CD133-positive Cells Research Articles

Interconnection of CD133 Stem Cell Marker with Autophagy and Apoptosis in Colorectal Cancer.

CD133 protein expression is observable in differentiated cells, stem cells, and progenitor cells within normal tissues, as well as in tumor tissues, including colorectal cancer cells. The CD133 protein is the predominant cell surface marker utilized to detect cancer cells exhibiting stem cell-like characteristics. CD133 alters common abnormal processes in colorectal cancer, such as the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) and Wnt/β-catenin pathways. Autophagy is a cellular self-digestion mechanism that preserves the intracellular milieu and plays a dual regulatory role in cancer. In cancer cells, apoptosis is a critical cell death mechanism that can impede cancer progression. CD133 can modulate autophagy and apoptosis in colorectal cancer cells via several signaling pathways; hence, it is involved in the regulation of these intricate processes. This can be an explanation for why CD133 expression is associated with enhanced cellular self-renewal, migration, invasion, and survival under stress conditions in colorectal cancer. The purpose of this review article is to explain the complex relationship between the CD133 protein, apoptosis, and autophagy. We also want to highlight the possible ways that CD133-mediated autophagy may affect the apoptosis of colorectal cancer cells. Targeting the aforementioned mechanisms may have a significant therapeutic role in eliminating CD133-positive stem cell-phenotype colorectal cancer cells, which can be responsible for tumor recurrence.

Role of CD44-Positive Extracellular Vesicles Derived from Highly Metastatic Mouse Mammary Carcinoma Cells in Pre-Metastatic Niche Formation.

Malignant breast cancers pose a notable challenge when it comes to treatment options. Recently, research has implicated extracellular vesicles (EVs) secreted by cancer cells in the formation of a pre-metastatic niche. Small clumps of CD44-positive breast cancer cells are efficiently transferred through CD44-CD44 protein homophilic interaction. This study aims to examine the function of CD44-positive EVs in pre-metastatic niche formation in vitro and to suggest a more efficacious EV formulation. We used mouse mammary carcinoma cells, BJMC3879 Luc2 (Luc2 cells) as the source of CD44-positive EVs and mouse endothelial cells (UV2 cells) as the recipient cells in the niche. Luc2 cells exhibited an enhanced secretion of EVs expressing CD44 and endothelial growth factors (VEGF-A, -C) under 20% O2 (representative of the early stage of tumorigenesis) compared to its expression under 1% O2 (in solid tumor), indicating that pre-metastatic niche formation occurs in the early stage. Furthermore, UV2 endothelial cells expressing CD44 demonstrated a high level of engulfment of EVs that had been supplemented with hyaluronan, and the proliferation of UV2 cells occurred following the engulfment of EVs. These results suggest that anti-VEGF-A and -C encapsulated, CD44-expressing, and hyaluronan-coated EVs are more effective for tumor metastasis.

Knocking down RAD51AP1 enhances chemosensitivity by inhibiting the self-renewal of CD133 positive ovarian cancer stem-like cells

PurposeThis study was designed to investigate the function of RAD51AP1 in the self-renewal and chemosensitivity of CD133 positive (CD133+) ovarian cancer (OC) stem-like cells.MethodsCD133+ (CD133 positive) OVCAR4 and CD133 negative (CD133−) OVCAR4 cells were separated from OVCAR4 by flow cytometry. Then, the separated CD133+OVCAR4 cells were divided into the following groups: Vector group; RAD51AP1 group; siNC group; si-RAD51AP1 group. Next, sphere-formation assay and colony forming assay were used to evaluate the self-renewal and proliferation ability of cells; western blot to detect the expression of RAD51AP1, transforming growth factor beta 1 (TGF-β1) and SMAD4 proteins in tissues and cells; qRT-PCR to assess the mRNA levels of sex-determining region Y-box 2 (SOX2), octamer-binding transcription factor 4 (OCT4), NANOG and Kruppel-like factor 4 (KLF4).ResultsThe performance of CD133+OVCAR4 cells was much better than that of CD133−OVCAR4 cells in sphere-formation assay and colony forming assay. Besides, compared with adjacent group and CD133−OVCAR4 cells, the expression level of RAD51AP1 increased significantly in OC group and CD133+OVCAR4 cells. Moreover, the over-expression of RAD51AP1 promoted the self-renewal and proliferation of CD133+OVCAR4 cells. On the contrary, knocking down the expression level of RAD51AP1 could inhibit the self-renewal and proliferation of CD133+OVCAR4 cells and improve the sensitivity of cells to chemotherapy drugs.ConclusionThe findings of this study showed that RAD51AP1 was highly expressed in OC tissue and CD133+OVCAR4 cells, and regulated the self-renewal and chemosensitivity of tumor cells through the TGF-β1/SMAD4 signaling pathway.

P.104: Isolation of CD133 positive cells from rat bone marrow by immune-magnetic beads and Fluorescence-activated cell sorting.

P.104: Isolation of CD133 positive cells from rat bone marrow by immune-magnetic beads and Fluorescence-activated cell sorting.

Anti-CD44 Variant 10 Monoclonal Antibody Exerts Antitumor Activity in Mouse Xenograft Models of Oral Squamous Cell Carcinomas.

CD44 regulates cell adhesion, proliferation, survival, and stemness and has been considered a tumor therapy target. CD44 possesses the shortest CD44 standard (CD44s) and a variety of CD44 variant (CD44v) isoforms. Since the expression of CD44v is restricted in epithelial cells and carcinomas compared to CD44s, CD44v has been considered a promising target for monoclonal antibody (mAb) therapy. We previously developed an anti-CD44v10 mAb, C44Mab-18 (IgM, kappa), to recognize the variant exon 10-encoded region. In the present study, a mouse IgG2a version of C44Mab-18 (C44Mab-18-mG2a) was generated to evaluate the antitumor activities against CD44-positive cells compared with the previously established anti-pan CD44 mAb, C44Mab-46-mG2a. C44Mab-18-mG2a exhibited higher reactivity compared with C44Mab-46-mG2a to CD44v3-10-overexpressed CHO-K1 (CHO/CD44v3-10) and oral squamous cell carcinoma cell lines (HSC-2 and SAS) in flow cytometry. C44Mab-18-mG2a exerted a superior antibody-dependent cellular cytotoxicity (ADCC) against CHO/CD44v3-10. In contrast, C44Mab-46-mG2a showed a superior complement-dependent cytotoxicity (CDC) against CHO/CD44v3-10. A similar tendency was observed in ADCC and CDC against HSC-2 and SAS. Furthermore, administering C44Mab-18-mG2a or C44Mab-46-mG2a significantly suppressed CHO/CD44v3-10, HSC-2, and SAS xenograft tumor growth compared with the control mouse IgG2a. These results indicate that C44Mab-18-mG2a could be a promising therapeutic regimen for CD44v10-positive tumors.

Histological evaluation of renal progenitor/stem cells, renal mesenchymal stem-like cells, and endothelial progenitor cells in chronic kidney disease and end-stage renal disease, and molecular docking analysis of drug-receptor interactions

Chronic kidney disease (CKD) and end-stage renal disease (ESRD) are prevalent and debilitating conditions with a significant impact on patients' quality of life. In this study, we conducted a comprehensive investigation into the histological characteristics of renal progenitor/stem cells (RPCs), renal mesenchymal stem-like cells, and endothelial progenitor cells (EPCs) in CKD and ESRD patients. Additionally, we performed a molecular docking analysis to explore potential drug-receptor interactions involving common medications prescribed to CKD patients. Our histological examination revealed a noteworthy increase in the number of CD24- and CD133-positive cells in CKD and ESRD patients, representing RPCs. These cells are implicated in kidney repair and regeneration, underscoring their potential role in CKD management. Moreover, we observed an elevation in the number of EPCs within the kidneys of CKD and ESRD patients, suggesting a protective role of EPCs in kidney preservation. The molecular docking analysis unveiled intriguing insights into potential drug interventions. Notably, digoxin exhibited the highest in-silico binding affinity to numerous receptors associated with the functions of RPCs, renal mesenchymal stem-like cells, and EPCs, emphasizing the potential multifaceted effects of this cardiac glycoside in CKD patients. Other drugs, including apixaban, glimepiride, and glibenclamide, also displayed strong in-silico affinities to specific receptors, indicating their potential influence on various renal cell functions. In conclusion, this study provides valuable insights into the histological alterations in renal cell populations in CKD and ESRD patients and underscores the potential roles of RPCs and EPCs in kidney repair and preservation. The molecular docking analysis reveals the complex interactions between common drugs and renal cells, suggesting the need for further in-vitro and in-vivo research to fully understand these relationships. These findings contribute to our understanding of CKD and offer new avenues for research into potential therapeutic interventions.

Chondroitin sulfate-tocopherol succinate modified exosomes for targeted drug delivery to CD44-positive cancer cells

Exosomes (Exos), natural nanovesicles released by various cell types, show potential as an effective drug delivery platform due to their intrinsic role as transporters of biomolecules between different cells. However, Exos functionalization with targeting ligands is a critical step to enhance their targeting capability, which could be challenging. In this study, Exos were modified to specifically bind to CD44-positive cells by anchoring chondroitin sulfate (CS) to their surface. Exo modification was facilitated with CS conjugation with alpha-tocopherol succinate (TOS) as an anchorage. The modified Exos were utilized for delivering curcumin (Cur) to pancreatic cancer (PC) cells. In vitro Cur release studies revealed that Exos play a crucial role in maintaining Cur within themselves, demonstrating their potential as effective carriers for drug delivery to targeted locations. Notably, Cur loaded into the modified Exos exhibited enhanced cytotoxicity compared to unmodified Exo-Cur. Meanwhile, Exo-Cur-TOS-CS induced apoptosis more effectively in AsPC-1 cells than unmodified Exos (70.2 % versus 56.9 %). It is worth mentioning that with CD44-mediated cancer-specific targeting, Exo-CS enabled increased intracellular accumulation in AsPC-1 cells, showing promise as a targeted platform for cancer therapy. These results confirm that Exo modification has a positive impact on enhancing the therapeutic efficacy and cytotoxicity of drugs.

Characterization of a human thyroid microtissue model for testing thyroid disrupting chemicals.

Perturbation of thyroid hormone (T4) synthesis is known to cause numerous developmental, metabolic, and cognitive disorders in humans. Due to species differences in sensitivity to chemical exposures, there is a need for human-based in vitro approaches that recapitulate thyroid cellular architecture and T4 production when screening. To address these limitations, primary human thyrocytes, isolated from healthy adult donor tissues and cryopreserved at passage one (p'1) were characterized for cellular composition, 3D follicular architecture, and thyroglobulin (TG)/T4 expression and inhibition by prototype thyroid disrupting chemicals (TDC). Flow analysis of the post-thaw cell suspension showed >80% EpCAM-positive cells with 10%-50% CD90-positive cells. When seeded onto 96-well Matrigel®-coated plates and treated with bovine thyroid stimulating hormone (TSH), thyrocytes formed 3D microtissues during the initial 4-5 days of culture. The microtissues exhibited a stable morphology and size over a 14-day culture period. TG and T4 production were highest in microtissues when the proportion of CD90-positive cells, seeding density and thyroid stimulating hormone concentrations were between 10%-30%, 6K-12Kcells per well, and 0.03-1 mIU/mL, respectively. At maximal TG and T4 production levels, average microtissue diameters ranged between 50 and 200µm. The T4 IC50 values for two prototype TPO inhibitors, 6-propyl-2-thiouracil and methimazole, were ∼0.7µM and ∼0.5 µM, respectively, in microtissue cultures treated between days 9 and 14. Overall, p'1 cryopreserved primary human thyrocytes in 3D microtissue culture represent a promising new model system to prioritize potential TDC acting directly on the thyroid as part of a weight-of-evidence hazard characterization.

The effect of <i>NOTCH1</i> knockdown on the phenotype of human lung and colon cancer stem cells

Introduction. Lung and colorectal cancers are the most common cancer types, characterized by a poor prognosis. Tumor progression is also caused by the aberrant activity of intercellular signaling pathways, which can arise due to mutations in genes encoding their components. In particular, the oncogenic role of NOTCH1 receptor of Notch signaling pathway has been proven for various cancer types, including lung and colorectal cancers. In this research, we delved deeper into the importance of NOTCH1 receptor expression for the progression of these malignancies. Aim. To investigate the importance of NOTCH1 expression in maintaining the cancer stem cell (CSC) pool and phenotype of human lung and colon cancers. Materials and methods. Experiments were performed on previously obtained NOTCH1 knockdown cell lines of human lung A549 and colon HCT116 carcinomas. First of all, we studied the effect of NOTCH1 knockdown on the metastatic ability of A549 cells and the tumorigenicity of A549 and HCT116 cells when injected to immunodeficient BALB/c nu/nu mice. Next, we carried out in vitro tests to determine CSC pool and phenotype in tumor cell culture: cytofluorimetric analysis of ABC-transporters activity to exclude dye to the external environment and analysis of colony formation in a semi-liquid medium. In conclusion, we assessed the proportion of cells in the culture producing the CSC marker – CD133 using flow cytometry and the expression level of some genes associated with CSC phenotype (NANOG, POU5F1, SOX2) using real-time polymerase chain reaction. Results. NOTCH1 knockdown decreased the number of experimental animals with metastases, the number of formed metastatic foci and increased in the minimum cell inoculation dose. The activity of ABC-transporters, the ability for unattached growth, the proportion of CD133-positive cells in culture, and the expression of genes associated with maintaining of CSC pool and phenotype decreased under NOTCH1 knockdown in both cell lines. Conclusion. NOTCH1 expression is important for maintaining CSC pool and phenotype of human lung and colon carcinomas. The obtained data may be valuable in the development of anticancer therapies.

Abnormal epigenetic memory of mesenchymal stem and progenitor cells caused by fetal malnutrition induces hypertension and renal injury in adulthood.

Fetal malnutrition has been reported to induce hypertension and renal injury in adulthood. We hypothesized that this hypertension and renal injury would be associated with abnormal epigenetic memory of stem and progenitor cells contributing to organization in offspring due to fetal malnutrition. We measured blood pressure (BP) for 60 weeks in offspring of pregnant rats fed a normal protein diet (Control), low-protein diet (LP), and LP plus taurine (LPT) in the fetal period. We used western blot analysis to evaluate the expression of αSMA and renin in CD44-positive renal mesenchymal stem cells (MSCs) during differentiation by TGF-β1. We measured kidney label-retaining cells (LRCs) at 11 weeks of age and formation of endothelial progenitor cells (EPCs) at 60 weeks of age from the offspring with fetal malnutrition. Epigenetics of the renal MSCs at 14 weeks were investigated by ATAC-sequence and RNA-sequence analyses. BP was significantly higher in LP than that in Control and LPT after 45-60 weeks of age. Numbers of LRCs and EPC colonies were significantly lower in LP than in Control. Renal MSCs from LP already showed expression of h-caldesmon, αSMA, LXRα, and renin before their differentiation. Epigenetic analyses identified PAR2, Chac1, and Tspan6 genes in the abnormal differentiation of renal MSCs. These findings suggested that epigenetic abnormalities of stem and progenitor cell memory cause hypertension and renal injury that appear in adulthood of offspring with fetal malnutrition.

Diallyl trisulfide induces pyroptosis and impairs lung CSC-like properties by activating the ROS/Caspase 1 signaling pathway

Lung cancer stem cells (CSCs) drive continuous cancer growth and metastatic dissemination; thus, there is an urgent requirement to acquire effective therapeutic strategies for targeting lung CSCs. Diallyl trisulfide (DATS), a garlic organosulfide, possesses suppressive potential in lung cancer; however, its underlying mechanism is still unclear. In this study, we identified DATS as a pyroptosis inducer in lung cancer cells. DATS-treated A549 and H460 cells exhibited pyroptotic cell death, with characteristic large bubbles appearing on their plasma membrane and LDH release. DATS induced cell death, arrested the cell cycle at the G2/M phase, and inhibited colony formation in lung cancer cells. Meanwhile, we found that DATS significantly suppressed the malignant features by impairing lung CSC-like properties, including sphere formation ability, CD133 positive cell number, and lung CSCs marker expression. Mechanistically, DATS induced cell pyroptosis via increasing the expression of NLRP3, ASC, Pro Caspase 1, Cleaved Caspase 1, GSDMD, GSDMD-N, and IL-1β. The verification experiments showed that the effects of DATS on pyroptosis and lung CSC-like properties were weakened after Caspase 1 inhibitor VX-765 treatment, indicating that DATS activated NLRP3 inflammasome-mediated pyroptosis by targeting Caspase 1 in lung cancer cells. Moreover, DATS increased ROS overproduction and mitochondrial dysfunction, which contributed to DATS-induced pyroptosis of lung cancer cells. NAC treatment reversed the effects of DATS on pyroptosis and CSC-like properties. In vivo experiment further confirmed that DATS restrained tumor growth. Together, our results suggest that DATS promotes pyroptosis and impairs lung CSC-like properties by activating ROS/Caspase 1 signaling pathway, thereby retarding lung cancer progression.

Evaluating the role of CD44 in oral squamous cell carcinoma and potentially malignant disorders – An immunohistochemical study

BackgroundOral squamous cell carcinoma (OSCC) accounts for 84–97 % of all cases of oral cancer. Oral submucous and leukoplakia are examples of potentially malignant disorders (PMDs) that are signs of the preclinical stage of oral cancerCD44 (Cluster of differentiation) is a cell surface glycoprotein that serves as a Cancer Stem Cell marker. AimThe purpose of the study was to look at the presence of CD44 immunohistochemically in oral epithelial dysplasia, oral submucous fibrosis, and OSCC and how it influences a patient's prognosis. Materials and methodsIn this retrospective study, the expression of CD44 were evaluated in 60 paraffin –embedded specimens (20 OSCC, 20 Dysplasia, 20 OSMF). This study was evaluated by spss 21. Differences in the mean number of CD 44-positive cells per field among epithelial dysplasia, oral submucous fibrosis and squamous cell carcinomas were assessed using one-way ANOVA and comparison between the groups were analysed by using Bonferroni post hoc test. Statistical significance was at P < 0.05. ResultsOn comparing the CD44 immuno-expression, the highest expression was seen in OSCC followed by dysplasia and OSMF. The OSCC group had a high mean total score of 7.90, followed by dysplasia at 5.40 and OSMF at 5.05. ConclusionAccording to our research, there was a discernible pattern of elevated CD44 expression in OSCC relative to OSMF and dysplasia. These findings set the premise for further research to better outline the role of CD44 in the diagnosis of OSCC.

Emodin is a Potential Drug Targeting CD44-positive Hepatocellular Cancer.

Liver cancer is one of the most prevalent forms of cancer of the digestive system in our country. The most common subtype of this disease is hepatocellular carcinoma (HCC). Currently, treatment options for HCC patients include surgical resection, liver transplantation, radiofrequency ablation, chemoembolization, and biologic-targeted therapy. However, the efficacy of these treatments is suboptimal, as they are prone to drug resistance, metastasis, spread, and recurrence. These attributes are closely related to cancer stem cells (CSCs). Therefore, the utilization of drugs targeting CSCs may effectively inhibit the development and recurrence of HCC. HepG2 and Huh7 cells were used to analyze the antitumor activity of emodin by quantifying cell growth and metastasis, as well as to study its effect on stemness. Emodin effectively suppressed the growth and movement of HCC cells. Emodin also significantly inhibited the proliferation of CD44-positive hepatoma cells. Emodin shows promise as a potential therapeutic agent for HCC by targeting CD44-- positive hepatoma cells.

Decorin suppresses stemness and migration potential of malignant peripheral nerve sheath tumor through inhibiting epidermal growth factor receptor signaling

Cancer stem cells (CSCs) play pivotal roles in the growth, invasion, metastasis, chemo-resistance in malignant peripheral nerve sheath tumor (MPNST). The current characterization of CSCs in MPNST is not complete. Decorin is a critical regulator of microenvironment, but its expression and function in CSCs of MPNST has not been studied. In the current study, Decorin levels and its relationship with lung and liver metastasis were determined in clinical specimens. Decorin expression in CD133-positive or CD44-positive CSCs was analyzed by RT-qPCR on cytospun MPNST cells after flow cytometry-based cell sorting. Decorin-positive cells were separated from Decorin-negative cells in transfected MPNST cell lines using a designed plasmid expressing red fluorescent protein (RFP) under a Decorin promoter. Tumor sphere formation, tumor growth, cell invasion, cell migration, and the resistance to chemotherapy-induced apoptosis were determined on Decorin-positive versus Decorin-negative MPNST cells. In vivo tumor growth was analyzed in mice receiving subcutaneous transplantation of Decorin-positive versus Decorin-negative MPNSTs. We found that Decorin levels were significantly downregulated in MPNST specimens, compared to non-tumorous adjacent tissue. Significantly lower Decorin levels were detected in MPNSTs with lung or liver metastasis compared to those without. Poorer patient survival was detected in Decorin-low MPNST, compared to Decorin-high subjects. More Decorin-negative cells were detected in CD133-positive MPNST cells than CD133-negative MPNST cells, and in CD44-positive MPNST cells than in CD44-negative MPNST cells. Compared to Decorin-positive MPNST cells, Decorin-negative MPNST cells generated significantly more tumor spheres in culture, were more invasive and migratory, and were more resistant to chemotherapy-induced apoptosis, likely due to the inhibition of epidermal growth factor receptor signaling by Decorin. Decorin-negative MPNST cells grew significantly larger tumor in vivo. Thus, depletion of Decorin may occur in CSCs in MPNSTs, serving possibly as a new therapeutic target.

Abstract LB452: Effects of AKT1, 2, and 3 knock-out by CRISPR-Cas9 in CD133+human melanoma stem cells treated with MEK and AKT inhibitors

Abstract Melanoma-initiating stem cells are involved in tumorigenesis, invasion, and drug resistance in malignant melanoma, and are characterized by increased expression of the stem cell marker CD133. We previously showed that CD133 knockout (KO) by CRISPR-Cas9 enhanced MEK inhibitor trametinib (T) -induced apoptosis in patient-derived BAKP melanoma cells by reducing anti-apoptotic p-AKT and p-BAD and increasing pro-apoptotic BAX. Dox-induced CD133 expression, in contrast, diminished apoptosis in T-treated cells, and exhibited elevated p-AKT, p-BAD, and decreased activation of BAX and caspases-3 and -9. This is the first time that CD133 is shown to activate a survival pathway wherein (1) CD133 increases AKT phosphorylation and activation, which induces (2) BAD phosphorylation and inactivation, and (3) reduces caspases-3 and -9 activity leading to apoptosis suppression, increased cell survival and drug resistance in melanoma. We investigated the effects of MEK inhibition with T in combination with the pan-AKT inhibitor capivasertib (AZD5363; C) in BAKP melanoma cells, with or without Dox-induced CD133 expression. Kinetic XTT cell viability and Annexin-Sytox Blue apoptosis assays revealed significantly reduced survival of BAKP cells exposed to a combination of T+C, compared to T alone. To determine the relative contribution of each of the three AKT genes to increased drug resistance of CD133-inducible melanoma stem cells, AKT 1, 2, and/or 3 were knocked out using sgRNAs specific to each of the three AKTs in a CRISPR-Cas9 lentiviral vector, followed by puromycin selection. Gene knockout was confirmed by immunoblot analysis using antibodies specific to each of the three AKTs, as well as by DNA sequence analysis. BAKP control or AKT KO cells were then exposed to T or T+C, and subjected to apoptosis assays, as well as immunoblot analysis with antibodies to apoptosis markers. Single KO of AKT 2 or 3, but not AKT 1, sensitized BAKP cells exposed to T alone, partially obviating the requirement for C to inhibit AKT. Triple KO of AKT 1, 2, and 3 rendered cells even more sensitive to T alone. Targeting nodes of the AKT and MAPK survival pathways with trametinib and AZD5363 highlights the potential for combination therapies for melanoma stem cells increasing the arsenal of more effective treatments for patients with high-risk melanoma. Targeting AKT 1, 2, and/or 3 might be effective in eliminating recalcitrant CD133-positive stem-like cells that may be responsible for tumor recurrence. Citation Format: Cynthia M. Simbulan-Rosenthal, Nusrat Islam, Ryyan Alobaidi, Alejandro Moreno, Veerupaxagouda Patil, Amal Alzharani, Peter Sykora, Dean S. Rosenthal. Effects of AKT1, 2, and 3 knock-out by CRISPR-Cas9 in CD133+human melanoma stem cells treated with MEK and AKT inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB452.

CD44 positive cancer stem cells express invasion and metastasis markers: Analysis of three tongue squamous cell carcinoma cell lines

CD44 positive cancer stem cells express invasion and metastasis markers: Analysis of three tongue squamous cell carcinoma cell lines

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 &lt; 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.

Development of a Mouse Experimental System for the In Vivo Characterization of Bioengineered Adipose-Derived Stromal Cells.

Human adipose-derived stromal cells (ADSCs) are an important resource for cell-based therapies. However, the dynamics of ADSCs after transplantation and their mechanisms of action in recipients remain unclear. Herein, we generated genetically engineered mouse ADSCs to clarify their biodistribution and post-transplantation status and to analyze their role in recipient mesenchymal tissue modeling. Immortalized ADSCs (iADSCs) retained ADSC characteristics such as stromal marker gene expression and differentiation potential. iADSCs expressing a fluorescent reporter gene were seeded into biocompatible nonwoven fabric sheets and transplanted into the dorsal subcutaneous region of neonatal mice. Transplanted donor ADSCs were distributed as CD90-positive stromal cells on the sheets and survived 1 month after transplantation. Although accumulation of T lymphocytes or macrophages inside the sheet was not observed with or without donor cells, earlier migration and accumulation of recipient blood vascular endothelial cells (ECs) inside the sheet was observed in the presence of donor cells. Thus, our mouse model can help in studying the interplay between donor ADSCs and recipient cells over a 1-month period. This system may be of value for assessing and screening bioengineered ADSCs in vivo for optimal cell-based therapies.

Abstract 1884: DXC006, Anti-CD56-CPT113 ADC displays favorable anti-tumor efficacy, pharmacokinetic and safety profiles in preclinical studies

Abstract CD56, also known as neural cell adhesion molecule 1 (NCAM1), is a type I plasma membrane glycoprotein involved in cell-cell and cell-matrix adhesion. It is overexpressed in almost all neuroblastomas, 95% of small cell lung cancers and 78% of multiple myeloma patients[1], whereas CD56 is less frequently or less abundantly expressed in normal tissues, making it a potential target for tumor therapy. So far, no CD56-targeting ADCs have entered late-stage clinical trials.DXC006 is an antibody-drug conjugate of a humanized anti-CD56 antibody (DXA006) linked to the topoisomerase I inhibitor CPT113 through a cleavable peptidyl linker and CROSSCONJU™ technology. The pharmacologic activity and mechanism of action of DXC006 were investigated in several human cancer cell lines and xenograft mouse models. Pharmacokinetic and safety profiles were also assessed in rats and cynomolgus monkeys.DXC006 binds specifically to CD56 and internalizes into tumor cells, after intracellular trafficking to lysosome and CPT113 releasing, induces DNA damage. In vitro cell based assays showed that DXC006 remarkably inhibited the cell growth of CD56-positive cell lines of IMR-32 (neuroblastoma), NCI-H526 (small cell lung cancer), and NCI-H929 (multiple myeloma), with IC50 of 0.0046~0.061 nmol/L. Although DXC006 also binds to CD56 expressed NK cells, we found that the inhibition of DXC006 to human NK cells was much less effective, with IC50 higher than 675 nmol/L, which is ten thousand-folds less potent than that of targeted abnormal cells. In vivo, DXC006 demonstrated good durable antitumor activities in three CD56-positive xenograft models of IMR-32, NCI-H526 and NCI-H929 cell lines, with a single dose of 2 mg/Kg, 3 mg/Kg, and 5 mg/Kg, respectively. No animal death or moribund outcome was observed in two months during preclinical safety studies in 150 mg/Kg Q2W dosed rats. For cynomolgus monkeys, the highest non-severely toxic dose (HNSTD) of DXC006 was 20 mg/Kg, in Q2W, four times of administration. PK profiles of both DXC006 the naked antibody DXA006 were comparable in both rats and monkeys. In summary, DXC006 demonstrated strong antitumor activity in various types of cancer cells both in vitro and in vivo, and good pharmacokinetic and safety profiles in rat and monkeys, a potential to be explored in clinical applications. [1] Yang Feng, Yanping Wang, Zhongyu Zhu, et al. Differential killing of CD56-expressing cells by drug-conjugated human antibodies targeting membrane-distal and membrane-proximal nonoverlapping epitopes. mAbs 2016; 799-810. Citation Format: Xiangfei Kong, Huihui Guo, Yong Du, Zhicang Ye, Yongxiang Chen, Zhixiang Guo, Lingli Zhang, Lu Bai, Junxiang Jia, Yunxia Zheng, Wei Zheng, Jun Zheng, Wenjun Li, Yuanyuan Huang, Zhongliang Fan, Mengmeng Liu, Binbin Chen, Meng Dai, Juan Wang, Fang Du, Miaomiao Chen, Qingliang Yang, Robert Y. Zhao. DXC006, Anti-CD56-CPT113 ADC displays favorable anti-tumor efficacy, pharmacokinetic and safety profiles in preclinical studies [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 1884.

Optimized Design of Hyaluronic Acid-Lipid Conjugate Biomaterial for Augmenting CD44 Recognition of Surface-Engineered NK Cells.

Triple-negative breast cancer (TNBC) presents treatment challenges due to a lack of detectable surface receptors. Natural killer (NK) cell-based adaptive immunotherapy is a promising treatment because of the characteristic anticancer effects of killing malignant cells directly by secreting cytokines and lytic granules. To maximize the cancer recognition ability of NK cells, biomaterial-mediated ex vivo cell surface engineering has been developed for sufficient cell membrane immobilization of tumor-targeting ligands via hydrophobic anchoring. In this study, we optimized amphiphilic balances of NK cell coating materials composed of CD44-targeting hyaluronic acid (HA)-poly(ethylene glycol) (PEG)-lipid to improve TNBC recognition and the anticancer effect. Changes in the modular design of our material by differentiating hydrophilic PEG length and incorporating lipid amount into HA backbones precisely regulated the amphiphilic nature of HA-PEG-lipid conjugates. The optimized biomaterial demonstrated improved anchoring into NK cell membranes and facilitating the surface presentation level of HA onto NK cell surfaces. This led to enhanced cancer targeting via increasing the formation of immune synapse, thereby augmenting the anticancer capability of NK cells specifically toward CD44-positive TNBC cells. Our approach addresses targeting ability of NK cell to solid tumors with a deficiency of surface tumor-specific antigens while offering a valuable material design strategy using amphiphilic balance in immune cell surface engineering techniques.