Cancer Stem-like Cells Markers Research Articles

Frizzled class receptor 5 contributes to ovarian cancer chemoresistance through aldehyde dehydrogenase 1A1

BackgroundChemoresistance is associated with tumor relapse and unfavorable prognosis. Multiple mechanisms underlying chemoresistance have been elucidated, including stemness and DNA damage repair. Here, the involvement of the WNT receptor, FZD5, in ovarian cancer (OC) chemoresistance was investigated.MethodsOC cells were analyzed using in vitro techniques including cell transfection, western blot, immunofluorescence and phalloidin staining, CCK8 assay, colony formation, flowcytometry, real-time PCR, and tumorisphere formation. Pearson correlation analysis of the expression levels of relevant genes was conducted using data from the CCLE database. Further, the behavior of OC cells in vivo was assessed by generation of a mouse xenograft model.ResultsFunctional studies in OC cells showed that FZD5 contributes to epithelial phenotype maintenance, growth, stemness, HR repair, and chemoresistance. Mechanistically, FZD5 modulates the expression of ALDH1A1, a functional marker for cancer stem-like cells, in a β-catenin-dependent manner. ALDH1A1 activates Akt signaling, further upregulating RAD51 and BRCA1, to promote HR repair.ConclusionsTaken together, these findings demonstrate that the FZD5-ALDH1A1-Akt pathway is responsible for OC cell survival, and targeting this pathway can sensitize OC cells to DNA damage-based therapy.

Abstract 7281: Targeting the YAP/TAZ-TEAD pathway with TEAD inhibitors synergizes with chemotherapy and blocks diffuse gastric cancer progression

Abstract Introduction: Diffuse gastric cancer (DGC) accounts for one third of gastric cancers and is associated with poor differentiation, discohesive growth, chemoresistance, and worse survival compared to intestinal-type tumors. DGC is typically genomically stable, and there are no approved DGC-targeted therapies. Recent studies have identified a potential tumor-promoting role for Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif (YAP/TAZ), which interact with TEAD transcription factors to regulate gene expression. Therefore, we evaluated the activity and mechanism of TEAD inhibitors in DGC preclinical models. Methods: Gastric cancer patient-derived organoids (PDOs) and cell lines were treated with TEAD inhibitors (K-975, VT103, VT104, VT107) and/or 5-fluorouracil (5-FU) for in vitro assays evaluating cell proliferation or invasion. Additional experiments were conducted to test the in vivo activity of TEAD inhibitors or shRNA-mediated knockdown of YAP in subcutaneous xenograft, orthotopic xenograft, and lung metastasis models using a genetically engineered mouse model of gastric cancer driven by oncogenic KrasG12D and loss of Trp53/Cdh1. Results: TEAD inhibitors K-975, VT103, VT104, and VT107 reduced proliferation, colony formation, and invasion in DGC cell lines and PDOs. We observed no effect of TEAD inhibitors on the viability of normal gastric organoids. Combining the TEAD1-selective inhibitor VT103 with 5-FU chemotherapy resulted in further reduction of DGC PDO viability relative to monotherapy, including in a PDO derived from a patient with locally advanced DGC that demonstrated intrinsic resistance to neoadjuvant chemotherapy. In flank tumors and orthotopic models, inhibition of the YAP/TAZ-TEAD pathway using YAP shRNA and VT103, either alone or in combination with 5-FU, abrogated primary tumor formation, with the strongest inhibition observed with combination treatment. Loss of YAP or VT103 treatment also significantly reduced lung metastasis after tail vein injection of mouse gastric cancer cells. We hypothesized that TEAD inhibition blocks an epithelial-to-mesenchymal transition (EMT) program and generation of cancer stem-like cells (CSCs). Consistent with this hypothesis, TEAD1 inhibition with VT103 or YAP knockdown reduced expression of the CSC marker CD44 in vitro and decreased expression of CD44 and the EMT marker Slug in tumor xenografts. Conclusions: The YAP/TAZ-TEAD pathway is an important driver of gastric cancer growth and metastasis, particularly in DGC where it may promote EMT and cancer stemness. TEAD inhibition reduces gastric cancer growth and invasion in vitro and in vivo in preclinical models and demonstrates synergistic activity with 5-FU chemotherapy. These results suggest a potential role for evaluating the efficacy of TEAD inhibitors in patients with DGC. Citation Format: Anastasiia Bulakhova, Jin Sun Cho, Jack T. Rifkin, Jaewon Kim, Thomas J. Ryan, Tracy T. Tang, Ryan H. Moy. Targeting the YAP/TAZ-TEAD pathway with TEAD inhibitors synergizes with chemotherapy and blocks diffuse gastric cancer progression [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 7281.

Abstract 5432: The role of nuclear factorκB-inducing kinase in ovarian cancer stem cells, chemoresistance, and relapse

Abstract Ovarian cancer is the most lethal gynecological cancer in the United States, largely due to its high relapse rates. Research suggests these high relapse rates are due to cancer stem-like cells (CSCs), a subpopulation of tumor cells that are resistant to chemotherapy and have enhanced tumor-initiation capacity. Post-treatment, CSCs self-renew to re-establish new tumors, leading to recurrent disease that is now resistant to chemotherapy. Given that over-expression of NF-κB has been linked to CSCs, cancer progression, and chemoresistance in various tumors, including ovarian, studies uncovering the mechanisms of this pathway are needed to develop novel therapeutics for ovarian cancer treatment. The upstream activator of the alternative NF-κB pathway, Nuclear Factor κB-inducing Kinase (NIK), could be an enticing target for eliminating CSCs, however its function in these cells is not established. In our current studies, we show that HGSOC cells grown as CSC-enriching 3D spheroids have increased NIK expression and activity relative to monolayer cultured cells. Moreover, cells expressing CSC markers (CD117+/CD133+), have increased NIK gene expression, further supporting an association between NIK and CSC maintenance. Upon treatment with chemotherapies carboplatin and paclitaxel, NIK gene expression increased in the surviving population, indicating a role in chemoresistance. Knockdown of NIK using siRNA led to changes in alternative NF-kB transcriptional activity and higher sensitivity to carboplatin and paclitaxel relative to vehicle controls. Current studies are evaluating the role of NIK in tumor-initiation capacity, asymmetric division, proliferation, and changes in expression of stemness genes. Lastly, an in vivo relapse model will test the feasibility of using a NIK inhibitor following chemotherapy to prevent relapse and prolong survival. Findings from this work will further define the role of NF-kB pathway components in facilitating CSCs survival and re-establishment of tumors and identify a new potential target for eliminating CSCs in ovarian cancer. Citation Format: Cassidy C. Lucht, Mikella Robinson, Samuel F. Gilbert, Omar Lujano Olazaba, Carrie D. House. The role of nuclear factorκB-inducing kinase in ovarian cancer stem cells, chemoresistance, and relapse [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 5432.

USP14 promotes the cancer stem-like cell properties of OSCC via promoting SOX2 deubiquitination.

USP14 (Ubiquitin-specific-processing protease 14) is a deubiquitinating enzyme with oncogenic effects in oral squamous cell carcinoma (OSCC). This study aims to identify new substrates of USP14 and elucidate their role in modulating cancer stem-like cells (CSCs) in OSCC. Bioinformatics prediction and docking were performed using UbiBrowser 2.0 and HDOCK, respectively. OSCC cell lines and patient-derived cells were used for experimental validation, employing co-immunoprecipitation, cycloheximide chase assays, and tumor sphere formation to evaluate the effects of USP14 on SOX2 stability, ubiquitination, and CSC phenotypes. USP14 upregulation was associated with worse overall survival and progression-free interval in OSCC. USP14 interacted with SOX2 with its ubiquitin carboxyl-terminal hydrolase domain. USP14 knockdown impaired SOX2 stability by increasing its polyubiquitination. Ectopic overexpression of wild-type USP14, but not the hydrolase-deficient-mutant USP14C114A , enhanced SOX2 stability by reducing polyubiquitination. USP14 knockdown suppressed OSCC cell proliferation, colony formation, and tumor sphere formation invitro and tumor growth invivo. However, the reduction of CSC markers following USP14 knockdown was mitigated by overexpressing SOX2. These findings were verified in OSCC patient-derived CSC cells. This study revealed a USP14-SOX2 axis regulating the CSC properties of OSCC.

CD24 induced cellular quiescence-like state and chemoresistance in ovarian cancer cells via miR-130a/301a-dependent CDK19 downregulation

Cancer stem-like cell (CSC) is thought to be responsible for ovarian cancer recurrence. CD24 serves as a CSC marker for ovarian cancer and regulates the expression of miRNAs, which are regulators of CSC phenotypes. Therefore, CD24-regulated miRNAs may play roles in manifesting the CSC phenotypes in ovarian cancer cells. Our miRNA transcriptome analysis showed that 94 miRNAs were up or down-regulated in a CD24-high clone from an ovarian cancer patient compared to a CD24-low one. The CD24-dependent expression trend of the top 7 upregulated miRNAs (miR-199a-3p, 34c, 199a-5p, 130a, 301a, 214, 34b*) was confirmed in other 8 clones (4 clones for each group). CD24 overexpression upregulated the expression of miR-199a-3p, 34c, 199a-5p, 130a, 301a, 214, and 34b* in TOV112D (CD24-low) cells compared to the control, while CD24 knockdown downregulated the expression of miR-199a-3p, 199a-5p, 130a, 301a, and 34b* in OV90 (CD24-high) cells. miR-130a and 301a targeted CDK19, which induced a cellular quiescence-like state (increased G0/G1 phase cell population, decreased cell proliferation, decreased colony formation, and decreased RNA synthesis) and resistance to platinum-based chemotherapeutic agents. CD24 regulated the expression of miR-130a and 301a via STAT4 and YY1 phosphorylation mediated by Src and FAK. miR-130a and 301a were positively correlated in expression with CD24 in ovarian cancer patient tissues and negatively correlated with CDK19. Our results showed that CD24 expression may induce a cellular quiescence-like state and resistance to platinum-based chemotherapeutic agents in ovarian cancer via miR-130a and 301a upregulation. CD24-miR-130a/301a-CDK19 signaling axis could be a prognostic marker for or a potential therapeutic target against ovarian cancer recurrence.

Abstract PR01: Identification and pharmacological targeting of treatment-resistant, stem-like breast cancer cells for combination therapy

Abstract Marker-based isolation of Cancer Stem-Like Cells (CSLCs), which can display preferential resistance to standard-of-care chemotherapy, has proven challenging, due to their very small frequency in breast cancer tissue, thus impeding their characterization, as well as identification and targeting of mechanistic dependencies. To address this challenge, we leveraged a systems biology framework for the protein-activity based characterization of CSLC biology at the single cell level and for the identification of drugs that could reprogram them to a differentiated, chemotherapy-sensitive state. To enrich for CSLCs we performed flow cytometry-based sorting of cells dissociated from 7 metastatic breast cancer patients, both TNBC and HR+, using the EpCAM and CD49f markers, followed by RNA-seq profiling. Protein activity-based clustering, using the VIPER algorithm (Alvarez et al. Nat Genet 2017), identified complementary cell states associated with high activity of either established CSLC markers (BMI1, NOTCH1, etc.) or markers of differentiated cells (GATA3, KRT18, etc.). VIPER-inferred Master Regulator (MR) proteins, identified by comparing highest versus lowest stemness score cells in each patient, were virtually identical across patients and independent of hormone receptor status. Following MR validation by pooled, CRISPR/Cas9-mediated KO followed by single cell RNA-seq profiling (CROP-seq) in cell lines comprising both CLSC and differentiated BRCA cells, we used the OncoTreat algorithm (Alvarez et al. Nat Genet 2018) to identify small molecule compounds capable of inverting their activity (MR-inverter drugs), thus potentially inducing differentiation or cell death. For this purpose, we leveraged gene expression profiles of BT-20 cells—which significantly recapitulate the CSLC MR signature—following treatment with a repertoire of 91 clinically relevant drugs. Albendazole, the top drug identified by OncoTreat, depleted CSLC population (p = 2.0 × 10−4, by Fisher’s exact test, w.r.t. CSLCs to differentiated cells ratio) without noticeable cytotoxicity in the experiment of single cell-based assays in a TNBC PDX model, at 14 days after treatment. In contrast, paclitaxel, a commonly used chemotherapeutic, significantly increase the ratio of CSLC to differentiated cells (p = 2.6 × 10−4, by Fisher’s exact test), suggesting that alternating treatment with the two drugs may abrogate the tumor-initiating potential of paclitaxel-resistant cells, while also preventing uncontrolled tumor growth. This rationale of combination therapy was confirmed by preclinical study, where treatment with multiple cycles of albendazole and paclitaxel displayed significant synergy (p = 0.00869), compared to the corresponding monotherapies. In conclusion, our systems biology framework is a proof-of-concept that drugs can be effectively repurposed to enhance the therapeutic activity of anti-tumor agents used in conventional chemotherapy, and this method can be generalizable to address the challenge of other cancers with heterogeneity. Citation Format: Jeremy Worley, Heeju Noh, Daoqi You, Mikko M Turunen, Hongxu Ding, Evan Paull, Aaron T Griffin, Adina Grunn, Kristina Guillan, Erin C Bush, Samantha J Brosius, Prabhjot S Mundi, Peter Sims, Piero Dalerba, Filemon S Dela Cruz, Andrew L Kung, Andrea Califano. Identification and pharmacological targeting of treatment-resistant, stem-like breast cancer cells for combination therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr PR01.

Potential markers of cancer stem-like cells in ESCC: a review of the current knowledge.

In patients with esophageal squamous cell carcinoma (ESCC), the incidence and mortality rate of ESCC in our country are also higher than those in the rest of the world. Despite advances in the treatment department method, patient survival rates have not obviously improved, which often leads to treatment obstruction and cancer repeat. ESCC has special cells called cancer stem-like cells (CSLCs) with self-renewal and differentiation ability, which reflect the development process and prognosis of cancer. In this review, we evaluated CSLCs, which are identified from the expression of cell surface markers in ESCC. By inciting EMTs to participate in tumor migration and invasion, stem cells promote tumor redifferentiation. Some factors can inhibit the migration and invasion of ESCC via the EMT-related pathway. We here summarize the research progress on the surface markers of CSLCs, EMT pathway, and the microenvironment in the process of tumor growth. Thus, these data may be more valuable for clinical applications.

RCC2 promotes prostate cancer cell proliferation and migration through Hh/GLI1 signaling pathway and cancer stem-like cells

BackgroundRegulator of chromosome condensation 2 (RCC2) was a telophase disk-binding protein on mitosis, and functions as an oncogene in many human cancers. However, its role on prostate cancer (PCa) was unknown. The goal of this study is to explore the function of RCC 2 on PCa development.MethodsThe expression of RCC2 and its methylation level, its correlation with lymph node metastasis or disease-free survival (DFS) was analyzed using TCGA database. The effect of RCC2 on PCa cell proliferation, migration and invasion were detected using CCK-8, cell colony formation, Transwell and wood healing assays. RNA-seq and GSEA analysis were used to search the downstream genes and pathways of RCC2 in mediated PCa progression. Western blot was used to detect the proteins in PCa cells transfected with indicated siRNAs or plasmids.ResultsRCC2 had high expression and low promoter methylation level in PCa, and its expression was correlated with regional node metastasis and disease-free survival. Cell proliferation, migration, invasion and EMT of PCa cells in vitro were greatly enhanced after RCC2 overexpression, while the RCC2 knockdown suppressed these processes. RNA-seq and GSEA results showed the Hedgehog signaling regulator Gli1 and Gli3 were involved in RCC2 knockdown DU145 cells. Gli1 was also a marker of cancer stem-like cells (CSCs). Mechanistically, RCC2 induced cell growth, EMT, CSCs markers through Gli1; inhibiting Gli1 expression using siGli1 or GLI inhibitor suppressed cell progression in vitro and tumor growth in vivo.ConclusionIn summary, RCC2 promoted PCa development through Hh/Gli1 signaling pathway via regulating EMT and CSCs.

Loss of p53-DREAM-mediated repression of cell cycle genes as a driver of lymph node metastasis in head and neck cancer

BackgroundThe prognosis for patients with head and neck cancer (HNC) is poor and has improved little in recent decades, partially due to lack of therapeutic options. To identify effective therapeutic targets, we sought to identify molecular pathways that drive metastasis and HNC progression, through large-scale systematic analyses of transcriptomic data.MethodsWe performed meta-analysis across 29 gene expression studies including 2074 primary HNC biopsies to identify genes and transcriptional pathways associated with survival and lymph node metastasis (LNM). To understand the biological roles of these genes in HNC, we identified their associated cancer pathways, as well as the cell types that express them within HNC tumor microenvironments, by integrating single-cell RNA-seq and bulk RNA-seq from sorted cell populations.ResultsPatient survival-associated genes were heterogenous and included drivers of diverse tumor biological processes: these included tumor-intrinsic processes such as epithelial dedifferentiation and epithelial to mesenchymal transition, as well as tumor microenvironmental factors such as T cell-mediated immunity and cancer-associated fibroblast activity. Unexpectedly, LNM-associated genes were almost universally associated with epithelial dedifferentiation within malignant cells. Genes negatively associated with LNM consisted of regulators of squamous epithelial differentiation that are expressed within well-differentiated malignant cells, while those positively associated with LNM represented cell cycle regulators that are normally repressed by the p53-DREAM pathway. These pro-LNM genes are overexpressed in proliferating malignant cells of TP53 mutated and HPV + ve HNCs and are strongly associated with stemness, suggesting that they represent markers of pre-metastatic cancer stem-like cells. LNM-associated genes are deregulated in high-grade oral precancerous lesions, and deregulated further in primary HNCs with advancing tumor grade and deregulated further still in lymph node metastases.ConclusionsIn HNC, patient survival is affected by multiple biological processes and is strongly influenced by the tumor immune and stromal microenvironments. In contrast, LNM appears to be driven primarily by malignant cell plasticity, characterized by epithelial dedifferentiation coupled with EMT-independent proliferation and stemness. Our findings postulate that LNM is initially caused by loss of p53-DREAM-mediated repression of cell cycle genes during early tumorigenesis.

Study of DNA Damage-Induced MSI1 Serine-347 Phosphorylation in Regulation of Radiosensitivity in Glioblastoma Multiforme

Despite the growing therapeutic techniques, Glioblastoma Multiforme (GBM) patients have limited survival due to the location and malignancies of tumor burdens. Radiotherapy is one of the common treatments with maximal surgery and chemotherapy. However, the often radioresistance occurred in GBM results in recurrence and marginal disease control. Therefore, the medical need remains unmet to treat GBM efficiently. Musashi-1 (MSI1) is one of the RNA-binding proteins known for its high oncogenicity in many cancers, including GBM. MSI1 is highly expressed in tumors and abundant in tumorigenic cancer cells. However, the relationship between MSI1 and GBM radioresistance is still largely unknown. Three human GBM cell lines, U87-MG, 05MG, and S1R1, were used in vitro, and an orthotopic nude mouse GBM model was employed in vivo. Phos-Tag blotting and phospho-MSI1 (Ser 347) antibody helped to detect the phosphorylation level of MSI1. Ultra-low attachment dishes and 3D cell culture were performed for the tumor initiation study. Flow cytometry, immunohistochemistry staining, and confocal microscope with immunofluorescent staining provide respective biomarker expressions. We initiated our study by inducing DNA damage on GBM cell lines using ionizing radiation and an alkylating agent. With Phos-Tag and NetPhos3.1 prediction, we hypothesized that a novel phosphorylation site on MSI1 serine-347 (S347) may sense DNA damage events. To unveil the molecular mechanism, we found DNA-dependent protein kinase catalytic subunits (DNA-PKcs), one of the DNA damage repair components, physically interacted with MSI1 in the role of a kinase. We suggest that activated DNA-PKcs may phosphorylate MSI1-S347, leading to enhanced oncogenicity and subsequent malignancies. Wild-type MSI1-overexpressing GBM cells (MSI1-WT) showed better colony and sphere-forming ability, whereas loss-of-function mutation S347A deprived the oncogenic behaviors. Besides, S347A limited the cancer initiation in the orthotopic animal model compared with MSI1-WT, suggesting that phosphorylation of MSI1-S347 promotes GBM tumorigenicity both in vitro and in vivo. In addition, we noticed that CD133 (Prominin-1), a well-characterized cancer stem-like cell marker, accompanied MSI1-WT introduction but was not seen in S347A cells. Intriguingly, we identified a few members of the let-7 microRNA family, including let-7b, 7e, and 7i, who were rescued by the S347A mutation and correspondingly expressed with CD133 levels and tumorigenic abilities. This study explains a vital relationship between GBM radioresistance and a novel oncogenic post-translational modification on MSI1 S347. IR-induced DNA damage triggers DNA-PKcs activation that subsequently phosphorylates MSI1-S347, enhancing GBM tumorigenicity via let-7 pathway. Further studies on the potential regulators, either inhibiting DNA-PKcs or antagonizing agents, may provide GBM patients with an optimistic outcome.

Isolation of tumor stem-like cells from primary laryngeal squamous cell carcinoma cells (FD-LS-6).

The development of efficient treatments for laryngeal squamous cell carcinoma (LSCC) is hindered by the lack of applicable tumor cell lines and animal models of the disease, especially those related to cancer stem-like cells (CSCs). CSCs play critical roles in tumor propagation and pathogenesis whereas no CSCs lines have been developed to date. In this study, we establish an LSCC cell line (FD-LS-6) from primary LSCC tumor tissue (not experienced single-cell cloning) and adapted a culturing condition for the expansion of potential stem cells (EPSCs) to isolate CSCs from FD-LS-6. We successfully derived novel CSCs and named them as LSCC sphere-forming cells (LSCSCs) which were subsequently characterized for their CSC properties. We showed that LSCSCs shared many properties of CSCs, including CSC marker, robust self-renewal capacity, tumorigenesis ability, potential to generate other cell types such as adipocytes and osteoblasts, and resistance to chemotherapy. Compared to parental cells, LSCSCs were significantly more potent in forming tumors in vivo in mice and more resistant to chemotherapy. LSCSCs have higher expressions of epithelial-mesenchymal transition proteins and chemotherapy resistance factors, and exhibit an activated COX2/PEG2 signaling pathway. Altogether, our work establishes the first CSCs of LSCC (FD-LS-6) and provides a tool to study tumorigenesis and metastasis of LSCC and help the development of anticancer therapies.

RETRACTED ARTICLE: KRAS activation in gastric cancer stem-like cells promotes tumor angiogenesis and metastasis

Our previous work showed that KRAS activation in gastric cancer cells leads to activation of an epithelial-to-mesenchymal transition (EMT) program and generation of cancer stem-like cells (CSCs). Here we analyze how this KRAS activation in gastric CSCs promotes tumor angiogenesis and metastasis. Gastric cancer CSCs were found to secrete pro-angiogenic factors such as vascular endothelial growth factor A (VEGF-A), and inhibition of KRAS markedly reduced secretion of these factors. In a genetically engineered mouse model, gastric tumorigenesis was markedly attenuated when both KRAS and VEGF-A signaling were blocked. In orthotropic implant and experimental metastasis models, silencing of KRAS and VEGF-A using shRNA in gastric CSCs abrogated primary tumor formation, lymph node metastasis, and lung metastasis far greater than individual silencing of KRAS or VEGF-A. Analysis of gastric cancer patient samples using RNA sequencing revealed a clear association between high expression of the gastric CSC marker CD44 and expression of both KRAS and VEGF-A, and high CD44 and VEGF-A expression predicted worse overall survival. In conclusion, KRAS activation in gastric CSCs enhances secretion of pro-angiogenic factors and promotes tumor progression and metastasis.

A Novel Anti-CD44 Variant 9 Monoclonal Antibody C44Mab-1 Was Developed for Immunohistochemical Analyses against Colorectal Cancers

Cluster of differentiation 44 (CD44) is a type I transmembrane glycoprotein and has been shown to be a cell surface marker of cancer stem-like cells in various cancers. In particular, the splicing variants of CD44 (CD44v) are overexpressed in cancers and play critical roles in cancer stemness, invasiveness, and resistance to chemotherapy and radiotherapy. Therefore, the understanding of the function of each CD44v is indispensable for CD44-targeting therapy. CD44v9 contains the variant 9-encoded region, and its expression predicts poor prognosis in patients with various cancers. CD44v9 plays critical roles in the malignant progression of tumors. Therefore, CD44v9 is a promising target for cancer diagnosis and therapy. Here, we developed sensitive and specific monoclonal antibodies (mAbs) against CD44 by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary-K1 (CHO/CD44v3-10) cells. We first determined their critical epitopes using enzyme-linked immunosorbent assay and characterized their applications as flow cytometry, western blotting, and immunohistochemistry. One of the established clones, C44Mab-1 (IgG1, kappa), reacted with a peptide of the variant 9-encoded region, indicating that C44Mab-1 recognizes CD44v9. C44Mab-1 could recognize CHO/CD44v3-10 cells or colorectal cancer cell lines (COLO201 and COLO205) in flow cytometric analysis. The apparent dissociation constant (KD) of C44Mab-1 for CHO/CD44v3-10, COLO201, and COLO205 was 2.5 × 10-8 M, 3.3 × 10-8 M, and 6.5 × 10-8 M, respectively. Furthermore, C44Mab-1 was able to detect the CD44v3-10 in western blotting and the endogenous CD44v9 in immunohistochemistry using colorectal cancer tissues. These results indicated that C44Mab-1 is useful for detecting CD44v9 not only in flow cytometry or western blotting but also in immunohistochemistry against colorectal cancers.

Abstract 2451: KRAS activation in gastric cancer stem-like cells promotes tumor angiogenesis and metastasis

Abstract Our previous work showed that KRAS activation in gastric cancer cells leads to activation of an epithelial-to-mesenchymal transition (EMT) program and generation of cancer stem-like cells (CSCs). Here we analyze how this KRAS activation in gastric CSCs promotes tumor angiogenesis and metastasis. Gastric cancer CSCs were found to secrete pro-angiogenic factors such as vascular endothelial growth factor A (VEGF-A), and inhibition of KRAS markedly reduced secretion of these factors. In a genetically engineered mouse model, gastric tumorigenesis was markedly attenuated when both KRAS and VEGF-A signaling were blocked. In orthotropic implant and experimental metastasis models, silencing of KRAS and VEGF-A using shRNA in gastric CSCs abrogated primary tumor formation, lymph node metastasis, and lung metastasis far greater than individual silencing of KRAS or VEGF-A. Analysis of gastric cancer patient samples using RNA sequencing revealed a clear association between high expression of the gastric CSC marker CD44 and expression of both KRAS and VEGF-A, and high CD44 and VEGF-A expression predicted worse overall survival. In conclusion, KRAS activation in gastric CSCs enhances secretion of pro-angiogenic factors and promotes tumor progression and metastasis. Citation Format: Changhwan Yoon, Jun Lu, Yukyung Jun, Bang- Jin Kim, Jacob Till, Jong Hyun Kim, Sara Keshavjee, Sandra Ryeom, Sam Yoon. KRAS activation in gastric cancer stem-like cells promotes tumor angiogenesis and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2451.

Abstract P5-14-08: Machine learning based histopathology images analysis reveals cancer stemness in TNBC patient with 17p loss

Abstract Background: The area of computational pathology has made huge progresses due to advances in artificial intelligence (AI) and machine learning technologies. It has been applied to many research and translational tasks which provide great improvement on medical diagnosis and treatment. Cancer stem-like cells (CSC) have been consistently reported for its key role in Triple negative breast cancer (TNBC) . Given the large amount of existing H&E stained histological slides of TNBC, digital identification of CSC could benefit the evaluation of tumor status and prediction of patients’ response to chemotherapy. Here we proposed an AI framework based on Convolutional Neural Network (CNN) to predict CSC from the histological images of TNBC patient. And our preliminary work suggested that chromosome 17p loss, a common genetic variations in breast cancer, is linked to cancer stemness. Methods: A modified GoogleNet model was adopted as our CNN classifier. Consecutive breast cancer tissue microarrays (TMA), which stained with H&E ,SOX2, OCT4 and NANOG antibodies respectively by IHC, were used as training dataset for the CNN model. Gene expression data from the TCGA and METABRIC datasets were used to identify gene signatures associated with CSC. The connectivity map (CMAP) and Cancer Cell Line Encyclopedia (CCLE) were used for screening compounds that target stemness in cancer cell lines with chromosomal 17p loss. HS578T and EO771 cells with or without heterozygous 17p loss (11b in EO771) were used for in vitro experiments. Female immunodeficient nude (Nu/J) mice were used for animal studies. Results: The well trained GoogleNet model was applied to TNBC patient diagnosis images in TCGA BRCA dataset. By analyzing patient genomic alteration on chromosomal level, we found that loss of chromosome 17p associate with high cancer stemness in TNBC. Flow cytometry assays also demonstrated higher ALDH1 activity and higher CD44+/CD24−/low cell population in HS578T cells with 17p loss. RNA-seq of HS578T cells revealed that most CSC marker genes were located in the unregulated differentially expressed genes (DEGs) of 17p loss cells. We next compared the cytotoxicity of chemotherapy drugs including doxorubicin, paclitaxel, docetaxel and cisplatin on 17p loss and 17p intact HS578T cells, 11b loss and 11b intact EO771 cells, in terms of IC50 value. The IC50 value of indicated drug on 17p loss HS578T cells with were 3-6 fold higher than their IC50 on 17p intact HS578T cells. Similar result was observed in EO771 cells. Next, 17p loss and 17p intact HS578T cells were orthotopically implanted into the Nu/J mice. Under the doxorubicin treatment, mice bearing 17p loss HS578T derived tumors had larger and heavier tumors in compare to mice bearing 17p intact tumors. Next, we did a computational drug screening to identify compounds that can target the cancer stemness in 17p loss cells. Screened out compounds were tested for their cytotoxicity on 17p loss and 17p intact HS578T cells and FK866 showed the most pronounced efficacy on inhibiting the viability of 17p loss cells, compared to 17p intact cells. Followup experiments demonstrated that FK866 can decrease the CSC features induced by doxorubicin, both in vitro and in vivo. FK866 also potentiates the effect of doxorubicin on treating TNBC cells with 17p loss, which provide a drug combination potential for TNBC patient with 17p loss. Conclusions: A CNN based model was developed to identify CSC from TNBC histopathology images. The images analysis combined with patient genomic data revealed that chromosome 17p loss associated with cancer stemness in TNBC. This result was confirmed using assays on TNBC cells with or without 17p loss. A computational drug screening was performed to identify candidates that targeting stemness in 17p loss cells. FK866 was identified and it potentiates the anti-tumor effect of doxorubicin on treating TNBC cells with 17p loss. Our study provides a novel strategy on applying AI to precision treatment for cancers. Citation Format: tianhan dong, jiannan liu, yuanzhang Fang, Ziyu Liu, Xiongbin Lu, kun huang. Machine learning based histopathology images analysis reveals cancer stemness in TNBC patient with 17p loss [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-14-08.

Eradication of Heterogeneous Tumors by T Cells Targeted with Combination Bispecific Chemically Self-assembled Nanorings.

Cancer stem-like cells (CSCs) are often the root cause of refractive relapse due to their inherent resistance to most therapies and ability to rapidly self-propagate. Recently, the antigen CD133 has been identified as a CSC marker on several cancer types and αCD133 therapies have shown selective targeting against CSCs with minimal off-target toxicity. Theoretically, by selectively eliminating CSCs, the sensitivity to bulk tumor-targeting therapies should be enhanced. Previously, our laboratory has developed bispecific chemically self-assembled nanorings (CSANs) that successfully induced T-cell eradication of EpCAM-positive (EpCAM+) tumors. We reasoned that targeting both CSCs [CD133-positive (CD133+)] and the bulk tumor (EpCAM+) simultaneously using our CSAN platform should produce a synergistic effect. We evaluated αCD133/αCD3 CSANs as both a single agent and in combination with αEpCAM/αCD3 CSANs to treat triple-negative breast cancer (TNBC) cells, which express a subpopulation of CD133+ cancer stem cells and EpCAM+ bulk tumor cells. Furthermore, an orthotopic breast cancer model validated the ability of αCD133 and αEpCAM targeting to combine synergistically in the elimination of TNBC MDA-MB-231 cells. Complete tumor eradication only occurred when EpCAM and CD133 were targeted simultaneously and lead to full remission in 80% of the test mice. Importantly, the depletion and enrichment of CD133 TNBCs highlighted the role of CD133+ cancer cells in regulating tumor growth and progression. Collectively, our results demonstrate that dual targeting with bispecific CSANs can be effective against heterogenous tumor cell populations and that elimination of primary and CD133+ CSCs may be necessary for eradication of at least a subset of TNBC.

Sevoflurane Does Not Promote the Colony-Forming Ability of Human Mesenchymal Glioblastoma Stem Cells In Vitro

Background and Objectives: Clinically used concentrations of sevoflurane, an inhaled anesthetic, have been reported to significantly inhibit tumor growth. We investigated the effects of sevoflurane on sphere formation and the proliferation of human glioblastoma stem cells (GSCs) to determine whether sevoflurane exerts short- and long-term effects on human tumor cells. Materials and Methods: High-grade patient-derived GSCs (MD13 and Me83) were exposed to 2% sevoflurane. To evaluate the effect of sevoflurane on viability, proliferation, and stemness, we performed a caspase-3/7 essay, cell proliferation assay, and limiting dilution sphere formation assays. The expression of CD44, a cell surface marker of cancer stem-like cells in epithelial tumors, was evaluated using quantitative reverse transcription PCR. Differences between groups were evaluated with a one-way analysis of variance (ANOVA). Results: Sevoflurane exposure for 4 days did not significantly promote caspase 3/7 activity in MD13 and Me83, and cell proliferation was not observed after 5 days of exposure. Furthermore, prolonged exposure to sevoflurane for 6 days did not promote the sphere-forming and proliferative potential of MD13 and Me83 cells. These results suggest that sevoflurane does not promote either apoptosis, proliferative capacity, or the colony-forming ability of human mesenchymal glioblastoma stem cells in vitro. Conclusions: Sevoflurane at clinically used concentrations does not promote the colony-forming ability of human mesenchymal glioblastoma stem cells in vitro. It is very important for neurosurgeons and anesthesiologists to know that sevoflurane, a volatile anesthetic used in surgical anesthesia, would not exacerbate the disease course of GSCs.

Targeting aldehyde dehydrogenase for prostate cancer therapies.

Prostate cancer (PCa) is the most common cancer in men in the United States. About 10 – 20% of PCa progress to castration-resistant PCa (CRPC), which is accompanied by metastasis and therapeutic resistance. Aldehyde dehydrogenase (ALDH) is famous as a marker of cancer stem-like cells in different cancer types, including PCa. Generally, ALDHs catalyze aldehyde oxidation into less toxic carboxylic acids and give cancers a survival advantage by reducing oxidative stress caused by aldehyde accumulation. In PCa, the expression of ALDHs is associated with a higher tumor stage and more lymph node metastasis. Functionally, increased ALDH activity makes PCa cells gain more capabilities in self-renewal and metastasis and reduces the sensitivity to castration and radiotherapy. Therefore, it is promising to target ALDH or ALDHhigh cells to eradicate PCa. However, challenges remain in moving the ALDH inhibitors to PCa therapy, potentially due to the toxicity of pan-ALDH inhibitors, the redundancy of ALDH isoforms, and the lack of explicit understanding of the metabolic signaling transduction details. For targeting PCa stem-like cells (PCSCs), different regulators have been revealed in ALDHhigh cells to control cell proliferation and tumorigenicity. ALDH rewires essential signaling transduction in PCa cells. It has been shown that ALDHs produce retinoic acid (RA), bind with androgen, and modulate diverse signaling. This review summarizes and discusses the pathways directly modulated by ALDHs, the crucial regulators that control the activities of ALDHhigh PCSCs, and the recent progress of ALDH targeted therapies in PCa. These efforts will provide insight into improving ALDH-targeted treatment.

Posaconazole inhibits the stemness of cancer stem-like cells by inducing autophagy and suppressing the Wnt/β-catenin/survivin signaling pathway in glioblastoma.

Posaconazole (POS) has been reported to present potential antitumor activity for glioblastoma (GBM). However, its molecular mechanisms remain unclear. In this study, we found that POS has potent cytotoxicity and inhibits cell viability and proliferation in GBM. In addition, we adopted a sphere formation assay to detect the self-renewal capacity, performed western blotting to measure cancer stem-like cells (CSCs) marker proteins (CD133, SOX2, Nanog and Oct4) and applied flow cytometry to monitor the subpopulation of CD144+/CD33+ cells, and the results all demonstrated that POS can remarkably weaken CSCs stemness. Furthermore, western blotting, immunoflurescence, transmission electron microscopy and acridine orange staining were performed to detect autophagy-related proteins (LC3, SQSTM1, Beclin 1 and Atg5), count the numbers of endogenous LC3 puncta, visually observe the ultrastructural morphology of autophagosomes and judge the formation of acidic vesicular organelles, respectively, and the results validated that POS promotes autophagy induction. Importantly, the suppressive effect of POS on CSCs stemness was partially relieved when autophagy was blocked by the autophagy inhibitor chloroquine (CQ) or Atg5 shRNA. Bioinformatic techniques, including weighted gene coexpression network analysis (WGCNA), gene set difference analysis (GSVA) and KEGG pathway analysis, combined with experimental validations showed that survivin, which is implicated in both autophagy and the stem cell index, is one of the target proteins of POS and that POS weakens CSCs stemness via suppressing the Wnt/β-catenin signaling pathway in GBM. Besides, POS-induced autophagy and the Wnt/β-catenin signaling pathway are negative regulators for each other. Finally, the antitumor activity of POS was confirmed in GBM xenograft models in vivo. Consistent with the in vitro conclusions, POS upregulated the expression of LC3 and decreased the expression of CD133, survivin and β-catenin, as shown by the immunohistochemistry analysis. In summary, this work provides an experimental foundation for exploiting POS as a CSCs-targeting antitumor drug for GBM treatment.

Tumor necrosis factor-α-inducing protein of Helicobacter pylori promotes epithelial-mesenchymal transition and cancer stem-like cells properties via activation of Wnt/β-catenin signaling pathway in gastric cancer cells.

Tumor necrosis factor-α-inducing protein (Tipα) is a newly identified toxin that promotes the inflammation and carcinogenesis caused by Helicobacter pylori. However, its mechanism of pathogenesis is still unclear. To investigate the carcinogenic mechanisms of Tipα, SGC7901 cells and SGC7901-derived cancer stem-like cells (CSCs) were stimulated by recombinant Tipα with or without Wnt/β-catenin signaling inhibitor XAV939. qRT-PCR and Western blotting were employed to detect expression of epithelial-mesenchymal transition (EMT), CSCs markers and downstream target genes of this signaling pathway. The cell migration ability was measured by wound healing assay and transwell assay. Our results indicated that Tipα promoted CSC properties of SGC7901 spheroids, including increased expression of CSC specific surface markers CD44, Oct4 and Nanog and an increased capacity for self-renewal. Tipα activated Wnt/β-catenin signaling in both SGC7901 cells or CSCs. Furthermore, Tipα induced the EMT and increased the expressions of downstream target genes of this signaling, including c-myc, cyclin D1 and CD44. However, XAV939 pretreatment inhibited Tipα-induced EMT and CSC properties in SGC7901 cells or CSCs. These results suggest that Tipα promotes EMT and CSC-like properties in gastric cancer cells through activation of Wnt/β-catenin signaling pathway, thereby accelerating the progression of gastric cancer.