanog) and display the hallmark CSC properties of self-renewal/clonal formation and the ability to generate heterogeneous cell populations. Importantly, hyaluronan (HA) stimulates the CD44v3 (an HA receptor) interaction with Oct4-Sox2-Nanog leading to both a complex formation and the nuclear translocation of three CSC transcription factors. Further analysis reveals that microRNA-302 (miR-302) is controlled by an upstream promoter containing Oct4-Sox2-Nanog-binding sites, whereas chromatin immunoprecipitation (ChIP) assays demonstrate that stimulation of miR-302 expression by HA-CD44 is Oct4-Sox2-Nanog-dependent in HNSCC-specific CSCs. This process results in suppression of several epigenetic regulators (AOF1/AOF2 and DNMT1) and the up-regulation of several survival proteins (cIAP-1, cIAP-2, and XIAP) leading to self-renewal, clonal formation, and cisplatin resistance. These CSCs were transfected with a specific anti-miR-302 inhibitor to silence miR-302 expression and block its target functions. Our results demonstrate that the anti-miR-302 inhibitor not only enhances the expression of AOF1/AOF2 and DNMT1 but also abrogates the production of cIAP-1, cIAP-2, and XIAP and HA-CD44v3-mediated cancer stem cell functions. Taken together, these findings strongly support the contention that the HA-induced CD44v3 interaction with Oct4-Sox2-Nanog signaling plays a pivotal role in miR-302 production leading to AOF1/AOF2/DNMT1 down-regulation and survival of protein activation. All of these events are critically important for the acquisition of cancer stem cell properties, including self-renewal, clonal formation, and chemotherapy resistance in HA-CD44v3-activated head and neck cancer.

Tumor suppressor p53 is frequently mutated or inactivated in colorectal cancer while p53 family member p73 is rarely mutated in cancer cells. Small molecules that activate p73 can elicit a p53-like tumor suppressive function and represent a novel approach for p53 pathway restoration. Colorectal tumors contain a small population of cancer stem cells (CSCs) capable of self-renewal that contributes to tumor maintenance and resistance to therapy. Targeting CSCs could improve treatment response and prolong patient survival. We tested the hypothesis that small molecule-mediated p53 pathway restoration via p73 activation can target CSCs. We have previously shown that small molecule Prodigiosin restores the p53 pathway in vitro and in vivo via activation of p73. We tested the effects of Prodigiosin on colonosphere formation, a functional in vitro model of CSC self-renewal. Prodigiosin significantly inhibits colonosphere formation and growth of multiple human colorectal cancer cell lines independent of p53 status. Aldefluor positive (Aldefluor(+)) cells represent the CSC population as they formed greater number of colonospheres and xenograft tumors upon passage, and were resistant to chemotherapeutic drugs 5-Fluorouracil and Irinotecan compared to Aldefluor negative cells. Prodigiosin prevented colonosphere formation and reduced the viability of sorted Aldefluor(+) HCT116, DLD1 and SW480 cells. Next, we tested the effects of Prodigiosin on CSC-mediated tumor growth and self-renewal in a tumor xenograft model. Prodigiosin significantly reduces the growth of tumors initiated with sorted Aldefluor(+) SW480 and DLD1 cells and prevents the passage of these tumors. No measurable toxic effects were observed in mice during the anti-CSC effects of Prodigiosin as determined by serum chemistry analysis and body weight measurement. Thus, Prodigiosin inhibits CSC self-renewal in vitro and in vivo. Mechanistic studies were performed to evaluate Prodigiosin-mediated p53-pathway restoration in CSCs. In a p53-reponsive luciferase reporter assay, Prodigiosin induced p53-pathway transcription in colonospheres, sorted Aldefluor(+) cells and tumor xenografts initiated with sorted Aldefluor(+) cells. Western blot analysis revealed that Prodigiosin elevated p73 protein levels in sorted CSCs. Prodigiosin elevated protein levels of p53/p73-regulated as well as p73-regulated p53-independent target genes in sorted CSCs. Sub-G1 analysis indicated that Prodigiosin induced cell death in colonospheres. Prodigiosin-mediated cell death in CSCs involves induction of Caspase-8 and PARP cleavage. In conclusion, Prodigiosin targets both non-CSCs and CSCs via p73-mediated p53 pathway restoration making it an attractive agent for colorectal cancer therapy. Our study has previously unrecognized implications for the role of p73 in cancer stem cell biology. Citation Format: Varun Vijay Prabhu, Bo Hong, Joshua E. Allen, Shengliang Zhang, David T. Dicker, Wafik S. El-Deiry. p53 pathway restoring small molecule Prodigiosin targets chemotherapy-resistant colorectal cancer stem cells in vitro and in vivo via p73 activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 202. doi:10.1158/1538-7445.AM2014-202

Ovarian cancer is a disease commonly complicated by the presence of ascites in the abdominal cavity which represents a major clinical problem. Currently, the treatment for ovarian cancer entails cytoreductive surgery followed by chemotherapy, mainly cisplatin or carboplatin combined with paclitaxel. Although this regimen is initially effective in a high percentage of cases, unfortunately within few months of initial treatment, tumor relapse occurs because of platinum-resistance. This is attributed mainly due to the presence of cancer stem cells (CSCs) present in ascites which are chemo-resistant and responsible for recurrence of cancer. In our preliminary studies, we show for the first time that withaferin A (WFA), a bioactive compound isolated from the plant Withania somnifera targets putative CSCs. Treatment of nude mice bearing orthotopic ovarian tumors with WFA resulted in a 70% to 80% reduction in tumor growth, complete inhibition of metastasis, and a significant elimination of the cells expressing CSCs markers - CD44, CD24, CD34, CD117, ALDH1 and Oct4 and down regulation of Notch1 and its downstream signaling genes (Hes1 and Hey1) reported to play crucial role for self-renewal and maintenance of CSCs. In addition, WFA also resulted in a significant inhibition of securin expression an “oncogene” important in ovarian tumorigenesis. In contrast, treatment of mice with CIS alone had the opposite effects, causing an increase in cells expressing CSC markers and Notch1 signaling pathway. However, combining of WFA with CIS showed enhanced effects on suppression of securin as well as elimination of CSCs. This may explain the development of platinum-resistance and recurrence of cancer in patients treated with first line chemotherapy. Based on this information, we hypothesize that a combination of WFA with CIS should target ovarian cancer cells as well as cancer stem cells by reducing securin and CSCs thereby reducing CIS resistance and recurrence of ovarian cancer. In our study, we used cells from ascites from patients with recurrent ovarian cancer and determine the effect of WFA alone and in combination with CIS on tumorigenic functions of ascites cells and CSCs from ascites in vitro and in vivo, and defined the molecular mechanisms associated with self-renewal, maintenance and recurrence of cancer in relation to expression of securin. Because the two agents act synergistically, this combination may minimize side effects as well as induction of cisplatin resistance and recurrence of cancer. In addition, this study will justify the initiation of phase I/phase II clinical trials to assess potential toxicity and efficacy of WFA/CIS combination in advanced/recurrent ovarian cancer patients. Citation Format: Sham S. Kakar, Mariusz Z. Ratajczak. Targeting cancer stem cells in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3332.

Downregulation of miRNA-200c Links Breast Cancer Stem Cells with Normal Stem Cells

Recent advances in experimental technologies and cancer models have made possible to demonstrate that the tumor is a dynamic system comprising heterogeneous populations of cancer cells organized in a hierarchical fashion with cancer stem cells (CSCs) at the apex. CSCs are immature cells characterized by self-renewal property and long-term repopulation potential. CSCs have been causally linked to cancer initiation, propagation, spreading, recurrence and relapse as well as to resistance to anticancer therapy. A growing body of evidence suggests that the function and physiology of CSCs may be influenced by genetic/epigenetic factors and tumor environment. In this context, macroautophagy is a lysosomal degradative process (herein referred to as autophagy) critical for the adaptive response to stress and the preservation of cellular and tissue homeostasis in all eukaryotes that may have a crucial role of in the origin, maintenance and invasiveness of CSCs. The activation of the autophagic machinery is also considered as an adaptive response of CSCs to perturbation of tumor microenvironment, caused for instance by anticancer therapy. Nevertheless, compelling preclinical and clinical evidence on the cytoprotective role of autophagy for CSCs is still missing. Here, we summarize the results on the contribution of autophagy in CSCs and how it impacts tumorigenesis and tumor progression. We also discuss the therapeutical potential of the modulation of autophagy as a means to eradicate CSCs.

Cancer stem cells (CSCs) can drive tumor growth, and their maintenance may rely on post-transcriptional regulation of gene expression, including that mediated by microRNAs (miRNAs). The let-7 miRNA family has been shown to induce differentiation by silencing stem cell programs. Let-7-mediated target gene suppression is prevented by LIN28A/B, which reduce let-7 biogenesis in normal embryonic and some cancer stem cells and ensure maintenance ofstemness. Here, we find that glioblastoma stem cells (GSCs) lack LIN28 and express both let-7 and their target genes, suggesting LIN28-independent protection from let-7 silencing. Using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP), we show that insulin-like growth factor 2 mRNA-binding protein 2 (IMP2) binds to let-7 miRNA recognition elements (MREs) and prevents let-7 target gene silencing. Our observations define the RNA-binding repertoire of IMP2 and identify a mechanism whereby it supports GSC and neural stem cell specification.

Cancer Stem Cell–Directed Therapies: Recent Data From the Laboratory and Clinic

Introduction: Despite recent advancements in treatment, pancreatic ductal adenocarcinoma (PDAC) continues to carry a very poor prognosis, which is largely attributable to drug resistance and early metastasis. PDAC cancer stem cells (CSCs) have been shown to play a role in these processes and are associated with worse median survival in patients. In order to better understand the mechanisms that regulate PDAC CSC maintenance and function, we have focused on their interactions with the extracellular matrix (ECM), a major component of the tumor microenvironment in PDAC. Methods: We used PDAC cell lines (Capan-1, MIA PaCa-2, and BxPC-3) and low passage human xenografts to evaluate the effects of a panel of ECM proteins (type I collagen, type IV collagen, fibronectin, and laminin) on CSC function and content. PDAC cells were plated on ECM protein coated plates (Fisher Scientific) and harvested at 72-96 hrs. We plated cells in 3% methylcellulose to assess clonogenic growth and on 8-µm transwell filters (Corning) to assess cell motility. We analyzed ECM-mediated activation of focal adhesion kinase (FAK) by western blot and the expression of phospho-tyrosine-FAK (pY397-FAK) in aldehyde dehydrogenase expressing (ALDH+) CSCs by flow cytometry. We created stable matched PDAC cell lines that overexpress full-length FAK (FAK-FL) or dominant-negative Y397F-FAK protein. We used these cell lines to study in vitro clonogenic growth and motility. We measured in vivo tumor-initiating cell frequency of these cell lines using a limiting dilution assay in NSG mice. We evaluated the efficacy of a small molecule inhibitor of pY397-FAK, PF573228, against PDAC CSCs in vitro. Results: Among the panel of ECM proteins, type I collagen consistently increased in vitro clonogenic growth and motility of all three PDAC cell lines, with a concurrent increase in ALDH+ cells. The effects of type I collagen were specific to ALDH+ cells, since culturing ALDH+ cells on type I collagen led to higher retention of the CSC phenotype compared to culture under normal conditions, whereas culturing ALDHneg cells on type I collagen had no effect. Since ECM proteins can induce FAK activation via Y397-FAK autophosphorylation, we examined the expression of pY397-FAK in ALDH+ cells from cell lines and low-passage xenografts, and found that ALDH+ cells are 6-10-fold enriched in pY397-FAK expression compared to bulk PDAC cells (P<.001). To investigate the role of FAK activity on CSC function, we overexpressed FAK-FL and Y397F-FAK in MIA-PaCa-2 and Capan-1 cells. Expression of pY397-FAK was 18-fold higher in FAK-FL-expressing cells but lost in Y397F-FAK-expressing cells. Compared to control cells, FAK-FL cells were 2-3 times (P<0.001) more clonogenic and 1.5-2 times (P<0.01) more motile, whereas Y397F-FAK cells were 1.6-5 times (P<0.01) less clonogenic and 1.7-3 times (P<0.01) less motile. FAK-FL expression enhanced the type I collagen-mediated increases in clonogenic growth and motility. In contrast, Y397F-FAK expression completely abrogated the effects of type I collagen. PF573228-treatment of PDAC cells inhibited clonogenic growth by 3-4-fold and motility by 3-fold and caused up to 40% loss of ALDH+ cells. Finally, we found that tumor-initiating cell frequency of MIAPaCa-2-control, MIAPaCa-2-FAK-FL, and MIAPaCa-2-pY397F-FAK cells was 1 in 350, 1 in 1, and 1 in 1150, respectively (P<0.01). Conclusion: Our studies show that interactions with the ECM enhance the number of PDAC CSCs, leading to increased clonogenic growth and cellular motility. We also show that FAK activation is necessary and sufficient for CSC function and may be a target of CSC-directed therapies. Citation Format: Asma Begum, Theodore Ewachiw, Ross McMillan, Vesselin Penchev, Clinton Jung, Ally Huang, Jessica Norberg, NV Rajeshkumar, Anirban Maitra, William Matsui, Zeshaan A. Rasheed. The extracellular matrix and focal adhesion kinase signaling regulate cancer stem cell function in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A70.

Many cancers contain cell subpopulations that display characteristics of stem cells. These cells are characterised by their ability to self-renew, form differentiated progeny and develop resistance to chemotherapeutic strategies. Cancer stem cells may utilise many of the same signalling pathways as normal stem cells including Wnt, Notch and Hedgehog. The dietary agent curcumin exerts a plethora of anti-carcinogenic effects both in vitro and in vivo, and can also inhibit many of the signalling pathways associated with stem cell biology. Emerging evidence suggests that curcumin can exert its anti-carcinogenic activity via targeting cancer stem cells through the disruption of stem cell signalling pathways. In this review we summarise the ability of curcumin to interfere with signalling pathways Wnt, Hedgehog, Notch, Signal Transducers and Activator (STAT) and interleukin-8, and report curcumin-induced changes in function and properties of cancer stem cells. We present evidence that the effects of curcumin on cancer stem cells mediate, or contribute to, its anti-carcinogenic activity.

Cancer stem cells (CSCs), which are operationally defined as tumor cell fractions that possess the selective capacity to drive tumorigenesis through self-renewal and differentiation, have been identified in a growing number of tumor entities. Importantly, CSCs have been linked to neoplastic progression and therapy resistance in cancer patients, and proof-of-concept has been established that CSC targeting can inhibit experimental tumor growth. However, the mechanisms by which CSCs fuel the tumorigenic process are only beginning to be unraveled. Recent evidence suggests that CSCs possess immunomodulatory capabilities that may enable them to evade host anticancer immunity to promote tumorigenicity. CSC immunological functions include evasion from immune clearance, induction of clonal anergy or deletion, and activation of regulatory immune cells. Strikingly, such novel CSC properties parallel established immunoregulatory pathways of physiologic stem cells and known mechanisms of tumor immunotherapy resistance. Accordingly, future cancer immunotherapeutic protocols that consider CSCs and their immunomodulatory functions hold the promise to achieve more durable responses in tumor patients.

Purpose: Tumours are composed of a heterogeneous cell population. Cancer stem cells, which make up a minor fraction of a tumour, may be the cells that initiate and sustain tumour growth. Cancer stem cells are believed to share many properties with normal stem cells that render them relatively insensitive to classical radio- and chemotherapy.Conclusions: We discuss what those (cancer) stem cell properties are and how the interactions with the microenvironment – ‘the niche’ – control those aspects of (cancer) stem cell biology. We also describe possible strategies to target cancer stem cells in order to prevent cancers from escaping therapy.

Post-treatment enrichment of the tumor initiating CD44+/CD24- breast cancer stem cell (CSC) population is believed to be responsible for breast tumor recurrence and metastasis. Among breast cancer subtypes, triple negative breast cancer (TNBC) particularly is known to have an abundant CSC population, and is characterized by frequent metastatic recurrence. Chloroquine (CQ), an anti-malarial drug, is a lysotropic reagent that inhibits autophagy. Recently, CQ has been shown to reduce the CSC population in various cancers and has been tested in clinical studies. Concurrently, CQ was identified as a potential CSC inhibitor discovered from gene expression signatures of the CD44+/CD24- CSC population. However, little, aside from inhibition of autophagy, is known about the working mechanism of chloroquine in reducing CSCs, particularly in TNBCs. Based on recent recommendations that low doses of CQ be used to limit toxicities in the heart and retina, we investigated how low doses of CQ enhance the antitumor effects of paclitaxel (PTX) and reduce the CSC population. CSC population changes and apoptosis were analyzed using flow cytometry analysis (CSC: CD44+/CD24-; apoptosis: annexin V) and western blot analysis for cleaved caspase 3, and CSC function was measured by mammosphere formation efficiency in SUM159PT, MDA-MB-231, MDA-MB-468, and Hs578T TNBC cell lines. We observed enhanced cytotoxic effects and significant reduction of the CSC population by combined treatment of PTX and CQ compared to single treatment of either PTX (5nM) or CQ (1 or 5 uM) in SUM159, Hs578T, and MDA-MB-231 cells. The enhanced cytotoxicity by co-treatment of CQ and PTX correlated well with inhibition of autophagy, as indicated by cleavage of LC3B, increased expression of p62, and accumulation of autophagosomes. Moreover, the combined treatment inhibited PTX induced STAT3 activation in CSCs and epigenetically regulated gene expression critical in maintenance of CSCs via repression of DNMT1 expression. Finally, we observed enhanced therapeutic efficacy in vivo with the combination of CQ (10-20 mg/kg, daily) and PTX (30 mg/kg, two times per week) when compared to either CQ or PTX alone (p< 0.05). Herein, we demonstrate effective reduction of the CD44+/CD24- CSC population by combined CQ and PTX treatment through autophagy inhibition. Moreover, we found that the low-dose combined treatment of CQ with PTX was able to regulate gene expression by altering DNA methylation, subsequently reducing CSCs in the TNBC cancer cells. Thus, a low dose treatment of CQ along with chemotherapy may be effective in treating TNBC patients, lend further support for in-depth studies on the mechanism of CQ among the subgroups of TNBC. Citation Format: Dong Soon Choi, Elvin Blanco, Sergio M. Granados-Principal, Bhuvanesh Dave, Melissa Landis, Helen Wong, Jenny Chang. Chloroquine inhibits cancer stem cells in triple negative breast cancer via regulation of DNA methylation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 237. doi:10.1158/1538-7445.AM2013-237

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that orchestrates cell signaling through integrins and growth factor receptors. FAK has been implicated in multiple steps of carcinogenesis including tumor initiation, growth and metastasis. Amplification and overexpression of FAK have been observed in multiple aggressive human cancers including breast and ovarian. VS-4718 is a potent and selective FAK kinase inhibitor that was previously shown by us to exhibit preferential inhibitory activities on breast cancer stem cells. We have further extended our investigation of the role of FAK on cancer stem cells to other solid tumors and report here that pharmacological attenuation of FAK activity by VS-4718 or RNAi-mediated depletion of FAK exhibits preferential inhibitory effects on cancer stem cells. To determine if FAK plays a role in the biology of cancer stem cells, we depleted FAK expression in breast, ovarian and mesothelioma cancer cell lines by RNAi. Our results indicated that shRNA-mediated knock-down of FAK inhibits tumorsphere formation in vitro. In parallel, VS-4718 was evaluated in a multitude of cancer stem cell assays both in vitro and in vivo. Pre-treatment of SUM159 cells with VS-4718 in matrigel reduced the percentage of ALDEFLUOR+ cancer stem cells and side population (SP). Similar effects were observed in ovarian cancer cell lines OVCAR-8 and OVCAR-5 where VS-4718 inhibited cancer stem cells as measured by multiple CSC assays. In an analogous fashion, VS-4718 also reduced the proportion of the ALDEFLUOR+ cells in H2052 human mesothelioma cells. In direct contrast, standard-of-care agents paclitaxel, carboplatin or pemetrexed increased the percentage of cancer stem cells, suggesting these agents do not effectively target cancer stem cells. Importantly combination of VS-4718 with standard-of-care agents attenuated chemotherapy-induced increases in the percentage of cancer stem cells in vitro in all three cancer models. The in vivo effect of VS-4718 on cancer stem cells was evaluated in SUM159 and MDA-MB-231 human triple negative breast cancer xenograft models. Following systemic administration, VS-4718 significantly reduced the proportion of cancer stem cells in tumors as evidenced by decreases in the percentage of ALDEFLUOR+ cells and tumorsphere-forming efficiency relative to vehicle-treated tumors and significantly abrogated tumor-initiating capabilities of cancer cells in a limiting dilution re-implantation assay. In summary, our results indicate the importance of FAK in the self-renewal of cancer stem cells in vitro and in vivo, and support the clinical development of FAK inhibitors to target cancer stem cells for the treatment of multiple cancers. Citation Format: Vihren Kolev, Quentin Wright, Christian Vidal, Irina Shapiro, Mahesh Pavdal, Mitchell Keegan, Qunli Xu, Jonathan Pachter. Pharmacological and genetic inhibition of FAK attenuates cancer stem cell function in vitro and in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 236. doi:10.1158/1538-7445.AM2013-236

Cancer stem cells (CSCs) have been shown to be markedly resistant to conventional cancer treatments such as chemotherapy and radiation therapy. Therefore, therapeutic strategies that selectively target CSCs will ultimately lead to better cancer treatments. Currently, accessible conventional therapeutic agents mainly eliminate the bulk tumor but do not eliminate CSCs. Therefore, the discovery and improvement of CSC-targeting therapeutic agents are necessary. Nanoparticles effectively inhibit multiple types of CSCs by targeting specific signaling pathways (Wnt/β-catenin, Notch, transforming growth factor-β, and hedgehog signaling) and/or specific markers (aldehyde dehydrogenases, CD44, CD90, and CD133) critically involved in CSC function and maintenance. In this review article, we summarized a number of findings to provide current information about their therapeutic potential of nanoparticles in various cancer cell types and CSCs.

Simple SummaryCancer stem cells (CSCs) represent a distinct cancer subpopulation that can influence the tumour microenvironment, in addition to cancer progression and relapse. A multitude of factors including CSC properties, long noncoding RNAs (lncRNAs), and autophagy play pivotal roles in maintaining CSCs. We discuss the methods of detection of CSCs and how our knowledge of regulatory and cellular processes, and their interaction with the microenvironment, may lead to more effective targeting of these cells. Autophagy and lncRNAs can regulate several cellular functions, thereby promoting stemness factors and CSC properties, hence understanding this triangle and its associated signalling networks can lead to enhanced therapy response, while paving the way for the development of novel therapeutic approaches. Cancer stem cells (CSCs) possess properties such as self-renewal, resistance to apoptotic cues, quiescence, and DNA-damage repair capacity. Moreover, CSCs strongly influence the tumour microenvironment (TME) and may account for cancer progression, recurrence, and relapse. CSCs represent a distinct subpopulation in tumours and the detection, characterisation, and understanding of the regulatory landscape and cellular processes that govern their maintenance may pave the way to improving prognosis, selective targeted therapy, and therapy outcomes. In this review, we have discussed the characteristics of CSCs identified in various cancer types and the role of autophagy and long noncoding RNAs (lncRNAs) in maintaining the homeostasis of CSCs. Further, we have discussed methods to detect CSCs and strategies for treatment and relapse, taking into account the requirement to inhibit CSC growth and survival within the complex backdrop of cellular processes, microenvironmental interactions, and regulatory networks associated with cancer. Finally, we critique the computationally reinforced triangle of factors inclusive of CSC properties, the process of autophagy, and lncRNA and their associated networks with respect to hypoxia, epithelial-to-mesenchymal transition (EMT), and signalling pathways.