Changes Induced by P2X7 Receptor Stimulation of Human Glioblastoma Stem Cells in the Proteome of Extracellular Vesicles Isolated from Their Secretome.

Extracellular vesicles (EVs) are secreted from many tumors, including glioblastoma multiforme (GBM), the most common and lethal brain tumor in adults, which shows high resistance to current therapies and poor patient prognosis. Given the high relevance of the information provided by cancer cell secretome, we performed a proteomic analysis of microvesicles (MVs) and exosomes (EXOs) released from GBM-derived stem cells (GSCs). The latter, obtained from the brain of GBM patients, expressed P2X7 receptors (P2X7Rs), which positively correlate with GBM growth and invasiveness. P2X7R stimulation of GSCs caused significant changes in the EV content, mostly ex novo inducing or upregulating the expression of proteins related to cytoskeleton reorganization, cell motility/spreading, energy supply, protection against oxidative stress, chromatin remodeling, and transcriptional regulation. Most of the induced/upregulated proteins have already been identified as GBM diagnostic/prognostic factors, while others have only been reported in peripheral tumors. Our findings indicate that P2X7R stimulation enhances the transport and, therefore, possible intercellular exchange of GBM aggressiveness-increasing proteins by GSC-derived EVs. Thus, P2X7Rs could be considered a new druggable target of human GBM, although these data need to be confirmed in larger experimental sets.

The Natural Product Secoemestrin C Inhibits Colorectal Cancer Stem Cells via p38-S100A8 Feed-Forward Regulatory Loop.

Cancer stem cells (CSCs) are closely associated with tumor initiation, metastasis, chemoresistance, and recurrence, which represent some of the primary obstacles to cancer treatment. Targeting CSCs has become an important therapeutic approach to cancer care. Secoemestrin C (Sec C) is a natural compound with strong anti-tumor activity and low toxicity. Here, we report that Sec C effectively inhibited colorectal CSCs and non-CSCs concurrently, mainly by inhibiting proliferation, self-renewal, metastasis, and drug resistance. Mechanistically, RNA-seq analysis showed that the pro-inflammation pathway of the IL17 axis was enriched, and its effector S100A8 was dramatically decreased in Sec C-treated cells, whose roles in the stemness of CSCs have not been fully clarified. We found that the overexpression of S100A8 hindered the anti-CSCs effect of Sec C, and S100A8 deficiency attenuated the stemness traits of CSCs to enhance the Sec C killing activity on them. Meanwhile, the p38 signal pathway, belonging to the IL17 downstream axis, can also mediate CSCs and counter with Sec C. Notably, we found that S100A8 upregulation increased the p38 protein level, and p38, in turn, promoted S100A8 expression. This indicated that p38 may have a mutual feedback loop with S100A8. Our study discovered that Sec C was a powerful anti-colorectal CSC agent, and that the positive feedback loop of p38-S100A8 mediated Sec C activity. This showed that Sec C could act as a promising clinical candidate in colorectal cancer treatment, and S100A8 could be a prospective drug target.

Bortezomib suppresses acute myelogenous leukaemia stem-like KG-1a cells via NF-κB inhibition and the induction of oxidative stress.

Acute myelogenous leukaemia (AML) originates and is maintained by leukaemic stem cells (LSCs) that are inherently resistant to antiproliferative therapies, indicating that a critical strategy for overcoming chemoresistance in AML therapy is to eradicate LSCs. In this work, we investigated the anti-AML activity of bortezomib (BTZ), emphasizing its anti-LSC potential, using KG-1a cells, an AML cell line with stem-like properties. BTZ presented potent cytotoxicity to both solid and haematological malignancy cells and reduced the stem-like features of KG-1a cells, as observed by the reduction in CD34- and CD123-positive cells. A reduction in NF-κB p65 nuclear staining was observed in BTZ-treated KG-1a cells, in addition to upregulation of the NF-κB inhibitor gene NFΚBIB. BTZ-induced DNA fragmentation, nuclear condensation, cell shrinkage and loss of transmembrane mitochondrial potential along with an increase in active caspase-3 and cleaved PARP-(Asp 214) level in KG-1a cells. Furthermore, BTZ-induced cell death was partially prevented by pretreatment with the pancaspase inhibitor Z-VAD-(OMe)-FMK, indicating that BTZ induces caspase-mediated apoptosis. BTZ also increased mitochondrial superoxide levels in KG-1a cells, and BTZ-induced apoptosis was partially prevented by pretreatment with the antioxidant N-acetylcysteine, indicating that BTZ induces oxidative stress-mediated apoptosis in KG-1a cells. At a dosage of 0.1 mg/kg every other day for 2 weeks, BTZ significantly reduced the percentage of hCD45-positive cells in the bone marrow and peripheral blood of NSG mice engrafted with KG-1a cells with tolerable toxicity. Taken together, these data indicate that the anti-LSC potential of BTZ appears to be an important strategy for AML treatment.

CMYC-initiated HNF1A-AS1 overexpression maintains the stemness of gastric cancer cells.

Cancer stem cells (CSCs) are believed to be responsible for cancer metastasis and recurrence due to their self-renewal ability and resistance to treatment. However, the mechanisms that regulate the stemness of CSCs remain poorly understood. Recently, evidence has emerged suggesting that long non-coding RNAs (lncRNAs) play a crucial role in regulating cancer cell function in different types of malignancies, including gastric cancer (GC). However, the specific means by which lncRNAs regulate the function of gastric cancer stem cells (GCSCs) are yet to be fully understood. In this study, we investigated a lncRNA known as HNF1A-AS1, which is highly expressed in GCSC s and serves as a critical regulator of GCSC stemness and tumorigenesis. Our experiments, both in vitro and in vivo, demonstrated that HNF1A-AS1 maintained the stemness of GC cells. Further analysis revealed that HNF1A-AS1, transcriptionally activated by CMYC, functioned as a competing endogenous RNA by binding to miR-150-5p to upregulate ß-catenin expression. This in turn facilitated the entry of ß-catenin into the nucleus to activate the Wnt/ß-catenin pathway and promote CMYC expression, thereby forming a positive feedback loop that sustained the stemness of GCSCs. We also found that blocking the Wnt/ß-catenin pathway effectively inhibited the function of HNF1A-AS1, ultimately resulting in the inhibition of GCSC stemness. Taken together, our results demonstrated that HNF1A-AS1 is a regulator of the stemness of GCSCs and could serve as a potential marker for targeted GC therapy.

Machine learning for identifying tumor stemness genes and developing prognostic model in gastric cancer.

Gastric cancer presents a formidable challenge, marked by its debilitating nature and often dire prognosis. Emerging evidence underscores the pivotal role of tumor stem cells in exacerbating treatment resistance and fueling disease recurrence in gastric cancer. Thus, the identification of genes contributing to tumor stemness assumes paramount importance. Employing a comprehensive approach encompassing ssGSEA, WGCNA, and various machine learning algorithms, this study endeavors to delineate tumor stemness key genes (TSKGs). Subsequently, these genes were harnessed to construct a prognostic model, termed the Tumor Stemness Risk Genes Prognostic Model (TSRGPM). Through PCA, Cox regression analysis and ROC curve analysis, the efficacy of Tumor Stemness Risk Scores (TSRS) in stratifying patient risk profiles was underscored, affirming its ability as an independent prognostic indicator. Notably, the TSRS exhibited a significant correlation with lymph node metastasis in gastric cancer. Furthermore, leveraging algorithms such as CIBERSORT to dissect immune infiltration patterns revealed a notable association between TSRS and monocytes and other cell. Subsequent scrutiny of tumor stemness risk genes (TSRGs) culminated in the identification of CDC25A for detailed investigation. Bioinformatics analyses unveil CDC25A's implication in driving the malignant phenotype of tumors, with a discernible impact on cell proliferation and DNA replication in gastric cancer. Noteworthy validation through in vitro experiments corroborated the bioinformatics findings, elucidating the pivotal role of CDC25A expression in modulating tumor stemness in gastric cancer. In summation, the established and validated TSRGPM holds promise in prognostication and delineation of potential therapeutic targets, thus heralding a pivotal stride towards personalized management of this malignancy.

LINC00909 up-regulates pluripotency factors and promotes cancer stemness and metastasis in pancreatic ductal adenocarcinoma by targeting SMAD4.

BACKGROUND: Pancreatic cancer stem cells are crucial for tumorigenesis and cancer metastasis. Presently, long non-coding RNAs were found to be associated with Pancreatic Ductal Adenocarcinoma stemness characteristics but the underlying mechanism is largely known. Here, we aim to explore the function of LINC00909 in regulating pancreatic cancer stemness and cancer metastasis. METHODS: The expression level and clinical characteristics of LINC00909 were verified in 80-paired normal pancreas and Pancreatic Ductal Adenocarcinoma tissues from Guangdong Provincial People's Hospital cohort by in situ hybridization. RNA sequencing of PANC-1 cells with empty vector or vector encoding LINC00909 was experimented for subsequent bioinformatics analysis. The effect of LINC00909 in cancer stemness and metastasis was examined by in vitro and in vivo experiments. The interaction between LINC00909 with SMAD4 and the pluripotency factors were studied. RESULTS: LINC00909 was generally upregulated in pancreatic cancer tissues and was associated with inferior clinicopathologic features and outcome. Over-expression of LINC00909 enhanced the expression of pluripotency factors and cancer stem cells phenotype, while knock-down of LINC00909 decreased the expression of pluripotency factors and cancer stem cells phenotype. Moreover, LINC00909 inversely regulated SMAD4 expression, knock-down of SMAD4 rescued the effect of LINC00909-deletion inhibition on pluripotency factors and cancer stem cells phenotype. These indicated the effect of LINC00909 on pluripotency factors and CSC phenotype was dependent on SMAD4 and MAPK/JNK signaling pathway, another downstream pathway of SMAD4 was also activated by LINC00909. Specifically, LINC00909 was localized in the cytoplasm in pancreatic cancer cells and decreased the stability the SMAD4 mRNA. Finally, we found over-expression of LINC00909 not only accelerated tumor growth in subcutaneous mice models, but also facilitated tumorigenicity and spleen metastasis in orthotopic mice models. CONCLUSION: We demonstrate LINC00909 inhibits SMAD4 expression at the post-transcriptional level, which up-regulates the expression of pluripotency factors and activates the MAPK/JNK signaling pathway, leading to enrichment of cancer stem cells and cancer metastasis in pancreatic cancer.

Modeling 5-FU-Induced Chemotherapy Selection of a Drug-Resistant Cancer Stem Cell Subpopulation.

(1) Background: Cancer stem cells (CSCs) are a subpopulation of cells in a tumor that can self-regenerate and produce different types of cells with the ability to initiate tumor growth and dissemination. Chemotherapy resistance, caused by numerous mechanisms by which tumor tissue manages to overcome the effects of drugs, remains the main problem in cancer treatment. The identification of markers on the cell surface specific to CSCs is important for understanding this phenomenon. (2) Methods: The expression of markers CD24, CD44, ALDH1, and ABCG2 was analyzed on the surface of CSCs in two cancer cell lines, MDA-MB-231 and HCT-116, after treatment with 5-fluorouracil (5-FU) using flow cytometry analysis. A machine learning model (ML)-genetic algorithm (GA) was used for the in silico simulation of drug resistance. (3) Results: As evaluated through the use of flow cytometry, the percentage of CD24-CD44+ MDA-MB-231 and CD44, ALDH1 and ABCG2 HCT-116 in a group treated with 5-FU was significantly increased compared to untreated cells. The CSC population was enriched after treatment with chemotherapy, suggesting that these cells have enhanced drug resistance mechanisms. (4) Conclusions: Each individual GA prediction model achieved high accuracy in estimating the expression rate of CSC markers on cancer cells treated with 5-FU. Artificial intelligence can be used as a powerful tool for predicting drug resistance.

Dynamic interplay of nuclear receptors in tumor cell plasticity and drug resistance: Shifting gears in malignant transformations and applications in cancer therapeutics.

Recent advances have brought forth the complex interplay between tumor cell plasticity and its consequential impact on drug resistance and tumor recurrence, both of which are critical determinants of neoplastic progression and therapeutic efficacy. Various forms of tumor cell plasticity, instrumental in facilitating neoplastic cells to develop drug resistance, include epithelial-mesenchymal transition (EMT) alternatively termed epithelial-mesenchymal plasticity, the acquisition of cancer stem cell (CSC) attributes, and transdifferentiation into diverse cell lineages. Nuclear receptors (NRs) are a superfamily of transcription factors (TFs) that play an essential role in regulating a multitude of cellular processes, including cell proliferation, differentiation, and apoptosis. NRs have been implicated to play a critical role in modulating gene expression associated with tumor cell plasticity and drug resistance. This review aims to provide a comprehensive overview of the current understanding of how NRs regulate these key aspects of cancer biology. We discuss the diverse mechanisms through which NRs influence tumor cell plasticity, including EMT, stemness, and metastasis. Further, we explore the intricate relationship between NRs and drug resistance, highlighting the impact of NR signaling on chemotherapy, radiotherapy and targeted therapies. We also discuss the emerging therapeutic strategies targeting NRs to overcome tumor cell plasticity and drug resistance. This review also provides valuable insights into the current clinical trials that involve agonists or antagonists of NRs modulating various aspects of tumor cell plasticity, thereby delineating the potential of NRs as therapeutic targets for improved cancer treatment outcomes.

Ascorbic acid predominantly kills cancer stem cell-like cells in the hepatocellular carcinoma cell line Li-7 and is more effective at low cell density and in small spheroids.

The development of therapies that target cancer stem cells (CSCs) is an important challenge in cancer research. The antioxidant system is enhanced in CSCs, which may lead to resistance to existing therapies. Ascorbic acid (AA) has the potential to act as both an antioxidant and a pro-oxidant agent, but its effects on CSCs are a subject of current research. Here, we investigated the effect of AA focusing specifically on CSCs with the hepatocellular carcinoma cell line Li-7. The Li-7 cell line is heterogenous consisting of CD166- and CD166+ cells; CD166- cells include CSC-like cells (CD13+CD166- cells) and CD13-CD166- cells that can revert to CD13+CD166- cells. The addition of AA to the culture medium caused cell death in both cell populations in CD166- cells in a concentration dependent manner. In contrast, AA administration had a limited effect on CD166+ non-CSC cells. The level of reactive oxygen species after AA treatment was elevated only in CD166- cells. The effect of AA only occurred at low cell densities in 2D and 3D cultures. In a mouse tumor model injected with Li-7 cells, intraperitoneal administration of AA failed to prevent tumor formation but appeared to delay tumor growth. Our findings shed light on why AA administration has not become a mainstream treatment for cancer treatment; however, they also show the possibility that AA can be used in therapies to suppress CSCs.

YTHDF1 promotes the viability and self­renewal of glioma stem cells by enhancing LINC00900 stability.

YTHDF1, an N6­methyladenosine (m6A)­binding protein, is significantly upregulated in glioma tissues. The present study investigated the molecular mechanism underlying the regulatory effects of YTHDF1 on the viability, invasion and self­renewal of glioma stem cells (GSCs). Glioma and normal brain tissues were collected, and reverse transcription­quantitative PCR and western blotting were used to measure the gene and protein expression levels, respectively. Methylated RNA immunoprecipitation­PCR was used to assess the m6A modification level of the target gene. Subsequently GSCs were induced, and YTHDF1 and LINC00900 gene regulation was carried out using lentiviral infection. The viability, invasion and self­renewal of GSCs were assessed by Cell Counting Kit­8, Transwell and sphere formation assays, respectively. Binding between YTHDF1 and LINC00900 was verified by RNA immunoprecipitation and RNA pull­down assays. The targeted binding of microRNA (miR)­1205 to the LINC00900/STAT3 3'­UTR was verified using a luciferase reporter assay. The results revealed that YTHDF1 and LINC00900 expression levels were significantly upregulated in glioma tissues, and a high m6A modification level in LINC00900 transcripts was detected in glioma tissues. Overexpression of YTHDF1 promoted GSC viability, invasion and self­renewal, whereas knockdown of YTHDF1 had the opposite effects. In addition, YTHDF1 maintained the stability of LINC00900 and upregulated its expression through binding to it, thereby promoting GSC viability, invasion and self­renewal. Furthermore, LINC00900 promoted GSC viability, invasion, self­renewal and tumor growth by regulating the miR­1205/STAT3 axis. In conclusion, YTHDF1 promotes GSC viability and self­renewal by regulating the LINC00900/miR­1205/STAT3 axis.

Breast cancer stem cells as novel biomarkers.

Breast cancer is the most common cancer and the leading cause of mortality worldwide. Despite advancements in detection and treatment, it remains a major cause of cancer-related deaths in women. Breast cancer stem cells (BCSCs) are a crucial group of cells responsible for carcinogenesis, metastasis, medication resistance, and tumor recurrence. Identifying and understanding their molecular pathways is essential for developing effective breast cancer therapy. BCSCs are responsible for tumor genesis, development, metastasis, treatment resistance, and recurrence. Biomarkers are essential tools for identifying high-risk patients, improving diagnostic accuracy, developing follow-up programs, assessing treatment susceptibility, and predicting prognostic outcomes. Stem cell intervention therapy can provide specialized tools for precision therapy. Biomarker analysis in cancer patients is crucial to identify cells associated with disease progression and post-therapeutic relapse. However, negative post-therapeutic impacts can enhance cancer stemness by boosting BCSCs plasticity phenotypes, activating stemness pathways in non-BCSCs, and promoting senescence escape, leading to tumor relapse and metastasis. Despite the advancements in precision medicine, challenges persist in identifying stem cell markers, limiting the number of eligible patients for treatment. The diversity of biomedical research hinders the development of individualization-based preventative, monitoring, and treatment strategies, especially in oncology. Integrating and interpreting clinical and scientific data remains challenging. The development of stem cell-related indicators could significantly improve disease precision, enabling stem cell-targeted therapy and personalized treatment plans, although BCSCs are promising for breast cancer treatment optimization, serving as biomarkers for current therapy modalities. This summary discusses recent advancements in breast cancer stem cell research, including biomarkers, identification methods, molecular mechanisms, and tools for studying their biological origin and lineage development for precision medicine.

Targeted immunotherapy to cancer stem cells: A novel strategy of anticancer immunotherapy.

Cancer is a major disease that endangers human health. Cancer drug resistance and relapse are the two main causes contributing to cancer treatment failure. Cancer stem cells (CSCs) are a small fraction of tumor cells that are responsible for tumorigenesis, metastasis, relapse, and resistance to conventional anticancer therapies. Therefore, CSCs are considered to be the root of cancer recurrence, metastasis, and drug resistance. Novel anticancer strategies need to face this new challenge and explore their efficacy against CSCs. Recently, immunotherapy has made rapid advances in cancer treatment, and its potential against CSCs is also an interesting area of research. Meanwhile, immunotherapy strategies are novel therapeutic modalities with promising results in targeting CSCs. In this review, we summarize the targeting of CSCs by various immunotherapy strategies such as monoclonal antibodies(mAb), tumor vaccines, immune checkpoint inhibitors, and chimeric antigen receptor-T cells(CAR-T) in pre-clinical and clinical studies. This review provides new insights into the application of these immunotherapeutic approaches to potential anti-tumor therapies in the future.

Reactivating PTEN to impair glioma stem cells by inhibiting cytosolic iron-sulfur assembly.

Glioblastoma, the most lethal primary brain tumor, harbors glioma stem cells (GSCs) that not only initiate and maintain malignant phenotypes but also enhance therapeutic resistance. Although frequently mutated in glioblastomas, the function and regulation of PTEN in PTEN-intact GSCs are unknown. Here, we found that PTEN directly interacted with MMS19 and competitively disrupted MMS19-based cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) machinery in differentiated glioma cells. PTEN was specifically succinated at cysteine (C) 211 in GSCs compared with matched differentiated glioma cells. Isotope tracing coupled with mass spectrometry analysis confirmed that fumarate, generated by adenylosuccinate lyase (ADSL) in the de novo purine synthesis pathway that is highly activated in GSCs, promoted PTEN C211 succination. This modification abrogated the interaction between PTEN and MMS19, reactivating the CIA machinery pathway in GSCs. Functionally, inhibiting PTEN C211 succination by reexpressing a PTEN C211S mutant, depleting ADSL by shRNAs, or consuming fumarate by the US Food and Drug Administration-approved prescription drug N-acetylcysteine (NAC) impaired GSC maintenance. Reexpressing PTEN C211S or treating with NAC sensitized GSC-derived brain tumors to temozolomide and irradiation, the standard-of-care treatments for patients with glioblastoma, by slowing CIA machinery-mediated DNA damage repair. These findings reveal an immediately practicable strategy to target GSCs to treat glioblastoma by combination therapy with repurposed NAC.

Cancer stem cells: The important role of CD markers, Signaling pathways, and MicroRNAs.

For the first time, a subset of small cancer cells identified in acute myeloid leukemia has been termed Cancer Stem Cells (CSCs). These cells are notorious for their robust proliferation, self-renewal abilities, significant tumor-forming potential, spread, and resistance to treatments. CSCs are a global concern, as it found in numerous types of cancer, posing a real-world challenge today. Our review encompasses research on key CSC markers, signaling pathways, and MicroRNA in three types of cancer: breast, colon, and liver. These factors play a critical role in either promoting or inhibiting cancer cell growth. The reviewed studies have shown that as cells undergo malignant transformation, there can be an increase or decrease in the expression of different Cluster of Differentiation (CD) markers on their surface. Furthermore, alterations in essential signaling pathways, such as Wnt and Notch1, may impact CSC proliferation, survival, and movement, while also providing potential targets for cancer therapies. Additionally, some research has focused on MicroRNAs due to their dual role as potential therapeutic biomarkers and their ability to enhance CSCs' response to anti-cancer drugs. MicroRNAs also regulate a wide array of cellular processes, including the self-renewal and pluripotency of CSCs, and influence gene transcription. Thus, these studies indicate that MicroRNAs play a significant role in the malignancy of various tumors. Although the gathered information suggests that specific CSC markers, signaling pathways, and MicroRNAs are influential in determining the destiny of cancer cells and could be advantageous for therapeutic strategies, their precise roles and impacts remain incompletely defined, necessitating further investigation.

Morusin suppresses the stemness characteristics of gastric cancer cells induced by hypoxic microenvironment through inhibition of HIF-1α accumulation.

Gastric cancer (GC) is a common, life-threatening malignancy that contributes to the global burden of cancer-related mortality, as conventional therapeutic modalities show limited effects on GC. Hence, it is critical to develop novel agents for GC therapy. Morusin, a typical prenylated flavonoid, possesses antitumor effects against various cancers. The present study aimed to demonstrate the inhibitory effect and mechanism of morusin on the stemness characteristics of human GC in vitro under hypoxia and to explore the potential molecular mechanisms. The effects of morusin on cell proliferation and cancer stem cell-like properties of the human GC cell lines SNU-1 and AGS were assessed by MTT assay, colony formation test, qRT-PCR, flow cytometry analysis, and sphere formation test under hypoxia or normoxia condition through in vitro assays. The potential molecular mechanisms underlying the effects of morusin on the stem-cell-like properties of human GC cells in vitro were investigated by qRT-PCR, western blotting assay, and immunofluorescence assay by evaluating the nuclear translocation and expression level of hypoxia-inducible factor-1α (HIF-1α). The results showed that morusin exerted growth inhibitory effects on SNU-1 and AGS cells under hypoxia in vitro. Moreover, the proportions of CD44+/CD24- cells and the sphere formation ability of SNU-1 and AGS reduced in a dose-dependent manner following morusin treatment. The expression levels of stem cell-related genes, namely Nanog, OCT4, SOX2, and HIF-1α, gradually decreased, and the nuclear translocation of the HIF-1α protein was apparently attenuated. HIF-1α overexpression partially reversed the abovementioned effects of morusin. Taken together, morusin could restrain stemness characteristics of GC cells by inhibiting HIF-1α accumulation and nuclear translocation and could serve as a promising compound for GC treatment.

Exploiting transcription factors to target EMT and cancer stem cells for tumor modulation and therapy.

Transcription factors (TFs) are essential in controlling gene regulatory networks that determine cellular fate during embryogenesis and tumor development. TFs are the major players in promoting cancer stemness by regulating the function of cancer stem cells (CSCs). Understanding how TFs interact with their downstream targets for determining cell fate during embryogenesis and tumor development is a critical area of research. CSCs are increasingly recognized for their significance in tumorigenesis and patient prognosis, as they play a significant role in cancer initiation, progression, metastasis, and treatment resistance. However, traditional therapies have limited effectiveness in eliminating this subset of cells, allowing CSCs to persist and potentially form secondary tumors. Recent studies have revealed that cancer cells and tumors with CSC-like features also exhibit genes related to the epithelial-to-mesenchymal transition (EMT). EMT-associated transcription factors (EMT-TFs) like TWIST and Snail/Slug can upregulate EMT-related genes and reprogram cancer cells into a stem-like phenotype. Importantly, the regulation of EMT-TFs, particularly through post-translational modifications (PTMs), plays a significant role in cancer metastasis and the acquisition of stem cell-like features. PTMs, including phosphorylation, ubiquitination, and SUMOylation, can alter the stability, localization, and activity of EMT-TFs, thereby modulating their ability to drive EMT and stemness properties in cancer cells. Although targeting EMT-TFs holds potential in tackling CSCs, current pharmacological approaches to do so directly are unavailable. Therefore, this review aims to explore the role of EMT- and CSC-TFs, their connection and impact in cellular development and cancer, emphasizing the potential of TF networks as targets for therapeutic intervention.

Isolating Cancer Stem Cells from Solid Tumors.

Over the past 20 years, there has been a lot of interest in the study and investigation of cancer stem cells (CSCs) or tumor-initiating cells (TICs). CSCs are rare, dormant cells and able to self-renew and maintain tumor development and heterogeneity. A new age of basic and clinical cancer research, reclassification of human tumors, and the development of novel therapeutic approaches will undoubtedly result from a better knowledge of CSCs. In order to develop effective and therapeutic strategies to treat cancer, it is crucial to understand the basic characteristics of CSCs, their importance to cancer therapy, and methodologies to isolate, detect, and characterize them. Here, we outline the main methods and protocols to identify, isolate, and culture CSCs from primary tumors.

Multicellular Tumoroids for Investigating Cancer Stem-Like Cells in the Heterogeneous Tumor Microenvironment.

Cancer stem-like cells (CSC) are a major contributing factor to chemoresistance, tumor recurrence, and poor survival outcomes in patients across cancer types. Signaling from non-tumor cells in the tumor microenvironment (TME) enriches for and supports CSC. This complex cell-cell signaling in the heterogeneous TME presents a challenge for patient survival; however, it also presents an opportunity to develop new targeted therapies that can inhibit survival of CSC. In this chapter, we report a multicellular tumoroid model which can be used to investigate the interactions between cancer cells and non-tumor cells in the TME to better understand the contribution of various cell types to cancer cell phenotypes, as well as the underlying mechanisms involved. The following methods allow for each cell type to be distinguished using FACS and studied individually. Gene expression can be analyzed for cancer cells, as well as the other non-tumor cells using qPCR following sorting. The response to chemotherapeutic agents and expression of stem markers can be determined for cancer cells using flow cytometry, excluding the other cell types to get an accurate view of the cancer cells. Furthermore, the viability of non-tumor cells can be analyzed as well to determine if there are cytotoxic effects of the drugs on non-tumor cells. Thus, the multicellular tumoroid model will reveal the interactions between the CSC and non-tumor cells in the heterogenous TME, resulting in discoveries in the fields of cancer biology, novel targeted therapies, and personalized drug screening for precision medicine.

Isolating Circulating Cancer Stem Cells (CCSCs) from Human Whole Blood.

Measuring circulating tumor cells (CTCs) or circulating cancer stem cells (CCSCs) in blood, which shed from primary tumors, is a noninvasive method to screen and/or diagnose patients with a high risk of developing metastatic cancers or relapse. Here, we describe an optimized and standardized laboratory method for isolating CCSCs from human blood samples, using cancer-specific stem cell biomarkers (Kantara et al., Lab Invest 95:100-112, 2015). When performing this technique, 0-1 circulating epithelial tumor cells/mL blood should be expected in patients free of malignant adenocarcinomas compared to over 3 circulating cancer stem cells/mL blood in patients positive for malignant adenocarcinomas (Kantara et al., Lab Invest 95:100-112, 2015).

Mimicking the Tumor Niche: Methods for Isolation, Culture, and Characterization of Cancer Stem Cells and Multicellular Spheroids.

Cancer stem cells (CSCs) play a significant role in driving several tumor hallmarks. Their behavior and tumor progression are strictly related to the tumor microenvironment (TME). The dynamic interplay between CSCs and TME drives metastasis, chemoresistance, and disease relapse. In this chapter, we describe different techniques and protocols for isolating, culturing, and characterizing CSCs and we explain the methodology for the culture of multicellular spheroids comprising CSCs.