Sensitivity Of Cancer Stem Cells Research Articles

Nebulized Therapy of Early Orthotopic Lung Cancer by Iron-Based Nanoparticles: Macrophage-Regulated Ferroptosis of Cancer Stem Cells.

Cancer stem cells (CSCs) within protumorigenic microlesions are a critical driver in the initiation and progression of early stage lung cancer, where immune cells provide an immunosuppressive niche to strengthen the CSC stemness. As the mutual interactions between CSCs and immune cells are increasingly recognized, regulating the immune cells to identify and effectively eliminate CSCs has recently become one of the most attractive therapeutic options, especially for abundant tumor-associated macrophages (TAMs). Herein, we developed a nebulized nanocatalytic medicine strategy in which iron-based nanoparticle-regulated TAMs effectively target CSC niches and trigger CSC ferroptosis in the early stage of lung cancer. Briefly, the iron-based nanoparticles can effectively accumulate in lung cancer microlesions (minimum 122 μm in diameter) through dextran-mediated TAM targeting by nebulization administration, and as a result, nanoparticle-internalized TAMs can play a predominant role of the iron factory in elevating the iron level surrounding CSC niches and destroying redox equilibrium through downregulating glucose-6-phosphate metabolite following their lysosomal degradation and iron metabolism. The altered microenvironment results in the enhanced sensitivity of CSCs to ferroptosis due to their high expression of the CD44 receptor mediating iron endocytosis. In an orthotopic mouse model of lung cancer, the initiation and progression of early lung cancer are significantly suppressed through ferroptosis-induced stemness reduction of CSCs by nebulization administration. This work presents a nebulized therapeutic strategy for early lung cancer through modulation of communications between TAMs and CSCs, which is expected to be a general approach for regulating primary microlesions and micrometastatic niches of lung cancer.

Ex Vivo Irradiation of Lung Cancer Stem Cells Identifies the Lowest Therapeutic Dose Needed for Tumor Growth Arrest and Mass Reduction In Vivo.

Radiotherapy represents a first-line treatment for many inoperable lung tumors. New technologies offer novel opportunities for the treatment of lung cancer with the administration of higher doses of radiation in smaller volumes. Because both therapeutic and toxic treatment effects are dose-dependent, it is important to identify a minimal dose protocol for each individual patient that maintains efficacy while decreasing toxicity. Cancer stem cells sustain tumor growth, promote metastatic dissemination, and may give rise to secondary resistance. The identification of effective protocols targeting these cells may improve disease-free survival of treated patients. In this work, we evaluated the existence of individual profiles of sensitivity to radiotherapy in patient-derived cancer stem cells (CSCs) using both in vitro and in vivo models. Both CSCs in vitro and mice implanted with CSCs were treated with radiotherapy at different dose intensities and rates. CSC response to different radiation doses greatly varied among patients. In vitro radiation sensitivity of CSCs corresponded to the therapeutic outcome in the corresponding mouse tumor model. On the other side, the dose administration rate did not affect the response. These findings suggest that in vitro evaluation of CSCs may potentially predict patients’ response, thus guiding clinical decision.

Preferential radiosensitization to glioblastoma cancer stem cell-like cells by a Hsp90 inhibitor, N-vinylpyrrolidone-AUY922.

The present study examined the radiosensitization induced by a heat shock protein 90 inhibitor, N-vinylpyrrolidone (NVP)-AUY922, in CD133-positive cells in a hypoxic area of T98G spheroids. CD133-positive cells that are induced in the hypoxic microenvironment of spheroids have previously been reported to exhibit cancer stem cell-like properties. The present study used CD133-positive cells from a glioblastoma cell line (T98G) as cancer stem cell-like cells. CD133-positive and negative cells were sorted from T98G spheroids using fluorescence-activated cell sorting and used for colony formation assay. Colony formation assay results indicated that NVP-AUY922 enhanced radiosensitivity more strongly in CD133-positive cells compared with CD133-negative cells. This result showed that NVP-AUY922 was a preferential radiosensitization candidate targeting glioblastoma cancer stem cells. The mechanisms underlying radiosensitization by NVP-AUY922 are discussed in relation to the properties of cancer stem cells. Overall, HIF-1α inhibition by NVP-AUY922 may induce higher sensitization of cancer stem cells to radiation.

Targeting the metabolism of cancer stem cells by energy disruptor molecules

Cancer stem cells (CSCs) have been identified in various tumor types. CSCs are believed to contribute to tumor metastasis and resistance to conventional therapy. So targeting these cells could be an effective strategy to eliminate tumors and a promising new type of cancer treatment. Alterations in metabolism play an essential role in CSC biology and their resistance to treatment. The metabolic properties pathways in CSCs are different from normal cells, and to some extent, are different from regular tumor cells. Interestingly, CSCs can use other nutrients for their metabolism and growth. The different metabolism causes increased sensitivity of CSCs to agents that disrupt cellular homeostasis. Compounds that interfere with the central metabolic pathways are known as energy disruptors and can reduce CSC survival. This review highlights the differences between regular cancer cells and CSC metabolism and discusses the action mechanisms of energy disruptors at the cellular and molecular levels.

Cancer Stem Cells and Somatic Stem Cells as Potential New Drug Targets, Prognosis Markers, and Therapy Efficacy Predictors in Breast Cancer Treatment

New drug targets, markers of disease prognosis, and more efficient treatment options are an unmet clinical need in breast cancer (BC). We have conducted a pilot study including patients with luminal B stage breast cancer IIA–IIIB. The presence and frequency of various populations of cancer stem cells (CSC) and somatic stem cells were assessed in the blood, breast tumor tissue, and normal breast tissue. Our results suggest that patients with BC can be divided into two distinct groups based on the frequency of aldehyde dehydrogenase positive cells (ALDH1+ cells) in the blood (ALDH1hi and ALDH1low). In the ALDH1hi cells group, the tumor is dominated by epithelial tumor cells CD44+CD24low, CD326+CD44+CD24−, and CD326−CD49f+, while in the ALDH1low cells group, CSCs of mesenchymal origin and epithelial tumor cells (CD227+CD44+CD24− and CD44+CD24−CD49f+) are predominant. In vitro CSCs of the ALDH1low cells group expressing CD326 showed high resistance to cytostatics, CD227+ CSCs of the ALDH1hi cells group are sensitive to cytostatics. Epithelial precursors of a healthy mammary gland were revealed in normal breast tissue of patients with BC from both groups. The cells were associated with a positive effect of chemotherapy and remission in BC patients. Thus, dynamic control of their presence in blood and assessment of the sensitivity of CSCs to cytostatics in vitro can improve the effectiveness of chemotherapy in BC.

STEM-21. CLINICAL RELEVANCE OF CANCER STEM CELL CYTOTOXICITY ASSAY IN GUIDING TREATMENT FOR GLIOBLASTOMA

Abstract BACKGROUND. The prognosis of glioblastoma (GBM) treated with standard-of-care regimens remains very poor with a median time to recurrence of 7-9 months, and a median survival of 15-18 months. GBMs contain a small, but resilient population of cancer stem cells (CSCs) that contribute to tumor initiation, maintenance, and therapy resistance. For these reasons, its is extremely important to determine the sensitivity of CSCs to chemotherapeutic agents, with the intent of identifying more effective treatment protocols which would then translate into improved clinical survival. METHODS. We have used a novel CLIA and CAP-accredited Cancer Stem Cell Cytotoxicity Assay (ChemoID) to guide treatment for 55 Grade III and Grade IV glioma patients with the most efficacious chemotherapy treatments from a panel of FDA approved drugs or their combinations. Patients were evaluated by MRI scans and response was assessed according to RANO criteria. RESULTS: We report here a prospective clinical investigation using the ChemoID assay to measure the sensitivity and resistance of CSCs and bulk of tumor cells cultured from 55 GBM clinical specimens challenged with several chemotherapy agents, which were also correlated to the clinical response of the treated patients, independently of other biomarkers. The median recurrence time was 15 months for patients with a sensitive (>40% cell kill) CSCs test versus only 6 months for patients with a resistant CSCs test. CONCLUSIONS. The data suggests that GBM patients treated with CSC Cytotoxicity Assay-guided responsive drugs have delayed time to recurrence and the assay has the potential to help tailor chemotherapy choices to improve clinical outcomes.

Increasing the colon cancer cells sensitivity toward radiation therapy via application of Oct4-Sox2 complex decoy oligodeoxynucleotides.

Low sensitivity of cancer stem cells toward regular cancer therapy strategies is an important issue in the field of cancer remedy. The concept of cancer stem cell elimination has been a topic of interest in the field of molecular medicine for a long time. At the current study, it was aimed to elevate the sensitivity of cancer stem-like cells toward radiotherapy by treating with Oct4-Sox2 complex decoy oligodeoxynucleotides (ODNs). After treating HT29 and HT29-ShE cells with Oct4-Sox2 complex decoy ODNs, and analyzing the cellular uptake and localization of decoys, treated cells and control groups were subjected to irradiation by fractionated 6MV X-ray with a final dose of 2Gy. Thereafter, the influence of radiotherapy on ODNs treated groups and control group was investigated on cell viability, cell cycle, apoptosis, colonosphere formation and scratch assay. Cellular uptake and localization assays demonstrated that decoy ODNs can efficiently be transfected to the cells and reside in subcellular compartment, where they pose their action on gene regulation. Post radiotherapy analysis indicated statistical significance in decoy ODNs treated cells by means of lower cell viability, cell cycle arrest in G2/M phase, increased cellular apoptosis,and reduced cell motility. Also, formed colonospheres were smaller in size and fewer in numbers. Considering the role of Oct4, and Sox2 transcription factors in signaling pathways of preserving stemness and inducing reverse EMT, application of decoy strategy could increasethe sensitivity of cancer cells toward irradiation, which has a potential to eliminate the cancerous cells fromtumors andsupport cancer treatment.

YB-1 interplays with ERα to regulate the stemness and differentiation of ER-positive breast cancer stem cells.

Background: Some stemness-associated transcription factors consistently play essential roles in the maintenance of pluripotency or induce the differentiation of cancer stem cells (CSCs). However, the regulatory mechanism of CSC stemness mediated by transcription factors has not been extensively explored. Here, we show that two transcription factors (YB-1 and ERα), which are simultaneously highly expressed in estrogen receptor (ER)-positive CSCs, interact with each other to regulate the stemness and differentiation of ER-positive CSCs.Methods: The expression of YB-1 was examined in ER-positive CSCs and patient specimens. Western blot, real-time PCR, cell viability analysis, tumorsphere formation assay and subcutaneous tumorigenesis assays were used to study the stemness functions of YB-1 and ERα in CSCs. The relationship between YB-1 and ERα in cells was studied by promoter activity analysis, the electrophoretic mobility shift assay (EMSA) and the Co-IP assay. The mechanisms and functional significance of YB-1 in the sensitivity of CSCs to tamoxifen were further investigated with both in vitro and in vivo models.Results: YB-1 was aberrantly upregulated in the cancerous tissue of ER-positive breast cancer patients and in CSCs. Knockdown of YB-1 in ER-positive CSCs significantly inhibited cell stemness and induced differentiation, and the expression of YB-1 could be regulated by estrogen signaling and ERα in ER-positive breast CSCs. The Co-IP results showed that YB-1 interacted directly with ERα specifically in ER-positive non-CSCs and that YB-1 induced ERα degradation by ubiquitination via direct interaction in differentiated cells. Cell differentiation induced by FBS could inhibit YB-1 phosphorylation and promote YB-1 protein transfer from the nucleus to the cytoplasm. Moreover, cell differentiation induced by targeting inhibited the expression of YB-1 in ER-positive CSCs, which increased the sensitivity of cells to tamoxifen in vitro and in vivo.Conclusion: The ERα/YB-1 axis has an important role in the regulation of ER-positive breast cancer stemness. The dephosphorylation of YB-1 and the interaction between YB-1 and ERα may be the switch that initiates the differentiation of ER-positive CSCs. Targeting YB-1 to sensitize ER-positive CSCs to antiestrogen therapy might represent a new therapeutic strategy that warrants further exploration.

Disulfiram modulates ROS accumulation and overcomes synergistically cisplatin resistance in breast cancer cell lines

The chemotherapeutic agent cisplatin typically induces apoptosis by inhibiting the cell cycle. Cancer Stem Cells (CSCs), which are a proliferative quiescent and slowly-cycling cell population, are less sensitive and therefore frequently spared from toxic effects. Thus, it remains a priority to increase the sensitivity of CSCs to cisplatin-based chemotherapy, or to specifically target CSCs to improve the therapeutic outcome in breast cancer. Disulfiram (DSF) is a drug used clinically for alcoholism treatment that has displayed promising anti-cancer activity in vitro and in cancer xenografts in breast cancer. Our study provides evidence that DSF inhibits Aldehyde dehydrogenase (ALDH) enzyme activity, inhibits the expression of stemness-related transcription factors (Sox, Nanog, Oct) in CSC derived from breast cancer cell lines, and modulates intracellular reactive oxygen species (ROS) generation. Importantly, our research proved that ALDH + stem-like cells play important roles in the resistance to the conventional chemotherapeutic agent cisplatin. DSF enhances the cytotoxic effect of cisplatin through inhibiting the stemness and by overcoming cisplatin resistance of ALDH + stem-like cells. A quantitative measurement showed the synergistic effect of DSF and cisplatin. Further, we show that ALDH + cancer stem-like cells and ALDH- bulk cancer cells have different intrinsic ROS levels, what may explain differences in susceptibility to cisplatin treatment. Importantly, this difference is eliminated by DSF treatment making both cell types similarly susceptible for cytotoxic effects by cisplatin. These findings may influence chemotherapeutic treatment approaches in the future.

Inhibiting Wee1 and ATR kinases produces tumor-selective synthetic lethality and suppresses metastasis.

We used the cancer-intrinsic property of oncogene-induced DNA damage as the base for a conditional synthetic lethality approach. To target mechanisms important for cancer cell adaptation to genotoxic stress and thereby to achieve cancer cell-specific killing, we combined inhibition of the kinases ATR and Wee1. Wee1 regulates cell cycle progression, whereas ATR is an apical kinase in the DNA-damage response. In an orthotopic breast cancer model, tumor-selective synthetic lethality of the combination of bioavailable ATR and Wee1 inhibitors led to tumor remission and inhibited metastasis with minimal side effects. ATR and Wee1 inhibition had a higher synergistic effect in cancer stem cells than in bulk cancer cells, compensating for the lower sensitivity of cancer stem cells to the individual drugs. Mechanistically, the combination treatment caused cells with unrepaired or under-replicated DNA to enter mitosis leading to mitotic catastrophe. As these inhibitors of ATR and Wee1 are already in phase I/II clinical trials, this knowledge could soon be translated into the clinic, especially as we showed that the combination treatment targets a wide range of tumor cells. Particularly, the antimetastatic effect of combined Wee1/ATR inhibition and the low toxicity of ATR inhibitors compared with Chk1 inhibitors have great clinical potential.

Salinomycin may inhibit the cancer stem-like populations with increased chemoradioresistance that nasopharyngeal cancer tumorspheres contain.

Tumor recurrence and metastasis of nasopharyngeal cancer (NPC) often result in the failure of treatment due to chemoradioresistance. Cancer stem cells (CSCs) have been observed to drive tumor initiation and tumor chemoradioresistance. Therefore, the poor prognosis of advanced NPC is likely to result from the failure to kill CSCs. Sphere formation may be used as an experimental method to enrich potential CSC subpopulations. At present, there are few reports on NPC tumorspheres. The present study focused on examining the cancer stem-like properties of NPC tumorspheres from NPC cell lines. Western blot analysis revealed that NPC tumorspheres had a higher expression of stem cell markers Nanog homeobox and SRY-box 2, compared with parental cells. It was additionally verified that NPC tumorspheres contained a high aldehyde dehydrogenase (ALDH) enzymatic activity compared with parental cells. ALDH+ cells were amplified by 9- to 10-fold in tumorspheres, compared with parental cells (1.8 vs. 16.9%). The tumorsphere cells exhibited an increased half maximal inhibitory concentration value of >10-fold with cisplatin compared with the control parental cells. Compared with the parental cells, the percentage of side population cells in the tumorsphere cell population increased significantly (10.3 vs. 2.3%; P<0.05). NPC tumorsphere cells demonstrated enhanced resistance to radiation. Further investigation verified that salinomycin inhibited NPC CSCs by selectively targeting its stem cells. Altogether, the data revealed that NPC tumorspheres contain cancer stem-like populations with increased chemoradioresistance. It was suggested that the serum-free culture of NPC cells may provide an appropriate model for researching the sensitivity of CSCs to therapeutic agents. It was additionally revealed that salinomycin is an efficient inhibitor of NPC CSCs, supporting the hypothesis that salinomycin may eliminate CSCs and imply a need for further clinical evaluation.

Expression and function of immune ligand-receptor pairs in NK cells and cancer stem cells: therapeutic implications.

The interplay between the immune system and cancer cells has come to the forefront of cancer therapeutics, with novel immune blockade inhibitors being approved for the treatment of an increasing list of cancers. However, the majority of cancer patients still display or develop resistance to these promising drugs. It is possible that cancer stem cells (CSCs) are contributing to this therapeutic resistance. Although CSCs usually represent a small percentage of the total number of cancer cells, they are endowed with the ability of self-renewal and to produce differentiated progeny. Additionally, they have shown the capacity to establish tumors after transplantation to animals, even in small numbers. CSCs have also been found to be resistant to various anti-cancer therapies, including chemotherapy, radiation therapy and, more recently, immunotherapy. This is true despite the sensitivity of CSCs to lysis in vitro by natural killer (NK) cells, the main effector cells of the innate immune system. In this paper the expression of ligands specific for NK cells on CSCs, the intracellular network responsible for the expression of the NK cytotoxicity receptors, and the status of activation of NK cells in the tumor micro-environment are reviewed. The aim of this review is to highlight potential strategies for overcoming CSC immune resistance, thereby enhancing the efficacy of current and future anti-cancer therapies. NK cell activation in the tumor micro-environment through drugs neutralizing inhibitory immune receptors, and combined with other drugs harnessing the potential of the adaptive immune system, could be the most effective approach for attacking both stemcell and non-stem cell cancer populations.

Connexin 43 SUMOylation improves gap junction functions between liver cancer stem cells and enhances their sensitivity to HSVtk/GCV.

Connexin43 (Cx43) can be modified and regulated by small ubiquitin-like modifier (SUMO)1; however, its role in liver cancer stem cells is poorly understood. In this study, we found a significant difference in the expression of Cx43 and SUMO1 between cancer stem cells and non-cancer stem cells in liver cancer. In liver cancer stem cells, Cx43 was almost absent, although the level of SUMO1 was significantly higher than that in non-cancer stem cells. Further experiments confirmed that the conjugated site of Cx43 by SUMO1 was located in Lys-144 and Lys-237, both of which are highly conserved among species. By the co-expression of Cx43 and SUMO1 in cancer stem cells, the gap junction intercellular communication(GJIC) of liver cancer stem cells was obviously improved. Using this feature, we verified whether it could effectively increase the sensitivity of cancer stem cells to the herpes simplex virus1 thymidine kinase (HSVtk) gene in combination with ganciclovir (GCV), a conventional chemotherapeutic drug, invitro and invivo. As expected, increasing the expression of Cx43 SUMOylation in liver cancer stem cells effectively enhanced their sensitivity to HSVtk/GCV. On the whole, this study revealed a novel method which may be used to effectively restore GJIC in cancer stem cells in liver cancer, which enhances their sensitivity to conventional chemotherapeutic drugs.

Экспериментальные основы применения гипертермии в онкологии

This review presents the results of experimental studies on the effects of hyperthermia, at a temperature of 40–45 °C, in normal and tumor tissues. These results show that there is a strong rationale for using hyperthermia in addition to radiotherapy and/or chemotherapy in patients with cancer. The following study results are summarized. Hyperthermia has cell killing effects which specifically occur in tumor regions with hypoxia and low pH. In well perfused and well oxygenated tissues, cell damage is usually not observed at temperatures of up to 43 °C. This cell killing effect of hyperthermia is complementary to the effects of radiotherapy and chemotherapy, which treatment modalities are less effective in hypoxic or insufficiently perfused areas. In combination with radio- or chemotherapy, hyperthermia further has sensitizing effects, partly through an increase in blood flow. An important feature in radiosensitization is the inhibition of DNA repair pathways. Radiosensitizing is stronger in tumor tissue than in normal tissue when radiation and hyperthermia are given with a time interval, leading to therapeutic gain. The interaction with various drugs is related to changes in pharmacokinetics, enhancement of drug accumulation and a decrease in intracellular detoxification and damage repair. It was also observed that hyperthermia can overcome chemoresistance associated with multidrug resistance, by influencing transmembrane transport, metabolism, and the functioning of involved proteins. The effects of hyperthermia are stronger with higher temperatures and longer exposure times. Treatment with hyperthermia induces heat shock proteins (HSP’s). These may lead to thermotolerance to the next treatment when that is applied too soon. On the other hand it was found that HSP’s induce an antitumor immune response which may further add to the therapeutic effect. A stimulating effect of hyperthermia on the development of metastases could not be demonstrated in most studies. Studies with drugs in thermosensitive liposomes show that the uptake of drugs in tumor tissues can be largely enhanced. Further, it has been shown that hyperthermia considerably increases the sensitivity of cancer stem cells to radiotherapy and chemotherapy. Sofar, there is limited experience with the combination of hyperthermia and targeted agents. Nanoparticles which can be used for local heat treatment and local drug delivery are under investigation.

Efficacy of ChemoID® guided drug selection for palliative chemotherapy in advanced recurrent high-grade ovarian adenocarcinoma: Case study

Notwithstanding tremendous advances in surgery, primary chemotherapy, and novel treatments for recurrent disease, the diagnosis of advanced epithelial ovarian cancer (EOC) in 2016 remains ultimately fatal in the majority of cases. Advanced therapy refractory EOC patients are often treated in hospice and with chemotherapy palliative care. The main goals of chemotherapy for recurrent/refractory ovarian cancer are the palliation of disease-related symptoms, and improvement of quality and quantity of life. Unfortunately, there is contradicting evidence suggesting that chemotherapy has a role in palliation of symptoms with an apparent improvement in quality of life. Anticancer drugs have a high rate of failure and chemotherapy response assays have been used to identify which drugs are more likely to be effective against those of gynecological origin. ChemoID® is a functional drug response assay which measures the sensitivity of cancer stem cells (CSCs) and bulk of tumor cells to chemotherapy to determine the most effective combination of anticancer drugs for solid tumors. We present a clinical case that demonstrates the utility of ChemoID® guidance to commonly available drugs with identified toxicity profiles and predictable cost profile available at POS, along with rapid response in correcting symptomatic disease features, and minimal treatment burden from toxicities. We observed in the reported case the survival and symptom management benefits of ChemoID® guided therapy even after plateau of response to cisplatin. Further studies are indicated to increase the clinical adoption of ChemoID® for gynecological malignancies in the palliative setting.

Suppression of Nrf2 Activity by Chestnut Leaf Extract Increases Chemosensitivity of Breast Cancer Stem Cells to Paclitaxel.

Due to metastatic potential and drug resistance, cancer stem cells (CSCs) have become a critical target for the development of chemotherapeutic agents. Recent studies showed that CSCs highly express NF-E2-related factor 2 (Nrf2)-mediated antioxidant enzymes and thereby retain relatively low levels of reactive oxygen species (ROS). Since anticancer agents usually utilize ROS as an arsenal for killing cancer cells, we hypothesized that inhibition of Nrf2 activity could increase the sensitivity of CSCs to anticancer drugs, and thus enhancing their therapeutic efficacy. We found that MCF-7-derived CSCs with a CD44high/CD24low phenotype formed mammospheres and highly expressed Nrf2 compared to the adherent parental MCF-7 cells. In a separate experiment, we screened 89 different edible plant extracts for inhibitory activity against the Nrf2 signaling pathway by using an antioxidant response element (ARE)-luciferase assay system. Among those extracts, Castanea crenata (chestnut) leaf extract significantly decreased the nuclear translocation of Nrf2 and protein expression of antioxidant enzymes in MCF-7-derived CSCs. The combined treatment of the CSCs with chestnut leaf extract and paclitaxel resulted in more effective cell death than the treatment with paclitaxel alone. These findings suggest that the chestnut leaf extract or its constituents could increase the susceptibility of breast CSCs to an anticancer drug, paclitaxel, through inhibition of the Nrf2 signaling pathway, and could be utilized as an adjuvant for chemotherapy.

Aryl hydrocarbon receptor/cytochrome P450\xa01A1 pathway mediates breast cancer stem cells expansion through PTEN inhibition and \u03b2-Catenin and Akt activation

BackgroundBreast cancer stem cells (CSCs) are small sub-type of the whole cancer cells that drive tumor initiation, progression and metastasis. Recent studies have demonstrated a role for the aryl hydrocarbon receptor (AhR)/cytochrome P4501A1 pathway in CSCs expansion. However, the exact molecular mechanisms remain unclear.MethodsThe current study was designed to a) determine the effect of AhR activation and inhibition on breast CSCs development, maintenance, self-renewal, and chemoresistance at the in vitro and in vivo levels and b) explore the role of β-Catenin, PI3K/Akt, and PTEN signaling pathways. To test this hypothesis, CSC characteristics of five human breast cancer cells; SKBR-3, MCF-7, and MDA-MB231, HS587T, and T47D treated with AhR activators or inhibitor were determined using Aldefluor assay, side population, and mammosphere formation. The mRNA, protein expression, cellular content and localization of the target genes were determined by RT-PCR, Western blot analysis, and Immunofluorescence, respectively. At the in vivo level, female Balb/c mice were treated with AhR/CYP1A1 inducer and histopathology changes and Immunohistochemistry examination for target proteins were determined.ResultsThe constitutive mRNA expression and cellular content of CYP1A1 and CYP1B1, AhR-regulated genes, were markedly higher in CSCs more than differentiating non-CSCs of five different human breast cancer cells. Activation of AhR/CYP1A1 in MCF-7 cells by TCDD and DMBA, strong AhR activators, significantly increased CSC-specific markers, mammosphere formation, aldehyde dehydrogenase (ALDH) activity, and percentage of side population (SP) cells, whereas inactivation of AhR/CYP1A1 using chemical inhibitor, α-naphthoflavone (α-NF), or by genetic shRNA knockdown, significantly inhibited the upregulation of ALDH activity and SP cells. Importantly, inactivation of the AhR/CYP1A1 significantly increased sensitization of CSCs to the chemotherapeutic agent doxorubicin. Mechanistically, Induction of AhR/CYP1A1 by TCDD and DMBA was associated with significant increase in β-Catenin mRNA and protein expression, nuclear translocation and its downstream target Cyclin D1, whereas AhR or CYP1A1 knockdown using shRNA dramatically inhibited β-Catenin cellular content and nuclear translocation. This was associated with significant inhibition of PTEN and induction of total and phosphorylated Akt protein expressions. Importantly, inhibition of PI3K/Akt pathway by LY294002 completely blocked the TCDD-induced SP cells expansion. In vivo, IHC staining of mammary gland structures of untreated and DMBA (30 mg/kg, IP)- treated mice, showed tremendous inhibition of PTEN expression accompanied with an increase in the expression p-Akt, β-Catenin and stem cells marker ALDH1.ConclusionsThe present study provides the first evidence that AhR/CYP1A1 signaling pathway is controlling breast CSCs proliferation, development, self-renewal and chemoresistance through inhibition of the PTEN and activation of β-Catenin and Akt pathways.

Enhanced Sensitivity of Cancer Stem Cells to Chemotherapy Using Functionalized Mesoporous Silica Nanoparticles.

Cancer stem cells (CSCs) are responsible for cancer drug resistance with high expression of ABCG2, which pumps the internalized chemotherapeutic out to escape drug-induced cytotoxicity. Here, we established a functionalized mesoporous silica nanoparticle (MSN) system to deliver shABCG2 and doxorubicin (Dox) synergistically. With excellent cell uptake and endosomal escape capacities, the dual-delivery carriers internalized shABCG2 and Dox into CSCs efficiently. ABCG2 depletion increased intracellular and intranuclear Dox enrichment, drove vigorous Dox-induced cell death, and impaired the self-renewal of CSCs. Additionally, the nanoparticles eliminated tumors efficiently and reduced tumor initiation by CSCs in vivo, with negligible side effects. Our findings suggest that well-designed delivery systems for conventional chemotherapeutic agents are promising for CSC therapy.

Hydrogel microstructure live-cell array for multiplexed analyses of cancer stem cells, tumor heterogeneity and differential drug response at single-element resolution.

Specific phenotypic subpopulations of cancer stem cells (CSCs) are responsible for tumor development, production of heterogeneous differentiated tumor mass, metastasis, and resistance to therapies. The development of therapeutic approaches based on targeting rare CSCs has been limited partially due to the lack of appropriate experimental models and measurement approaches. The current study presents new tools and methodologies based on a hydrogel microstructure array (HMA) for identification and multiplex analyses of CSCs. Low-melt agarose integrated with type I collagen, a major component of the extracellular matrix (ECM), was used to form a solid hydrogel array with natural non-adhesive characteristics and high optical quality. The array contained thousands of individual pyramidal shaped, nanoliter-volume micro-chambers (MCs), allowing concomitant generation and measurement of large populations of free-floating CSC spheroids from single cells, each in an individual micro-chamber (MC). The optical live cell platform, based on an imaging plate patterned with HMA, was validated using CSC-enriched prostate and colon cancer cell lines. The HMA methodology and quantitative image analysis at single-element resolution clearly demonstrates several levels of tumor cell heterogeneity, including morphological and phenotypic variability, differences in proliferation capacity and in drug response. Moreover, the system facilitates real-time examination of single stem cell (SC) fate, as well as drug-induced alteration in expression of stemness markers. The technology may be applicable in personalized cancer treatment, including multiplex ex vivo analysis of heterogeneous patient-derived tumor specimens, precise detection and characterization of potentially dangerous cell phenotypes, and for representative evaluation of drug sensitivity of CSCs and other types of tumor cells.

Human cancer cells with stem cell-like phenotype exhibit enhanced sensitivity to the cytotoxicity of IL-2 and IL-15 activated natural killer cells

Tumors harbor a population of cancer stem cells (CSCs) which can drive tumor progression and therapeutical resistance. Nature killer (NK) cells are best known for their ability to directly recognize and kill malignant cells. However, the susceptibility of cancer stem cells to NK cells is not fully understood. Here we demonstrated that human CD44+CD24− breast CSCs were shown enhanced sensitivity to IL-2 and IL-15 activated NK cells. CD44+CD24− CSCs expressed higher levels of NKG2D ligands ULBP1, ULBP2 and MICA. Blockade assay showed that the sensitivity of CSCs to NK cells-mediated lysis was mainly dependent on NKG2D. Furthermore, redox oxygen species (ROS)-low tumor cells were more sensitive to NK cells. The presence of antioxidant enzymes inhibitor L-S,R-buthionine sulfoximine or H2O2 retarded the cytotoxicity of NK cells to CD44+CD24− CSCs. In addition, NK cells could readily target CD133+ colonal CSCs. Our findings provide novel targets for NK cells-based immunotherapy and are of great importance for translational medicine.