Subpopulation Of Cancer Stem Cells Research Articles

Case Study: Heterogeneity of and CD44+/CD24- Cancer Stem Cell Subpopulation of Breast Cancer Patients in Bandung, Indonesia

Breast cancer (BC) cells characteristics have played a crucial role in clinical strategies decision-making and patient outcome prediction. Although there have been several studies examining this, there are still relatively few that analyze stem cell subpopulations or protein biomarkers. The conducted research sought to elucidate breast cancer subtype characteristics based on immunohistochemical analysis and cancer stem cell presence based on flow cytometry analysis in three breast cancer samples from Bandung, Indonesia. A flow cytometry analysis was conducted to determine how much CD44/CD24 was expressed as a marker of cancer stem cell in isolated BC cells using various cell references, such as cell lines of SKBR-3, MDA-MB-468, and Hs587T. Immunohistochemistry analysis was carried out to investigate the expression of p53, HER2, PGR, Ki67, and ER in sample tissue sections. According to histological analysis, 2 samples were solid mammary carcinoma, and 1 sample was mammary fibroadenoma. Immunohistochemistry and histological analysis showed that all BC tissues are classified as Luminal B. In addition, a large percentage of CD44+/CD24- subpopulation was found in isolated BC cells (> 90 % in patients 1 and 3; > 60 % in patient 2). All samples showed similarity to Hs587T cell line characteristic. This study has successfully characterized solid mammary tumors and mammary fibroadenoma with luminal B characteristics. All specimens contained a high proportion of cancer stem cells. HIGHLIGHTS The characteristics of breast cancer (BC) cells are essential factors in clinical strategy decision-making and patient outcome prediction Solid mammary tumor and mammary fibroadenoma were the cancer subtypes of isolated BC tissues from 3 different patients. The cancer subtypes found in 3 different patient’s isolated BC were solid mammary tumor and mammary fibroadenoma BC tissues from all 3 patients expressed different level of HER2, p53, Ki67, PGR and ER. Based on markers expressed, those BC tissues were classified as luminal B tumors Based on flow cytometry analysis, all BC tissues contained a high proportion of cancer stem cells indicated by the high proportion of CD44+/CD24- subpopulation GRAPHICAL ABSTRACT

Isolation and phenotypic characterization of cancer stem cells from metastatic oral cancer cells.

To isolate cancer stem cells (CSC) from a metastatic oral squamous cell carcinoma (OSCC) cell line and investigate their invitro and invivo phenotypic characteristics. Subpopulations with individual staining intensities for CD44 and CD326 were isolated from the OSCC cell line LN-1A by FACS: CD44Low/CD326- (CSC-M1), CD44Low/CD326High (CSC-E), and CD44High/CD326- (CSC-M2). Proliferation, clonogenic potential, adhesion, migration, epithelial-mesenchymal transition markers, and sensitivity to cisplatin and TVB-3166 were analyzed invitro. Tumor formation and metastasis were assessed by subcutaneous and orthotopic inoculations into BALB/c mice. E-cadherin levels were higher in CSC-E cells while vimentin and Slug more produced by CSC-M2 cells. CSC-M1 and CSC-M2 subpopulations showed higher proliferation, produced more colonies, and have stronger adhesion to the extracellular matrix. All cell lines established tumors; however, CSC-E and CSC-M2 formed larger masses and produced more metastases. The CSC subpopulations here described show increased cancer capabilities invitro, tumorigenic and metastatic potential invivo, and may be exploited in the search for novel therapeutic targets for OSCC.

Machine Learning Model for Prediction of Development of Cancer Stem Cell Subpopulation in Tumurs Subjected to Polystyrene Nanoparticles.

Cancer stem cells (CSCs) play a key role in tumor progression, as they are often responsible for drug resistance and metastasis. Environmental pollution with polystyrene has a negative impact on human health. We investigated the effect of polystyrene nanoparticles (PSNPs) on cancer cell stemness using flow cytometric analysis of CD24, CD44, ABCG2, ALDH1 and their combinations. This study uses simultaneous in vitro cell lines and an in silico machine learning (ML) model to predict the progression of cancer stem cell (CSC) subpopulations in colon (HCT-116) and breast (MDA-MB-231) cancer cells. Our findings indicate a significant increase in cancer stemness induced by PSNPs. Exposure to polystyrene nanoparticles stimulated the development of less differentiated subpopulations of cells within the tumor, a marker of increased tumor aggressiveness. The experimental results were further used to train an ML model that accurately predicts the development of CSC markers. Machine learning, especially genetic algorithms, may be useful in predicting the development of cancer stem cells over time.

Abstract PO2-24-06: Cancer-Associated Fibroblasts Promote Tumor Stemness in Aggressive Breast Cancers via NDRG1

Abstract Background: Inflammatory breast cancer (IBC) and triple negative breast cancer (TNBC) have the lowest 5-year survival rates among breast cancers: 40% for IBC and 77% for TNBC. Effective treatments for these aggressive tumors are urgently needed. To develop better treatments, it is crucial to understand the underlying mechanisms driving their aggressive biology. Our recent research has identified N-myc downstream regulated gene 1 (NDRG1) as a crucial driver of tumor progression and metastasis in aggressive breast cancers. We have also found that NDRG1 expression in tumors is correlated with poorer outcomes in IBC patients. Additionally, we have observed that cancer-associated fibroblasts (CAFs), derived from breast cancer biopsies, stimulate NDRG1 expression and phosphorylation as well as tumor stemness. Based on these findings, we hypothesize that NDRG1 is a key regulator of tumor stemness and progression, and secreted stromal factors regulate tumor stemness through NDRG1. Methods: To assess tumor stemness, we conducted in vitro experiments by quantifying the CD44+/CD24- subpopulation and performing mammosphere assays in NDRG1 control and depleted cells (SUM149, BCX010, MDA-IBC3). In vivo, we employed a limiting dilution transplantation assay, where NDRG1 control and knockdown SUM149 cells were transplanted into the mammary fat pad at different dilutions. We treated NDRG1 control and knockdown breast cancer cells with different patient-derived CAF-conditioned media (CAFs-CM, 50%, 24h) or control medium. The expression of NDRG1/pNDRG1 and CD44+/CD24- cells was evaluated by immunoblotting and flow cytometry. qPCR array was performed to evaluate tumor stemness markers. Results: NDRG1 depletion significantly reduced the CD44+/CD24- subpopulation (p< 0.001) and the efficiency of mammosphere formation (p=0.001). In vivo limiting dilution experiments demonstrated a substantial reduction in the frequency of breast cancer stem cells in NDRG1 knockdown cells (p= 1 x 10-12). Treatment of IBC cells with CAF-CM derived from breast cancer patients stimulated the cancer stem cell subpopulation [CD44+/CD24-: Control medium (43.1 ± 0.1) vs CAF-CM (55.1 ± 0.7); p=0.004] and increased NDRG1 and phospho-NDRG1 expression. However, the stimulation of NDRG1 knockdown cells with CAF-CM did not significantly alter the cancer stem cell population (0.2 ± 0.2 in Control medium versus 0.4 ± 0.3 in CAF-CM) and the expression of NDRG1/phospho-NDRG1. Additionally, qPCR array analysis revealed the upregulation of several cancer stem cell and self-renewal markers in CAF-CM stimulated cells compared to control-medium treated cells. Conclusions: Our data strongly support the role of NDRG1 as a critical regulator of tumor stemness. Moreover, we have discovered that conditioned media from patient-derived CAFs induces tumor stemness in breast cancer cells, and this effect is dependent on the expression NDRG1. Citation Format: Emilly Villodre, Jenny M Hogstrom, Xiaoding Hu, Nina Kozlova, Debu Tripathy, Taru Muranen, Bisrat Debeb. Cancer-Associated Fibroblasts Promote Tumor Stemness in Aggressive Breast Cancers via NDRG1 [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-06.

Chordoma cancer stem cell subpopulation characterization may guide targeted immunotherapy approaches to reduce disease recurrence.

Cancer stem cells (CSCs), a group of tumor-initiating and tumor-maintaining cells, may be major players in the treatment resistance and recurrence distinctive of chordoma. Characterizing CSCs is crucial to better targeting this subpopulation. Using flow cytometry, six chordoma cell lines were evaluated for CSC composition. In vitro, cell lines were stained for B7H6, HER2, MICA-B, ULBP1, EGFR, and PD-L1 surface markers. Eighteen resected chordomas were stained using a multispectral immunofluorescence (mIF) antibody panel to identify CSCs in vivo. HALO software was used for quantitative CSC density and spatial analysis. In vitro, chordoma CSCs express more B7H6, MICA-B, and ULBP1, assessed by percent positivity and mean fluorescence intensity (MFI), as compared to non-CSCs in all cell lines. PD- L1 percent positivity is increased by >20% in CSCs compared to non-CSCs in all cell lines except CH22. In vivo, CSCs comprise 1.39% of chordoma cells and most are PD-L1+ (75.18%). A spatial analysis suggests that chordoma CSCs cluster at an average distance of 71.51 mm (SD 73.40 mm) from stroma. To our knowledge, this study is the first to identify individual chordoma CSCs and describe their surface phenotypes using in vitro and in vivo methods. PD-L1 is overexpressed on CSCs in chordoma human cell lines and operative tumor samples. Similarly, potential immunotherapeutic targets on CSCs, including B7H6, MICA-B, ULBP1, EGFR, and HER2 are overexpressed across cell lines. Targeting these markers may have a preferential role in combating CSCs, an aggressive subpopulation likely consequential to chordoma's high recurrence rate.

POLQ inhibition attenuates the stemness and ferroptosis resistance in gastric cancer cells via downregulation of dihydroorotate dehydrogenase

Gastric cancer (GC) contains subpopulations of cancer stem cells (CSCs), which are described as the main contributors in tumor initiation and metastasis. It is necessary to clarify the molecular mechanism underlying CSCs phenotype and develop novel biomarkers and therapeutic targets for gastric cancer. Here, we show that POLQ positively regulates stem cell-like characteristics of gastric cancer cells, knockdown of POLQ suppressed the stemness of GC cells in vitro and in vivo. Further mechanistic studies revealed that POLQ knockdown could downregulate the expression of dihydroorotate dehydrogenase (DHODH). DHODH overexpression rescued the reduced stemness resulted by POLQ knockdown. Furthermore, we found that POLQ expression correlated with resistance to ferroptosis, and POLQ inhibition renders gastric cancer cells more vulnerable to ferroptosis. Further investigation revealed that POLQ regulated DHODH expression via the transcription factors E2F4, thereby regulating ferroptosis resistance and stemness of gastric cancer cells. Given the importance of POLQ in stemness and ferroptosis resistance of GC, we further evaluated the therapeutic potential of POLQ inhibitor novobiocin, the results show that novobiocin attenuates the stemness of GC cells and increased ferroptosis sensitivity. Moreover, the combination of POLQ inhibitor and ferroptosis inducer synergistically suppressed MGC-803 xenograft tumor growth and diminished metastasis. Our results identify a POLQ-mediated stemness and ferroptosis defense mechanism and provide a new therapeutic strategy for gastric cancer.

Anti-ENO1 antibody combined with metformin against tumor resistance: a novel antibody-based platform.

Antibody-based platforms (i.e., ADC) have emerged as one of the most encouraging tools for the cancer resistance caused by cancer stem cells (CSCs) enrichment. Our study might provide a promising therapeutic direction against drug resistance and serve as a potential precursor platform for screening ADC. The cell migration, invasion, drug resistance, and self-renewal were assessed by the cell invasion and migration assay, wound healing assay, CCK-8 assay, colony formation assay, and sphere formation assay, respectively. The expression profiles of CSCs (ALDH+ and CD44+) subpopulations were screened by flow cytometry. The western blot and cell immunofluorescence assay were used to evaluate pathway-related protein expression in both anti-ENO1 antibody, MET combined with DPP/CTX-treated CSCs. In the present study, western blot and flow cytometry verified that anti-ENO1 antibody target the CD44+ subpopulation by inhibiting the PI3K/AKT pathway, while metformin might target the ALDH+ subpopulation through activation of the AMPK pathway and thus reverse drug resistance to varying degrees. Subsequently, in vitro investigation indicated that anti-ENO1 antibody, metformin combined with cisplatin/cetuximab could simultaneously target ALDH+ and CD44+ subpopulations. The combination also inhibited the CSCs proliferation, migration, invasion, and sphere formation; which may result in overcoming the drug resistance. Then, molecular mechanism exploration verified that the anti-ENO1 antibody, metformin combined with cisplatin/cetuximab inhibited the Wnt/β-catenin signaling. The study preliminarily revealed anti-ENO1 antibody combined with metformin could overcome drug resistance against CSCs by inhibiting the Wnt//β-catenin pathway and might serve as a potential precursor platform for screening ADC. More importantly, it is reasonably believed that antibody-based drug combination therapy might function as an encouraging tool for oncotherapy.

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.

Bimetallic Gold/Silver and Bioactive Camptothecin Hybrid Nanoparticles for Eradication of Cancer Stem Cells in a Combination Manner.

The defeat of cancer is still a challenge due to the existence of cancer stem cells (CSCs) because they resist conventional chemotherapy via multifactor regulated mechanisms. Consequently, one-dimensional action toward CSCs cannot work. Herein, we used rationally designed hybrid nanoparticles as a combined cancer therapy, hoping to form a multidimensional control network. In this paper, gold/silver alloy nanoparticle decorated camptothecin nanocrystals were formulated according to complementary anti-CSC mechanisms from gold, silver, and organic drug. This smart drug formulation could combine chemotherapy and thermotherapy, target different tumor sites, and demonstrate versatile toxicity profiles from each component. Major results indicated that this nanosystem demonstrated indiscriminately effective cytotoxic/proapoptotic/necrotic activity against bulk MCF-7 cells and their CSC subpopulation, in particular under laser ablation. Moreover, this nanosystem displayed enhanced antineoplastic activity against CSC spheroids, resulting in a significant reduction in their number and size, that is, their self-renewal capacity. All the results indicated that CSCs upon treatment of these new hybrid nanoparticles underwent reduced stemness and conversion from the original quiescent state and recovered their sensitivity toward chemotherapy. The relevant anticancer mechanism was ascribed to NIR-pH dual responsive drug release, synergistic/combined thermo-chemotherapy of organic drug and inorganic alloy nanoparticles, enhanced cellular uptake mediated by alloy nanoparticles, and Ag+-induced biomembrane damage. This thermo-chemotherapy platform provides a new combinatorial strategy for inorganic and organic agents in the complete elimination of CSCs.

Affinity-Based Magnetic Nanoparticle Development for Cancer Stem Cell Isolation.

Cancer is still the leading cause of death in the world despite the developing research and treatment opportunities. Failure of these treatments is generally associated with cancer stem cells (CSCs), which cause metastasis and are defined by their resistance to radio- and chemotherapy. Although known stem cell isolation methods are not sufficient for CSC isolation, they also bring a burden in terms of cost. The aim of this study is to develop a high-efficiency, low-cost, specific method for cancer stem cell isolation with magnetic functional nanoparticles. This study, unlike the stem cell isolation techniques (MACS, FACS) used today, was aimed to isolate cancer stem cells (separation of CD133+ cells) with nanoparticles with specific affinity and modification properties. For this purpose, affinity-based magnetic nanoparticles were synthesized and characterized by providing surface activity and chemical reactivity, as well as making surface modifications necessary for both lectin affinity and metal affinity interactions. In the other part of the study, synthesized and characterized functional polymeric magnetic nanoparticles were used for the isolation of CSC from the human osteosarcoma cancer cell line (SAOS-2) with a cancer stem cell subpopulation bearing the CD133 surface marker. The success and efficiency of separation after stem cell isolation were evaluated via the MACS and FACS methods. As a result, when the His-graft-mg-p(HEMA) nanoparticle was used at a concentration of 0.1 µg/mL for 106 and 108 cells, superior separation efficiency to commercial microbeads was obtained.

Single-cell transcriptome sequencing reveals aberrantly activated inter-tumor cell signaling pathways in the development of clear cell renal cell carcinoma

BackgroundAberrant intracellular or intercellular signaling pathways are important mechanisms that contribute to the development and progression of cancer. However, the intercellular communication associated with the development of ccRCC is currently unknown. The purpose of this study was to examine the aberrant tumor cell-to-cell communication signals during the development of ccRCC.MethodsWe conducted an analysis on the scRNA-seq data of 6 ccRCC and 6 normal kidney tissues. This analysis included sub clustering, CNV analysis, single-cell trajectory analysis, cell–cell communication analysis, and transcription factor analysis. Moreover, we performed validation tests on clinical samples using multiplex immunofluorescence.ResultsThis study identified eleven aberrantly activated intercellular signaling pathways in tumor clusters from ccRCC samples. Among these, two of the majors signaling molecules, MIF and SPP1, were mainly secreted by a subpopulation of cancer stem cells. This subpopulation demonstrated high expression levels of the cancer stem cell markers POU5F1 and CD44 (POU5F1hiCD44hiE.T), with the transcription factor POU5F1 regulating the expression of SPP1. Further research demonstrated that SPP1 binds to integrin receptors on the surface of target cells and promotes ccRCC development and progression by activating potential signaling mechanisms such as ILK and JAK/STAT.ConclusionAberrantly activated tumor intercellular signaling pathways promote the development and progression of ccRCC. The cancer stem cell subpopulation (POU5F1hiCD44hiE.T) promotes malignant transformation and the development of a malignant phenotype by releasing aberrant signaling molecules and interacting with other tumor cells.

Transforming Growth Factor-β/Smad Signaling Inhibits Melanoma Cancer Stem Cell Self-Renewal, Tumor Formation and Metastasis.

The secreted protein transforming growth factor-beta (TGFβ) plays essential roles, ranging from cell growth regulation and cell differentiation in both normal and cancer cells. In melanoma, TGFβ acts as a potent tumor suppressor in melanoma by blocking cell cycle progression and inducing apoptosis. In the present study, we found TGFβ to regulate cancer stemness in melanoma through the Smad signaling pathway. We discovered that TGFβ/Smad signaling inhibits melanosphere formation in multiple melanoma cell lines and reduces expression of the CD133+ cancer stem cell subpopulation in a Smad3-dependent manner. Using preclinical models of melanoma, we further showed that preventing Smad3/4 signaling, by means of CRISPR knockouts, promoted both tumorigenesis and lung metastasis in vivo. Collectively, our results define new functions for the TGFβ/Smad signaling axis in melanoma stem-cell maintenance and open avenues for new therapeutic approaches to this disease.

In Vitro Tumorigenic Assay: A Tumor Sphere Assay for Cancer Stem Cells.

Cancer stem cells (CSCs) represent a subpopulation of tumor cells that are thought to be responsible for therapy resistance, recurrence, and metastasis through their capacity to self-renew and differentiate into heterogeneous downstream lineages of cancer cells. Understanding the features of CSCs is crucial for managing cancer disease and establishing potential targeted therapeutics. Tumor sphere formation assay is a widely used in vitro method that selects and enriches the CSC subpopulation from the total population of cancer cells, based on their inherent ability to grow and clonally expand in serum-free and nonadherent culture conditions. Here we provide a detailed methodology to generate and propagate spheres from isolated cell suspensions of tumor tissues and cell lines using a semisolid MatrigelTM-based three-dimensional (3D) culture system.

SOX2 promotes a cancer stem cell-like phenotype and local spreading in oral squamous cell carcinoma

Emerging evidence shows that oral squamous cell carcinoma (OSCC) invasiveness can be attributed to a small subpopulation of cancer stem cells (CSCs) in the bulk of the tumor. However, the presence of CSCs in the OSCC close resection margins is still poorly unexplored. Here, we found that BMI1, CD44, SOX2, OCT4, UBE2C, CXCR4 CSCs marker genes are significantly upregulated, while IGF1-R, KLF4, ALDH1A1, CD133, FAM3C are downregulated in the tumor core vs healthy mucosa of 24 patients with OSCC. Among these, SOX2 appears also upregulated in the tumor close margin vs healthy mucosa and this significantly correlates with tumor size and lymph node compromise. In vitro analyses in CAL27 and SCC15 tongue squamous cell carcinoma cell lines, show that SOX2 transient knockdown i) promotes the mesenchymal-to-epithelial transition, ii) smooths the invasiveness, iii) attenuates the 3D tumor sphere-forming capacity, and iv) partially increases the sensitivity to cisplatin treatment. Overall, our study highlights that the OSCC close margins can retain CSC-specific markers. Notably, SOX2 may represent a useful CSCs marker to predict a more aggressive phenotype and a suitable target to prevent local invasiveness.

Can CD133 Be Regarded as a Prognostic Biomarker in Oncology: Pros and Cons.

The CD133 cell membrane glycoprotein, also termed prominin-1, is expressed on some of the tumor cells of both solid and blood malignancies. The CD133-positive tumor cells were shown to exhibit higher proliferative activity, greater chemo- and radioresistance, and enhanced tumorigenicity compared to their CD133-negative counterparts. For this reason, CD133 is regarded as a potential prognostic biomarker in oncology. The CD133-positive cells are related to the cancer stem cell subpopulation in many types of cancer. Recent studies demonstrated the involvement of CD133 in the regulation of proliferation, autophagy, and apoptosis in cancer cells. There is also evidence of its participation in the epithelial-mesenchymal transition associated with tumor progression. For a number of malignant tumor types, high CD133 expression is associated with poor prognosis, and the prognostic significance of CD133 has been confirmed in a number of meta-analyses. However, some published papers suggest that CD133 has no prognostic significance or even demonstrate a certain correlation between high CD133 levels and a positive prognosis. This review summarizes and discusses the existing evidence for and against the prognostic significance of CD133 in cancer. We also consider possible reasons for conflicting findings from the studies of the clinical significance of CD133.

Interleukin-6 Induces Stem Cell Propagation through Liaison with the Sortilin-Progranulin Axis in Breast Cancer.

Unraveling the complex network between cancer cells and their tumor microenvironment is of clinical importance, as it might allow for the identification of new targets for cancer treatment. Cytokines and growth factors secreted by various cell types present in the tumor microenvironment have the potential to affect the challenging subpopulation of cancer stem cells showing treatment-resistant properties as well as aggressive features. By using various model systems, we investigated how the breast cancer stem cell-initiating growth factor progranulin influenced the secretion of cancer-associated proteins. In monolayer cultures, progranulin induced secretion of several inflammatory-related cytokines, such as interleukin (IL)-6 and -8, in a sortilin-dependent manner. Further, IL-6 increased the cancer stem fraction similarly to progranulin in the breast cancer cell lines MCF7 and MDA-MB-231 monitored by the surrogate mammosphere-forming assay. In a cohort of 63 patient-derived scaffold cultures cultured with breast cancer cells, we observed significant correlations between IL-6 and progranulin secretion, clearly validating the association between IL-6 and progranulin also in human-based microenvironments. In conclusion, the interplay between progranulin and IL-6 highlights a dual breast cancer stem cell-promoting function via sortilin, further supporting sortilin as a highly relevant therapeutic target for aggressive breast cancer.

FXYD3 functionally demarcates an ancestral breast cancer stem cell subpopulation with features of drug-tolerant persisters.

The heterogeneity of cancer stem cells (CSCs) within tumors presents a challenge in therapeutic targeting. To decipher the cellular plasticity that fuels phenotypic heterogeneity, we undertook single-cell transcriptomics analysis in triple-negative breast cancer (TNBC) to identify subpopulations in CSCs. We found a subpopulation of CSCs with ancestral features that is marked by FXYD domain-containing ion transport regulator 3 (FXYD3), a component of the Na+/K+ pump. Accordingly, FXYD3+ CSCs evolve and proliferate, while displaying traits of alveolar progenitors that are normally induced during pregnancy. Clinically, FXYD3+ CSCs were persistent during neoadjuvant chemotherapy, hence linking them to drug-tolerant persisters (DTPs) and identifying them as crucial therapeutic targets. Importantly, FXYD3+ CSCs were sensitive to senolytic Na+/K+ pump inhibitors, such as cardiac glycosides. Together, our data indicate that FXYD3+ CSCs with ancestral features are drivers of plasticity and chemoresistance in TNBC. Targeting the Na+/K+ pump could be an effective strategy to eliminate CSCs with ancestral and DTP features that could improve TNBC prognosis.

Cytotoxic Imidazolyl-Mesalazine Ester-Based Ru(II) Complexes Reduce Expression of Stemness Genes and Induce Differentiation of Oral Squamous Cell Carcinoma.

The aggressiveness and recurrence of cancer is linked to cancer stem cells (CSCs), but drugs targeting CSCs may not succeed in the clinic due to the lack of a distinct CSC subpopulation. Clinical Pt(II) drugs can increase stemness. We screened 15 RuII or IrIII complexes with mesalazine or 3-aminobenzoate Schiff bases of the general formulas [Ru(p-cym)L]+, [Ru(p-cym)L], and [Ir(Cp*)L]+ (L = L1-L9) and found three complexes (2, 12, and 13) that are active against oral squamous cell carcinoma (OSCC) CSCs. There is a putative oncogenic role of transcription factors (viz. NOTCH1, SOX2, c-MYC) to enhance the stemness. Our work shows that imidazolyl-mesalazine ester-based RuII complexes inhibit growth of CSC-enriched OSCC 3D spheroids at low micromolar doses (2 μM). Complexes 2, 12, and 13 reduce stemness gene expression and induce differentiation markers (Involucrin, CK10) in OSCC 3D cultures. The imidazolyl-mesalazine ester-based RuII complex 13 shows the strongest effect. Downregulating c-MYC suggests that RuII complexes may target c-MYC-driven cancers.

MUC1-C integrates aerobic glycolysis with suppression of oxidative phosphorylation in triple-negative breast cancer stem cells

Activation of the MUC1-C protein promotes lineage plasticity, epigenetic reprogramming, and the cancer stem cell (CSC) state. The present studies performed on enriched populations of triple-negative breast cancer (TNBC) CSCs demonstrate that MUC1-C is essential for integrating activation of glycolytic pathway genes with self-renewal and tumorigenicity. MUC1-C further integrates the glycolytic pathway with suppression of mitochondrial DNA (mtDNA) genes encoding components of mitochondrial Complexes I-V. The repression of mtDNA genes is explained by MUC1-C-mediated (i)downregulation of the mitochondrial transcription factor A (TFAM) required for mtDNA transcription and (ii) induction of the mitochondrial transcription termination factor 3 (mTERF3). In support of pathogenesis that suppresses mitochondrial ROS production, targeting MUC1-C increases (i)mtDNA gene transcription, (ii) superoxide levels, and (iii) loss of self-renewal capacity. These findings and scRNA-seq analysis of CSC subpopulations indicate that MUC1-C regulates self-renewal and redox balance by integrating activation of glycolysis with suppression of oxidative phosphorylation.

ΔNp63 is regulated by insulin/IGF-1 signaling in normal basal/progenitor mammary cells and in luminal-type breast cancer cells.

Breast cancers are a heterogeneous group of tumors classified according to their histological growth patterns and receptor expression characteristics. Intratumor heterogeneity also exists, with subpopulations of cells with different phenotypes found in individual cancers, including cells with stem or progenitor cell properties. At least two types of breast cancer stem cells (CSCs) exist, the epithelial and the basal/mesenchymal subtypes, although how these phenotypes are controlled is unknown. ΔNp63 is a basal cell marker and regulator of stem/progenitor cell activities in the normal mammary gland and is expressed in the basal-like CSC subpopulation in some estrogen receptor-positive (ER+) and/or human epidermal growth factor receptor 2-positive (HER2+) breast adenocarcinomas. Whilst p63 is known to directly impart CSC properties in luminal breast cancer cells, how p63 is regulated and induced in these cells is unknown. We initially confirmed the existence of a small subpopulation of ΔNp63+ cells in lymph node metastases of ER+ human ductal adenocarcinomas, indicating together with previous reports that ΔNp63+ tumor cells are present in approximately 40% of these metastases. Notably, ΔNp63+ cells show a preferential location at the edge of tumor areas, suggesting possible regulation of ΔNp63 by the tumor microenvironment. Subsequently, we showed that the high levels of ΔNp63 in basal non-transformed MCF-10A mammary epithelial cells rely on insulin in their culture medium, whilst ΔNp63 levels are increased in MCF-7 ER+ luminal-type breast cancer cells treated with insulin or insulin-like growth factor 1 (IGF-1). Mechanistically, small molecule inhibitors and siRNA gene knockdown demonstrated that induction of ΔNp63 by IGF-1 requires PI3K, ERK1/2, and p38 MAPK activation, and acts through FOXO transcriptional inactivation. We also show that metformin inhibits ΔNp63 induction. These data reveal an IGF-mediated mechanism to control basal-type breast CSCs, with therapeutic implications to modify intratumor breast cancer cell heterogeneity and plasticity.