Called Cancer Stem Cells Research Articles

Acid-sensing ion channel 3 is a new potential therapeutic target for the control of glioblastoma cancer stem cells growth

Glioblastoma (GBM) is the most common malignant primary brain cancer that, despite recent advances in the understanding of its pathogenesis, remains incurable. GBM contains a subpopulation of cells with stem cell-like properties called cancer stem cells (CSCs). Several studies have demonstrated that CSCs are resistant to conventional chemotherapy and radiation thus representing important targets for novel anti-cancer therapies. Proton sensing receptors expressed by CSCs could represent important factors involved in the adaptation of tumours to the extracellular environment. Accordingly, the expression of acid-sensing ion channels (ASICs), proton-gated sodium channels mainly expressed in the neurons of peripheral (PNS) and central nervous system (CNS), has been demonstrated in several tumours and linked to an increase in cell migration and proliferation. In this paper we report that the ASIC3 isoform, usually absent in the CNS and present in the PNS, is enriched in human GBM CSCs while poorly expressed in the healthy human brain. We propose here a novel therapeutic strategy based on the pharmacological activation of ASIC3, which induces a significant GBM CSCs damage while being non-toxic for neurons. This approach might offer a promising and appealing new translational pathway for the treatment of glioblastoma.

Anti-Cancer Stem Cell Cobalt(III)-Polypyridyl Complexes Containing Salicylic Acid

Metal-containing compounds are an important class of chemotherapeutics used to treat various manifestations of cancer. Despite the widespread clinical use and success of metallopharmaceuticals, they are ineffective towards a sub-population of tumours called cancer stem cells (CSCs). CSCs evade current chemotherapeutic regimens (including metallopharmaceuticals) and promote cancer relapse and metastasis. Here, we report the synthesis, characterisation and anti-breast CSCs properties of a series of cobalt(III)-polypyridyl complexes with salicylic acid. The lead cobalt(III) complex 6 (containing 3,4,7,8-tetramethyl-1,10-phenanthroline) displayed low micromolar potency towards breast CSCs, significantly lower than the gold-standard anti-breast CSC agent, salinomycin, and the clinically used metallodrug, cisplatin. Mechanistic studies indicate that the cobalt(III) complex 6 induces its anti-breast CSC effect by entering breast CSCs, penetrating the nuclei, damaging nuclear DNA and triggering caspase-dependent apoptosis. The cytotoxic mechanism of action of the cobalt(III) complex 6 is also dependent on the modulation of cyclooxygenase-2 (COX-2) expression. This work highlights the anti-breast CSC properties of cobalt(III) coordination complexes with non-steroidal anti-inflammatory drugs (NSAIDs) and more widely spotlights the importance of metallopharmaceuticals in the development of new anticancer agents that can tackle chemotherapeutic-resistant sub-populations.

Effect of Melatonin on Chemoresistance Exhibited by Spheres Derived from Canine Mammary Carcinoma Cells.

Mammary cancer is a frequent disease in female dogs, where a high proportion of cases correspond to malignant tumors that may exhibit drug resistance. Within the mammary tumor microenvironment, there is a cell subpopulation called cancer stem cells (CSCs), which are capable of forming spheres in vitro and resisting anti-tumor treatments, partly explaining the recurrence of some tumors. Previously, it has been described that spheres derived from canine mammary carcinoma cells CF41.Mg and REM 134 exhibit stemness characteristics. Melatonin has shown anti-tumor effects on mammary tumor cells; however, its effects have been poorly evaluated in canine mammary CSCs. This study aimed to analyze the effect of melatonin on the chemoresistance exhibited by stem-like neoplastic cells derived from canine mammary carcinoma to cytotoxic drugs such as doxorubicin and mitoxantrone. CF41.Mg and REM 134 cells were cultured in high-glucose DMEM supplemented with fetal bovine serum and L-glutamine. The spheres were cultured in ultra-low attachment plates in DMEM/F12 medium without fetal bovine serum and with different growth factors. The CD44+/CD24-/low phenotype was analyzed by flow cytometry. The viability of sphere-derived cells (MTS reduction) was studied in the presence of melatonin (0.1 or 1 mM), doxorubicin, mitoxantrone, and luzindole. In addition, the gene (RT-qPCR) of the multidrug resistance bombs MDR1 and ABCG2 were analyzed in the presence of melatonin. Both cell types expressed the MT1 gene, which encodes the melatonin receptor MT1. Melatonin 1 mM does not modify the CD44+/CD24-/low phenotype; however, the hormone reduced viability (p < 0.0001) only in CF41.Mg spheres, without inducing an additive effect when co-incubated with cytotoxic drugs. These effects were independent of the binding of the hormone to its receptor MT1, since, by pharmacologically inhibiting them, the effect of melatonin was not blocked. In CF41.Mg spheres, the relative gene expression of ABCG2 and MDR1 was decreased in response to the hormone (p < 0.001). These results indicate that melatonin negatively modulates the cell survival of spheres derived from CF41.Mg cells, in a way that is independent of its MT1 receptor. These effects did not counteract the resistance to doxorubicin and mitoxantrone, even though the hormone negatively regulates the gene expression of MDR1 and ABCG2.

Abstract 4468: Elucidating the phosphoinositide-modulatory function of INPP5B in regulating cancer stemness and tumorigenesis in liver cancer

Abstract Hepatocellular carcinoma (HCC) represents a major global health challenge, characterized by its heterogeneity and aggressive nature. Solid evidence has now suggested that tumor growth is driven by subpopulations of tumor cells with less differentiated cell states, called cancer stem cells (CSCs), which govern therapeutic resistance and tumor recurrence. Therefore, characterization of CSCs and their associated oncogenic signaling pathways is crucial to the development of more specific and sensitive HCC prognostic markers and to the design of novel targeted therapies against CSCs for HCC treatment. In search of the altered genes and pathways that are tightly associated with cancer stemness trait in HCC, we exploited the stemness and oncogenic dedifferentiation signature to mine the HCC dataset of the Cancer Genome Atlas. Phosphoinositide signaling was identified to be enriched and downregulated in the transcriptomes of HCC patients with strong stemness and dedifferentiated signature. Subsequent clinical correlation analysis found inositol polyphosphate-5-phosphatase B (INPP5B) to be the top downregulated gene in phosphoinositide signaling with its expression correlated with poor survival in HCC patients. Functional studies showed that INPP5B overexpression reversed the oncogenic and stemness properties while INPP5B knockout promoted tumor growth in immunocompetent mice. Subsequent RNA sequencing and molecular assays revealed INPP5B deregulation to be associated with lipid metabolism remodeling. Collectively, our results suggest an intricate relationship between phosphoinositide signaling and lipid metabolism through the regulation of INPP5B in HCC. The findings may open new avenue for future research aimed at devising targeted therapies to interfere with liver CSCs and enhance the prognosis of HCC patients. Citation Format: Linglin Liu, Yifei Wang, Yunong Xie, Man Tong. Elucidating the phosphoinositide-modulatory function of INPP5B in regulating cancer stemness and tumorigenesis in liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4468.

Mechanical constraints in tumor guide emergent spatial patterns of glioblastoma cancer stem cells

The mechanical constraints in the overcrowding glioblastoma (GBM) microenvironment have been implicated in the regulation of tumor heterogeneity and disease progression. Especially, such mechanical cues can alter cellular DNA transcription and give rise to a subpopulation of tumor cells called cancer stem cells (CSCs). These CSCs with stem-like properties are critical drivers of tumorigenesis, metastasis, and treatment resistance. Yet, the biophysical and molecular machinery underlying the emergence of CSCs in tumor remained unexplored. This work employed a two-dimensional micropatterned multicellular model to examine the impact of mechanical constraints arisen from geometric confinement on the emergence and spatial patterning of CSCs in GBM tumor. Our study identified distinct spatial distributions of GBM CSCs in different geometric patterns, where CSCs mostly emerged in the peripheral regions. The spatial pattern of CSCs was found to correspond to the gradients of mechanical stresses resulted from the interplay between the cell-ECM and cell–cell interactions within the confined environment. Further mechanistic study highlighted a Piezo1-RhoA-focal adhesion signaling axis in regulating GBM cell mechanosensing and the subsequent CSC phenotypic transformation. These findings provide new insights into the biophysical origin of the unique spatial pattern of CSCs in GBM tumor and offer potential avenues for targeted therapeutic interventions.

Tumor heterogeneity from the viewpoint of pathologists.

Morphological and functional heterogeneity are found in tumors, with the latter reflecting the different levels of resistance against antitumor therapies. In a therapy-resistant subpopulation, the expression levels of differentiation markers decrease, and those of immature markers increase. In addition, this subpopulation expresses genes involved in drug metabolism, such as aldehyde dehydrogenase 1A1 (ALDH1A1). Because of their similarity to stem cells, cells in the latter therapy-resistant subpopulation are called cancer stem cells (CSCs). Like normal stem cells, CSCs were originally thought not to arise from non-CSCs, but this hierarchical model is too simple. It is now believed that CSCs are generated from non-CSCs. The plasticity of tumor phenotypes between CSCs and non-CSCs causes difficulty in completely curing tumors. In this review, focusing on ALDH1A1 as a marker for CSCs or immature tumor cells, the dynamics of ALDH1A1-expressing tumor cells and their regulatory mechanisms are described, and the plausible regulatory mechanisms of plasticity of ALDH1A1 expression phenotype are discussed. Genetic mutations are a significant factor for tumorigenesis, but non-mutational epigenetic reprogramming factors yielding tumor heterogeneity are also crucial in determining tumor characteristics. Factors influencing non-mutational epigenetic reprogramming in tumors are also discussed.

The Role of Hedgehog Signaling Pathway in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the sixth leading malignancy worldwide, with a poor prognosis and limited treatment options. Molecularly targeted therapies for HNSCC are still lacking. However, recent reports provide novel insights about many molecular alterations in HNSCC that may be useful in future therapies. Therefore, it is necessary to identify new biomarkers that may provide a better prediction of the disease and promising targets for personalized therapy. The poor response of HNSCC to therapy is attributed to a small population of tumor cells called cancer stem cells (CSCs). Growing evidence indicates that the Hedgehog (HH) signaling pathway plays a crucial role in the development and maintenance of head and neck tissues. The HH pathway is normally involved in embryogenesis, stem cell renewal, and tissue regeneration. However, abnormal activation of the HH pathway is also associated with carcinogenesis and CSC regulation. Overactivation of the HH pathway was observed in several tumors, including basal cell carcinoma, that are successfully treated with HH inhibitors. However, clinical studies about HH pathways in HNSCC are still rare. In this review, we summarize the current knowledge and recent advances regarding the HH pathway in HNSCC and discuss its possible implications for prognosis and future therapy.

Abstract 2444: RAB27B drives a cancer stem cell phenotype in non-small cell lung cancer cells through enhanced extracellular vesicle secretion

Abstract Non-small cell lung cancer (NSCLC), a major subtype of lung cancer, accounts for ~85% of all lung cancer diagnoses. A sub-population of cells within human NSCLC tumors called cancer stem cells (CSCs) have been found to exhibit distinct properties that contribute to factors associated with poor outcomes in NSCLC patients including metastasis, relapse, and therapeutic resistance. We previously performed RNAseq analysis on bulk cancer cells (BCCs) and CSCs from a panel of primary NSCLC tumors and cell lines to identify signaling mechanisms involved in the tumorigenic behavior of NSCLC CSCs. Our analysis revealed that expression of RAB27B, a small GTPase that plays a critical role in extracellular vesicle (EV) formation and secretion, was significantly upregulated in CSCs when compared to BCCs. Interestingly, RAB27B is overexpressed and predicts patient survival in NSCLC. The goal of this study was to assess the role of RAB27B in the tumorigenic behavior of NSCLC CSCs and determine the mechanism by which RAB27B promotes an aggressive stem-cell like phenotype in NSCLC cells an aspect of RAB27B function that has not been investigated in NSCLC. QPCR and immunoblot analysis was used to compare RAB27B mRNA and protein expression in a panel of NSCLC BCCs and CSCs. Two independent shRNA constructs were used to knockdown (KD) RAB27B in NSCLC CSCs. The effects of RAB27B KD on CSC expansion, transformed growth and invasion were analyzed by clonal expansion, soft agar colony formation and Boyden chamber assays, respectively. Xenograft and tail vein mouse models were used to assess the effects of RAB27B KD on CSC tumor growth and lung colonization. EVs were isolated from the conditioned media of BCCs, CSCs and RAB27B KD CSCs using differential ultracentrifugation, and characterized by nanoparticle tracking analysis, transmission electron microscopy and immunoblotting according to MISEV guidelines. Our analysis revealed that RAB27B is significantly upregulated in NSCLC CSCs compared to BCCs. RAB27B KD cells exhibited a significant decrease in clonal expansion, transformed growth and invasion when compared to non-target shRNA control CSCs. In vivo studies demonstrated that RAB27B is important for the tumorigenicity of CSCs and promotes tumor cell proliferation, and angiogenesis. EVs were positive for EV-specific markers and negative for an intracellular vesicle marker. CSCs secrete significantly more EVs when compared to BCCs and KD of RAB27B in CSCs led to a decrease in the levels of EVs secreted. Furthermore, delivery of CSC-derived, but not BCC-derived EVs to BCCs induced spheroid growth, clonal expansion, and invasion in BCCs. Taken together, our results indicate that RAB27B is required for maintenance of a highly tumorigenic, cancer-initiating and invasive stem-like cell population in NSCLC and RAB27B is involved in propagating EV-mediated communication from NSCLC CSCs to BCCs. Citation Format: Kayleah M. Meneses, Jennifer A. Lindemann, Prita Pandya, Dania S. Al-Qasrawi, Ryan Argo, Celeste Weems, Danielle J. Beetler, Irene K. Yan, Joy Wolfram, Tushar C. Patel, Verline Justilien. RAB27B drives a cancer stem cell phenotype in non-small cell lung cancer cells through enhanced extracellular vesicle secretion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2444.

The presence of stem cells in ovarian cancer: a review

Ovarian cancer is one of the deadly gynecological malignancies. The disease has usually difficult to detect at the early stage as there are only a few specific symptoms and not many screening methods, thus patients usually come in an advanced and metastatic stage of the disease. Initial therapy for the disease includes surgery to remove the tumor, then followed by chemotherapy. This therapy shows a promising result at first until some sign of reoccurrence starts to show. One of the main reasons is thought to be the presence of a small subset of cells called cancer stem cells (CSCs). These cells may escape from the initial therapy, and due to the CSCs' ability to self-renew, grow into various lineages, and multiply widely. There are multiple efforts done to isolate and identify this subset cell, such as identifying the specific antigen in the cell surface, using Side Population, the ability of CSCs to form floating spheres in serum-free media, and the level of ALDH expression. Such methods have been performed together to identify purely cancer stem cells from the malignancy. The ability of the cell to produce new tumors in immuno-deficient animals, however, is the gold standard for identifying CSC, according to the current study. This review will discuss several methods that have been used to isolate cancer stem cells and prove the stemness of these cells by injecting them into immuno-deficient mice. Cancer stem cells also can avoid the effect of chemotherapy. Several things contribute to this ability. This review there will discuss methods that have been several things that make it possible to be able to escape chemotherapy drugs. By understanding these mechanisms, hopefully, new therapy modalities will be developed and specifically targeted, so that can be used to treat malignancies more effectively.

A Therapeutic Vaccine Targeting Rat BORIS (CTCFL) for the Treatment of Rat Breast Cancer Tumors.

Cancer testis antigens are ideal for tumor immunotherapy due to their testis-restricted expression. We previously showed that an immunotherapeutic vaccine targeting the germ cell-specific transcription factor BORIS (CTCFL) was highly effective in treating aggressive breast cancer in the 4T1 mouse model. Here, we further tested the therapeutic efficacy of BORIS in a rat 13762 breast cancer model. We generated a recombinant VEE-VRP (Venezuelan Equine Encephalitis-derived replicon particle) vector-expressing modified rat BORIS lacking a DNA-binding domain (VRP-mBORIS). Rats were inoculated with the 13762 cells, immunized with VRP-mBORIS 48 h later, and then, subsequently, boosted at 10-day intervals. The Kaplan-Meier method was used for survival analysis. Cured rats were re-challenged with the same 13762 cells. We demonstrated that BORIS was expressed in a small population of the 13762 cells, called cancer stem cells. Treatment of rats with VRP-BORIS suppressed tumor growth leading to its complete disappearance in up to 50% of the rats and significantly improved their survival. This improvement was associated with the induction of BORIS-specific cellular immune responses measured by T-helper cell proliferation and INFγ secretion. The re-challenging of cured rats with the same 13762 cells indicated that the immune response prevented tumor growth. Thus, a therapeutic vaccine against rat BORIS showed high efficacy in treating the rat 13762 carcinoma. These data suggest that targeting BORIS can lead to the elimination of mammary tumors and cure animals even though BORIS expression is detected only in cancer stem cells.

Abstract B034: CXCR4 targeting endogenous human peptides eliminate migrating cancer stem cells by disrupting tumor-stroma crosstalk in pancreatic ductal adenocarcinomas

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer characterized by late diagnosis, lack of early symptoms and extensive metastasis. One of the foremost reasons for such startling statistics is the presence of a subpopulation of highly plastic stem-like cells within the tumor called cancer stem cells (CSCs). We have previously identified a distinct subset of these CSCs within the invasive front of patient tumors. This subset, called migrating cancer stem cells (miCSCs), is characterized by CD133+CXCR4+ expression and determines the metastatic phenotype of pancreatic cancer. Therefore, targeting CXCR4 may represent a potential therapeutic approach to lower metastatic burden in PDAC. Here, we examined the effect of endogenous human peptides EPI-X4 and other derivatives thereof as CXCR4 antagonist on (i) patient-derived primary pancreatic cancer cells and (ii) tumor-stroma crosstalk by using a dual culture system with pancreatic stellate cells. We established these peptides as novel therapeutic strategy for combating the metastatic activity of pancreatic cancer using combinatorial therapeutic approaches and testing different in vivo delivery system such as peptide fatty-acid (FA) conjugates and silica nanoparticles (Si-NP). Our results show that EPI-X4 as well as its derivatives (e.g., JM#21) strongly inhibited migratory capacity of primary pancreatic cancer cells towards the CXCR4 ligand CXCL12 in vitro. Thereby, JM#21 was identified as the most potent EPI-X4 derivate. Mechanistical analysis by western blot, gene expression and immunofluorescence revealed that JM#21 increased Cadherin-1 expression by suppression of Snail1 via inactivation of SHH pathway. Moreover, JM#21 decreased CXCL12-induced phosphorylation of AKT and IKBa as well as NANOG expression, which further suppressed self-renewal capacity and EMT in the tumor cells. Strikingly, JM#21 sensitized selected cell lines towards gemcitabine and paclitaxel. Furthermore, FA conjugated and Si-NP encapsulated JM#21 restricted miCSCs maintenance which was predominantly regulated via stellate cell secreted CXCL12. In serum conditions, both FA conjugated, and Si-NP encapsulated JM#21 was found to be stable and active, proving as a valuable delivery system for in vivo studies. In conclusion, our study reveals that targeting CXCR4/CXCL12 signaling axis using human endogenous EPI-X4 derivates particularly JM#21 inhibits tumor-stroma crosstalk which is paramount for the propagation and maintenance of miCSC. Particularly, we demonstrate, in both mechanistic and preclinical set up, that these peptides abrogate the metastatic capacity of patient-derived pancreatic cancer cells by selective targeted elimination of miCSCs. Moreover, tumor cells show increased susceptibility towards conventional treatment strategies enforcing EPI-X4 derivate as a novel combinatory therapy to treat metastatic pancreatic cancer. Citation Format: Kanishka Tiwary, Mirja Harms, Bastian Beitzinger, Roman Schmid, Syeda Inaas, Karolin Walter, Alexander Kleger, Mika Lindén, Thomas Seufferlein, Jan Münch, Patrick Christian Hermann. CXCR4 targeting endogenous human peptides eliminate migrating cancer stem cells by disrupting tumor-stroma crosstalk in pancreatic ductal adenocarcinomas [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B034.

Silibinin exhibits anti-tumor effects in a breast cancer stem cell model by targeting stemness and induction of differentiation and apoptosis.

Introduction: Malignant breast cancer (BC) frequently contains a rare population of cells called cancer stem cells which underlie tumor relapse and metastasis, and targeting these cells may improve treatment options and outcomes for patients with BC. The aim of the present study was to determine the effect of silibinin on the self-renewal capacity, tumorgenicity, and metastatic potential of mammospheres. Methods: The effect of silibinin on viability and proliferation of MCF-7, MDA-MB-231 mammospheres, and MDA-MB-468 cell aggregation was determined after 72-120 hours of treatment. Colony and sphere formation ability, and the expression of stemness, differentiation, and epithelial-mesenchymal-transition (EMT)-associated genes were assessed by reverse transcription-quantitative polymerase chain reaction (qRT-PCR) in mammospheres treated with an IC50 dose of silibinin. Additionally, the antitumor capacity of silibinin was assessed in vivo, in mice. Results: The results of the present study showed that silibinin decreased the viability of all mammospheres derived from MCF-7, MDA-MB-231, and MDA-MB-468 cell aggregation in a dose-dependent manner. Colony and sphere-forming ability, as well as the expression of genes associated with EMT were reduced in mammospheres treated with silibinin. Additionally, the expression of genes associated with stemness and metastasis was also decreased and the expression of genes associated with differentiation were increased. Intra-tumoral injection of 2 mg/kg silibinin decreased tumor volumes in mice by 2.8 fold. Conclusion: The present study demonstrated that silibinin may have exerted its anti-tumor effects in BC by targeting the BC stem cells, reducing the tumorgenicity and metastasis. Therefore, silibinin may be a potential adjuvant for treatment of BC.

Heparanase regulates EMT and cancer stem cell properties in prostate tumors.

Prostate cancer displays a certain phenotypic plasticity that allows for the transition of cells from the epithelial to the mesenchymal state. This process, known as epithelial–mesenchymal transition (EMT), is one of the factors that give the tumor cells greater invasive and migratory capacity with subsequent formation of metastases. In addition, many cancers, including prostate cancer, are derived from a cell population that shows the properties of stem cells. These cells, called cancer stem cells (CSCs) or tumor-initiating cells, not only initiate the tumor process and growth but are also able to mediate metastasis and drug resistance. However, the impact of EMT and CSCs in prostate cancer progression and patient survival is still far from fully understood. Heparanase (HPSE), the sole mammalian endoglycosidase capable of degrading heparan sulfate (HS), is also involved in prostate cancer progression. We had previously proved that HPSE regulates EMT in non-cancerous pathologies. Two prostate cancer cell lines (DU145 and PC3) were silenced and overexpressed for HPSE. Expression of EMT and stemness markers was evaluated. Results showed that the expression of several EMT markers are modified by HPSE expression in both the prostate cancer cell lines analyzed. In the same way, the stemness markers and features are also modulated by HPSE expression. Taken together, the present findings seem to prove a new mechanism of action of HPSE in sustaining prostate cancer growth and diffusion. As for other tumors, these results highlight the importance of HPSE as a potential pharmacological target in prostate cancer treatment.

Mitochondrial Elongation and OPA1 Play Crucial Roles during the Stemness Acquisition Process in Pancreatic Ductal Adenocarcinoma.

Simple SummaryPancreatic ductal adenocarcinoma (PDAC) is a highly lethal neoplasia and the currently used treatments are not effective in a wide range of patients. Presently, the evidence points out that cancer stem cells (CSCs) are key players during tumor development, metastasis, chemoresistance, and tumor relapse. The study of the metabolism of CSCs, specifically the mitochondrial alterations, could pave the way to the discovery of new therapeutical targets. In this study, we show that during progressive de-differentiation, pancreatic CSCs undergo changes in mitochondrial mass, dynamics, and function. Interestingly, the silencing of OPA1, a protein involved in mitochondrial fusion, significantly inhibits the formation of CSCs. These results reveal new insight into mitochondria and stemness acquisition that could be useful for the design of novel potential therapies in PDAC.Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer with an overall 5-year survival rate of less than 9%. The high aggressiveness of PDAC is linked to the presence of a subpopulation of cancer cells with a greater tumorigenic capacity, generically called cancer stem cells (CSCs). CSCs present a heterogeneous metabolic profile that might be supported by an adaptation of mitochondrial function; however, the role of this organelle in the development and maintenance of CSCs remains controversial. To determine the role of mitochondria in CSCs over longer periods, which may reflect more accurately their quiescent state, we studied the mitochondrial physiology in CSCs at short-, medium-, and long-term culture periods. We found that CSCs show a significant increase in mitochondrial mass, more mitochondrial fusion, and higher mRNA expression of genes involved in mitochondrial biogenesis than parental cells. These changes are accompanied by a regulation of the activities of OXPHOS complexes II and IV. Furthermore, the protein OPA1, which is involved in mitochondrial dynamics, is overexpressed in CSCs and modulates the tumorsphere formation. Our findings indicate that CSCs undergo mitochondrial remodeling during the stemness acquisition process, which could be exploited as a promising therapeutic target against pancreatic CSCs.

Cancer Stem Cells: From an Insight into the Basics to Recent Advances and Therapeutic Targeting.

Cancer is characterized by an abnormal growth of the cells in an uncontrolled manner. These cells have the potential to invade and can eventually turn into malignancy, leading to highly fatal forms of tumor. Small subpopulations of cancer cells that are long-lived with the potential of excessive self-renewal and tumor formation are called cancer stem cells (CSCs) or cancer-initiating cells or tumor stem cells. CSCs can be found in tissues, such as breast, brain, lung, liver, ovary, and testis; however, their origin is still a matter of debate. These cells can differentiate and possess self-renewal capacity maintained by numerous intracellular signal transduction pathways, such as the Wnt/β-catenin signaling, Notch signaling, transforming growth factor-β signaling, and Hedgehog signaling. They can also contribute to numerous malignancies and are an important reason for tumor recurrence and metastasis because they are resistant to the known therapeutic strategies that mainly target the bulk of the tumor cells. This review contains collected and compiled information after analyzing published works of the last three decades. The goal was to gather information of recent breakthroughs related to CSCs, strategies to target CSCs' niche (e.g., nanotechnology with tumor biology), and their signaling pathways for cancer therapy. Moreover, the role of metformin, an antidiabetic drug, acting as a chemotherapeutic agent on CSCs by inhibiting cellular transformation and its selective killing is also addressed.

Abstract 3458: Novel anti-NME7antibody inhibits metastasis of solid tumor cancers

Abstract Purpose: To develop an antibody therapeutic that disrupts interaction between onco-embryonic growth factor NME7AB and its cognate growth factor receptor, MUC1*, for the treatment of solid tumors and inhibition of their metastases. Normally, NME7 is only expressed and secreted by cells of a Day 3 to Day 5 human blastocyst. NME7 expression is then turned off and expression of self-regulating, adult forms of NME proteins is turned on. However, we found that NME7 expression is mistakenly turned on again in cancer cells. Cleavage of NME7 to NME7AB enables secretion from the cell, where it then binds to and dimerizes the MUC1* extracellular domain, which activates the MAP kinase signaling cascade. Methods: Because NME7AB shares homology to other adult forms of NME proteins, which are required for normal cellular function, we needed to develop a monoclonal antibody that binds to NME7AB, but not to other NME proteins, and disrupts its interaction with the MUC1* growth factor receptor as well. To investigate the effects of NME7AB on cancer growth and metastasis, we generated populations of cancer cells that had been cultured in a serum-free media to which was added a recombinant NME7AB. The parent cells, the NME7AB grown progeny, or mixed populations were implanted into NSG mice to test the effect of novel anti-NME7AB antibodies on tumor growth and metastasis. Parent tumor cells and the NME7AB-grown cells were engineered to emit light at different wavelengths so that growth of each population could be separately tracked in live animals. Results: Cancer cells that were cultured in a serum-free media containing recombinant NME7AB for 10 days acquired characteristics of more metastatic cells, often called cancer stem cells (referred to here as NME7AB-CSCs). They upregulated metastatic markers by up to 100-fold, became non-adherent, entered dormancy and formed tumors from injection of as few as 10,000 cells. The addition of an anti-NME7AB antibody during the 10-day culture period blocked this transition. NME7AB-CSCs that were injected into the tail vein of NSG mice induced widespread metastasis in 6-10 days, after which an anti-NME7AB antibody was i.v. administered. The anti-NME7AB antibody greatly reduced or cleared the metastasis. Animals implanted sub-cutaneously with NME7AB-CSCs, then administered anti-NME7AB antibody, showed a significant reduction in the spread of breast cancer cells to the liver. Mixed populations of parent cells plus NME7AB-CSCs, which emitted light at different wavelengths, were implanted sub-cutaneously and allowed to become established. The percent of the implanted tumor that was NME7AB-CSCs determined the growth rate of the entire tumor. An anti-NME7AB antibody inhibited growth of the parent cells as well as the NME7AB-CSCs. Conclusion: These data support further pre-clinical development to allow for a clinical candidate to be tested in a metastatic as well as an adjuvant setting. Citation Format: Cynthia Carol Bamdad, Benoit J. Smagghe, Mark G. Carter, Trevor J. Grant, Danica M. Page, Laura M. Reale, Michael J. Nash, Jac-Leen S. Nash, Andrew K. Stewart. Novel anti-NME7antibody inhibits metastasis of solid tumor cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3458.

High CD44 Immunoexpression Correlates with Poor Overall Survival: Assessing the Role of Cancer Stem Cell Markers in Oral Squamous Cell Carcinoma Patients from the High-Risk Population of Pakistan.

Oral squamous cell carcinoma (OSCC) is a top-ranked cancer in the Pakistani population, and patient survival has remained unchanged at ∼50% for several decades. Recent advances have claimed that a subset of tumour cells, called cancer stem cells (CSCs), are responsible for tumour progression, treatment resistance, and metastasis, which leads to a poor prognosis. This study investigated the impact of CSC markers expression on overall survival (OS) and disease-free survival (DFS) of OSCC patients. Materials and Methods. Immunohistochemistry was used to evaluate CD44, CD133, L1CAM, and SOX2 expression in a well-characterized cohort of 100 Pakistani patients with primary treatment naïve OSCC. The immunoreactivity for each marker was correlated with patient clinicopathologic characteristics, oral cancer risk chewing habits, and survival. The minimum follow-up time for all patients was five years, and survival estimates were calculated using the Kaplan–Meier method and Cox proportional hazards model. Results. In this cohort of 100 patients, there were 57 males and 43 females. The median OS and DFS time durations observed were 64 and 52.5 months, respectively. Positive expression for CD44, CD133, L1CAM, and SOX2 was observed in 33%, 23%, 41%, and 63% of patients. High CD44 expression correlated with decreased OS (P=0.047) but did not influence DFS. However, CD133, L1CAM, and SOX2 had no effect on either OS or DFS. Tonsils, nodal involvement, and AJCC stage were independent predictors of worse OS and DFS both. Conclusion. Of the CSC markers investigated here, only CD44 was a predictor for poor OS. CD44 was also associated with advanced AJCC and T stages. Interestingly, CD133 was significantly lower in patients who habitually consumed oral cancer risk factors.

Piperlongumine loaded PLGA nanoparticles inhibit cancer stem-like cells through modulation of STAT3 in mammosphere model of triple negative breast cancer

TNBC exhibits higher rate of chemoresistance, metastasis, and relapse among all subtypes of breast cancer. This malignant statein TNBC is due to self-renewing sub-population of cells called cancer stem cells (CSCs). They are major caveats in TNBC treatment and need to be obliterated. In this regard, we explored piperlongumine (PL) that has remarkable anti-cancerous property but poor pharmacokinetics limits its application. So, to enhance its biological activity we developed PLGA based nanoformulation for PL (PL-NPs) and examined anti-CSCs effects of PL and PL-NPs in mammospheres. Results indicated that PL-NPs have higher cellular uptake than PL in mammospheres. Further, we demonstrated that PL-NPs remarkably inhibit various characteristics of CSCs like expression of ALDH, self-renewability, chemoresistance, and EMT in mammopsheres. We next investigated the possible mechanism underlying these multi-modal effects, and found that inhibition of STAT3 might be the driving force. In order to confirm this, we used colivelin a potent synthetic peptide activator of STAT3 in combination with treatments and found that anti-CSCs effects of PL and PL-NPs were reversed. Taken together, our data indicates that PL-NPs show enhanced inhibition of CSCs through downregulation of STAT3 and provides insight into development of PL based nanomedicine for targeting CSCs in TNBC.

Emerging roles of epithelial-mesenchymal plasticity in invasion-metastasis cascade and therapy resistance.

Strong association of cancer incidence and its progression with mortality highlights the need to decipher the cellular and molecular mechanisms that drive tumor cells to rapidly progress to metastatic disease and therapy resistance. Epithelial-mesenchymal plasticity (EMP) emerged as a key regulator of metastatic outgrowth. It allows neoplastic epithelial cells to delaminate from their neighbors either individually or collectively, traverse the extracellular matrix (ECM) barrier, enter into the circulation, and establish distal metastases. Plasticity between epithelial and mesenchymal states and the existence of hybrid epithelial/mesenchymal (E/M) phenotypes are increasingly being reported in different tumor contexts. Small subset of cancer cells with stemness called cancer stem cells (CSCs) exhibit plasticity, possess high tumorigenic potential, and contribute to high degree of tumoral heterogeneity. EMP characterized by the presence of dynamic intermediate states is reported to be influenced by (epi)genomic reprograming, growth factor signaling, inflammation, and low oxygen generated by tumor stromal microenvironment. EMP alters the genotypic and phenotypic characteristics of tumor cells/CSCs, disrupts tissue homeostasis, induces the reprogramming of angiogenic and immune recognition functions, and renders tumor cells to survive hostile microenvironments and resist therapy. The present review summarizes the roles of EMP in tumor invasion and metastasis and provides an update on therapeutic strategies to target the metastatic and refractory cancers.

Determining The Role of MicroRNAs in Self-Renewal, Metastasis and Resistance to Drugs in Human Gastric Cancer Based on Data Mining Approaches: A Systematic Review.

Gastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide. The major problems of patients with GC are the lack of proper response to the treatment, drug resistance and metastasis attributed to the presence of a subpopulation of cells inside the tumour that are called cancer stem cells (CSCs). In addition, deregulation of microRNAs (miRNAs) has been reported in different stages of GC. The aim of the present study is to determine and introduce miRNAs that contribute to regulation of stemness, metastasis and drug resistance in GC. A systematic review, we conducted data mining of available datasets and a review of previous studies to select miRNAs that target stemness, epithelial-mesenchymal transition (EMT) and drug resistance. All selected miRNAs were analysed by R software to find a common miRNA target for all three processes. Then, the target prediction of miRNAs and their related signalling pathways were obtained by using bioinformatics tools, ONCO.IO and KEGG databases, respectively. We identified seven miRNAs (miR-34a, miR-23a, miR-27a, miR-30a, miR-19b, miR-107, miR-100) from our searching approach. These miRNAs regulate pathways that contribute to stemness, EMT and drug resistance in GC. Four (miR- 34a, miR-23a, miR-30a, and miR-100) had significant interactions with each other and 52 target genes among them, from which MYC, CDK6, NOTCH1, NOTCH2, SIRT1, CD44, CD24, and AXL were involved in the regulation of several biological processes. These data suggest that the three significant properties can be regulated by common miRNAs (hsa-miR-34a, hsa-miR-23a, hsa-miR-30a and hsa-miR-100). Hence, targeting selected miRNAs or their targets might be helpful to stop tumour growth and metastasis development, and increase tumour sensitivity to chemotherapy agents. This signature can also be assumed for early detection of metastasis or drug resistance. However, there should be additional experimentation to validate these results.