Enrichment Of Cancer Stem Cells Research Articles

Effect of aerobic exercise on acquired gefitinib resistance in lung adenocarcinoma

Lung adenocarcinoma patients with epidermal growth factor receptor (EGFR)-activating mutations respond well to tyrosine kinase inhibitors but typically develop resistance. Current therapies mainly target differentiated cells, not cancer stem cells (CSCs), but CSCs affect the occurrence, invasion, metastasis and treatment sensitivity of malignant tumours. Recently, aerobic exercise has emerged as adjuvant therapy for cancer. Aerobic exercise can accelerate blood circulation, improve tissue oxygen supply, reduce the stress level of patients, improve the antioxidant capacity of the body, and facilitate the degradation of hypoxia-inducible factor-1 (HIF-1) in tumour tissues, thus weakening its maintenance effect on CSCs. In this study, we successfully established lung adenocarcinoma cell lines with gefitinib resistance. Long-term gefitinib induction could increase the level of oxidative stress in lung adenocarcinoma cells and reduce the antioxidant capacity, resulting in the high expression of HIF-1 and ALDH1 and leading to the enrichment of CSCs, and a decreased response to gefitinib. This may be one of the important reasons for gefitinib-acquired resistance in lung adenocarcinoma. In the case of drug resistance, effective aerobic exercise could reduce ROS, activate SOD, inhibit HIF-1 and ALDH1, and cause a reduction in CSCs to sensitise cells to gefitinib again and ultimately inhibit the malignant proliferation of tumours. Therefore, in the treatment of lung adenocarcinoma, the inhibitory effect of aerobic exercise on oxidative stress can enhance the response of drug-resistant cells to gefitinib and can be used as an effective adjunct measure in the treatment of lung adenocarcinoma.

Phenotypic changes on central nervous system (CNS) tumor cell lines cultured in vitro 2D and 3D models and treated with cisplatin

Despite aggressive therapy, most patients with brain tumors present disease relapse due to the cellular and molecular nature of these tumors. One of the models that best explains the heterogeneity observed in CNS tumors is the presence of cancer stem cells (CSCs). In this paper, we evaluated platinum-based response in brain tumor U-87 MG, LN-18, and KELLY cell lines cultured in monolayer (2D) and neurosphere (CSC enrichment– 3D) models. We evaluated mRNA expression of heat shock proteins (HSPA1A, HSPB1, HSPA1AL, TRAP1, and HSPD1), and DNA repair gene ERCC1. Changes in cell cycle and glycosylation profile were assessed by flow cytometry. After treatment with cisplatin, we found that the mRNA expression of HSPs markedly increased in the U-87 MG and LN-18 neurosphere cells. In KELLY monolayer cells, cisplatin induced upregulation of all genes. In KELLY neurosphere cells, only the HSPA1A, HSPB1, TRAP1, and HSPD1 genes were upregulated. The proportion of cells in the G0/G1 phase was significantly higher in U-87 MG neurosphere cisplatin-treated cells. A trend towards a greater proportion of cells in the S phase of U-87 MG monolayer cisplatin-treated cells was also observed. On the other hand, a significant decrease in the number of cells in the S phase and an increase in G2/M was observed in LN-18 monolayer cisplatin-treated cells. Glycosylation analysis using lectins showed a higher surface binding for PNA in the U-87 MG treated monolayer and a lower binding for Concanavalin A in the treated neurospheres. The binding of Isolectin GS-IB4, GSII, and SBA in KELLY monolayer cisplatin-treated cells was lower whereas the proportion of cells labeled with Concanavalin A was higher. In the KELLY neurosphere cisplatin-treated cells, the binding of Concanavalin A was lower than nontreated cells. Thus, our findings strongly supported the idea that definitions of phenotypic characteristics may help to establish better therapeutic strategies for brain tumors.

Triple Negative Breast Cancer: A Mountain Yet to Be Scaled Despite the Triumphs.

Simple SummaryTriple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer that cannot be treated with endocrine therapy and Her2-targeted therapy. Although its prevalence among newly diagnosed breast cancers is approximately 12.7%, it accounts for 40% of breast cancer-related mortality. Higher mortality rates among TNBC is partly because of the lack of targeted therapies and the development of resistance to chemotherapy. We discuss several important mechanisms that lead to chemoresistance and focus on important pathways and biological features that can be potentially exploited to develop therapies for TNBC. TNBC is currently defined by the absence of ER, PR, and Her2 and the greatest leap for TNBC would be our ability to characterize them with the presence of ‘x’ proteins. With this review, we intend to highlight the key nodes of TNBC and push the field towards connecting the dots between key features of TNBC and novel drug(s).Metastatic progression and tumor recurrence pertaining to TNBC are certainly the leading cause of breast cancer-related mortality; however, the mechanisms underlying TNBC chemoresistance, metastasis, and tumor relapse remain somewhat ambiguous. TNBCs show 77% of the overall 4-year survival rate compared to other breast cancer subtypes (82.7 to 92.5%). TNBC is the most aggressive subtype of breast cancer, with chemotherapy being the major approved treatment strategy. Activation of ABC transporters and DNA damage response genes alongside an enrichment of cancer stem cells and metabolic reprogramming upon chemotherapy contribute to the selection of chemoresistant cells, majorly responsible for the failure of anti-chemotherapeutic regime. These selected chemoresistant cells further lead to distant metastasis and tumor relapse. The present review discusses the approved standard of care and targetable molecular mechanisms in chemoresistance and provides a comprehensive update regarding the recent advances in TNBC management.

Abstract 2110: Platinum-inducedBRCA1 promoter DNA hypermethylation and altered metabolic profile promotes chemoresistance in ovarian cancer

Abstract High grade serous ovarian cancer (HGSOC) is the most common and aggressive type of ovarian cancer (OC). Platinum resistance is a common occurrence in HGSOC leading to tumor relapse and patient mortality. Multiple mechanisms contribute to platinum resistance in OC, including enrichment of ovarian cancer stem cells (OCSCs), which is observed in relapsed tumors. Platinum treatment activates the DNA damage response (DDR) and the DNA repair protein BRCA1 is an OC susceptibility factor. In our study, we demonstrate that platinum treatment induces a decrease in BRCA1 levels that leads to an increase in activity of ALDH, an OCSC marker. In a parallel pathway that is associated with G2/M arrest, platinum treatment also induces an increase in NAMPT expression and NAD+ levels, the cofactor required for ALDH activity. BRCA1 is repressed through platinum-induced promoter DNA hypermethylation. Importantly, combination of platinum with decitabine, a DNA methyltransferase (DNMT) inhibitor, prevents the platinum-induced increase in OCSCs. This study uniquely provides preclinical support for the use of DNMT inhibitors in the neoadjuvant setting in combination with platinum treatment for OC patients. Citation Format: Riddhi Sood, Sikai Xiao, Shruthi Sriramkumar, Christiane Hassel, Kenneth Nephew, Heather O'Hagan. Platinum-inducedBRCA1 promoter DNA hypermethylation and altered metabolic profile promotes chemoresistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2110.

Abstract 3104: EMT and CSC Enrichment Induced by ERK Inhibition in NSCLC

Abstract The activation of Mitogen-activated protein kinase pathway (MAPK/ERK pathway) has been found to play an important role in many cancers including lung cancer, and multiple inhibitors targeting this pathway have been developed. Several ERK inhibitors (ERKi, e.g., Ulixertinib, SCH772984) have been shown to be active against different cancers harboring RAS, BRAF, or MEK mutations, as well as cancers with developed BRAF/MEK inhibitor resistance. Nevertheless, sustained inhibition of ERK can lead to resistance and is likely to cause tumor relapse as well. Studies over the years have shown that a small portion of cells within tumors, which can reproduce themselves and sustain cancer, being called “cancer stem cells (CSCs)”, is the cause of tumor initiation and progression. Many reports have implicated the association of Epithelial-to-Mesenchymal Transition (EMT) program with CSCs and identified EMT-transcription factors (TFs) as CSC regulators. Here, we demonstrated that ERKi treatment induces EMT in non-small cell lung cancer cells (NSCLC), probably by inducing Slug expression. We also found that ERKi treatment can enrich the cancer stem cell (CSC) like population in NSCLC cells, evidenced by increased ALDH+ cells, enhanced in vitro sphere formation capability, and enhanced tumorigenicity in immunodeficient mice. Moreover, in NSCLC cells, we proved ERK knockdown or ERKi treatment can reduce C/EBPα expression, which is believed to be a master epithelial “gatekeeper” whose expression is required to prevent unwarranted mesenchymal transition. Our findings reveal a novel mechanism underlying the tumor relapse after ERKi treatment in NSCLC, and ultimately will help us improve current NSCLC therapeutic methods. Citation Format: Shurui Cai, Xuetao Bai, Na Li, Ananya Banerjee, Kousalya Lavudi, Tejinder Pal. EMT and CSC Enrichment Induced by ERK Inhibition in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3104.

Bortezomib-Activated Macrophages Contribute to Multiple Myeloma Progression Via Cancer Stem Cell Enrichment

Introduction: Multiple myeloma (MM) is a progressive malignancy of plasma cells. Bortezomib, a novel proteosome inhibitor, was approved for MM treatment, demonstrating revolutionary clinical results, including a significant improvement in patient outcome. However, mechanisms involved in bortezomib resistance in case of treatment failure require investigation. We previously showed that similar to chemotherapeutic agents, bortezomib induced host effects which could contribute to MM aggressive course and relapse. Mice primed with bortezomib and subsequently implanted with MM cells exhibited increased mortality rate compared to mice injected with the vehicle control. We found that bortezomib promoted the activation of pro-inflammatory macrophages, that in turn, affected MM cell aggressiveness. The current study has explored the mechanisms through which bortezomib-activated macrophages could contribute to MM aggressiveness, with the focus on MM cancer stem cells (CSC), known to be the subset of MM cells with therapy resistance properties.Methods: CAG and RPMI human cell lines were cultured with plasma and macrophage-conditioned medium obtained from mice, 24 hours after bortezomib administration. MM CSCs were phenotypically defined as CD138-/CD20+ cells and functionally defined by increased aldehyde dehydrogenase activity and presence of side population.Results: A 5-day exposure of MM cells to plasma from bortezomib-treated mice resulted in enriched CSC population, as compared to the cells cultured with plasma from control mice. Similarly, the medium conditioned with bortezomib-activated macrophages promoted enrichment of the CSCs in MM cell lines tested. Furthermore, this medium exhibited significantly increased IL-1 β levels compared to those observed in the medium conditioned with control macrophages, suggesting a role of this pro-inflammatory cytokine in MM CSC-mediated effects. To test this ability, RPMI cells were cultured with IL-1β at escalating concentrations. The resultant distinct upsurge in the CSC population could indicate the impact of IL-1β on MM CSC enrichment. As CD47 expressed by various hematopoietic cells is known to contribute to phagocytosis and macrophage killing evasion, the present study assessed CD47 expression on MM CSCs cultured with IL-1β. A significant expression of CD47 was demonstrated in MM-IL1β-enriched CSCs when compared to control MM CSCs.Conclusion: Overall, the results of the current study demonstrate that along with beneficial therapeutic effects in MM, bortezomib, interacting with host, is capable of promoting resistance to therapy by enriching the MM CSC population. This may be partially attributed to secretion of IL-1β from bortezomib-activated pro-inflammatory macrophages and enhanced CD47 expression by CSCs. Blocking such bortezomib pro-tumorigenic effects could improve therapy outcome in MM patients. DisclosuresNo relevant conflicts of interest to declare.

PCGF1 promotes epigenetic activation of stemness markers and colorectal cancer stem cell enrichment

Colorectal cancer (CRC) stem cells are resistant to cancer therapy and are therefore responsible for tumour progression after conventional therapy fails. However, the molecular mechanisms underlying the maintenance of stemness are poorly understood. In this study, we identified PCGF1 as a crucial epigenetic regulator that sustains the stem cell-like phenotype of CRC. PCGF1 expression was increased in CRC and was significantly correlated with cancer progression and poor prognosis in CRC patients. PCGF1 knockdown inhibited CRC stem cell proliferation and CRC stem cell enrichment. Importantly, PCGF1 silencing impaired tumour growth in vivo. Mechanistically, PCGF1 bound to the promoters of CRC stem cell markers and activated their transcription by increasing the H3K4 histone trimethylation (H3K4me3) marks and decreasing the H3K27 histone trimethylation (H3K27me3) marks on their promoters by increasing expression of the H3K4me3 methyltransferase KMT2A and the H3K27me3 demethylase KDM6A. Our findings suggest that PCGF1 is a potential therapeutic target for CRC treatment.

Epigenetic Mechanism of Enrichment of A549 Lung Cancer Stem Cells with 5-Fu.

BackgroundThe influence of 5-fluorouracil (5-Fu) and cisplatin (CDDP) on the A549 and NCI-H226 cells was studied, and the epigenetic mechanism of enrichment of A549 lung cancer stem cells with 5-Fu was explored.Materials and MethodsThe cell proliferation of both A549 and NCI-H226 was detected by BrdU assay, and apoptosis condition was measured by flow cytometric analysis. The expressions of OCT3/4 and Nanog in cells treated with 5-Fu or CDDP were measured by immunofluorescence, Western blot and qPCR. qPCR was also performed to determine the relative expression of methyltransferase genes and miRNA. Sequencing after bisulfite treatment (BSP) was employed to detect the methylation of OCT3/4 promoter in A549 cells. And ChIP was conducted to detect the expression of H3K9Me3 and H3K9Ace.ResultsBoth 5-Fu and CDDP result in the apoptosis of A549 and NCI-H226 cells and improve the expressions of has-miR-134 and has-miR-296. However, 5-Fu enhances the expression of OCT3/4 in A549 cells, and the change of methyltransferase genes and BSP results suggested some genetic differences between CDDP and 5-Fu treatment in A549 cells. ChIP assay showed that the expression of H3K9Me3 significantly decreased and H3K9Ace significantly increased in A549 cells.ConclusionThe enrichment effect of CDDP on A549 and NCI-H226 carcinoma stem cells is inconsistent with the enrichment effect of 5-Fu. The enrichment of A549 lung cancer stem cells with 5-Fu might be related to the methylation of OCT3/4 promoter and the expression of H3K9Me3 and H3K9Ace.

Effect of prolonged exposition to growth factors differently on proliferation of CRC cell cultures encapsulated in alginate.

e15610 Background: Cancer stem cells (CSCs) are promising targets in modern oncology. A lot of CSCs enrichment and cultivation techniques are being currently developed. Most of the approaches deploy high doses of growth factors in culture media, - EGF and FGF2 above all, - without prior testing. Since prolonged exposition to growth factors may cause paradoxical growth inhibition it is worth developing test systems to evaluate culturing conditions before establishing the main experiment. Encapsulation in alginate may serve as a promising approach to develop such test-systems due to alginate reduced ability to adsorb proteins and maintain proper attachment to the ECM of non-stem cell even in the presence of serum. It is important to notice that serum addition may be crucial during initial stages of primary CSCs culture establishment. The aim of the study was to test EGF and FGF2 effects on anoikis resistant cells growth in permanent colorectal cancer cell lines. Methods: The cells of Caco-2, HT-29, and HCT 116 cultures were encapsulated in 1% alginate beads at a concentration of 100 000 cells/ml. Subsequently encapsulated cells were kept in 2 variants of culture media: DMEM/F12 + 10%FBS with and without addition of growth factors (EGF 20 ng/ml, FGF2 10 ng/ml). The cultivation was carried out for 10 days, after that the colonies area (A) was measured. Data are given as: Mean ± 95% confidence interval. Results: The average area of Caco-2 culture colonies increased significantly with the addition of growth factors (FBS only A = 1755.16±195.87 μm2, with additional growth factors Af = 3270.57±274.91 μm2), the difference was significant (t = 8.83, n = 300, p < 0,05). The area of HCT116 colonies after growth factors addition increased only slightly (A = 2844.89±461.57 μm2, Af = 3530.31±503.85 μm2), the difference nevertheless did not reach significant values (t = 1.94, n = 300, p > 0.05). At the same time, the area of HT-29 colonies significantly decreased in the culture medium containing additional growth factors (A = 4605.10±324.02 μm2, Af = 3167.85±249.07 μm2), the difference was significant (t = 6.92, n = 300, p < 0.05). Conclusions: Combining alginate encapsulation and colonies size measurement enabled us to evaluate culturing conditions of anoikis resistant cells in permanent CRC cultures and proved growth factors addition to be an ambiguous practice in CSCs research. This tactics can be applied in a broad spectrum of tasks concerning more effective CSCs medium formulations development.

Aspartame induces cancer stem cell enrichment through p21, NICD and GLI1 in human PANC-1 pancreas adenocarcinoma cells

This study aimed to investigate the molecular effects of the common natural sugar glucose and artificial sweetener aspartame on cancer stem cell (CSC) population and cancer aggressiveness of PANC-1 human pancreas adenocarcinoma cells. According to our findings while aspartame exposure significantly increased the CSC population, high glucose had no effect on it. The epithelial-mesenchymal transition marker N-cadherin increased only in the aspartame group. The findings indicate that a high level of glucose exposure does not effect the invasion and migration of PANC-1 cells, while aspartame increases both of these aggressiveness criteria. The findings also suggest that a high concentration of glucose maintains CSC population through induction of nuclear Oct3/4 and differentiation to parental cells via increasing cytoplasmic c-myc. Aspartame exposure to PANC-1 cells activated AKT and deactivated GSK3β by increasing levels of ROS and cytoplasmic Ca+2, respectively, through T1R2/T1R3 stimulation. Then p-GSK3β(Ser9) boosted the CSC population by increasing pluripotency factors Oct3/4 and c-myc via NICD, GLI1 and p21. In the aspartame group, T1R1 silencing further increased the CSC population but decreased cell viability and suppressed the p21, NICD and GLI activation. The presence and amount of T1R subunits in the membrane fraction of PANC-1 cells are demonstrated for the first time in this study, as is the regulatory effect of T1R1's on CSC population. In conclusion, the present study demonstrated that long-term aspartame exposure increases CSC population and tumor cell aggressiveness through p21, NICD, GLI1. Moreover, while aspartame had no tumorigenic effect, it could potentially advance an existing tumor.

Mesenchymal Epithelial Transition Factor Signaling in Pediatric Nervous System Tumors: Implications for Malignancy and Cancer Stem Cell Enrichment.

Malignant nervous system cancers in children are the most devastating and worrisome diseases, specifically due to their aggressive nature and, in some cases, inoperable location in critical regions of the brain and spinal cord, and the impermeable blood-brain barrier that hinders delivery of pharmaco-therapeutic compounds into the tumor site. Moreover, the delicate developmental processes of the nervous system throughout the childhood years adds another limitation to the therapeutic modalities and doses used to treat these malignant cancers. Therefore, pediatric oncologists are charged with the daunting responsibility of attempting to deliver effective cures to these children, yet with limited doses of the currently available therapeutic options in order to mitigate the imminent neurotoxicity of radio- and chemotherapy on the developing nervous system. Various studies reported that c-Met/HGF signaling is affiliated with increased malignancy and stem cell enrichment in various cancers such as high-grade gliomas, high-risk medulloblastomas, and MYCN-amplified, high-risk neuroblastomas. Therapeutic interventions that are utilized to target c-Met signaling in these malignant nervous system cancers have shown benefits in basic translational studies and preclinical trials, but failed to yield significant clinical benefits in patients. While numerous pre-clinical data reported promising results with the use of combinatorial therapy that targets c-Met with other tumorigenic pathways, therapeutic resistance remains a problem, and long-term cures are rare. The possible mechanisms, including the overexpression and activation of compensatory tumorigenic mechanisms within the tumors or ineffective drug delivery methods that may contribute to therapeutic resistance observed in clinical trials are elaborated in this review.

Orai3-Mediates Cisplatin-Resistance in Non-Small Cell Lung Cancer Cells by Enriching Cancer Stem Cell Population through PI3K/AKT Pathway.

Simple SummaryLung cancer is recognized for having a very poor prognosis with an overall survival rate of 5-years not exceeding 15%. Platinum-doublet therapy is the most current chemotherapeutic treatment used to treat lung tumors. However, resistance to such drugs evolves rapidly in patients with non-small cell lung cancer (NSCLC) and is one of the major reasons behind therapy failure. Tumor recurrence due to chemoresistance is mainly attributed to the presence of cancer stem cells (CSCs) subpopulations. Thus, the identification of resistance actors and markers is necessary. The Orai3 channel has been recently identified as a predictive marker of metastasis and survival in resectable NSCLC tumors. Our results show, for the first time, that the Orai3 channel is able to induce chemoresistance by enriching CSCs population. Our findings present Orai3 as a promising predictive biomarker which could help with selecting chemotherapeutic drugs.The development of the resistance to platinum salts is a major obstacle in the treatment of non-small cell lung cancer (NSCLC). Among the reasons underlying this resistance is the enrichment of cancer stem cells (CSCs) populations. Several studies have reported the involvement of calcium channels in chemoresistance. The Orai3 channel is overexpressed and constitutes a predictive marker of metastasis in NSCLC tumors. Here, we investigated its role in CSCs populations induced by Cisplatin (CDDP) in two NSCLC cell lines. We found that CDDP treatment increased Orai3 expression, but not Orai1 or STIM1 expression, as well as an enhancement of CSCs markers. Moreover, Orai3 silencing or the reduction of extracellular calcium concentration sensitized the cells to CDDP and led to a reduction in the expression of Nanog and SOX-2. Orai3 contributed to SOCE (Store-operated Calcium entry) in both CDDP-treated and CD133+ subpopulation cells that overexpress Nanog and SOX-2. Interestingly, the ectopic overexpression of Orai3, in the two NSCLC cell lines, lead to an increase of SOCE and expression of CSCs markers. Furthermore, CD133+ cells were unable to overexpress neither Nanog nor SOX-2 when incubated with PI3K inhibitor. Finally, Orai3 silencing reduced Akt phosphorylation. Our work reveals a link between Orai3, CSCs and resistance to CDDP in NSCLC cells.

The Molecular Basis of Different Approaches for the Study of Cancer Stem Cells and the Advantages and Disadvantages of a Three-Dimensional Culture.

Cancer stem cells (CSCs) are a rare tumor subpopulation with high differentiation, proliferative and tumorigenic potential compared to the remaining tumor population. CSCs were first discovered by Bonnet and Dick in 1997 in acute myeloid leukemia. The identification and isolation of these cells in this pioneering study were carried out through the flow cytometry, exploiting the presence of specific cell surface molecular markers (CD34+/CD38−). In the following years, different strategies and projects have been developed for the study of CSCs, which are basically divided into surface markers assays and functional assays; some of these techniques also allow working with a cellular model that better mimics the tumor architecture. The purpose of this mini review is to summarize and briefly describe all the current methods used for the identification, isolation and enrichment of CSCs, describing, where possible, the molecular basis, the advantages and disadvantages of each technique with a particular focus on those that offer a three-dimensional culture.

Bcl3 Couples Cancer Stem Cell Enrichment With Pancreatic Cancer Molecular Subtypes

The existence of different subtypes of pancreatic ductal adenocarcinoma (PDAC) and their correlation with patient outcome have shifted the emphasis on patient classification for better decision-making algorithms and personalized therapy. The contribution of mechanisms regulating the cancer stem cell (CSC) population in different subtypes remains unknown. Using RNA-seq, we identified B-cell CLL/lymphoma 3 (BCL3), an atypical nf-κb signaling member, as differing in pancreatic CSCs. To determine the biological consequences of BCL3 silencing invivo and invitro, we generated bcl3-deficient preclinical mouse models as well as murine cell lines and correlated our findings with human cell lines, PDX models, and 2 independent patient cohorts. We assessed the correlation of bcl3 expression pattern with clinical parameters and subtypes. Bcl3 was significantly down-regulated in human CSCs. Recapitulating this phenotype in preclinical mouse models of PDAC via BCL3 genetic knockout enhanced tumor burden, metastasis, epithelial to mesenchymal transition, and reduced overall survival. Fluorescence-activated cell sorting analyses, together with oxygen consumption, sphere formation, and tumorigenicity assays, all indicated that BCL3 loss resulted in CSC compartment expansion promoting cellular dedifferentiation. Overexpression of BCL3 in human PDXs diminished tumor growth by significantly reducing the CSC population and promoting differentiation. Human PDACs with low BCL3 expression correlated with increased metastasis, and BCL3-negative tumors correlated with lower survival and nonclassical subtypes. We demonstrate that bcl3 impacts pancreatic carcinogenesis by restraining CSC expansion and by curtailing an aggressive and metastatic tumor burden in PDAC across species. Levels of BCL3 expression are a useful stratification marker for predicting subtype characterization in PDAC, thereby allowing for personalized therapeutic approaches.

ETMM-01. CANCER STEM CELL ENRICHMENT AND METABOLIC SUBSTRATE ADAPTABILITY ARE DRIVEN BY HYDROGEN SULFIDE SUPPRESSION IN GLIOBLASTOMA

Glioblastoma (GBM) remains among the deadliest of human malignancies. The emergence of the cancer stem cell (CSC) phenotype represents a major challenge to disease management and durable treatment response. The extrinsic, environmental, and lifestyle factors that result in CSC enrichment are not well understood. The CSC state endows cells with a fluid metabolic profile, enabling the utilization of multiple nutrient sources. Therefore, to test the impact of diet on CSC enrichment, we evaluated disease progression in tumor-bearing mice fed an obesity-inducing high-fat diet (HFD) versus an energy-balanced, low-fat control diet. HFD consumption resulted in hyper-aggressive disease that was accompanied by CSC enrichment and shortened survival. HFD consumption also drove intracerebral accumulation of saturated fats, which in turn inhibited the production and signaling of the gasotransmitter hydrogen sulfide (H2S). H2S is an endogenously produced bio-active metabolite derived from sulfur amino acid catabolism. It functions principally through protein S-sulfhydration and regulates a variety of programs including mitochondrial bioenergetics and cellular metabolism. Inhibition of H2S synthesis resulted in increased proliferation and chemotherapy resistance, whereas treatment with H2S donors led to cytotoxicity and death of cultured GBM cells. Compared to non-cancerous controls, patient GBM specimens were reduced in overall protein S-sulfhydration, which was primarily lost from proteins regulating cellular metabolism. These findings support the hypothesis that diet-regulated H2S signaling serves to suppress GBM by restricting metabolic adaptability, while its loss triggers CSC enrichment and disease acceleration. Interventions augmenting H2S bioavailability concurrent with GBM standard of care may improve outcomes for GBM patients.

Targeted nanoformulation of C1 inhibits the growth of KB spheroids and cancer stem cell-enriched MCF-7 mammospheres

C1, a synthetic analog of curcumin, has been reported to show potent antiproliferative effects against a variety of cancer cells. Here, we report a strong anticancer activity of the folate receptor-targeted lipid nanoparticle formulation of C1 against cancer cells and cancer stem cells both in 2D culture and 3D spheroids. The size of the C1 encapsulated folic acid functionalized nanoliposomes (Lipos-C1) was determined to be 83 ± 17 nm. Lipos-C1 nanoparticles displayed sustained C1 release kinetics at both pH 7.4 and 5.5. The folate receptor (FR) targeted nanoliposomes were internalized into FR-positive KB cells via the folate receptor-mediated endocytosis process. Lipos-C1 killed KB cells much more efficiently than C1. Lipos-C1 depolymerized microtubules, generated ROS, caused DNA damage, and induced apoptosis in KB cells. Importantly, Lipos-C1 strongly inhibited the growth of the 3D KB spheroids than C1. Interestingly, Lipos-C1 also suppressed cancer stem cells (CSCs) enriched MCF-7 mammosphere growth by impeding breast cancer stem cells (BCSCs) enrichment, growth, and proliferation. The results suggested that Lipos-C1 could be a promising nanoformulation for cancer chemotherapy.

Cucurbitacin B exhibits antitumor effects on CD133+ HepG2 liver cancer stem cells by inhibiting JAK2/STAT3 signaling pathway.

Cancer stem cells (CSCs), a crucial cancer cell subpopulation, possess stemness phenotypic characteristics. Cucurbitacin B (CuB), a tetracyclic triterpenoid isolated from Cucurbitaceae, exerts widely pharmacological activities in many diseases. The aim of this study was to enrich, identify liver CSCs and investigate antitumor effects of CuB as well as explore the underlying molecular mechanisms in these liver CSCs. HepG2 cell lines were used for the enrichment of liver CSCs by serum-free medium culture and magnetic-activated cell sorting. The CSC characteristics were analyzed by immunofluorescent staining, sphere-forming, western blot and xenograft tumorigenicity assay. CuB' antitumor effects and underlying molecular mechanism were measured by cell counting kit-8, colony formation, sphere-forming, cell cycle, xenograft and western blot assay. Our results showed that we could enrich 97.29% CD133+ HepG2 cells, which possessed CSC characteristics including re-renewal capacity, proliferative ability, sorafenib resistance, overexpressed stemness-related molecules and enhanced tumorigenic potential. Furthermore, we also found that CuB inhibited cell viability, sphere formation, colony formation and arrested cell cycle at G2/M phase as well as sensitized CD133+ HepG2 cells to sorafenib in vitro and in vivo. Western blot assay indicated that CuB inhibited expression levels of cyclin B1, CDK1, CD133, p-JAK2 and p-STAT3. In conclusion, our findings indicated that CuB could exhibit antitumor effects on CD133+ HepG2 CSCs by inhibiting the Janus kinase 2/signal transducers and activators of transcription-3 signaling pathway, expanding basic and preclinical investigations on liver CSCs.

Abstract LT020: Cancer associated fibroblasts in the tumor microenvironment maintain ovarian cancer stem cells through non-canonical Wnt5a signaling

Abstract Frequent relapse and development of chemoresistance are major causes of poor survival in ovarian cancer. Cancer stem cells (CSCs) consist of a small subpopulation in the tumor that are capable of initiation and maintenance. CSCs are typically resistant to cytotoxic chemotherapy and are a potential cause of relapse and chemoresistance. Most studies on CSCs focus on cancer cells alone while CSCs exist in a complex microenvironment in tumors. This microenvironment or CSC niche provides optimal conditions to maintain their tumor initiating potential. Hence, understanding the crosstalk between CSCs and the tumor microenvironment can potentially provide effective therapeutic targets to prevent chemoresistance and relapse. Cancer associated fibroblasts (CAFs) are a major constituent of the ovarian cancer tumor microenvironment and are highly enriched in the residual tumors following chemotherapy. Therefore, we studied the role of CAFs in promoting ovarian cancer chemoresistance and disease relapse through providing an optimal microenvironment for CSCs. CAFs isolated from ovarian cancer patient tumors were used in heterotypic 3D or 2D coculture systems with high grade serous ovarian cancer cell lines or with patient-derived ovarian cancer cells to study their effect on CSCs and chemoresistance. Matched pre- and post-chemotherapy patient tumors were used to confirm our findings. CAFs significantly increased resistance to carboplatin and enriched CSCs by increasing their symmetric division as well as dedifferentiation of bulk ovarian cancer cells. Pre-coculture with CAFs increased in vivo tumor initiation capacity of the ovarian cancer cells 10-fold. The CSC-CAF crosstalk responsible for CSC induction was found to be mediated by Wnt signaling. Inhibition of Wnt secretion by CAFs could block their effect on CSCs. Wnt5a was the most highly expressed Wnt in CAFs, which was further induced by ovarian cancer cells. CRISPR knockdown of Wnt5a in CAFs or treatment with a specific Wnt5a inhibitor abrogated the induction of CSCs by CAFs. Wnt5a was found to signal through a non-canonical Wnt pathway in the CSCs involving the coreceptor ROR2, protein kinase C (PKC), and CAMP Responsive Element Binding Protein 1 (CREB1). Inhibition of each of them prevented CSC induction and functional rescue experiments confirmed the sequence of the Wnt5a-ROR2-PKC-CREB1 axis. Treatment of mouse xenografts established by co-injection of CAFs and ovarian cancer cells, with the Wnt5a inhibitor sensitized them to carboplatin, and eliminated the CSCs in the residual tumors. Our results indicate that CAF-derived Wnt5a is instrumental in ovarian cancer CSC growth and maintenance. In the long term, our studies will broaden the understanding of the mechanism of CSC maintenance by the tumor microenvironment and contribute towards the development of novel therapeutic approaches to prevent ovarian cancer chemoresistance and relapse. Citation Format: Yiming Fang, Xue Xiao, Ji Wang, Subramanyam Dasari, Kenneth P. Nephew, Dmitriy Zamarin, Anirban K. Mitra. Cancer associated fibroblasts in the tumor microenvironment maintain ovarian cancer stem cells through non-canonical Wnt5a signaling [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT020.

Dendritic cells loaded with exosomes derived from cancer stem cell-enriched spheroids as a potential immunotherapeutic option.

Cancer stem cells (CSCs) are responsible for therapeutic resistance and recurrence in colorectal cancer. Despite advances in immunotherapy, the inability to specifically eradicate CSCs has led to treatment failure. Hence, identification of appropriate antigen sources is a major challenge in designing dendritic cell (DC)‐based therapeutic strategies against CSCs. Here, in an in vitro model using the HT‐29 colon cancer cell line, we explored the efficacy of DCs loaded with exosomes derived from CSC‐enriched colonospheres (CSCenr‐EXOs) as an antigen source in activating CSC‐specific T‐cell responses. HT‐29 lysate, HT‐29‐EXOs and CSCenr lysate were independently assessed as separate antigen sources. Having confirmed CSCs enrichment in spheroids, CSCenr‐EXOs were purified and characterized, and their impact on DC maturation was investigated. Finally, the impact of the antigen‐pulsed DCs on the proliferation rate and also spheroid destructive capacity of autologous T cells was assessed. CSCenr‐EXOs similar to other antigen groups had no suppressive/negative impacts on phenotypic maturation of DCs as judged by the expression level of costimulatory molecules. Notably, similar to CSCenr lysate, CSCenr‐EXOs significantly increased the IL‐12/IL‐10 ratio in supernatants of mature DCs. CSCenr‐EXO‐loaded DCs effectively promoted T‐cell proliferation. Importantly, T cells stimulated with CSCenr‐EXOs disrupted spheroids' structure. Thus, CSCenr‐EXOs present a novel and promising antigen source that in combination with conventional tumour bulk‐derived antigens should be further explored in pre‐clinical immunotherapeutic settings for the efficacy in hampering recurrence and metastatic spread.

Enriched cancer stem cells, dense stroma, and cold immunity: Interrelated events in pancreatic cancer.

Cold tumors generally show low mutational burden and low infiltration of effector T cells. The pancreas, prostate, ovary, breast, and colon are placed into the category of cold tumors. In such tumors, effector T cells are either excluded from the tumor area or taken away from being in contact with tumor cells. The stromal reaction in the form of desmoplasia is important for the pathogenesis of tumors like the pancreas. Besides acting as a barrier for the penetration of drugs into the tumor area, the dense stroma presumably creates an immunosuppressive tumor microenvironment (TME), which accounts for low responses from tumor to immunotherapy. Cancer stem cells (CSCs) are an important part of the immunosuppressive complex within the TME. The presence of CSCs within the TME is related negatively to the activity of the antitumor immune system. Here, the question is how desmoplastic aggregates can influence the functionality of CSCs for promoting a cold pancreatic tumor immunity? This review is aimed at responding to this question, the disruption of which can be an effective strategy for improving responses from cold tumors to immunotherapy.