Cancer Stem Cell Markers Research Articles

The Combination of PD-L1 and CTLA-4 Suppression Significantly Decreased the Expression Levels of Cancer Stem Cell Factors in the Pancreatic Cancer Cell Line

Background: It is thought that a limited number of aberrant cancer stem cells (CSCs), which encourage carcinogenesis, tumor metastasis, and treatment resistance, are the cause of pancreatic cancer, a disease with a high mortality rate. Increasing evidence shows that CSCs use immunosuppressive properties to avoid immune system identification. In Pancreatic cancer, the therapeutic consequences of the relationship between the expression of immune checkpoints such as programmed death receptor ligand-1 (PD-L1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and the presence of CSCs are poorly known. As a result, in the present investigation, we examined how the expression levels of CSCs were affected by PD-L1 and CTLA-4 knockdown using specific siRNA. Methods: Independently and together, PD-L1 and CTLA-4 siRNA were transfected into MIA PaCa-2 cells. RNA extraction and cDNA synthesis were then performed. Finally, using qRT-PCR, the gene expression levels of CSCs markers, including Nanog, CD133, CD44, and Oct-4, in transfected groups were measured. Results: In the MIA PaCa-2 cell line, siRNA-mediated inhibition of PD-L1 and CTLA-4 decreased the expression of CSC factors. Moreover, combined suppression of these two stemness-related immune checkpoints significantly decreased Nanog, CD133, CD44, and Oct-4 expression compared to inhibition of PD-L1 and CTLA-4 separately. Conclusion: Considering that the combination of PD-L1 and CTLA-4 suppression using siRNA significantly decreased the expression levels of CSCs factors, this approach may be regarded as an effective therapy for this cancer.

Combinations of PRI-724 Wnt/β-Catenin Pathway Inhibitor with Vismodegib, Erlotinib, or HS-173 Synergistically Inhibit Head and Neck Squamous Cancer Cells.

The Wnt/β-catenin, EGFR, and PI3K pathways frequently undergo upregulation in head and neck squamous carcinoma (HNSCC) cells. Moreover, the Wnt/β-catenin pathway together with Hedgehog (Hh) signaling regulate the activity of cancer stem cells (CSCs). The aim of this study was to investigate the effects of the combinatorial use of the Wnt/β-catenin and Hh pathway inhibitors on viability, cell cycle progression, apoptosis induction, cell migration, and expression of CSC markers in tongue (CAL 27) and hypopharynx (FaDu) cancer cells. Co-inhibition of Wnt signaling with EGFR or PI3K pathways was additionally tested. The cells were treated with selective inhibitors of signaling pathways: Wnt/β-catenin (PRI-724), Hh (vismodegib), EGFR (erlotinib), and PI3K (HS-173). Cell viability was evaluated by the resazurin assay. Cell cycle progression and apoptosis induction were tested by flow cytometric analysis after staining with propidium iodide and Annexin V, respectively. Cell migration was detected by the scratch assay and CSC marker expression by the R-T PCR method. Mixtures of PRI-724 and vismodegib affected cell cycle distribution, greatly reduced cell migration, and downregulated the transcript level of CSC markers, especially POU5F1 encoding OCT4. Combinations of PRI-724 with erlotinib or HS-173 were more potent in inducing apoptosis.

Establishment of a Novel Anti-CD44 Variant 10 Monoclonal Antibody C44Mab-18 for Immunohistochemical Analysis against Oral Squamous Cell Carcinomas.

Head and neck squamous cell carcinoma (HNSCC) is the most common type of head and neck cancer, and has been revealed as the second-highest expression of CD44 in cancers. CD44 has been investigated as a cancer stem cell marker of HNSCC and plays a critical role in tumor malignant progression. Especially, splicing variant isoforms of CD44 (CD44v) are overexpressed in cancers and considered a promising target for cancer diagnosis and therapy. We developed monoclonal antibodies (mAbs) against CD44 by immunizing mice with CD44v3-10-overexpressed PANC-1 cells. Among the established clones, C44Mab-18 (IgM, kappa) reacted with CHO/CD44v3-10, but not with CHO/CD44s and parental CHO-K1 using flow cytometry. The epitope mapping using peptides that cover variant exon-encoded regions revealed that C44Mab-18 recognized the border sequence between variant 10 and the constant exon 16-encoded sequence. These results suggest that C44Mab-18 recognizes variant 10-containing CD44v, but not CD44s. Furthermore, C44Mab-18 could recognize the human oral squamous cell carcinoma (OSCC) cell line, HSC-3, in flow cytometry. The apparent dissociation constant (KD) of C44Mab-18 for CHO/CD44v3-10 and HSC-3 was 1.6 × 10-7 M and 1.7 × 10-7 M, respectively. Furthermore, C44Mab-18 detected CD44v3-10 but not CHO/CD44s in Western blotting, and endogenous CD44v10 in immunohistochemistry using OSCC tissues. These results indicate that C44Mab-18 is useful for detecting CD44v10 in flow cytometry and immunohistochemistry.

Targeting liver cancer stem cell through EpCAM therapy targeted with chemotherapy endorse enhanced progression in hepatocellular carcinoma

BackgroundTwo chief hurdles in most cancer treatments are chemoresistance and tumor recurrence, especially counting hepatocellular carcinoma (HCC). Most conformist chemotherapy fails to completely cure HCC patients because of its susceptibility to develop multidrug resistance (MDR) through factors such as hypoxia, cancer stem cells, and drug efflux mechanism cancer stem cells (CSC) which are significant factors involved in chemoresistance. It has been exposed that targeting liver cancer stem cells and chemotherapeutic drugs have a better selected, overall survival rate for hepatocellular carcinoma patients.AimThis study aims to investigate the effectiveness of targeting stem cells for liver cancer using a therapy that targets EpCAM in combination with chemotherapy and how this approach can enhance the treatment outcomes in hepatocellular carcinoma, the most prevalent kind of liver cancer.ResultsThe outcome was studied by flow cytometry, Western blot, RT-PCR, and cytotoxicity assays. EpCAM gene silenced and XAV939-treated cells showed decreased expression of CD133, a liver cancer stem cell (LCSC) marker in flow cytometry analysis, and reduced expression of ABCG2 gene, which is a reliable marker for chemoresistance in RT-PCR and western blot analysis; it was also unable to form colonies in colony forming assay. Similarly, in the spheroid formation assay, EpCAM gene silenced cells and XAV939-treated cells in combinations with cisplatin treatment were powerless to appear spheroid, whereas cisplatin alone-treated cells showed spheroids. In the cytotoxicity assay, cisplatin alone and combined with EpCAM silenced and XAV939-treated cells showed more lactate dehydrogenase (LDH) release than EpCAM silenced arm XAV939 treated components.ConclusionThese findings confirm our hypothesis that conventional chemotherapy kills cancer cells but not cancer stem cells. We believe EpCAM-targeted therapy enhances chemosensitivity and decreases relapsed chances. This approach might be the best option for a better prognosis for hepatocellular carcinoma patients.

Nε-(1-Carboxymethyl)-L-lysine, an advanced glycation end product, exerts malignancy on chondrosarcoma via the activation of cancer stemness.

Despite epidemiological evidence that suggests diabetes mellitus is a risk factor for cancer, the link between diabetes mellitus and primary bone cancer is rarely discussed. Chondrosarcomas are primary malignant cartilage tumors with poor prognosis and high metastatic potential. It remains unclear whether hyperglycemia affects the stemness and malignancy of chondrosarcoma cells. Nε-(1-Carboxymethyl)-L-lysine (CML), an advanced glycation end product (AGE), is a major immunological epitope detected in the tissue proteins of diabetic patients. We hypothesized that CML could enhance cancer stemness in chondrosarcoma cells. CML enhanced tumor-sphere formation and the expression of cancer stem cell markers in human chondrosarcoma cell lines. Migration and invasion ability and the epithelial-mesenchymal transition (EMT) process were also induced by CML treatment. Moreover, CML increased the protein expression levels of the receptor for AGE (RAGE), phosphorylated NFκB-p65, and decreased the phosphorylation of AKT and GSK-3. We also found that hyperglycemia with high CML levels facilitated tumor metastasis, whereas tumor growth was not affected in the streptozotocin (STZ)-induced diabetic NOD/SCID tumor xenograft mouse models. Our results indicate that CML enhances chondrosarcoma stemness and metastasis, which may reveal the relationship between AGE and bone cancer metastasis.

CAR-NK Cells Targeting HER1 (EGFR) Show Efficient Anti-Tumor Activity against Head and Neck Squamous Cell Carcinoma (HNSCC)

(1) Background: HNSCC is a highly heterogeneous and relapse-prone form of cancer. We aimed to expand the immunological tool kit against HNSCC by conducting a functional screen to generate chimeric antigen receptor (CAR)-NK-92 cells that target HER1/epidermal growth factor receptor (EGFR). (2) Methods: Selected CAR-NK-92 cell candidates were tested for enhanced reduction of target cells, CD107a expression and IFNγ secretion in different co-culture models. For representative HNSCC models, patient-derived primary HNSCC (pHNSCC) cell lines were generated by employing an EpCAM-sorting approach to eliminate the high percentage of non-malignant cells found. (3) Results: 2D and 3D spheroid co-culture experiments showed that anti-HER1 CAR-NK-92 cells effectively eliminated SCC cell lines and primary HNSCC (pHNSCC) cells. Co-culture of tumor models with anti-HER1 CAR-NK-92 cells led to enhanced degranulation and IFNγ secretion of NK-92 cells and apoptosis of target cells. Furthermore, remaining pHNSCC cells showed upregulated expression of putative cancer stem cell marker CD44v6. (4) Conclusions: These results highlight the promising potential of CAR-NK cell therapy in HNSCC and the likely necessity to target multiple tumor-associated antigens to reduce currently high relapse rates.

Cancer stem cell isolation and characterization from lung cancer cells

Lung cancer is one of the most common cancers in the world and a leading cause of death. Cancer stem cells (CSCs) are the cells responsible for tumor initiation. CSCs features include self-renewal capacity, differentiation, high invasion-migration, and resistance to chemotherapy. CSCs are characterized by specific surface markers. CD133 is widely used as a marker of CSCs in many cancers, including lung cancer. CD133-positive cells are an important marker of lung cancer stem cells because they show characteristics of lung cancer CSCs. CSCs are less sensitive and/or resistant to conventional treatments, remaining viable and regenerating tumors after treatment. Treatments that can target CSCs will be able to destroy CSCs more effectively, leading to the disappearance of tumors. Currently, attempts are being made to develop therapeutics that can effectively target CSCs. These studies concern the isolation and characterization of CSCs. In this study, CD133-positive CSCs and CD133-negative cells were isolated from H460 cells, a large cell lung cancer model, by MACS method. CSCs properties were investigated by tumorSphere formation assay (2% agar plate), hanging drop assay and qPCR method. CD133 positivity of H460 cells was determined as 0.3%. It has been observed that CD133-positive cells show CSCs-specific self-renewal properties. It was determined that the CD133 gene expression difference between CD133-positive CSCs and CD133-negative cells was approximately 25-fold. It has been observed that CD133-positive CSCs express more pluripotency genes (such as OCT-4, SOX-2 and KLF-4) compared to CD133-negative cells.

Constructing a cancer stem cell related prognostic model for predicting immune landscape and drug sensitivity in colorectal cancer.

Background: Colorectal cancer (CRC) ranks the second malignancy with high incidence and mortality worldwide. Cancer stem cells (CSCs) function critically in cancer progression and metastasis via the interplay with immune cells in tumor microenvironment. This study aimed to identify important CSC marker genes and parsed the role of these marker genes in CRC. Materials and methods: CRC samples' single-cell RNA sequencing data and bulk transcriptome data were utilized. Seurat R package annotated CSCs and identified CSC marker genes. Consensus clustering subtyped CRC samples based on CSC marker genes. Immune microenvironment, pathway and oxidative stress analysis was performed using ESTIMATE, MCP-counter analysis and ssGSEA analysis. A prognostic model was established by Lasso and stepAIC. Sensitivity to chemotherapeutic drugs was determined by the biochemical half maximal inhibitory concentration with pRRophetic R package. Results: We identified a total of 29 CSC marker genes related to disease-specific survival (DSS). Two clusters (CSC1 and CSC2) were determined, and CSC2 showed shorter DSS, a larger proportion of late-stage samples, and higher oxidative stress response. Two clusters exhibited differential activation of biological pathways associated with immune response and oncogenic signaling. Drug sensitivity analysis showed that 44 chemotherapy drugs were more sensitive to CSC2 that those in CSC1. We constructed a seven-gene prognostic model (DRD4, DPP7, UCN, INHBA, SFTA2, SYNPO2, and NXPH4) that was effectively to distinguish high-risk and low-risk patients. 14 chemotherapy drugs were more sensitive to high-risk group and 13 chemotherapy drugs were more sensitive to low-risk group. Combination of higher oxidative stress and risk score indicated dismal prognosis. Conclusion: The CSC marker genes we identified may help to further decipher the role of CSCs in CRC development and progression. The seven-gene prognostic model could serve as an indicator for predicting the response to immunotherapy and chemotherapy as well as prognosis of CRC patients.

FAP, CD10, and GPR77-labeled CAFs cause neoadjuvant chemotherapy resistance by inducing EMT and CSC in gastric cancer

ObjectiveA significant proportion of patients can not benefit from neoadjuvant chemotherapy (NCT) due to drug resistance. Cancer-associated fibroblasts (CAFs) influence many biological behaviours of tumors, including chemo-resistance. This study aims to explore whether CAFs expressing FAP, CD10, and GPR77 affect the efficacy of NCT and the prognosis of patients with gastric cancer, and its mechanism.MethodsOne hundred seventy-one patients with locally progressive gastric adenocarcinoma who had undergone NCT and radical surgery were collected. Immunohistochemistry was used to detect the expression of FAP, CD10, and GPR77 in CAFs; the EMT markers (N-cadherin, Snail1, and Twist1) and the CSC markers (ALDH1, CD44, and LGR5) in gastric cancer cells. The χ2 test was used to analyze the relationship between the expression of CAF, EMT, and CSC markers and the clinicopathological factors, as well as the relationship between CAF markers and EMT, and CSC markers. Logistic regression and Cox risk regression were used to analyze the relationship between the expression of CAF, EMT, and CSC markers and TRG grading and OS; Kaplan-Meier analysis was used for survival analysis and plotting the curves.ResultsThe expression of CAF markers FAP, CD10, and GPR77 was closely associated with that of EMT markers; FAP and CD10 were closely related to CSC markers. In the univariate analysis of pathological response, CAF markers (FAP, CD10, GPR77), EMT markers (N-cadherin, Snail1, Twist1), and CSC markers (ALDH1, LGR5, CD44), were all closely associated with pathological response (all p < 0.05). Only Twist1 was an independent factor affecting pathological response in multifactorial analysis (p = 0.001). In a univariate analysis of OS, expression of FAP and CD10 in CAF, as well as expression of EMT biomarkers (N-cadherin, Snail1), were significant factors influencing patient prognosis (all p < 0.05). Multifactorial analysis revealed N-cadherin (p = 0.032) and Snail1 (p = 0.028), as independent prognostic factors affecting OS.ConclusionFAP, CD10, and GPR77 labeled CAF subgroup may lead to NCT resistance and poor prognosis by inducing EMT and CSC of gastric cancer cells in locally advanced gastric cancer patients.

Structural modification of resveratrol analogue exhibits anticancer activity against lung cancer stem cells via suppression of Akt signaling pathway

BackgroundCompound with cancer stem cell (CSC)-suppressing activity is promising for the improvement of lung cancer clinical outcomes. Toward this goal, we discovered the CSC-targeting activity of resveratrol (RES) analog moscatilin (MOS). With slight structural modification from RES, MOS shows dominant cytotoxicity and CSC-suppressive effect.MethodsThree human lung cancer cell lines, namely H23, H292, and A549, were used to compare the effects of RES and MOS. Cell viability and apoptosis were determined by the MTT assay and Hoechst33342/PI double staining. Anti-proliferative activity was determined by colony formation assay and cell cycle analysis. Intracellular reactive oxygen species (ROS) were measured by fluorescence microscopy using DCFH2-DA staining. CSC-rich populations of A549 cells were generated, and CSC markers, and Akt signaling were determined by Western blot analysis and immunofluorescence. Molecular docking and molecular dynamics (MD) simulations were used to predict the possible binding of the compound to Akt protein.ResultsIn this study, we evaluated the effects of RES and MOS on lung cancer and its anti-CSC potential. Compared with RES, its analog MOS more effectively inhibited cell viability, colony formation, and induced apoptosis in all lung cancer cell lines (H23, H292, and A549). We further investigated the anti-CSC effects on A549 CSC-rich populations and cancer adherent cells (A549 and H23). MOS possesses the ability to suppress CSC-like phenotype of lung cancer cells more potent than RES. Both MOS and RES repressed lung CSCs by inhibiting the viability, proliferation, and lung CSC-related marker CD133. However, only MOS inhibits the CSC marker CD133 in both CSC-rich population and adherent cells. Mechanistically, MOS exerted its anti-CSC effects by inhibiting Akt and consequently restored the activation of glycogen synthase kinase 3β (GSK-3β) and decreased the pluripotent transcription factors (Sox2 and c-Myc). Thus, MOS inhibits CSC-like properties through the repression of the Akt/GSK-3β/c-Myc pathway. Moreover, the superior inhibitory effects of MOS compared to RES were associated with the improved activation of various mechanism, such as cell cycle arrest at G2/M phase, production of ROS-mediated apoptosis, and inhibition of Akt activation. Notably, the computational analysis confirmed the strong interaction between MOS and Akt protein. MD simulations revealed that the binding between MOS and Akt1 was more stable than RES, with MM/GBSA binding free energy of − 32.8245 kcal/mol at its allosteric site. In addition, MOS interacts with Trp80 and Tyr272, which was a key residue in allosteric inhibitor binding and can potentially alter Akt activity.ConclusionsKnowledge about the effect of MOS as a CSC-targeting compound and its interaction with Akt is important for the development of drugs for the treatment of CSC-driven cancer including lung cancer.

Quinacrine inhibits HIF-1α/VEGF-A mediated angiogenesis by disrupting the interaction between cMET and ABCG2 in patient-derived breast cancer stem cells

BackgroundBreast cancer stem cells (BCSCs) have a critical role in progression of breast cancer by inducing angiogenesis. Several therapeutic strategies have been designed for the treatment of breast cancer by specifically preventing angiogenesis. But there is a dearth of study regarding the treatment procedure which can specifically target and kill the BCSCs and cause lesser harm to healthy cells of the body. A plant-based bioactive compound Quinacrine (QC) specifically kills cancer stem cells (CSCs) without harming healthy cells and also inhibits cancer angiogenesis but the detailed mechanistic study of its anti-CSCs and anti-angiogenic activity is yet to explore. HypothesisEarlier report showed that both cMET and ABCG2 play an essential role in cancer angiogenesis. Both are present on the cell surface of CSCs and share an identical ATP-binding domain. Interestingly, QC a plant based and bioactive compound which was found to inhibit the function of CSCs marker cMET and ABCG2. These relevant evidence led us to hypothesize that cMET and ABCG2 may interact with each other and induce the production of angiogenic factors, resulting in activation of cancer angiogenesis and QC might disrupt the interaction between them to stop this phenomena. MethodsCo-immunoprecipitation assay, immunofluorescence assay, and western blotting were performed by using ex vivo patient-derived breast cancer-stem-cells (PDBCSCs) and human umbilical vein endothelial cells (HUVECs). In silico study was carried out to check the interaction between cMET and ABCG2 in presence or absence of QC. Tube formation assay using HUVECs and in ovo Chorioallantoic membrane (CAM) assay using chick fertilized eggs were performed to monitor angiogenesis. In vivo patient-derived xenograft (PDX) mice model was used to validate in silico and ex vivo results. ResultsData revealed that in a hypoxic tumor microenvironment (TME), cMET and ABCG2 interact with each other and upregulate HIF-1α/VEGF-A axis to induce breast cancer angiogenesis. In silico and ex vivo study showed that QC disrupted the interaction between cMET and ABCG2 to inhibit the angiogenic response in endothelial cells by reducing the secretion of VEGF-A from PDBCSCs within the TME. Knockdown of cMET, ABCG2 or both, significantly downregulated the expression of HIF-1α and reduced the secretion of pro-angiogenic factor VEGF-A in the TME of PDBCSCs. Additionally, when PDBCSCs were treated with QC, similar experimental results were obtained. ConclusionIn silico, in ovo, ex vivo and in vivo data confirmed that QC inhibited the HIF-1α/VEGF-A mediated angiogenesis in breast cancer by disrupting the interaction between cMET and ABCG2.

A combination of circulating tumor cell (CTC) and cancer stem cell (CSC) markers to predict the prognosis of breast cancer.

e13007 Background: CTCs are tumor cells that shed from either primary tumor or metastatic foci and can provide more information about the tumor's biological entity than circulating tumor DNA (ctDNA), or exosomes do. The current FDA-approved CTC assay misses the CSC phenotypes, which are pivotal in driving tumorigenesis, metastasis, resistance to treatment, and cancer recurrence. Therefore, we combine CTC and CSC markers to determine the prognosis. We hypothesized that the CSC markers on CTC correlate with poorer outcomes. Methods: Peripheral blood samples of qualified breast cancer patients were collected at diagnosis and every 6 months during follow-up. EpCAM, MUC-1, and HER2 are used as CTC markers, while TWIST, AKT, PI3K, and ALDH-1 are used to detect CSC. According to the expression level of the above markers, patients were categorized into four groups: NN negative with both CTC and CSC markers, NP expressing CSC markers, PN expressing CTC markers, and PP positive with both CTC and CSC markers. The progression-free survival (PFS) and overall survival (OS) of all four groups were calculated by Kaplan-Meier model from the date of surgery until the disease progression and death/the last follow-up of the patients, respectively. Results: We screened 100 patients aged 18 years or older with confirmed stage II to stage IV breast cancer and selected 81 patients for the study. Fifteen patients dropped out of the study due to loss of follow-up or incomplete clinical information leaving 66 evaluable patients. There are 55 Caucasians (83%), 5 African Americans (7.5%), 5 Hispanics (7.5%), and 1 Asian (1.5%). The median age of the patients is 52.5 years old (29-81), and the median follow-up is 22.6 months (2.5-65). Most patients have stage III disease (60 and 91%) followed by 4 stage IV (6%) and 2 Stage II (3%). The median time between the first sample date and surgery is 172 days (-148 to 270). The NN group has the most patients (41 and 62%) compared to NP group (16 and 24%) and PN group (6 and 9%). The PP group has the least number of patients (2 and 3%) and is not observed in patients with positive hormonal receptors (HR). The PN group has a higher ratio of HR-HER2+ patients than NP group does. The HR and HER2 statuses were checked in the four groups. Interestingly, the NN and NP groups are mostly HR+HER2- patients, while the PN group is mainly HR- patients regardless of the HER2 status. The PP group only has HR- patients. The PFS curves start to flatten at 12 months and separate among the four groups, with the highest PFS percentage in the NP (81.2%) and NN (77.8%) followed by the PN (62.5%) and PP (50%) group. The OS percentage is 70% in NN, 66% in PN, 60% in NP, and 50% in PP, similar to the PFS pattern. Conclusions: Checking both CTC and CSC markers help triage the prognosis of breast cancer patients. CTC and CSC markers observed in breast cancer patients with different HR or HER2 status can be further investigated for potential correlations.

Identification of Significant RNA-Binding Proteins in the Process of CD44 Splicing Using the Boosted Beta Regression Algorithm.

The expression of RNA-binding proteins and their interaction with the spliced pre-mRNA are the key factors in determining the final isoform profile. Transmembrane protein CD44 is involved in differentiation, invasion, motility, growth and survival of tumor cells, and is also a commonly accepted marker of cancer stem cells and epithelial-mesenchymal transition. However, the functions of the isoforms of this protein differ significantly. In this paper, we developed a method based on the boosted beta regression algorithm for identification of the significant RNA-binding proteins in the splicing process by modeling the isoform ratio. The application of this method to the analysis of CD44 splicing in colorectal cancer cells revealed 20 significant RNA-binding proteins. Many of them were previously shown as EMT regulators, but for the first time presented as potential CD44 splicing factors.

A phase 1/2a, multicenter, open-label study of CNA3103 (LGR5-targeted, autologous CAR-T cells) in patients with metastatic colorectal cancer (mCRC).

TPS3632 Background: Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a cancer stem cell (CSC) marker that is abundant in solid tumors, highly expressed in colorectal cancer and involved in tumor initiation, growth, and metastasis. Its expression is linked to poor survival and poor patient response to therapy in mCRC, and presents an attractive opportunity for selectively targeting a highly specific, functional CSC receptor in this disease. CNA3103 is a first-in-class cell product consisting of autologous T cells transduced to express a CAR to target LGR5. It contains the antigen-biding domain of BNC101, a humanized monoclonal LGR5 antibody previously tested in Phase 1 in mCRC subjects and demonstrated to be safe and well tolerated with no off-target binding activity. Methods: This is a first in human, multicenter, open label, Phase 1/2a dose escalation and expansion study to determine the safety of and overall best response to CNA3103 in subjects with mCRC. Up to 44 subjects (24 in Phase 1 and 20 in Phase 2a) will be enrolled. The trial follows a BOIN design with a minimum of 3 consecutively enrolled subjects per cohort, and a 28-day DLT evaluation period. Four cell dose levels (5 × 107, 1.5 × 108, 4.5 × 108, 1.5 × 109) of CNA3103 will be evaluated. A Safety Monitoring Committee (SMC) will oversee the conduct of the study. The pharmacokinetic (PK) profile of CNA3103 in blood will be assessed. Exploratory objectives include evaluation of LGR5+ cells and CNA3103 in tumor biopsies, immune cellular profile, cytokine levels and circulating tumor DNA in blood, and immunogenicity of CNA3103. Main inclusion criteria: mCRC subjects positive for LGR5 expression and failing prior treatment with 5-FU, oxaliplatin and irinotecan-based regimens for metastatic disease; &gt;1 radiologically measurable lesion per RECISTv1.1; &gt;1 biopsiable lesion at baseline; ECOG 0 or 1; serum albumin &gt;3 g/dL; and normal organ and marrow function. Main exclusion criteria: BRAF-mutated disease, clinical ascites/ pleural effusions; active autoimmune/ connective tissue disease; history of inflammatory bowel disease or active peptic ulcer disease. The trial is sponsored by Carina Biotech. Phase 1 enrolment is limited to Australian sites. Phase 2a enrolment will take place in Australia and the US. For information, contact Lina Jablonskis lina@carinabiotech.com or +61 437 891 563.

IFNγ-induced stem-like state of cancer cells as a driver of metastatic progression following immunotherapy

Despite the remarkable success of immune checkpoint blockade (ICB) therapy, most cancer patients still do not respond. We now find that immunotherapy can induce stem-like properties in tumors. Using mouse models of breast cancer, we observe that cancer stem cells (CSCs) show not only enhanced resistance to Tcell cytotoxicity, but that interferon gamma (IFNγ) produced by activated Tcells directly converts non-CSCs to CSCs. IFNγ enhances several CSC phenotypes, such as resistance to chemo- and radiotherapy and metastasis formation. We identified the branched-chain amino acid aminotransaminase 1 (BCAT1) as a downstream mediator of IFNγ-induced CSC plasticity. Targeting BCAT1 invivo improved cancer vaccination and ICB therapy by preventing IFNγ-induced metastasis formation. Breast cancer patients treated with ICB exhibited a similar increase in CSC markers expression indicating comparable responses to immune activation in humans. Collectively, we discover an unexpected, pro-tumoral role for IFNγ that may contribute to cancer immunotherapy failure.

Ardisia gigantifolia ethanolic extract inhibits cell proliferation and targets cancer stem cells in gastric cancer

Objective: To evaluate the effects of ethanol extract from Ardisia gigantifolia leaves on cell proliferation and cancer stem cell (CSC) number in gastric cancer. Methods: The inhibitory effect of Ardisia gigantifolia extract on the proliferation of MKN45 and MKN74 gastric cancer cells was assessed using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay. Non-adherent culture (3D) model was used to evaluate the effect of the extract on tumorsphere size and number. Moreover, the expression of CD44, ALDH, and p21 was determined by immunofluorescence analysis. Flow cytometric analysis was performed to evaluate cell cycle arrest and the expression of gastric CSC markers CD44 and ALDH. Real-time PCR analysis was also carried out to assess the effect of the extract on the expression of cell cycle-regulated genes. Results: Ardisia gigantifolia extract effectively inhibited cell proliferation with an IC50 of 55.7 µg/mL in MKN45 cells and 123.6 µg/mL in MKN74 cells. The extract also arrested cell cycle in the G0/G1 phase as well as significantly reduced the size and number of tumorspheres. The markedly increased expression of p21 was observed at both mRNA and protein levels in the extract-treated adherent cells and tumorspheres. In addition, Ardisia gigantifolia extract significantly reduced the number of CD44- and/or ALDH-expressing gastric CSC. Conclusions: The development of gastric CSC can be inhibited by the ethanol extract of Ardisia gigantifolia.

Transformation of tissue-resident, very small embryonic-like stem cells (VSELs) into cancer stem cells (CSCs) and its role in cancer initiation via epigenetic changes.

e15084 Background: CSCs are postulated to initiate cancer but still no consensus on their surface markers and origin. Pluripotent VSELs exist in adult tissues, express receptors for steroid and gonadotropin hormones and are possible ‘embryonic remnants’ that could initiate cancer. Methods: Neonatal exposure to diethylstilbestrol (DES) or estradiol (E2) resulted in tumor-like changes in uterus and testes of 100 days old mice. Various studies included histological changes, VSELs enumeration by flow cytometry, and a carefully selected panel of transcripts by qRT-PCR to delineate various cellular events. Mice neonatally exposed to endocrine disruption were administered XAR (a nano-formulation of resveratrol) or transplanted niche-providing mesenchymal stromal cells on D60 and studied later on D100 for reversal/prevention of cancer phenotype. Results: Both E2 and DES resulted in increased numbers of VSELs in testis (Ctrl:0.2%; E2:0.4%; DES:1.3%) and uterus (Ctrl:18.52%; E2:35.42%; DES:40.57%). An up-regulation of pluripotent transcripts ( Oct-4, Sox-2, Nanog) was detected. Oct4A (specific marker for pluripotent VSELs) was upregulated after both DES (testis &gt; 8 folds; uterus &gt; 15 folds) and E2 (testis &gt; 3 folds and uterus &gt; 10 folds) treatment. Excessive self-renewal of VSELs was associated with dysregulated expression of DNA methyltransferases (Dnmts). Dnmt1 was downregulated while Dnmt3a, Dnmt3b, and Dnmt3L were upregulated in the testis. In the uterus, Dnmts showed increased expression after both E2 and DES treatment. Marked reduction of 5mC suggested global hypomethylation and increased Igf2 and Dlk-1 suggested affected/loss of imprinting at IGF2/H19 and DLK-1/MEG3 loci. VSELs acquired CSCs phenotype with increased expression of CD166 and ALDH1. Genomic instability in CSCs was suggested by dysregulated mismatch repair axis (DNMT-1/NP95/PCNA) and repair enzymes ( Brca-1, Rad-51, Mlh-1). Reduced expression of Pten and p53 was associated with blocked differentiation. Both reversal strategies resulted in normalization of stem cells compartment, improved differentiation (spermatogenesis/adenogenesis), reduced CSC markers, and increased p53, Pten, Np95, Sirt-1 expression. Interestingly, XAR treatment resulted in downregulation of Igf2 and Dlk1. Stem cells niche was also improved as evident by increased expression of SOX9 in testis and CD90, VIMENTIN in endometrium. Conclusions: Only immortal VSELs can transmit developmental insults to adult cells. Altered epigenetic state of VSELs resulted in cancer initiation and it was possible to prevent/reverse cancer by normalizing epigenetic state of VSELs and their niche. Targeting CSCs and pushing them back into quiescent VSELs and facilitating their normal differentiation offers novel method to win the war against cancer.

High ALDH-1 Expression Predicts Non-Complete Response of Radiotherapy in Stage III Squamous Cell Cervical Carcinoma Patients.

ALDH1 is a cervical cancer stem cell marker that has radioresistance profile. Recurrence and metastasis following radiotherapy are still being problems of most patients. This study aimed to determine the correlation between ALDH1 and radiotherapy response in stage III squamous cell cervical carcinoma (SCCC) of the cervix. A total 58 of 360 patients of stage III SCCC who received external beem radiation and brachytherapy (2016-2021) at Cipto Mangunkusumo Hospital met the eligibility criteria of this study. Pre- and post-irradiation MRI examinations and ALDH expression with immunohistochemistry (Santa Cruz®) were performed on formalin-fixed paraffin-embedded of pre-treatment cervical tissue biopsy taken from RSCM pathological anatomy laboratory. Patients were divided into two groups, complete responders vs non-complete responders. ALDH-1 scores were compared between two groups to assess ALDH-1 expression. The statistical analyses were carried out by SPSS 24. The optimal ALDH-1 score cut-off point on the radiation response was 166.05 pg/mL which was obtained from the analysis of the ROC curve. The AUC value was 0.682 with sensitivity and specificity, 63,6% and 64%, respectively. ALDH score ≥166.05 increased the risk by 3.127 times for not achieving complete response (adj OR 3.127, 95% CI 1.034 - 9.456, p = 0.043). Pre-radiation tumor size (p = 0.593), degree of differentiation (p = 0.161), renal abnormalities (p = 0.114), and keratinization (p = 0.477) were not associated with radiation response. High ALDH expression was associated with non-complete radiation response in stage III squamous cell cervical carcinoma.<br />.

Proportion of CD44+ Subset of Tumour Cells in Single Cell Suspension Prepared from FFPET Sections Directly Correlates with Histological Subtyping of Head and Neck Squamous Cell Carcinoma

CD44 expression in tumours imparts potential to progress, metastasize, recurrence, and resistance against antineoplastic therapy. In this study, we sought to describe the variation in the immuno-expression and numeration of MDR1+ and CD44+ potential cancer stem cells in different histological grades and subtypes of head and neck squamous cell carcinoma (HNSCC). Flow-cytometric analysis was performed on single cell suspension prepared from formalin fixed paraffin embedded tissue (FFPET) sections of HNSCC using anti-CD44 and anti-MDR1/ABCB-1 primary monoclonal antibodies. Immunohistochemical (IHC) staining was also carried out using both of these antibodies on HNSCC tissue sections mounted on super frosted glass slides. On immunohistochemical analysis, the mean IRS for CD44 and MDR1 were 8.6364 ±3.02114 and 1.5909 ±1.27674 respectively. When mean immune-expression scores of CD44 antibody and MDR1/ABCB-1 were compared with histological grades and subtypes of HNSCC, the relationship was found to be statistically insignificant. Interestingly, a strong statistical difference (p = 0.000) was observed when the mean score of subset of dysplastic squamous epithelial cells with characteristics of cell stemness (CD326+CD44+) was compared among different histological subtypes of HNSCC using flowcytometric analysis. While no statistically significant association was observed when the mean score for subset of dysplastic cells with potential of drug resistance (CD44+MDR1+) was compared among different histological subtypes of HNSCC. Although potential cancer stem cell marker CD44 and the multidrug resistance maker ABCB-1/MDR1 co-expressed in HNSCC but the proportion of CD326+CD44+ subset of tumour cells (potential cancer stem cells/CSCs) significantly correlates with least aggressive to more aggressive tumour subtypes.

Patchouli alcohol induces G0 /G1 cell cycle arrest and apoptosis in vincristine-resistant non-small cell lung cancer through ROS-mediated DNA damage.

Lung cancer, especially non-small cell lung cancer (NSCLC), is one of the leading causes of cancer-related deaths worldwide. Vincristine (VCR) is a chemotherapeutic agent for lung cancers; however, its effectiveness is limited by side effects and the development of drug resistance. Patchouli alcohol (PA), from Pogostemon cablin extract, is known to possess anti-inflammatory and anticancer properties. In this study, we investigated the role of PA in inducing reactive oxygen species (ROS)-mediated DNA damage in A549 and VCR-resistant A549/V16 NSCLC cells. The anticancer potential of PA was studied using the MTT assay, colony formation, flow cytometry analysis, western blotting, DCFDA staining, immunofluorescence staining, and TUNEL assay techniques. The intracellular ROS levels were enhanced in PA-treated cells, activating the CHK1 and CHK2 signaling pathways. PA further inhibited proliferation and colony-forming abilities and induced cell cycle arrest at the G0 /G1 phase by regulating p53/p21 and CDK2/cyclin E1 expression. Moreover, PA increased the percentage of cells in the subG1 phase and induced apoptosis by activating the Bax/caspase-9/caspase-3 intrinsic pathway. In addition, drug resistance (p-glycoprotein) and cancer stem cell (CD44 and CD133) markers were downregulated after PA treatment. Furthermore, combining PA and cisplatin exhibited synergistic inhibitory activity in A549 and A549/V16 cells. PA induced ROS-mediated DNA damage, triggered cell cycle arrest and apoptosis, attenuated drug resistance and cancer stem cell phenotypes, and synergistically inhibited proliferation in combination with cisplatin. These findings suggest that PA has the potential to be used for the treatment of NSCLC with or without VCR resistance.