Sasa quelpaertensis Leaf Extract Inhibits Colon Cancer by Regulating Cancer Cell Stemness in Vitro and in Vivo

A rare subpopulation of cancer cells, termed cancer stem cells (CSCs), may be responsible for tumor relapse and resistance to conventional chemotherapy. The development of a non-toxic, natural treatment for the elimination of CSCs is considered a strategy for cancer treatment with minimal side effects. In the present study, the potential for Sasa quelpaertensis leaf extract (SQE) and its two bioactive compounds, tricin and p-coumaric acid, to exert anti-CSC effects by suppressing cancer stemness characteristics were evaluated in colon cancer cells. CD133⁺CD44⁺ cells were isolated from HT29 and HCT116 cell lines using flow-activated cell sorting (FACs). SQE treatment was found to significantly suppress the self-renewal capacity of both cell lines. SQE treatment was also associated with the down-regulation of β-catenin and phosphorylated GSK3β, while significantly enhancing cell differentiation by up-regulating CK20 expression and blocking the expression of several stem cell markers, including DLK1, Notch1, and Sox-2. In vivo, SQE supplementation suppressed tumor growth in a xenograft model by down-regulating stem cell markers and β-catenin as well as HIF-1α signaling. Compared with two bioactive compounds of SQE, SQE exhibited the most effective anti-CSC properties. Taken together, these results provide evidence that SQE inhibits colon cancer by regulating the characteristics of CSCs.     

microRNA-21 Regulates Stemness in Pancreatic Ductal Adenocarcinoma Cells

Pancreatic ductal adenocarcinoma (PDAC) is the most common and aggressive type of pancreatic cancer (PCa) with a low survival rate. microRNAs (miRs) are endogenous, non-coding RNAs that moderate numerous biological processes. miRs have been associated with the chemoresistance and metastasis of PDAC and the presence of a subpopulation of highly plastic “stem”-like cells within the tumor, known as cancer stem cells (CSCs). In this study, we investigated the role of miR-21, which is highly expressed in Panc-1 and MiaPaCa-2 PDAC cells in association with CSCs. Following miR-21 knockouts (KO) from both MiaPaCa-2 and Panc-1 cell lines, reversed expressions of epithelial–mesenchymal transition (EMT) and CSCs markers were observed. The expression patterns of key CSC markers, including CD44, CD133, CX-C chemokine receptor type 4 (CXCR4), and aldehyde dehydrogenase-1 (ALDH1), were changed depending on miR-21 status. miR-21 (KO) suppressed cellular invasion of Panc-1 and MiaPaCa-2 cells, as well as the cellular proliferation of MiaPaCa-2 cells. Our data suggest that miR-21 is involved in the stemness of PDAC cells, may play roles in mesenchymal transition, and that miR-21 poses as a novel, functional biomarker for PDAC aggressiveness.     

In Ovarian Cancer Multicellular Spheroids,Platelet Releasate Promotes Growth,Expansion of ALDH+ and CD133+ Cancer Stem Cells,and Protection against the Cytotoxic Effects of Cisplatin,Carboplatin and Paclitaxel

A high platelet count is associated with a poor prognosis in ovarian cancer (OvCa). Despite good clinical responses with platinating agents in combination with taxanes, numerous OvCa patients relapse due to chemotherapy resistance. Here, we report that treatment of OvCa cells A2780, OVCAR5 and MDAH with releasate from activated platelets (PR) promoted multicellular tumor spheroid (MCTS) formation. These OvCa-MCTSs had increased percentages of CD133+ and aldehyde dehydrogenase (ALDH)+ cells, bona fide markers of OvCa cancer stem cells (CSCs). PR increased OVCAR5- and MDAH-MCTS viability and decreased the cytotoxic and pro-apoptotic effects of paclitaxel, cisplatin and carboplatin. PR increased the volume of spontaneously formed OVCAR8-MCTSs and counteracted their size reduction due to cisplatin, carboplatin and paclitaxel treatment. PR promoted the survival of ALDH+ and CD133+ OvCa cells during cisplatin, carboplatin and paclitaxel treatment. In conclusion, molecules and growth factors released by activated platelets (EGF, PDGF, TGF-β, IGF and CCL5) may protect tumor cells from chemotherapy by promoting the expansion of ALDH+ and CD133+ OvCa-CSCs, favoring drug resistance and tumor relapse.     

Cancer stem-like cells enriched in panc-1 spheres possess increased migration ability and resistance to gemcitabine

Pancreatic cancer is one of the most lethal malignancies with poor prognosis. Previously, we found that a subpopulation of cancer stem cells (CSCs) in the Panc-1 pancreatic cancer cell line could propagate to form spheres. Here we characterized the malignant phenotypes of the pancreatic cancer stem CD44+/CD24+ cells, which were enriched under sphere forming conditions as analyzed by flow cytometry. These cells demonstrated increased resistance to gemcitabine and increased migration ability. Moreover, these cells exhibited epithelial to mesenchymal transition characterized by a decreased level of the epithelial marker E-cadherin and an increased level of the mesenchymal marker vimentin. Notably, abnormal expression of Bmi-1, ABCG2, Cyclin D1 and p16 were found in Panc-1 CSCs. Our results suggest that targeted inhibition of CSCs represents a novel therapeutic approach to overcome chemoresistance and metastasis of pancreatic cancer.     

CD73/Adenosine Pathway Involvement in the Interaction of Non-Small Cell Lung Cancer Stem Cells and Bone Cells in the Pre-Metastatic Niche

Adenosinergic signaling is an important regulator of tissue homeostasis and extracellular accumulation of adenosine (Ado) and is associated with different pathologies, such as cancer. In non-small-cell lung cancer (NSCLC), a subset of CD133/CXCR4+ cancer stem cell (CSCs) has been demonstrated to initiate bone metastases. Here we investigated how NSCLC CSCs interact with osteoclasts (OCs) and osteoblasts (OBs) by modulating Ado production and OC activity. We proved that CSC-spheres, generated in vitro from NSCLC cell lines, express CD38, PC-1, and CD73, enzymes of the non-canonical adenosinergic pathway, produce high level of Ado, and down-regulate A1R and A3R inhibitory receptors, while expressing A2AR and A2BR. To address the Ado role and modulation of the in-bone pre-metastatic niche, we performed co-cultures of CSC-spheres with OCs and OBs cells. Firstly, we verified that active OCs do not activate non-canonical the adenosinergic pathway, conversely to OBs. OCs co-cultured with CSC-spheres increase Ado production that is related to the OC resorption activity and contributes to T-cell suppression. Finally, we proved the efficacy of anti-CD73 agents in blocking NSCLC cell migration. Overall, we assessed the importance of adenosinergic signaling in the interaction between CSCs and OCs at the pre-metastatic niche, with therapeutic implications related to Ado production.     

The Chick Chorioallantoic Membrane Model: A New In Vivo Tool to Evaluate Breast Cancer Stem Cell Activity

The high plasticity of cancer stem-like cells (CSCs) allows them to differentiate and proliferate, specifically when xenotransplanted subcutaneously into immunocompromised mice. CSCs are highly tumorigenic, even when inoculated in small numbers. Thus, in vivo limiting dilution assays (LDA) in mice are the current gold standard method to evaluate CSC enrichment and activity. The chick embryo chorioallantoic membrane (CAM) is a low cost, naturally immune-incompetent and reproducible model widely used to evaluate the spontaneous growth of human tumor cells. Here, we established a CAM-LDA assay able to rapidly reproduce tumor specificities—in particular, the ability of the small population of CSCs to form tumors. We used a panel of organotropic metastatic breast cancer cells, which show an enrichment in a stem cell gene signature, enhanced CD44⁺/CD24^−/low cell surface expression and increased mammosphere-forming efficiency (MFE). The size of CAM-xenografted tumors correlate with the number of inoculated cancer cells, following mice xenograft growth pattern. CAM and mice tumors are histologically comparable, displaying both breast CSC markers CD44 and CD49f. Therefore, we propose a new tool for studying CSC prevalence and function—the chick CAM-LDA—a model with easy handling, accessibility, rapid growth and the absence of ethical and regulatory constraints.     

HDAC Class I Inhibitor Domatinostat Preferentially Targets Glioma Stem Cells over Their Differentiated Progeny

Cancer stem cells (CSCs) are in general characterized by higher resistance to cell death and cancer therapies than non-stem differentiated cancer cells. However, we and others have recently revealed using glioma stem cells (GSCs) as a model that, unexpectedly, CSCs have specific vulnerabilities that make them more sensitive to certain drugs compared with their differentiated counterparts. We aimed in this study to discover novel drugs targeting such Achilles’ heels of GSCs as anti-GSC drug candidates to be used for the treatment of glioblastoma, the most therapy-resistant form of brain tumors. Here we report that domatinostat (4SC-202), a class I HDAC inhibitor, is one such candidate. At concentrations where it showed no or minimal growth inhibitory effect on differentiated GSCs and normal cells, domatinostat effectively inhibited the growth of GSCs mainly by inducing apoptosis. Furthermore, GSCs that survived domatinostat treatment lost their self-renewal capacity. These results suggested that domatinostat is a unique drug that selectively eliminates GSCs not only physically by inducing cell death but also functionally by inhibiting their self-renewal. Our findings also imply that class I HDACs and/or LSD1, another target of domatinostat, may possibly have a specific role in the maintenance of GSCs and therefore could be an attractive target in the development of anti-GSC therapies.     

New Insights into Cancer Targeted Therapy: Nodal and Cripto-1 as Attractive Candidates

The transforming growth factor beta (TGF-β) signaling is fundamental for correct embryonic development. However, alterations of this pathway have been correlated with oncogenesis, tumor progression and sustaining of cancer stem cells (CSCs). Cripto-1 (CR-1) and Nodal are two embryonic proteins involved in TGF-β signaling. Their expression is almost undetectable in terminally differentiated cells, but they are often re-expressed in tumor cells, especially in CSCs. Moreover, cancer cells that show high levels of CR-1 and/or Nodal display more aggressive phenotypes in vitro, while in vivo their expression correlates with a worse prognosis in several human cancers. The ability to target CSCs still represents an unmet medical need for the complete eradication of certain types of tumors. Given the prognostic role and the selective expression of CR-1 and Nodal on cancer cells, they represent archetypes for targeted therapy. The aim of this review is to clarify the role of CR-1 and Nodal in cancer stem populations and to summarize the current therapeutic strategy to target CSCs using monoclonal antibodies (mAbs) or other molecular tools to interfere with these two proteins.     

Cancer Stem Cell-Associated Pathways in the Metabolic Reprogramming of Breast Cancer

Metabolic reprogramming of cancer is now considered a hallmark of many malignant tumors, including breast cancer, which remains the most commonly diagnosed cancer in women all over the world. One of the main challenges for the effective treatment of breast cancer emanates from the existence of a subpopulation of tumor-initiating cells, known as cancer stem cells (CSCs). Over the years, several pathways involved in the regulation of CSCs have been identified and characterized. Recent research has also shown that CSCs are capable of adopting a metabolic flexibility to survive under various stressors, contributing to chemo-resistance, metastasis, and disease relapse. This review summarizes the links between the metabolic adaptations of breast cancer cells and CSC-associated pathways. Identification of the drivers capable of the metabolic rewiring in breast cancer cells and CSCs and the signaling pathways contributing to metabolic flexibility may lead to the development of effective therapeutic strategies. This review also covers the role of these metabolic adaptation in conferring drug resistance and metastasis in breast CSCs.     

Brain Metastasis-Initiating Cells: Survival of the Fittest

Brain metastases (BMs) are the most common brain tumor in adults, developing in about 10% of adult cancer patients. It is not the incidence of BM that is alarming, but the poor patient prognosis. Even with aggressive treatments, median patient survival is only months. Despite the high rate of BM-associated mortality, very little research is conducted in this area. Lack of research and staggeringly low patient survival is indicative that a novel approach to BMs and their treatment is needed. The ability of a small subset of primary tumor cells to produce macrometastases is reminiscent of brain tumor-initiating cells (BTICs) or cancer stem cells (CSCs) hypothesized to form primary brain tumors. BTICs are considered stem cell-like due to their self-renewal and differentiation properties. Similar to the subset of cells forming metastases, BTICs are most often a rare subpopulation. Based on the functional definition of a TIC, cells capable of forming a BM could be considered to be brain metastasis-initiating cells (BMICs). These putative BMICs would not only have the ability to initiate tumor growth in a secondary niche, but also the machinery to escape the primary tumor, migrate through the circulation, and invade the neural niche.     

NK Cells Lose Their Cytotoxicity Function against Cancer Stem Cell-Rich Radiotherapy-Resistant Breast Cancer Cell Populations

Cancer stem cells (CSCs) can be induced from differentiated cancer cells in the tumor microenvironment or in response to treatments and exhibit chemo- and radioresistance, leading to tumor recurrence and metastasis. We previously reported that triple negative breast cancer (TNBC) cells with acquired radioresistance exhibited more aggressive features due to an increased CSC population. Therefore, here, we isolated CSCs from radiotherapy-resistant (RT-R)-TNBC cells and investigated the effects of these CSCs on tumor progression and NK cell-mediated cytotoxicity. Compared to MDA-MB-231 and RT-R-MDA-MB-231 cells, CD24^−/low/CD44⁺ cells isolated from RT-R-MDA-MB-231 cells showed increased proliferation, migration and invasion abilities, and induced expression of tumor progression-related molecules. Moreover, similar to MDA-MB-231 cells, CD24^−/low/CD44⁺ cells recruited NK cells but suppressed NK cell cytotoxicity by regulating ligands for NK cell activation. In an in vivo model, CD24^−/low/CD44⁺ cell-injected mice showed enhanced tumor progression and lung metastasis via upregulation of tumor progression-related molecules and altered host immune responses. Specifically, NK cells were recruited into the peritumoral area tumor but lost their cytotoxicity due to the altered expression of activating and inhibitory ligands on tumors. These results suggest that CSCs may cause tumor evasion of immune cells, resulting in tumor progression.     

肿瘤相关巨噬细胞与肿瘤干细胞相互作用对肿瘤化疗耐药性影响的研究进展

肿瘤细胞对化疗药物产生耐药性是肿瘤化疗治疗失败的主要原因。目前研究普遍认为,在肿瘤治疗过程中,肿瘤相关巨噬细胞(tumor-associated macrophage,TAM)可与肿瘤干细胞(cancer stem cell,CSC)相互作用促进化疗耐药性的产生,即CSC能刺激TAM释放细胞因子,从而降低化疗对CSC的作用。本文就近年来二者相互作用对肿瘤化疗耐药性影响的相关研究进展作一综述。     

Extracellular Matrix Composition Modulates the Responsiveness of Differentiated and Stem Pancreatic Cancer Cells to Lipophilic Derivate of Gemcitabine

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease. Gemcitabine (GEM) is used as the gold standard drug in PDAC treatment. However, due to its poor efficacy, it remains urgent to identify novel strategies to overcome resistance issues. In this context, an intense stroma reaction and the presence of cancer stem cells (CSCs) have been shown to influence PDAC aggressiveness, metastatic potential, and chemoresistance. Methods: We used three-dimensional (3D) organotypic cultures grown on an extracellular matrix composed of Matrigel or collagen I to test the effect of the new potential therapeutic prodrug 4-(N)-stearoyl-GEM, called C18GEM. We analyzed C18GEM cytotoxic activity, intracellular uptake, apoptosis, necrosis, and autophagy induction in both Panc1 cell line (P) and their derived CSCs. Results: PDAC CSCs show higher sensitivity to C18GEM treatment when cultured in both two-dimensional (2D) and 3D conditions, especially on collagen I, in comparison to GEM. The intracellular uptake mechanisms of C18GEM are mainly due to membrane nucleoside transporters’ expression and fatty acid translocase CD36 in Panc1 P cells and to clathrin-mediated endocytosis and CD36 in Panc1 CSCs. Furthermore, C18GEM induces an increase in cell death compared to GEM in both cell lines grown on 2D and 3D cultures. Finally, C18GEM stimulated protective autophagy in Panc1 P and CSCs cultured on 3D conditions. Conclusion: We propose C18GEM together with autophagy inhibitors as a valid alternative therapeutic approach in PDAC treatment.     

MicroRNAs at the Crossroad of the Dichotomic Pathway Cell Death vs. Stemness in Neural Somatic and Cancer Stem Cells: Implications and Therapeutic Strategies

Stemness and apoptosis may highlight the dichotomy between regeneration and demise in the complex pathway proceeding from ontogenesis to the end of life. In the last few years, the concept has emerged that the same microRNAs (miRNAs) can be concurrently implicated in both apoptosis-related mechanisms and cell differentiation. Whether the differentiation process gives rise to the architecture of brain areas, any long-lasting perturbation of miRNA expression can be related to the occurrence of neurodevelopmental/neuropathological conditions. Moreover, as a consequence of neural stem cell (NSC) transformation to cancer stem cells (CSCs), the fine modulation of distinct miRNAs becomes necessary. This event implies controlling the expression of pro/anti-apoptotic target genes, which is crucial for the management of neural/neural crest-derived CSCs in brain tumors, neuroblastoma, and melanoma. From a translational point of view, the current progress on the emerging miRNA-based neuropathology therapeutic applications and antitumor strategies will be disclosed and their advantages and shortcomings discussed.     

Targeting Cancer Stem Cells with Small Molecules

Cancers arise as a result of physiological imbalances and subsequent uncontrolled cell division. Cancer initiation requires a set of biochemical alterations, including some occurring at the genetic and epigenetic levels. Thus, tumors are heterogeneous in nature making it challenging to selectively target different cancer cells by means of small molecule intervention. The paradigm of cancer stem cells (CSCs) describes subpopulations of cells with high self-renewal and tumor-seeding capacity. These cells, typically refractory to conventional therapies, can give rise to relapse after treatment. Combinatorial strategies, including drugs that selectively target this population of cells, have emerged in recent years. Here, we review how discovery-based – unbiased – screening approaches 1 have helped identify small molecules that specifically target CSCs. We also highlight biological pathways characteristic of CSCs that can potentially be selectively targeted in a hypothesis-driven manner by small molecules. We describe molecules that effectively target CSCs and emphasize what is known about their biological modes of action. The diversity and complexity of biochemical processes that CSCs may be addicted to, raises the question of how selective targeting of these pathways can be achieved. This challenge may be addressed by the continuing production of structurally complex and diverse small molecules using target and diversity-oriented synthesis approaches. 2     

Molecular Radiotherapy with 177Lu-Immunoliposomes Induces Cytotoxicity in Mesothelioma Cancer Stem Cells In Vitro

Malignant mesothelioma (MM) is a lethal tumor originating in the mesothelium with high chemotherapeutic resistance. Cancer stem cells (CSCs) persist in tumors and are critical targets responsible for tumor resistance and recurrence. The identification and characterization of CSCs may help develop effective treatment for MM. The objective of this study was to evaluate the therapeutic effect of molecular targeted radiotherapy by ¹⁷⁷Lu-labeled immunoliposomes (¹⁷⁷Lu-ILs) on CSCs of mesothelioma. MM CSCs were sorted based on CD26/CD24 expression level and their functional significances were established by small interference RNA. CSC potential of MM was evaluated for drug resistance, cell invasion, and cell growth rate in vitro. CSC metabolism was evaluated with the uptake of ¹⁸F-FDG. Therapeutic effects of ¹⁷⁷Lu-labeled immunoliposomes targeting CD26 and CD24 were evaluated in vitro through proliferation and apoptotic assays. CSCs sorted from H28 cells exhibited significant drug resistance and enhanced proliferative activity as well as increased metabolism indicated by higher ¹⁸F-FDG uptake. Treatment with ¹⁷⁷Lu-ILs, compared with ¹⁷⁷Lu-CL and ILs, showed enhanced therapeutic effects on inhibition of proliferation, up-regulation of apoptosis, and suppression of CD26 and CD24 expression. Thus, our results suggest that molecular radiotherapy targeting both CD26 and CD24 could be a promising approach for CSC-targeting therapy for MM.     

FOXM1-CD44 Signaling Is Critical for the Acquisition of Regorafenib Resistance in Human Liver Cancer Cells

Regorafenib is a multikinase inhibitor that was approved by the US Food and Drug administration in 2017. Cancer stem cells (CSCs) are a small subset of cancer-initiating cells that are thought to contribute to therapeutic resistance. The forkhead box protein M1 (FOXM1) plays an important role in the regulation of the stemness of CSCs and mediates resistance to chemotherapy. However, the relationship between FOXM1 and regorafenib resistance in liver cancer cells remains unknown. We found that regorafenib-resistant HepG2 clones overexpressed FOXM1 and various markers of CSCs. Patients with hepatocellular carcinoma also exhibited an upregulation of FOXM1 and resistance to regorafenib, which were correlated with a poor survival rate. We identified a close relationship between FOXM1 expression and regorafenib resistance, which was correlated with the survival of patients with hepatocellular carcinoma. Thus, a strategy that antagonizes FOXM1–CD44 signaling would enhance the therapeutic efficacy of regorafenib in these patients.     

Long Noncoding RNA MALAT-1 Enhances Stem Cell-Like Phenotypes in Pancreatic Cancer Cells

Cancer stem cells (CSCs) play a vital role in tumor initiation, progression, metastasis, chemoresistance, and recurrence. The mechanisms that maintain the stemness of these cells remain largely unknown. Our previous study indicated that MALAT-1 may serve as an oncogenic long noncoding RNA in pancreatic cancer by promoting epithelial-mesenchymal transition (EMT) and regulating CSCs markers expression. More significantly, there is emerging evidence that the EMT process may give rise to CSCs, or at least cells with stem cell-like properties. Therefore, we hypothesized that MALAT-1 might enhance stem cell-like phenotypes in pancreatic cancer cells. In this study, our data showed that MALAT-1 could increase the proportion of pancreatic CSCs, maintain self-renewing capacity, decrease the chemosensitivity to anticancer drugs, and accelerate tumor angiogenesis in vitro. In addition, subcutaneous nude mouse xenografts revealed that MALAT-1 could promote tumorigenicity of pancreatic cancer cells in vivo. The underlying mechanisms may involve in increased expression of self-renewal related factors Sox2. Collectively, we for the first time found the potential effects of MALAT-1 on the stem cell-like phenotypes in pancreatic cancer cells, suggesting a novel role of MALAT-1 in tumor stemness, which remains to be fully elucidated.