Cancer Stem Cell Markers Research Articles

The Combination of Afatinib With Dasatinib or Miransertib Results in Synergistic Growth Inhibition of Stomach Cancer Cells.

Of various human epidermal growth factor receptor (HER) inhibitors, only the anti-HER2 monoclonal antibody (mAb) Herceptin/trastuzumab and the antibody-drug conjugate trastuzumab deruxtecan (T-Dxd) has been approved for the treatment of patients with stomach cancer. However, the duration of response may be short in many patients, with tumor heterogeneity being one contributing factor. We investigated the effect of various types of targeted agents on growth in vitro and migration of a panel of human stomach cancer cells (HSCCLs) and the impact of cell proliferation rate on the anti-tumor activities of these agents. We also investigated the association between the cell surface expression of the HER family members, hepatocyte growth factor receptor (c-Met), anaplastic lymphoma kinase (ALK)7 and cancer stem cell markers CD44 and CD133, and the response to the targeted agents. Of the 18 agents examined, the cyclin dependent kinase (CDK) 1/2/5/9 inhibitor dinaciclib was the most effective and inhibited the growth of all human HSCCLs at 50% inhibitory concentration (IC50) values between 9 nM to 23 nM. Of various HER inhibitors, the irreversible pan-HER family inhibitors (e.g., afatinib) were more effective than the reversible dual epidermal growth factor receptor (EGFR)/HER2 tyrosine kinase inhibitor (TKI) lapatinib and the EGFR-specific TKI erlotinib in inhibiting the growth of HSCCLs. Of agents targeting different downstream cell signaling molecules, dasatinib targeting Ab1/Src/C-Kit, trametinib targeting MERK1/2 and miransertib targeting AKT1/2/3 inhibited growth of majority of HSCCLs, with the IC50 values ranging from 2 nM to 7 µM. Many of these agents were more effective in inhibiting the growth of HSCCLs when they were proliferating at a slower rate. Treatment with neratinib, afatinib, dinaciclib, dasatinib, stattic, miransertib and paclitaxel significantly inhibited migration of stomach cancer cells. Interestingly, treatment with a combination of afatinib and dasatinib or afatinib and miransertib resulted in synergistic and additive growth inhibition of stomach cancer cells. These results suggest that treatment with a combination of these agents may be of therapeutic value in stomach cancer and warrants further investigations.

Correlation of prognosis of oral squamous cell carcinoma with CD44 expression: A retrospective immunohistochemical analysis

ABSTRACT Background: Cluster of differentiation (CD44) is a cancer stem cell marker responsible for angiogenesis, invasion, and migration in oral squamous cell carcinoma (OSCC). Objectives: The primary objective of the study was to analyze the difference in expression of CD44 among different grades of OSCC. The secondary objective was to correlate the prognosis with the expression. Materials and Methods: Based on histopathological diagnosis, we retrieved OSCC paraffin-embedded blocks from the archives of the Department of Oral Pathology, Sri Ramaswamy Memorial (SRM) Dental College, Chennai, India, reported from January 2012 to May 2022. We evaluated sections from 15 tissues of normal oral mucosa, 15 paraffin blocks of well-differentiated OSCC, 15 moderately differentiated OSCC, and 15 poorly differentiated OSCC for CD44 immunostaining. Immunohistochemical expression of CD44 was studied in these tissues and graded based on the intensity of staining. The difference in immune expression of CD44 between different grades was quantitatively analyzed using image analysis by Fiji image analysis software. Analysis of variance (ANOVA) was used to compare the mean difference in expression between different groups by using the Statistical Package for Social Sciences (SPSS) software version 16. Statistical significance was set at P < 0.05. Results: We identified that 10% (n = 6) of the normal mucosa showed strong expression of CD44, whereas 13.3% (n = 8) of well-differentiated OSCC, 10% (n = 6) of moderately differentiated OSCC, and 0% (n = 0) of poorly-differentiated OSCC were strongly positive for CD44 expression. The difference in comparison was statistically significant (P, 0.003). By quantitative analysis, the mean expression of CD44-positive cells in well-differentiated OSCC was 38258 ± 4762, moderately-differentiated OSCC was 27353 ± 1357, and poorly-differentiated OSCC was 10265 ± 1685. Upon comparison, the difference in mean was found to be significant (P, 0.002). Conclusion: The expression of CD44 was noted to decrease from well-differentiated to poorly-differentiated OSCC.

Enhancing the anticancer effect of androgen deprivation therapy by monocarboxylate transporter 1 inhibitor in prostate cancer cells.

Tumor initiation and progression necessitate a metabolic shift in cancer cells. Consequently, the progression of prostate cancer (PCa), a leading cause of cancer-related deaths in males globally, involves a shift from lipogenic to glycolytic metabolism. Androgen deprivation therapy (ADT) serves as the standard treatment for advanced-stage PCa. However, despite initial patient responses, castrate resistance emerges ultimately, necessitating novel therapeutic approaches. Therefore, in this study, we aimed to investigate the role of monocarboxylate transporters (MCTs) in PCa post-ADT and evaluate their potential as therapeutic targets. PCa cells (LNCaP and C4-2 cell line), which has high prostate-specific membrane antigen (PSMA) and androgen receptor (AR) expression among PCa cell lines, was used in this study. We assessed the expression of MCT1 in PCa cells subjected to ADT using charcoal-stripped bovine serum (CSS)-containing medium or enzalutamide (ENZ). Furthermore, we evaluated the synergistic anticancer effects of combined treatment with ENZ and SR13800, an MCT1 inhibitor. Short-term ADT led to a significant upregulation in folate hydrolase 1 (FOLH1) and solute carrier family 16 member 1 (SLC16A1) gene levels, with elevated PSMA and MCT1 protein levels. Long-term ADT induced notable changes in cell morphology with further upregulation of FOLH1/PSMA and SLC16A1/MCT1 levels. Treatment with ENZ, a nonsteroidal anti-androgen, also increased PSMA and MCT1 expression. However, combined therapy with ENZ and SR13800 led to reduced PSMA level, decreased cell viability, and suppressed expression of cancer stem cell markers and migration indicators. Additionally, analysis of human PCa tissues revealed a positive correlation between PSMA and MCT1 expression in tumor regions. Our results demonstrate that ADT led to a significant upregulation in MCT1 levels. However, the combination of ENZ and SR13800 demonstrated a promising synergistic anticancer effect, highlighting a potential therapeutic significance for patients with PCa undergoing ADT.

A meta-analysis evaluating the role of calcium channel alpha-2 delta-1 subunit in carcinogenesis

There is hardly found any study accumulating all the experiments reported with the expression of alpha-2 delta-1 (α2δ-1) in cancer cells. This meta-analysis aimed to advance our knowledge about the role of calcium channel alpha2 delta-1 subunit in carcinogenesis in the present time. PubMed searches for peer-reviewed articles were conducted using the keywords “α2δ-1 protein in oncogenesis”, “α2δ-1 protein expression in cancer cells”, and “α2δ-1 protein as cancer cell marker”. The databases were developed in accordance with PRISMA guidelines. Seventeen studies out of 80 citations met the inclusion criteria pertaining to α2δ-1 expression in different cancer cells. The cancer patterns were hepatocellular carcinoma in 41%, non-small cell lung carcinoma in 12% and laryngeal squamous cell carcinoma in 12%. The remaining studies included small-cell lung cancer (6%), gastric cancer (6%), pancreatic cancer (6%), hypopharyngeal squamous cell carcinoma (6%), breast cancer (6%) and glioblastoma multiforme (6%). α2δ-1+ cells had a higher sphere-forming and tumorigenic efficiency in 76.5% of experiments. 58.8% experiments explored mechanistically in self-renewal efficiency and tumorigenesis of α2δ-1+ cancer cells. The cancer cells expressing α2δ-1 have the potential to serve as cell surface markers for tumour-initiating cells and cancer stem cells. These intriguing findings open up a promising avenue for future research, focusing on the targeting of α2δ-1 as a potential therapeutic strategy for cancer treatment.

Hsa_circTCF25 Facilitates Oncogenesis in Gastric Cancer Cells by Modulating miR-149 Expression.

Circular RNA (circRNA) is related to gastric carcinogenesis and progression. This study explored the effects of circTCF25 on gastric cancer cell proliferation, migration, invasion, and cancer stem cell markers, as well as the potential network of circTCF25-miR and miR-149. circTCF25 expression was detected in tissue specimens and cells by real-time quantitative reverse transcription polymerase chain reaction. Cell Counting Kit-8 and transwell assays were used to measure the effects of circTCF25 knockdown on proliferation, migration and invasion. The potential network of circTCF25 was analyzed using bioinformatic analysis. circTCF25 was overexpressed in human gastric cancer tissues, and a series of cancer cell lines, and was associated with shorter overall survival. Interfering with circTCF25 reduced gastric cancer cell proliferation, migration, invasion and expression of cancer stem cell markers. CircTCF25 reduced expression of miR-149, apparently by acting as a miR-149 sponge. A new circTCF25-miR-149 competitive endogenous RNA network in gastric cancer was constructed, and most core genes were associated with the malignant growth and metastatic behavior of gastric cancer. circTCF25 may have prognostic value and an oncogenic role in gastric cancer. A circTCF25-miR-149 RNA regulatory network was established which may provide novel biomarkers or potential therapeutic targets for treating gastric cancer.

Selected markers of ovarian cancer and their relation to targeted therapy (Review).

Despite advances in surgical treatment techniques and chemotherapy-including anti-angiogenic and immune poly (ADP-ribose) polymerase inhibitors, the 5-year survival rate in ovarian cancer (OC) remains low. The reasons for this are the diagnosis of cancer in advanced clinical stages, chemoresistance and cancer recurrence. New therapeutic approaches are being developed, including the search for new biomarkers that are also targets for targeted therapy. The present review describes new molecular markers with relevance to targeted therapy, which to date have been studied only in experimental research. These include the angiogenic protein angiopoietin-2, the transmembrane glycoprotein ectonucleotide pyrophosphatase/phosphodiesterase 1, the adhesion protein E-cadherin, the TIMP metallopeptidase inhibitor 1 and Kruppel-like factor 7. Drugs affecting cancer stem cells (CSCs) in OC, such as metformin and salinomycin, as well as inhibitors of CSCs markers aldehyde dehydrogenase 1 (with the drug ATRA) and the transcription factor Nanog homeobox (microRNA) are also discussed. A new approach to prevention and possible therapies under investigation such as development of vaccines containing a subpopulation of CD117(+) and CD44(+) stem cells with a promising option for use in women with OC was described.

Anti-IL-8 monoclonal antibodies inhibits the autophagic activity and cancer stem cells maintenance within breast cancer tumor microenvironment.

Breast cancer tumor microenvironment (TME) is a promising target for immunotherapy. Autophagy, and cancer stem cells (CSCs) maintenance are essential processes involved in tumorigenesis, tumor survival, invasion, and treatment resistance. Overexpression of angiogenic chemokine interleukin-8 (IL-8) in breast cancer TME is associated with oncogenic signaling pathways, increased tumor growth, metastasis, and poor prognosis. Thus, we aimed to investigate the possible anti-tumor effect of neutralizing antibodies against IL-8 by evaluating its efficacy on autophagic activity and breast CSC maintenance. IL-8 monoclonal antibody supplemented tumor tissue culture systems from 15 females undergoing mastectomy were used to evaluate the expression of LC3B as a specific biomarker of autophagy and CD44, CD24 as cell surface markers of breast CSCs using immunofluorescence technique. Our results revealed that anti-IL-8 mAb significantly decreased the level of LC3B in the cultured tumor tissues compared to its non-significant decrease in the normal breast tissues.Anti-IL-8 mAb also significantly decreased the CD44 expression in either breast tumors or normal cultured tissues. While it caused a non-significant decrease in CD24 expression in cultured breast tumor tissue and a significant decrease in its expression in the corresponding normal ones. Anti-IL-8 monoclonal antibody exhibits promising immunotherapeutic properties through targeting both autophagy and CSCs maintenance within breast cancer TME.

Shrimp Lipids Inhibit Migration, Epithelial-Mesenchymal Transition, and Cancer Stem Cells via Akt/mTOR/c-Myc Pathway Suppression.

Shrimp is a rich source of bioactive molecules that provide health benefits. However, the high cholesterol content in shrimp oil may pose a risk. We utilized the cholesterol elimination method to obtain cholesterol-free shrimp lipids (CLs) and investigated their anticancer potential, focusing on cancer stem cells (CSCs) and epithelial-to-mesenchymal transition (EMT). Our study focused on CSCs and EMT, as these factors are known to contribute to cancer metastasis. The results showed that treatment with CLs at doses ranging from 0 to 500 µg/mL significantly suppressed the cell migration ability of human lung cancer (H460 and H292) cells, indicating its potential to inhibit cancer metastasis. The CLs at such concentrations did not cause cytotoxicity to normal human keratinocytes. Additionally, CL treatment was found to significantly reduce the levels of Snail, Slug, and Vimentin, which are markers of EMT. Furthermore, we investigated the effect of CLs on CSC-like phenotypes and found that CLs could significantly suppress the formation of a three-dimensional (3D) tumor spheroid in lung cancer cells. Furthermore, CLs induced apoptosis in the CSC-rich population and significantly depleted the levels of CSC markers CD133, CD44, and Sox2. A mechanistic investigation demonstrated that exposing lung cancer cells to CLs downregulated the phosphorylation of Akt and mTOR, as well as c-Myc expression. Based on these findings, we believe that CLs may have beneficial effects on health as they potentially suppress EMT and CSCs, as well as the cancer-potentiating pathway of Akt/mTOR/c-Myc.

CD44 and CD133 protein expression might serve as a prognostic factor for early occurrence castration-resistant prostate cancer.

The occurrence of castration-resistant prostate cancer (CRPC) varies in patients with advanced prostate cancer (PCa) undergoing androgen deprivation therapy (ADT). The rate of occurrence of CRPC may be related to the presence of prostate cancer stem cells (CSC). Thus, this study aims to evaluate the presence of CSC markers (CD44 and CD133) in histopathology tissue at the time of diagnosis and their correlation with the occurrence of CRPC in patients with advanced PCa within 2 years of ADT. A retrospective case-control study was conducted to evaluate the incidence of CRPC within 2 years. The inclusion criteria were patients with PCa who had received treatment with ADT and a first-generation anti-androgen (AA) for 2 years. We classified patients based on whether they developed CRPC within 2 years (CRPC) of the therapy or did not experience CRPC within 2 years (non-CRPC) of the therapy. We performed immunohistochemical (IHC) staining for CD44 and CD133 on the prostate biopsy tissue samples. Data were collected from records spanning 2011-2019. We analyzed a total of 65 samples, including 22 patients with CRPC and 43 patients with non-CRPC who had received treatment with LHRH agonists and AA for up to 2 years. Our findings showed a significant H-score difference in CD44 protein expression between CRPC prostate adenocarcinoma samples 869 (200-1329) and non-CRPC 524 (154-1166) (p = 0.033). There was no significant difference in CD133 protein expression between the two groups (p = 0.554). However, there was a significant difference in the nonoccurrence of CRPC between the high expressions of both CD44 and CD133 groups with other expressions of CD44/CD133 groups (25% vs. 75%; p = 0.011; odds ratio = 4.29; 95% confidence interval [1.34, 13.76]). This study found a low expression of at least one CD44/CD133 protein in the patients without early occurrence of CRPC. This result might suggest that CD44/CD133 may function as a potential prognostic marker for PCa, especially in a low expression, to identify patients who have a better prognosis regarding the occurrence of early CRPC.

Osteopontin promotes tumor growth and metastasis and GPX4-mediated anti-lipid peroxidation in triple-negative breast cancer by activating the PI3k/Akt/mTOR pathway

PurposeTriple-negative breast cancer (TNBC) features high aggressiveness, metastasis rate, drug resistance as well as poor prognosis. Osteopontin (OPN) is a key protein in the process of osteogenesis and has emerged as a new tumor marker in recent years.MethodsCell viability was tested with the CCK-8 kit. Transwell and wound healing were adopted to test cell invasive and migratory abilities. Tumor sphere formation was detected by tumor sphere formation assay. Human umbilical vein endothelial cell (HUVEC) tube formation assay was used to measure the angiogenesis of tumor cells. Western blot was applied for the estimation of the expression of cancer stem cell markers, angiogenesis-, signaling pathway-related proteins as well as OPN. Bioinformatics tools predicted OPN expression in breast cancer tissues. The levels of oxidative stress-related markers were assessed with ELISA. Following the overexpression of OPN in MD-MB-436 cells and the addition of the PI3K/AKT/mTOR pathway inhibitor LY294002, the aforementioned functional experiments were implemented again to investigate the mechanism. Finally, in vivo experiments of tumor-bearing mice were performed for further verification. ResultsThe proliferative, invasive, migratory and tumor sphere formation capabilities as well as angiogenesis of TNBC cells were conspicuously increased in contrast to non-TNBC cell lines. OPN expression in TNBC tissues and cells was dramatically enhanced. OPN upregulation significantly elevated cell proliferative, invasive and migratory capabilities as well as tumor sphere formation and angiogenesis. The mechanism might be achieved by activating PI3K/AKT/mTOR signaling to regulate glutathione peroxidase 4 (GPX4)-mediated anti-lipid peroxidation.ConclusionOPN promoted tumor sphere formation and angiogenesis in TNBC by activating the PI3K/AKT/mTOR pathway to regulate GPX4-mediated anti-lipid peroxidation levels.

Single-cell and spatial analyses revealed the co-location of cancer stem cells and SPP1+ macrophage in hypoxic region that determines the poor prognosis in hepatocellular carcinoma

In hepatocellular carcinoma (HCC), classical cancer stem cells (CSC) markers were shared by normal stem cells, targeting which may hinder hepatic regeneration and cause liver failure. Additionally, the spatial structure of CSC still remained elusive. To address these limitations, we undertook a comprehensive study combining single-cell data (56,022 cells from 20 samples) and spatial data (38,191 spots from eight samples) to obtain CSC signature and uncover its spatial structure. Utilizing the CytoTRACE algorithm, we discretely identified CSC, which displayed upregulated proliferation pathways regulated by HIF1A. A CSC signature of 107 genes was then developed using Weighted Gene Co-expression Network Analysis (WGCNA). Notably, HCC patients with high CSC levels exhibited an accumulation of SPP1+ macrophages (Macro_SPP1) expressing metalloproteinases (MMP9, MMP12, and MMP7) regulated by HIF1A, suggesting a hypoxic tumor region connecting Macro_SPP1 and CSC. Both CSC and Macro_SPP1 correlated with worse prognosis and undesirable immunotherapy response. Spatial analysis revealed the co-location of CSC and Macro_SPP1, with CD8 T cells excluded from the tumor region. The co-location area and non-tumor area of boundary exhibited a high level of hypoxia, with the HAVRC2 checkpoint highly expressed. Within the co-location area, the SPP1 signaling pathway was most active in cell-cell communication, with SPP1-CD44 and SPP1-ITGA/ITGB identified as the main ligand-receptor pairs. This study successfully constructed a CSC signature and demonstrated the co-location of CSC and Macro_SPP1 in a hypoxic region that exacerbates the tumor microenvironment in HCC.

Abstract 5439: Metastasis-enhancing protein KITENIN confers temozolomide resistance in glioblastoma with unmethylated MGMT via upregulation of cancer stem cell makers

Abstract Background: Elevated expression of cancer stem cell (CSC) markers is linked with progression, poor survival, and treatment resistance in glioblastoma (GBM). Our prior work identified KAI1 COOH-terminal interacting tetraspanin (KITENIN) as key to GBM progression. We investigated its influence on temozolomide (TMZ) resistance in GBM through modulation of CSC markers. Methods: We examined KITENIN and CSC markers in TMZ-resistant GBM cells and patient-derived GBM cells, and assessed therapeutic responses to TMZ in mice implanted with KITENIN-modulated GBM cells. The correlation of these markers with clinical outcomes was analyzed using our cohort and The Cancer Genome Atlas (TCGA) data. Results: High KITENIN expression correlated positively with elevated CSC marker levels in human GBM samples and KITENIN-modulated GBM cells. Inhibition of KITENIN using usnic acid reduced tumor progression by decreasing CSC marker expression. Our data and TCGA analyses linked co-directional increase of these factors with tumor recurrence and shorter survival in GBM patients with unmethylated MGMT. This increase was also observed in TMZ-resistant cell lines with unmethylated MGMT. Implantation of KITENIN-overexpressing cells led to TMZ resistance in our mouse model, associated with co-directionally increased CD44 and ALDH1A1 expression. The link between KITENIN and CD44/ALDH1A1 could be inhibited by DKC2511, a novel KITENIN inhibitor, and T5244, a c-Fos/AP-1 inhibitor. Conclusion: KITENIN might contribute to TMZ resistance in GBM patients with unmethylated MGMT, potentially through AP-1 activation and subsequent upregulation of CSC markers. Hence, targeting KITENIN could provide a novel strategy to overcome TMZ resistance in GBM patients with unmethylated MGMT. Citation Format: Eun-Jeong Ahn, Yeong Jin Kim, Tae-Young Jung, Jae-Hyuk Lee, Joon Haeng Rhee, Kyung-Keun Kim, Sung Sun Kim, Nah Ihm Kim, Hangun Kim, Kyung-Sub Moon, Kyung-Hwa Lee. Metastasis-enhancing protein KITENIN confers temozolomide resistance in glioblastoma with unmethylated MGMT via upregulation of cancer stem cell makers [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 5439.

Abstract 6311: From bench to bedside: GMP manufacturing and testing of an LGR5-targeting CAR-T against colorectal cancer

Abstract Colorectal cancer is the third most diagnosed cancer worldwide, and is responsible for the second largest number of cancer related deaths, warranting the need for new treatments. The successful use of chimeric antigen receptor T (CAR-T) cells against B-cell malignancies has inspired the search for CAR-T cell therapies against solid tumours such as colorectal cancer. Cancer stem cells (CSC) are a small population of cells within a tumour with the capacity to self-renew, differentiate, and initiate new tumours. CSC are resistant to chemotherapy and radiotherapy and are enriched in residual disease. Targeting CSC is a strategy to reduce the tumours’ ability to generate new cells. Leucine-rich G protein-coupled receptor 5 (LGR5) is a marker of CSC and LGR5+ cancer cells are implicated in tumour progression and metastasis in colorectal cancer. We designed and tested a chimeric antigen receptor (CAR) targeting the extracellular domain of LGR5. LGR5-targeting CAR-T cells showed significant antigen-specific cytotoxicity and IFNγ release against colorectal cancer (CRC) cell lines in vitro, and in vivo efficacy in a CRC xenograft mouse model. We confirmed the specificity and safety of the LGR5 targeting CAR-T cells by testing against normal human tissues. A GMP ready protocol for clinical scale manufacture of LGR5-targeting CAR-T cells have been developed using CD3+ T-cells from healthy donors. We have successfully tested this protocol using CRC patient-derived CD3+ T-cells and were able to generate clinically relevant cell numbers (80-fold expansion) with high CAR expression. Cells manufactured using this GMP protocol expressed markers of self-renewing naïve-like and central memory phenotype and showed very low percentage of cells that were triple-positive for PD1, TIM3 and LAG3. This protocol has been successfully tested and validated in a GMP manufacturing facility for the production of LGR5 targeting CAR-T cells, in preparation for an FDA approved first in man clinical trial against metastatic colorectal cancer, starting in 2023 (NCT05759728). Citation Format: Veronika Bandara, Batjargal Gundsambuu, Silvana Napoli, Jade Foeng, Dylan McPeake, Timona Tylis, Emma Thompson, Stuart Mills, Allison Cowin, Claudine Bonder, Timothy Sadlon, Shaun McColl, Simon Barry. From bench to bedside: GMP manufacturing and testing of an LGR5-targeting CAR-T against 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 6311.

Abstract 1634: Cyclin D1 induces Akt1 activity in RB deficient prostate cancer

Abstract Background: Prostate cancer (PCa), the second leading cause of death in American men, is a genetically heterogeneous disease. The aberrant activation of AKT is one of the most frequent alterations in human cancers, and an elevated level of phosphorylated AKT at Ser473 site (pSer473) is associated with metastatic cancers (1). In prostate cancer (PCa), PI3K/AKT activation induces tumorigenesis (2), androgen receptor (AR)-mediated signaling, enzalutamide resistance and stemness, synergizing with other pathways including the notch intracellular domain (NICD). The cyclin D1 gene (CCND1), which is overexpressed in PCa, conveys canonical function by encoding the regulatory subunit of a holoenzyme that phosphorylates the retinoblastoma gene (RB) product, and non-canonical functions through phosphorylating other substrates and transcriptional activities. Cyclin D1 expression augments growth of some castrate-resistant PCa (CRPC) and clinical trials are targeting the cyclin D1 related CDK kinase activity in pRB+ PCa. Several Rb-independent mechanisms of action have been described (3). In recent studies the effect of CDK inhibitors was similar in patients with Rb+ vs. Rb− PCa (4). Whether Rb expression is required for the antiproliferative effects of CKD4/6 inhibition in PCa remains to be established. Methods: The kinetics of Akt1 induction by mitogens was assessed using the phosphorylation of the dichromic fluorescent (DCF) dye substrate LS456. We used cyclin D1 shRNA to show endogenous cyclin D1 augmented AKT1 signaling in response to mitogens. Results: Cyclin D1 reintroduction into cyclin D1−/− cells augmented Akt1 phosphorylation, requiring the cyclin D1 K112 residue. CDK inhibitors reduced Akt activity in RB+ and RB− human prostate cancer cell lines. Prostate-specific epithelial cell deletion of cyclin D1 (probasin-CRE-cyclin D1fl/fl) reduced prostate epithelial cell Akt1 activity assessed by immunohistochemical (IHC) staining. pAktSer473 was reduced in cell nuclei and the cytoplasm of both epithelial cells and stromal fibroblasts without a significant change in the abundance of Akt1. The downstream targets of Akt signaling (pTSC2Ser939, and pFKHRSer319) and ARSer213P were reduced in both epithelial cells and stromal fibroblasts. Genetic deletion of both epithelial cell and stromal cyclin D1 further reduced Akt1 activity, prostate epithelial cell proliferation, and markers of cancer stem cells. Proteomic analysis of cyclin D1-expressing stroma identified heterotypic chemokine signals that augment PCa AKT activity. Conclusions: Cyclin D1 induces Akt1 activity in both RB+ and RB− PCa cells which may contribute to tumor progression and therapy resistance. Reference: (1) Chen YL, Law PY, Loh HH. Curr Med Chem Anticancer Agents 2005;5:575-892. (2) Stoyanova T, et al. Proc Natl Acad Sci U S A 2013;110:20111-63. (3) Chen K, et al. Cell Rep 2020;32:1081514. (4) de Kouchkovsky I, et al. Clin Cancer Res 2022;28:1531-9 Citation Format: Xuanmao Jiao, Anthony W. Ashton, Zhiping Li, Ritika Harish, Danni Li, Gordon Robertson, Duanwen Shen, Xiaoming Ju, Ke Chen, Wei Zhang, Samuel Archilefu, Kenneth A. Iczkowski, Hallgeir Rui, Beatrice Knudsen, Paul Timpson, Max Nobis, Richard G. Pestell. Cyclin D1 induces Akt1 activity in RB deficient 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 1634.

Abstract 2520: CD133 expression is associated with less DNA repair, better response to chemotherapy, and survival in ER-positive/ER-negative breast cancer

Abstract Introduction: Cancer stem cells (CSC) constitute only 0.1-1% of all the cells in a bulk tumor, yet they play a crucial role in treatment resistance and relapses. Its role in breast cancer (BC) has been studied but a consensus remains elusive. We focused on gene expression of a CSC marker CD133 and studied its clinical significance in ER-positive/HER2-negative (ER+/HER2-) BC, the most prevalent subtype. We utilized multiple independent large patient cohorts of gene expression and clinical variables for transcriptome analysis. Methods: A total of 2969 patients from The Cancer Genome Atlas (TCGA, n=1065) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC, n=1904) BC cohorts, as well as BC patients who underwent neoadjuvant chemotherapy (NAC; GSE25066, GSE20194, GSE32646 cohorts) were analyzed. Results: By analyzing SCP1039 and SCP1106 single-cell sequencing cohorts, we found that epithelial cells were the predominant source of CD133 gene expression in the tumor microenvironment. High expression of CD133 in ER+/HER2- BC correlated with elevated gene expressions of the other CSC markers; CD24, NOTCH1, DLL1, and ALDH1A1 (all p<0.001), and significantly enriched WNT/β-Catenin, Hedgehog, and Notch signaling (all FDR<0.25), which are all known pathways related with CSC in TCGA and validated in METABRIC cohort. Consistent with CSC phenotype, CD133-high BC was negatively enriched with Hallmark cell proliferation-related gene sets, such as E2F Targets, G2M Checkpoint, MYC Targets V1, Ki67 gene expression and proliferation score (FDR<0.25 and p<0.001, respectively) in both cohorts. CD133-high BC was negatively associated with DNA repair genes, such as BRCA1, E2F7 in both cohorts (all p<0.001). In agreement, CD133-high BC was associated with less homologous recombination deficiency, lower silent and non-silent mutation rates. Surprisingly, CD133-high BC exhibited a proinflammatory microenvironment, as represented by higher cytolytic activity in the METABRIC and higher lymphocyte infiltrations, TGF-beta response, and TCR richness in TCGA (both p<0.001), but less Th1 cells, M1 macrophages and dendritic cell infiltrations consistently in both cohorts. It also enriched signaling pathways related to inflammation and immune responses such as inflammatory response, allograft rejection, TNF-alpha, IL-6, TGF-beta signaling, and complement signaling (all FDR<0.25). CD133-high tumors had better pathological complete response after NAC in GSE25066 cohort and improved Disease-Free Survival and Overall Survival in both TCGA and METABRIC cohorts (all p<0.05). Conclusion: CD133-high ER+/HER2- BC is associated with CSC phenotype, reduced cell proliferation, and impaired DNA repair, but enhanced inflammation, potentially explaining the better response to NAC and favorable patient prognosis. Citation Format: Takumi Sato, Masanori Oshi, Jing Lee, Arya M. Roy, Kohei Chida, Itaru Endo, Masato Obayashi, Kazuaki Takabe. CD133 expression is associated with less DNA repair, better response to chemotherapy, and survival in ER-positive/ER-negative breast 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 2520.

Abstract 1566: Cancer metastasis and stemness: The metastasis-inducer MACC1 regulates LGR5 to promote cancer stem cell-like properties in colorectal cancer

Abstract Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. The high mortality is directly associated with metastatic disease which is thought to be initiated by colon cancer stem cells according to cancer stem cell (CSC) model. Consequently, early identification of those patients who are at high risk for metastasis is crucial for improved treatment and patient outcome. Metastasis-associated in colon cancer 1 (MACC1) is a novel prognostic biomarker for tumor progression and metastasis formation independent of tumor stage. We previously showed an involvement of MACC1 in cancer stemness in the mouse intestine of our transgenic mouse models. However, the expression of MACC1 in human CSCs and possible implications remain elusive. Here, we explored the molecular mechanisms by which MACC1 regulates stemness and CSC-associated invasive phenotype based on patient-derived 3D cell culture models (PD3D), patient-derived xenografts (PDX) and human CRC cell lines. We showed that CD44-enriched CSCs from PD3D models express significantly higher levels of MACC1 and display higher tumorigenicity in immunocompromised mice. Similarly, RNA sequencing performed on PD3D and PDX models demonstrated significantly increased MACC1 expression in ALDH1(+) CSCs, highlighting its involvement in cancer stemness. We further showed the correlation of MACC1 with CSC markers CD44, NANOG and LGR5 in PD3D models as well as established cell lines. Additionally, MACC1 increased stem cell gene expression, clonogenicity and sphere formation. Strikingly, we showed that MACC1 binds as a transcription factor to the LGR5 gene promoter uncovering the long-known CSC marker LGR5 as novel essential signaling mediator used by MACC1 to induce CSC-like properties in human CRC patients. Our in vitro findings were further substantiated by significant positive correlation of MACC1 and LGR5 in CRC cell lines as well as CRC patient tumors. Taken together, this study indicates that the metastasis-inducer MACC1 act as a cancer stem cell-associated marker. Interventional approaches targeting MACC1 would potentially improve further targeted therapies for colorectal cancer patients to eradicate CSCs, prevent cancer recurrence and distant metastasis formation. Citation Format: Müge Erdem, Kyung Hwan Lee, Markus Hardt, Joseph Regan, Dennis Kobelt, Wolfgang Walther, Christian Regenbrecht, Ulrike S. Stein. Cancer metastasis and stemness: The metastasis-inducer MACC1 regulates LGR5 to promote cancer stem cell-like properties 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 1566.

Abstract 3642: Phenotypic biomarkers of aqueous humor extracellular vesicles from retinoblastoma eyes

Abstract Purpose: Retinoblastoma (RB) is the most common pediatric intraocular cancer, yet tumor biopsies are contraindicated. This challenge emphasizes the need for investigation of non-tissue biomarkers. Extracellular vesicles (EVs), which are secretory vesicles containing bioactive molecules, have emerged as promising biomarker candidates due to their ability to be phenotyped using known tetraspanin markers. Aqueous humor (AH) provides an exciting liquid biopsy source for intraocular tumor information, with EVs having recently been identified in AH. Our laboratory has previously proposed that CD63/81+EVs are associated with retinoblastoma, but specific tumor-derived markers have not been linked to AH EVs using traditional tetraspanin assays. In this study, we employed MACSPlex, a high-dimensional magnetic bead-based immunoassay, to further explore RB-EV. Methods: 7 AH samples consisting of two congenital glaucoma (GLC) samples and five RB samples taken at the time of primary enucleation were analyzed. We utilized Macsplex, a multiplex bead-based flow cytometry assay, to analyze the expression profiles of surface markers found on EVs. With 39 capture beads coated with monoclonal antibodies for 37 different EV surface antigens and two negative controls, the mean fluorescence intensity (MFI) of APC conjugated antibody cocktail from three tetrapanins (CD9, CD63 and CD81) on EVs captured by each surface antigen could be measured by flow cytometry. Enrichment ratios were then calculated by normalizing the fluorescence of each signal to the mean fluorescence of CD9, CD63, and C81. Results: Macsplex analysis of the 2 GLC AH samples demonstrated a strong CD63 dominance compared with RB AH. The mean CD63 enrichment ratio for GLC samples was 2.6 (S.D. = 0.34), and 1.3 (S.D. = 0.27) in RB samples, with a p-value of 0.069. In comparison to the GLC patients, the analysis of EVs in the AH of the 5 RB samples exhibited a greater co-dominance of CD63/CD81 positivity consistent with past research on RB EVs. The mean CD81 enrichment ratio for the 5 RB samples was 1.5 (S.D. = 0.26), in contrast to 0.3 (S.D. = 0.24) in GLC AH, with a p-value of 0.03. Interestingly, 4 out of the 5 RB AH samples demonstrated very strong CD133 signals, which is a known cancer marker. Deeper investigation into one RB AH sample utilizing single tetraspanin detection revealed higher coexpression of CD63 and CD81 with CD133 than seen with CD9. The CD133 enrichment ratio for the CD81, CD63, and CD9 single channel experiments were 6.6, 3.4, and 1.5 respectively. Conclusions: Comparison between this study and previous work done by our group demonstrate a high concordance between results utilizing Macsplex and Exoview. However, this is the first study identifying the expression of CD133, a cancer stem cell marker, on retinoblastoma-derived EVs in AH. Macsplex technology illustrates an incredible clinical potential of AH EVs for characterizing retinoblastoma phenotype in vivo. Citation Format: Anne Amacker, Chen-Ching Peng, Bibiana J. Reiser, Jesse L. Berry, Liya Xu. Phenotypic biomarkers of aqueous humor extracellular vesicles from retinoblastoma eyes [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 3642.

Abstract 6773: Tissue engineered colorectal cancer microspheres for drug screening

Abstract Three-dimensional (3D) disease models have garnered widespread interest for use in later stages of the drug discovery process, such as preclinical efficacy and toxicology studies, due to their pathophysiologically relevant properties. However, there is a need and opportunity for 3D cancer models to be used earlier in the drug screening process. To meet this need, the 3D models must strike a balance between throughput, which includes scalability and uniformity, and physiological relevance, such as the ability to modulate key attribute of the tumor microenvironment. Here we report the creation of 3D colorectal cancer (CRC) tissue models, referred to as VivoSpheres, and demonstrated their relevance to cancer drug screening. The VivoSphere production platform couples tissue engineering toolkits with microfluidics, enabling the scalable production of engineered cancer microspheres. The model supports the long-term maintenance of the cancer cell phenotype. In a preliminary study, we were able to generate more physiologically relevant drug responses. We formed CRC VivoSpheres by encapsulating HT-29 CRC cells within poly(ethylene glycol)-fibrinogen hydrogel microspheres using our previously developed microfluidic platform. CRC VivoSpheres were rapidly produced with high cell densities (20 × 106 cells/ml) and high uniformity on day 0 with a coefficient of variation (COV) < 7%. This high uniformity was maintained for 15 days (COV ≤ 10%), which is critical for long-term dose studies. The cells maintained high viability and showed high proliferative capability with a significant increase in colony size and expression of Ki67 up to day 29. The encapsulated cells maintained the CRC phenotype over time with the expression of CD44 (cancer stem cell marker) and CK20 (CRC marker). After establishing shipping conditions that maintained cell viability for remote use, the HT-29 VivoSpheres were shipped to the oncology team at Southern Research for drug testing. The CRC VivoSpheres were treated with DMSO, GANT61, and SRI-38832, the latter two of which are GLI1 inhibitors. Phase contrast images and western blot were used to assess the response of CRC VivoSpheres to the treatments. Oncogenic GLI1 transcription activity and NBS1 overexpression have been found to contribute to chemotherapeutic resistance, negating the anti-tumor effects of 5-fluorouracil. While 2D cultured HT-29s responded to treatment with GANT61, HT-29 VivoSpheres continued to express NBS1 following GANT1 treatment, but downregulated NBS1 in response to the GLI1 inhibitor SRI-38832, which is the same response Southern Research has seen in in vivo tumor models. In conclusion, we have developed tissue-engineered 3D CRC models that hold promise for use in drug screening. These models have demonstrated an initial capability to reproduce the CRC phenotype and mimic in vivo drug response. Citation Format: Yuan Tian, Mohammadjafar Hashemi, Benjamin Anbiah, Ruowen Zhang, Iman Hassani, Rebecca Boohaker, Elizabeth Lipke. Tissue engineered colorectal cancer microspheres for drug screening [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 6773.

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 122: Fusobacterium nucleatum in saliva of oral cancer patients induces tumor stemness defense phenotype in cancer stem cells

Abstract Background: Oral squamous cell carcinoma (OSCC) is a highly aggressive cancer. Cancer Stem Cells (CSCs) having cell surface markers CD44, ALDH, and EpCAM+ have been identified in OSCC. However, the host pathogen interaction between CSCs and oral microbiome has not yet been studied. A number of studies have recently demonstrated the presence of specific oral bacteria populations and their lipopolysaccharides (LPS) in the tumor microenvironment (1). We previously reported pathogenic bacterial internalization in the CSCs (2). Based on the findings, we have developed an in-vitro model to investigate how oral microbiota may integrate into the tumor microenvironment's CSC population and control its activity. Here, we investigated the host/pathogen interaction between CSCs and FN. Methods: Oral bacteria FN was selectively isolated using FEA agar plates from oral saliva of oral cancer patients (n=10) at stage 3 or 4. The isolated bacteria was co cultured with SCC 25 cells at MOI of 1:50. After co culturing the stemness genes such as SOX 2, OCT 4, Nanog,LPS and ABCG2+ cancer stem cell markers were analysed. ABCG2+ CSCs and ABCG2- cells were immunomagnetically sorted and lysed followed by RNA extraction and RT QPCR was performed to observe if FN has selectively internalised in the CSCs. Also ABCG2+ and ABCG 2- cells were used to perform clonogenic assay. Results: Coculture of FN with SCC 25 cell line induced high expression of stemness genes SOX, OCT 4 and Nanog along with CSC markers ABCG2+. Moreover high expression of LPS was observed indicating the presence of FN in the coculture. Importantly live bacteria was found internalized in ABCG2+ cells but not in ABCG2- cells. High clonogenicity was observed in ABCG2+ CSCs compared to ABCG2- cells. Notably, we found that live bacteria and their LPS, mostly Fusobacterium nucleatum isolated (FN) from clinical subjects, were capable of invading CSCs in the in-vitro setting. Post the host-pathogen interaction; it enabled the activation of a niche modulatory tumor stemness defense (TSD) phenotype in the CSCs. These aggressive CSCs with the TSD phenotype have been found to have a critical role in the progression and relapse of oral cancer. Conclusion: Expression of stemness genes infers the niche defence mechanism of the CSCs of the tumour microenvironment due to infection of FN. Importantly presence of live bacteria in ABCG2+ CSCs indicates that FN selectively infects ABCG2+ CSCs but not ABCG2- cells. High clonogenicity of ABCG2+ CSCs confirms the TSD phenotypic attributes of the CSCs.