Tumor Stem Cells Research Articles

In Situ Hydrogel ModulatescDC1-Based Antigen Presentation and Cancer Stemness to Enhance Cancer Vaccine Efficiency.

Effective presentation of antigens by dendritic cells (DC) is essential for achieving a robust cytotoxic T lymphocytes (CTLs) response, in which cDC1 is the key DC subtype for high-performance activation of CTLs. However, low cDC1 proportion, complex process, and high cost severely hindered cDC1 generation and application. Herein, the study proposes an in situ cDC1 recruitment and activation strategy with simultaneous inhibiting cancer stemness for inducing robust CTL responses and enhancing the anti-tumor effect. Fms-like tyrosine kinase 3 ligand (FLT3L), Poly I:C, and Nap-CUM (NCUM), playing the role of cDC1 recruitment, cDC1 activation, inducing antigen release and decreasing tumor cell stemness, respectively, are co-encapsulated in an in situ hydrogel vaccine (FP/NCUM-Gel). FP/NCUM-Gel is gelated in situ after intra-tumoral injection. With the near-infrared irradiation, tumor cell immunogenic cell death occurred, tumor antigens and immunogenic signals are released in situ. cDC1 is recruited to tumor tissue and activated for antigen cross-presentation, followed by migrating to lymph nodes and activating CTLs. Furthermore, tumor cell stemness are inhibited by napabucasin, which can help CTLs to achieve comprehensive tumor killing. Collectively, the proposed strategy of cDC1 in situ recruitment and activation combined with stemness inhibition provides great immune response and anti-tumor potential, providing new ideas for clinical tumor vaccine design.

FPR3 reprograms glycolytic metabolism and stemness in gastric cancer via calcium-NFATc1 pathway

Aerobic glycolysis accelerates tumor proliferation and progression, and inhibitors or drugs targeting abnormal cancer metabolism have been developing. Cancer stem-like cells (CSCs) significantly contribute to tumor initiation, metastasis, therapy resistance, and recurrence. Formyl peptide receptor 3 (FPR3), a member of FPR family, involves in inflammation, tissue repair, and angiogenesis. However, studies in exploring the regulatory mechanisms of aerobic glycolysis and CSCs by FPR3 in gastric cancer (GC) remain unknown. Here, we demonstrated that overexpressed FPR3 suppressed glycolytic capacity and stemness of tumor cells, then inhibited GC cells proliferation. Mechanistically, FPR3 impeded cytoplasmic calcium ion flux and hindered nuclear factor of activated T cells 1 (NFATc1) nuclear translocation, leading to the transcriptional inactivation of NFATc1-binding neurogenic locus notch homolog protein 3 (NOTCH3) promoter, subsequently obstructing NOTCH3 expression and the AKT/mTORC1 signaling pathway, and ultimately downregulating glycolysis. Additionally, NFATc1 directly binds to the sex determining region Y-box 2 (SOX2) promoter and modifies stemness in GC. In conclusion, our work illustrated that FPR3 played a negative role in GC progression by modulating NFATc1-mediated glycolysis and stemness in a calcium-dependent manner, providing potential insights into cancer therapy.

Establishing Genetic and Molecular Markers of Sensitivity to a Novel and Potent Tetracyclic Terpenoid Therapy Against High-Grade Gliomas

INTRODUCTION: High-grade gliomas have the worst prognosis of all intracranial tumors, and therapeutic options are very limited. We have previously described a novel, highly selective and potent agonist of ERβ called ent-28 that inhibits the proliferation of patient-derived glioma cells in vitro. In addition, ent-28 significantly decreases the sizes of orthotopically implanted high-grade glioma patient-derived xenografts and induces apoptosis of tumor-derived cells with varying sensitivity. METHODS: ent-28 induced cell death of patient-derived tumor adherent- and stem-cells was analyzed in combination with the histopathologic, molecular, and genetic characteristics of each primary resected tumor for respective cell lines. GI50 values and apoptotic death induced by ent-28 were compared to IDH1 and ATRX mutational status as well as MGMT methylation. RESULTS: Compared to both untreated and control groups, ent-28-treated tumor-derived MGMT unmethylated cells demonstrated sensitivity. Additionally, tumor adherent cells underwent apoptosis at a higher rate compared to tumor stem cells. CONCLUSIONS: We have completed pre-clinical testing, safety, pharmacokinetics, metabolic stability, in vivo efficacy, and species rationalization for developing a novel compound, ent-28, as an anti-glioma therapy. To further enhance the efficacy of ent-28 against high-grade gliomas, we have used histopathologic characteristics of resected primary tumors to identify specific molecular and genetic markers that correlate to drug-sensitivity. Our findings suggest that specific molecular markers of sensitivity to ent-28 can be used to stratify response in patient derived cells, target tumor stem cells specifically, and further support the development of a potent therapy against high-grade gliomas.

Identifying a Novel Eight-NK Cell-related Gene Signature for Ovarian Cancer Prognosis Prediction.

Ovarian cancer (OVC) is the most common and costly tumor in the world with unfavorable overall survival and prognosis. This study is aimed to explore the prognostic value of natural killer cells related genes for OVC treatment. RNA-seq and clinical information were acquired from the TCGA-OVC dataset (training dataset) and the GSE51800 dataset (validation dataset). Genes linked to NK cells were obtained from the immPort dataset. Moreover, ConsensusClusterPlus facilitated the screening of molecular subtypes. Following this, the risk model was established by LASSO analysis, and immune infiltration and immunotherapy were then detected by CIBERSORT, ssGSEA, ESTIMATE, and TIDE algorithms. Based on 23 NK cell-related genes with prognosis, TCGA-OVC samples were classified into two clusters, namely C1 and C2. Of these, C1 had better survival outcomes as well as enhanced immune infiltration and tumor stem cells. Additionally, it was more suitable for immunotherapy and was also sensitive to traditional chemotherapy drugs. The eight-gene prognosis model was constructed and verified via the GSE51800 dataset. Additionally, a high infiltration level of immune cells was observed in low-risk patients. Low-risk samples also benefited from immunotherapy and chemotherapy drugs. Finally, a nomogram and ROC curves were applied to validate model accuracy. The present study identified a RiskScore signature, which could stratify patients with different infiltration levels, immunotherapy, and chemotherapy drugs. Our study provided a basis for precisely evaluating OVC therapy and prognosis.

Clinical Analysis of PEG-rhG-CSF in Mobilization of Autologous Hematopoietic Stem Cells in Hematological Tumors

To investigate the efficiency and optimal time of stem cell apheresis mobilized by pegylated recombinant human granulocyte colony stimulating factor (PEG-rhG-CSF) in autologous stem cell transplantation (ASCT) for hematological malignancies without monitoring pre-collection CD34+ cells. Forty-six patients underwent stem cell mobilization were retrospectively analyzed between August 2017 and January 2022 at the First Affiliated Hospital of Fujian Medical University. 27 patients using high dose chemotherapy combined with PEG-rhG-CSF mobilization were enrolled in the PEG-rhG-CSF group, and other 19 patients mobilized with recombinant human granulocyte colony stimulating factor (G-CSF) were enrolled in G-CSF group. The mobilization and collection effects of the patients in two groups were compared. A total of 46 patients underwent 86 apheresis procedures, the median amount of mononuclear cell (MNC) in the PEG-rhG-CSF group and G-CSF group was 6.54（3.85-12.61）×108/kg and 6.15（1.13-11.58）×108/kg, respectively (P >0.05), the total CD34+ cells of the grafts were 11.44(1.33-65.02)×106/kg and 4.95(0.30-24.02)×106/kg (P < 0.05), with harvest timing of 14(10-20) days and 14(4-22) days, respectively (P >0.05). In the PEG-rhG-CSF group, there was a significant difference between the number of CD34+ cells collected when white blood cells (WBC) ≥10×109/L and WBC<10×109 /L, 19.04(2.85-65.02)×106/kg and 6.22(0.81-34.86)×106/kg, respectively (P < 0.05). Stem cells mobilization with PEG-rhG-CSF was highly efficient with a median mobilization time of 14 days. In the absence of peripheral blood CD34 monitoring, peripheral blood WBC≥10×109/L can be considered as a threshold for a single stem cell apheresis to collect sufficient stem cells.

Unboxing the network among long non-coding RNAs and TGF-β signaling in cancer.

Deeper analysis of molecular mechanisms arising in tumor cells is an unmet need to provide new diagnostic and therapeutic strategies to prevent and treat tumors. The transforming growth factor β (TGF-β) signaling has been steadily featured in tumor biology and linked to poor prognosis of cancer patients. One pro-tumorigenic mechanism induced by TGF-β is the epithelial-to-mesenchymal transition (EMT), which can initiate cancer dissemination, enrich the tumor stem cell population, and increase chemoresistance. TGF-β signals via SMAD proteins, ubiquitin ligases, and protein kinases and modulates the expression of protein-coding and non-coding RNA genes, including those encoding larger than 500 nt transcripts, defined as long non-coding RNAs (lncRNAs). Several reports have shown lncRNAs regulating malignant phenotypes by directly affecting epigenetic processes, transcription, and post-transcriptional regulation. Thus, this review aims to update and summarize the impact of TGF-β signaling on the expression of lncRNAs and the function of such lncRNAs as regulators of TGF-β signaling, and how these networks might impact specific hallmarks of cancer.

Abstract 1542: Epithelial cell adhesion molecule induces canonical Wnt pathway to promote stemness in colorectal cancer

Abstract Epithelial cell adhesion molecule (EpCAM) is a pleiotropic type-1 transmembrane glycoprotein and known cancer stem cell marker, yet its tumorigenic mechanisms remain elusive. Cancer stem cells (CSC) have been implicated in cancer progression and recurrence, but targeting them with therapeutics is a major challenge. It is known that EpCAM is highly expressed in CSCs of different cancer types. Here, we studied the role of EpCAM in colorectal cancer (CRC) stemness. First, we show that the extracellular domain of EpCAM (EpEX) functions as a ligand for Wnt receptors, frizzled6/7 and LRP5/6, while the intracellular domain (EpICD) upregulates transcription of these receptors. Further, EpEX-induced Wnt signaling activates TACE and γ-secretase enzymes to augment shedding of EpEX and EpICD, establishing a positive feedback loop. Importantly, we show that the EpCAM-neutralizing antibody, EpAb2-6, targets the Wnt pathway to attenuate stemness characteristics in colorectal cancer (CRC). Our EpCAM-neutralizing antibody (EpAb2-6) and a porcupine inhibitor (LGK974) each partially attenuated cancer stemness, while their combination abolished stemness-related endpoints, induced apoptosis in vitro and markedly diminished tumor progression in animal models of human CRC. From these findings, we conclude that EpCAM stimulates Wnt signaling to promote cancer stemness, and the combination of EpAb2-6 and porcupine inhibitors may represent an effective CRC treatment, including for KRAS-mutant cancers. Our findings reveal an opportunity to combat drug resistance in tumor stem cells. Thus, the mechanistic insights gained from our study may be useful to improve existing treatments or to develop novel anticancer therapeutics. Citation Format: Han-Chung Wu, Sushree Shankar Panda, Kai-Chi Chen, Chi-Chiu Lee, Hsing-Pang Hsieh, Yi-Jen Su. Epithelial cell adhesion molecule induces canonical Wnt pathway to promote stemness in colorectal 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 1542.

Abstract 5849: Integrated multi-omics of hydroxychloroquine evolved cancer cells reveal survival requires transcriptional upregulation of glycolysis, exocytosis, and chromosome fidelity, but not autophagy

Abstract Repurposed drugs are undergoing a renaissance in the era of genomics, particularly in oncology. Over 2,300 FDA-approved and natural product small molecules now exist, but challenges remain in identifying which cancer patients may best benefit from such drugs. To best position repurposed drugs, mechanisms of action and resistance to the drug in a cancer cell setting must be determined. Hydroxychloroquine (HCQ) and chloroquine are repurposed drugs reported to disrupt autophagy, a molecular recycling pathway which is essential for tumor cell survival, chemotherapeutic resistance, and stem cell phenotypes. However, oncology trials with chloroquine have no biomarker or other genetic test to categorize patients who may best respond to HCQ therapy. It is unclear how tumors mechanistically evolve resistance to HCQ. We pursued a multi-omic strategy in OVCAR3 ovarian cancer and CCL218 colorectal cancer cells. Two genome-scale screens were performed. In the forward genetic screen, cell populations were passaged in 93-independent cultures for 15 drug pulse-chases with HCQ or vehicle control. Evolved cells were collected and processed for bulk RNA-seq, exome-seq, and single cell RNA-seq (scRNA-seq). In the reverse genetic screen, a pooled Brunello CRISPR-Cas9 library of 19,114 genes targeted by multiple sgRNAs was used in cells over three pulse-chases of HCQ or vehicle control treatments. HCQ evolved cells displayed remarkably few mutational differences, but substantial transcriptional differences. Bulk transcriptomes revealed multiple broad-spectrum pathways associated with resistance to HCQ, including upregulation of glycolysis, exocytosis, and chromosome condensation/segregation, as well as downregulation of translation and apoptosis. Analysis of scRNA-seq data indicated cell clusters were differentially reliant on cytoskeletal regulation, DNA repair, and metabolism. The genome-scale Cas9 screen identified remarkably few autophagy genes. However, an integrated analysis of Cas9 hits with RNA-seq upregulated genes confirmed glycolytic genes KDM3A, ENO2, and PDK3 were essential for HCQ resistance. Additional integrated hits were NEK2, involved in exocytosis, and chromosome condensation/segregation genes CDK1 and CENPA. Transcriptional plasticity was the primary mechanism by which cells evolved resistance to HCQ. Contrary to the widely presumed autophagy-related mode of cell death, the multi-omic analysis instead prioritized glycolytic metabolism, exocytosis, and chromosome condensation/segregation as most involved in cancer cell survival with HCQ. Integrated multi-omic analysis defined high-value biomarkers of HCQ sensitivity and resistance. Our analysis may serve as a model for how to better define the effects and positioning of repurposed drugs in oncology. Citation Format: Madison Clark, Silvia G. Vaena, Joe R. Delaney. Integrated multi-omics of hydroxychloroquine evolved cancer cells reveal survival requires transcriptional upregulation of glycolysis, exocytosis, and chromosome fidelity, but not autophagy [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 5849.

Abstract 2092: Genomic and proteomic analysis of glioblastoma recurrences during TTFields exposure

Abstract Glioblastoma (GB) is the most aggressive type of brain tumor, and current treatments are generally ineffective in preventing recurrence. Tumor Treating Fields (TTFields) are an innovative treatment that has been shown to improve patients' life expectancy in the last two decades. TTFields therapy has an antitumoral effect that involves different mechanisms and is an optional add-on to standard maintenance temozolomide. Although TTFields therapy initially slows tumor progression, it does not prevent tumor relapse in most patients. In the present investigation, we run a genomic and proteomic analysis of surgical material from 10 patients treated with TTFields. GB tissues were obtained from the primary tumor mass and from a second surgery after tumor relapse. All the patients, in addition to brain tumor standard therapies, were treated with TTFields for eighteen hours a day, the time span of treatment application was dependent on GB recurrence. Control samples were obtained from primary and secondary surgeries of 5 patients treated with standard therapy alone. The GB tissue extracts were analyzed with an RNAseq routine, and the results from the secondary surgery were compared with the data obtained from tumor specimens of the primary surgery. A common feature of transcripts among all the patients was the alterations of several pathways promoting the increase of intracellular oxidation and cell cycle regulation. In parallel with the analysis of patient specimens, we developed an in vitro GB cell relapse model. This was instrumental in investigating the beneficial contribution of TTFields action during tumor relapse, but also in uncovering the limitation of this therapeutic procedure. GB primary cultures obtained from patients' surgery were exposed for up to 12 days with 200 kHz TTFields stimulation using the inovitro™ system. After 12 days of continuous TTFields application, GB cells showed an average depolarization of the resting membrane potential, increasing oxidation, and acidification of the cytoplasm. Genomic analysis of treated cells compared with wild-type primary culture, denoted an increase of tumor stem cell markers, activation of several metabolic pathways, and upregulation of different ion channel protein transcripts. Our final goal is to identify specific molecular features enhanced by TTFields treatment to be used as a parallel therapy to fight GB recurrence. Citation Format: Francesca Cianci, Guido Rey, Dietmar Krex, Davide Ceresa, Paolo Malatesta, Michele Mazzanti. Genomic and proteomic analysis of glioblastoma recurrences during TTFields exposure [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 2092.

Abstract 264: Differentiation therapy: Esculetin as a potent agent to alter cellular plasticity of leukemic &amp; solid tumor stem cells

Abstract Background The “Differentiation therapy” has been emerging as a promising and more effective strategy against acute leukemia relapses. Objective In extension to the revolutionising therapeutic outcomes of All Trans Retinoic Acid (ATRA) to induce terminal differentiation of Acute Promyelocytic Leukemic (APL) blast cells, we decipher the potential effect of a natural compound “Esculetin” to serve as a differentiating agent in Acute Myeloid Leukemia (AML) as well as solid tumour cells. Underlaying role of Wnt signaling pathways in esculetin mediated blast cell differentiation was also evaluated. Methods Human acute myeloid leukemic cells (Kasumi-1) with t(8;21/AML-ETO) translocation were used as a model system. Growth inhibitory and cytotoxic activity of esculetin were analysed using growth kinetics and MTT assay. Morphological alterations, cell scatter characteristics, NBT reduction assay and cell surface marker expression pat- terns were analysed to detect terminally differentiated phenotypes. We employed RT2profiler PCR array system for the analysis of transcriptome profile of Wnt signaling components. Calcium inhibitors (TMB8 and Amlodipine) and Transforming growth factor beta (TGF-β) were used to modulate the Wnt signaling axes. To investigate whether esculetin exerts the similar effects on the invasive solid tumour cancer stem cells subset, we used a cellular model system of colon carcinoma HCT116 cells reflecting EMT phenotype. Results We illustrate cytotoxic as well as blast cell differentiation potential of esculetin on Kasumi-1 cells. Morphological alterations akin to neutrophilic differentiation as well as the corresponding acquisition of myeloid lineage markers indicate terminal differentiation potential of esculetin in leukemic blast cells. Exposure to esculetin also resulted in downregulation of canonical Wnt axis while upto ~ 21 fold upregulation of non-canonical axis associated genes. Esculetin also showed potential to revert the CSC marker expressions and concurrent EMT consistent with reduced functional aggressiveness in colon cancer cells. Conclusions Our study highlights the importance of selective use of calcium pools as well as “axis shift” of the canonical to non-canonical Wnt signaling upon esculetin treatment which might abrogate the inherent proliferation to release maturation arrest and induce the differentiation in leukemic blast cells as well as CSC stem cells. The current findings provide further therapeutic interventions to consider esculetin as a potent differentiating agent to counteract relapses. Citation Format: Ankit Mathur, Daman Saluja. Differentiation therapy: Esculetin as a potent agent to alter cellular plasticity of leukemic &amp; solid tumor stem cells [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 264.

Abstract 458: Nanoparticle-mediated delivery of miR-590-3p decreases recurrent GBM tumor growth by inhibits multiple oncogenic nodes

Abstract Glioblastoma (GBM) is the most lethal and aggressive form of brain cancer. Despite aggressive therapy consisting of surgery followed by radio/chemotherapy GBM recurs in almost all patients and, currently, there are no proven therapies to treat recurrent GBM (rGBM). Recent developments in nanomedicine provide new and promising opportunities to develop new targeted therapeutics to treat brain tumors. We have recently developed PBAE nanoparticle formulations optimized for in vivo miRNA delivery to brain tumor stem cells, or GSCs. Carefully selecting one miRNA to target several mRNAs dysregulated within a pathway can allow the targeting of multiple nodes using a single agent, mimicking combinatorial therapies. To test this hypothesis, we performed bioinformatic analysis of RNA sequencing from GSCs and clinical rGBM specimens combined with gene set enrichment analysis (GSEA) and identified TGFBR2 signaling as a targetable pathway in rGBM. We show that SMAD2 signaling, a downstream effector of TGFBR2, is enriched in rGBM and that patients with elevated TGFBR2 transcripts respond poorly to current standard of care. Utilizing patient-derived xenograft lines from rGBM, we show that blocking TGFBR2 signaling using siRNAs or ITD1, a selective TGFBR2 inhibitor, decreases the stem cell capacity, cell viability and re-sensitizes clinical rGBM cells to temozolomide (TMZ) in-vitro. In vivo, we show that ITD1 is modestly effective in subcutaneous and ineffective in orthotopic rGBM models. Not surprisingly, these tumors exhibited classical growth patterns associated with acquisition of resistance. To overcome this obstacle, we performed a miRNA-based network analysis in rGBM and identified miR-590-3p as a putative tumor suppressor that targets SMAD2 signaling at multiple nodes in rGBM. In-vitro, transgenic miR-590-3p robustly decreased the expression of 37 putative oncogenes simultaneously concurrent with decreasing stem cell capacity, cell viability and re-sensitizing rGBM PDX cells to TMZ. In vivo delivery of miR-590-3p to established PDX-derived rGBM orthotopic tumors using novel PBAE nanocarriers robustly decreased tumor sizes and increased tumor necrosis. These results show that the promiscuous nature of miRNAs, in combination with cutting-edge nucleic acid delivery vehicles, can be leveraged as advanced targeted therapeutics for rGBM bypasses the resistance that is developed to standard of care and available small molecules. Citation Format: Hernando Lopez-Bertoni, Sophie Sall, Harmon Khela, Jack Korleski, Katherine Luly, Maya Johnson, Bachchu Lal, Amanda Johnson, Stephany Tzeng, Jordan Green, John Laterra. Nanoparticle-mediated delivery of miR-590-3p decreases recurrent GBM tumor growth by inhibits multiple oncogenic nodes [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 458.

Abstract 250: Sex differences in cancer stemness among individuals with early onset colorectal cancer

Abstract Since 1990, the overall incidence and mortality rates of late onset colorectal cancer (LOCRC ≥50 years) have been decreasing due to improvements in access to screening and available treatment options. However, the rates for early onset CRC (EOCRC ≤ 49 years) have steadily increased in men and women. Less than 30% of LOCRC patients present with late-stage tumors (clinical stages III or IV) at the time of diagnosis compared to 70% of EOCRC patients. Despite this, the cellular and molecular mechanisms underlying the incidence and stage are unclear, as are appropriate strategies for prevention of recurrence in men and women with EOCRC. Effective prognostic markers are therefore urgently needed. To address this issue, we performed a detailed bioinformatics analysis based on Gene Expression Omnibus database (GSE39582; n=585 patients), wherein we identified 7 prognostic biomarkers for EOCRC: KRAS, LGR5, CTNNB1, MYC, BMI1, CD44 and ASNS. Importantly, Kaplan Meier analysis revealed tumor onset at a younger age was significantly (p = 0.0082) associated with poorer 5-year survival in men with EOCRC only, after adjusting for KRAS status. CTNBB1 and CD44 transcript levels were higher in men with EOCRC versus men with LOCRC. Conversely, female EOCRC had a higher frequency of KRAS mutations (52.6%) compared to their LOCRC counterpart (38.8%), and an increased expression of markers of tumor stem cell and metabolic reprogramming including LGR5, MYC, BMI, and ASNS. A single CRISPR-Cas9 deletion of ASNS in HCT116 cell lines, a pre-clinical model of EOCRC, caused an inhibition of 3D spheroid formation compared to HCT116 ASNS wild type cells. In vivo study using a cell line derived R2G2 mouse xenograft model showed that ASNS disruption caused a reduction in tumor burden that was accompanied by low Lgr5 and Myc transcripts compared to the ASNS wild type group, indicating that ASNS may regulate cancer stemness. Together, this data suggests a unique molecular fingerprint for EOCRC and highlights sex-specific influences on cellular metabolism during EOCRC progression. Citation Format: Oladimeji Aladelokun, Kaelyn Sumigray, Sajid A. Khan, Caroline Johnson. Sex differences in cancer stemness among individuals with early onset colorectal 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 250.

Abstract 983: Epigenomic analysis of plasma cell-free DNA identifies stemness features associated with worse prognosis in AR-altered lethal metastatic castration resistant prostate cancer

Abstract Background: Metastatic castration-resistant prostate cancer (mCRPC) resistant to androgen receptor (AR)-targeted agents is often lethal. Unfortunately, biomarkers for this deadly disease remain under investigation, and underpinning mechanisms are ill-understood. Here, we applied epigenomic approaches to identify cell-free (cf) DNA features associated with these lethal AR-resistant mCRPC patients. Methods: Plasma from 99 mCRPC patients was collected from two independent institutions, either prior to first-line AR-targeted therapy (n = 63) or during treatment (n = 36). We applied EnhanceAR-Seq to detect alterations in the AR locus including the upstream enhancer. We then applied genome-wide cfDNA epigenomics to 43 pre-treatment plasma samples and performed both fragmentomics and methylation sequencing to delineate the biology of AR/enhancer-altered lethal mCRPC. Finally, we identified stemness genes by applying CytoTRACE to external single-cell RNA-seq data from mCRPC, and applied these to cfDNA via promoter-level methylation analysis. We then assessed these stem-associated genes in tumor bulk RNA-seq data from an independent mCRPC cohort. Results: AR/enhancer alterations were detected in 44% of pre-treatment cfDNA samples and correlated with significantly worse progression-free survival (PFS) (HR = 2.12, P = 0.01) and overall survival (OS) (HR = 2.48; P = 0.02). AR/enhancer alterations were detected in 19% of on-treatment cfDNA samples and were also associated with profoundly worse PFS (HR = 15.38, P = 0.0003) and OS (HR = 15.53, P = 0.002). Further, genome-wide cfDNA fragmentomic analysis revealed that binding sites for developmentally relevant transcription factors such as FOXA1, NKX-3 and HOXB13 were significantly more accessible in AR/enhancer-altered lethal mCRPC patients. Gene set enrichment analysis of the 20 most accessible transcription factors revealed several pathways associated with stem cell development. Interestingly, the 10 most stem-associated genes obtained using CytoTRACE applied to an external cohort of single-cell RNA-seq data from mCRPC (He et al. 2021) were promoter-hypomethylated in the plasma cfDNA of AR/enhancer-altered lethal mCRPC patients (P = 0.005). Further, metagene analysis of these stem-associated genes revealed worse PFS by Kaplan-Meier analysis (P = 0.04), which was confirmed by multivariate cox proportional hazards modeling. Our stemness metagene further stratified overall survival (P = 0.03) when applied to an external cohort of 80 mCRPC patients from Abida et al. 2019. Conclusions: Alterations in AR including the upstream enhancer are remarkably prognostic in mCRPC with pre-treatment predictive potential. The mechanism can be inferred via cfDNA epigenomic analysis and appears to involve increased tumor cell stemness in AR-altered lethal mCRPC. Citation Format: Pradeep Singh Chauhan, Irfan Alahi, Savar Sinha, Ryan Mueller, Alexander L. Shiang, Jace Webster, Ha X. Dang, Debanjan Saha, Lilli Greiner, Breanna Yang, Elisa M. Ledet, Ramandeep K. Babbra, Wenjia Feng, Peter K. Harris, Faridi Qaium, Ellen B. Jaeger, Patrick J. Miller, Sydney A. Caputo, Oliver A. Sartor, Russell K. Pachynski, Christopher A. Maher, Aadel A. Chaudhuri. Epigenomic analysis of plasma cell-free DNA identifies stemness features associated with worse prognosis in AR-altered lethal metastatic castration resistant prostate 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 983.

Sulfarotene Inhibits Colorectal Cancer via Mitigating Natural-Killer-Cell-Induced Stemness.

Tumor cell stemness stands out as a pivotal factor driving tumor recurrence or metastasis and significantly contributes to the mortality of patients with colorectal cancer (CRC). Recent research has unveiled a link between immune-active cells and the induction of tumor cell stemness, ultimately leading to heightened resistance to treatment. In this study, stemness in CRC cell lines was assessed after co-culture with natural killer (NK) cells, both with and without sulfarotene administration. Furthermore, a CRC xenograft model was utilized to scrutinize the in vivo efficacy of sulfarotene in overcoming stemness induced by NK cell activation. As a result, CRC cells exhibited significant stemness after NK cell co-culture, as evidenced by the upregulation of several stemness markers associated with cancer stem cells. Moreover, these cells demonstrated remarkable resistance to commonly used chemotherapy agents for CRC, such as oxaliplatin and irinotecan. Importantly, sulfarotene effectively reversed the altered stemness of CRC cells in both in vitro and in vivo assays. In conclusion, sulfarotene emerges as a promising therapeutic strategy for overcoming colorectal cancer resistance to NK cells by effectively inhibiting stemness remodeling. This study underscores the potential of sulfarotene in augmenting NK-cell-mediated immune surveillance, proposing a novel immunotherapeutic approach against colorectal cancer.

Development and clinical validation of a seven-gene signature based on tumor stem cell-related genes to predict ovarian cancer prognosis

ObjectiveTumors are highly heterogeneous, and within their parenchyma, a small population of tumor-stem cells possessing differentiation potential, high oncogenicity, and self-renewal capabilities exists. These cells are pivotal in mediating tumor development, chemotherapy resistance, and recurrence. Ovarian cancer shares characteristics with tumor stem cells, making it imperative to investigate molecular markers associated with these cells.MethodsStem cell-related genes were collected, and molecular subtypes were established based on gene expression profiles from The Cancer Genome Atlas using the R package tool “ConsensusClusterPlus.” Multi-gene prognostic markers were identified using LASSO regression analysis. Gene set enrichment analysis was employed to gain insights into the potential molecular mechanisms of these identified markers. The robustness of these prognostic markers was analyzed across different cohorts, and their clinical independence was determined through multivariate Cox analysis. A nomogram was constructed to assess the model’s clinical applicability. Immunohistochemistry was performed to validate the expression of hub genes.ResultsUtilizing 49 tumor stem cell-related genes associated with prognosis, 362 ovarian cancer samples were divided into two distinct clusters, revealing significant prognostic disparities. A seven-gene signature (GALP, CACNA1C, COL16A1, PENK, C4BPA, PSMA2, and CXCL9), identified through LASSO regression, exhibited stability and robustness across various platforms. Multivariate Cox regression analysis confirmed the signature’s independence in predicting survival in patients with ovarian cancer. Furthermore, a nomogram combining the gene signature demonstrated strong predictive abilities. Immunohistochemistry results indicated significantly elevated GALP, CACNA1C, COL16A1, PENK, C4BPA, PSMA2, and CXCL9 expression in cancer tissues.ConclusionThe seven-gene signature holds promise as a valuable tool for decision-making and prognosis prediction in patients with ovarian cancer.

Dynamic interplay of nuclear receptors in tumor cell plasticity and drug resistance: Shifting gears in malignant transformations and applications in cancer therapeutics.

Recent advances have brought forth the complex interplay between tumor cell plasticity and its consequential impact on drug resistance and tumor recurrence, both of which are critical determinants of neoplastic progression and therapeutic efficacy. Various forms of tumor cell plasticity, instrumental in facilitating neoplastic cells to develop drug resistance, include epithelial-mesenchymal transition (EMT) alternatively termed epithelial-mesenchymal plasticity, the acquisition of cancer stem cell (CSC) attributes, and transdifferentiation into diverse cell lineages. Nuclear receptors (NRs) are a superfamily of transcription factors (TFs) that play an essential role in regulating a multitude of cellular processes, including cell proliferation,differentiation, and apoptosis. NRs have been implicated to play a critical role in modulating gene expression associated with tumor cell plasticity and drug resistance. This review aims to provide a comprehensive overview of the current understanding of how NRs regulate these key aspects of cancer biology. We discuss the diverse mechanisms through which NRs influence tumor cell plasticity, including EMT, stemness, and metastasis. Further, we explore the intricate relationship between NRs and drug resistance, highlighting the impact of NR signaling on chemotherapy, radiotherapy andtargeted therapies. We also discuss the emerging therapeutic strategies targeting NRs to overcome tumor cell plasticity and drug resistance. This review also provides valuable insights into the current clinical trials that involve agonists or antagonists of NRs modulating various aspects of tumor cell plasticity, thereby delineating the potential of NRs as therapeutic targets for improved cancer treatment outcomes.

Identification of TMZ resistance-associated histone post-translational modifications in glioblastoma using multi-omics data.

Glioblastoma multiforme (GBM) is among the most aggressive cancers, with current treatments limited in efficacy. A significant hurdle in the treatment of GBM is the resistance to the chemotherapeutic agent temozolomide (TMZ). The methylation status of the MGMT promoter has been implicated as a critical biomarker of response to TMZ. To explore the mechanisms underlying resistance, we developed two TMZ-resistant GBM cell lines through a gradual increase in TMZ exposure. Transcriptome sequencing of TMZ-resistant cell lines revealed that alterations in histone post-translational modifications might be instrumental in conferring TMZ resistance. Subsequently, multi-omics analysis suggests a strong association between histone H3 lysine 9 acetylation (H3K9ac) levels and TMZ resistance. We observed a significant correlation between the expression of H3K9ac and MGMT, particularly in the unmethylated MGMT promoter samples. More importantly, our findings suggest that H3K9ac may enhance MGMT transcription by facilitating the recruitment of the SP1 transcription factor to the MGMT transcription factor binding site. Additionally, by analyzing single-cell transcriptomics data from matched primary and recurrent GBM tumors treated with TMZ, we modeled the molecular shifts occurring upon tumor recurrence. We also noted a reduction in tumor stem cell characteristics, accompanied by an increase in H3K9ac, SP1, and MGMT levels, underscoring the potential role of H3K9ac in tumor relapse following TMZ therapy. The increase in H3K9ac appears to enhance the recruitment of the transcription factor SP1 to its binding sites within the MGMT locus, consequently upregulating MGMT expression and driving TMZ resistance in GBM.

Advances in the biological characteristics and stem maintenance mechanisms of tumor stem cells

Tumor is one of the most serious diseases that threaten human health and social development, and it is the second most common cause of death worldwide. The latest statistics show that malignant tumors have surpassed cardiovascular disease as the leading cause of death in developed countries. Drug resistance, metastasis, and recurrence of tumors continue to present urgent challenges in clinical treatment. Tumor stem cells (TSCs) are a specific subset of cells that possess high capabilities of self-renewal, differentiation potential, tumorigenicity and drug resistance. They are resistant to non-specific treatment methods such as chemotherapy and radiotherapy, and play a crucial role in tumor initiation, metastasis, drug resistance, and recurrence. The surface markers, stemness maintenance mechanisms, microenvironment, and metabolic reprogramming of TSCs have become areas of intense research focus. The latest research results provide novel targets and strategies for the identification of TSCs and targeted therapy. This paper reviews the surface markers (CD133, CD44, etc.), self-renewal and epithelial mesenchymal transition (EMT) signaling pathways (Wnt/β-catenin, Hedgehog, etc.), microenvironment characteristics, metabolic reprogramming (glycolysis, oxidative phosphorylation, etc.) and their roles in the initiation, development, metastasis and drug resistance of TSCs.

Tumor cell stemness in gastrointestinal cancer: regulation and targeted therapy.

The cancer stem cells are a rare group of self-renewable cancer cells capable of the initiation, progression, metastasis and recurrence of tumors, and also a key contributor to the therapeutic resistance. Thus, understanding the molecular mechanism of tumor stemness regulation, especially in the gastrointestinal (GI) cancers, is of great importance for targeting CSC and designing novel therapeutic strategies. This review aims to elucidate current advancements in the understanding of CSC regulation, including CSC biomarkers, signaling pathways, and non-coding RNAs. We will also provide a comprehensive view on how the tumor microenvironment (TME) display an overall tumor-promoting effect, including the recruitment and impact of cancer-associated fibroblasts (CAFs), the establishment of an immunosuppressive milieu, and the induction of angiogenesis and hypoxia. Lastly, this review consolidates mainstream novel therapeutic interventions targeting CSC stemness regulation.

IL-24 improves efficacy of CAR-T cell therapy by targeting stemness of tumor cells.

Cancer stem cells (CSCs) induce therapeutic resistance and may be an important barrier to cancer immunotherapy. Chimeric antigen receptor T (CAR-T) cell therapy has demonstrated remarkable efficacy in clinical settings. However, CAR-T cell therapy fails in a large proportion of patients, especially in those with solid tumors. It is unclear how CSCs mediate resistance to CAR-T cells, and whether CAR-T cells can more effectively eradicate CSCs. In this study, the effect of CSCs on CAR-T cell therapy was determined using in vitro and in vivo assays. Subsequently, Interleukin-24 (IL-24) was expressed along with CAR in T cells. Further in vitro and in vivo tests were performed to determine the effects of IL-24 on CSCs and CAR-T cell therapy. IL-24 induced apoptosis in CSCs and contributed to T cell activation, differentiation, and proliferation. CAR.IL-24-T cells inhibited CSC enrichment and exhibited stronger antitumor activity in vitro and in vivo. IL-24 helps eliminate CSCs and endows CAR-T cells with improved antitumor reactivity.