Efficacy of Whole Cancer Stem Cell-Based Vaccines: A Systematic Review of Preclinical and Clinical Studies.

BACKGROUND: Despite the conventional cancer therapeutic, cancer treatment remains a medical challenge due to neoplasm metastasis and cancer recurrence; therefore, new approaches promoting therapeutic strategies are highly desirable. As a new therapy, the use of whole neoplastic stem cells or cancer stem cell (CSC)-based vaccines is one strategy to overcome these obstacles. We investigated the effects of whole CSC-based vaccines on the solid tumor development, metastasis, and survival rate. METHODS: Primary electronic databases (PubMed/MEDLINE, Scopus, Embase, and Web of Science) and a major clinical registry were searched. Interventional studies of whole CSC-based vaccines in rodent cancer models (38 studies) and human cancer patients (11 studies) were included; the vaccine preparation methodologies, effects, and overall outcomes were evaluated. RESULTS: Preclinical studies were divided into 4 groups: CSC-lysates/ inactivated-CSC-based vaccines, CSC-lysate-loaded dendritic cell (CSC-DC) vaccines, cytotoxic T-cell (CTL) vaccines generated with CSC-DC (CSC-DC-CTL), and combinatorial treatments carried out in the prophylactic and therapeutic experimental models. The majority of preclinical studies reported a promising effect on tumor growth, survival rate, and metastasis. Moreover, whole CSC-based vaccines induced several antitumor immune responses. A small number of clinical investigations suggested that the whole CSC-based vaccine treatment is beneficial; however, further research is required. CONCLUSIONS: This comprehensive review provides an overview of the available methods for assessing the efficacy of whole CSC-based vaccines on tumor development, metastasis, and survival rate. In addition, it presents a set of recommendations for designing high-quality clinical studies that may allow to determine the efficacy of whole CSC-based-vaccines in cancer therapy.

Novel neutralizing SARS-CoV-2-specific mAbs offer detection of RBD linear epitopes.

BACKGROUND: To stop the spread of the COVID-19 disease, it is crucial to create molecular tools to investigate and diagnose COVID-19. Current efforts focus on developing specific neutralizing monoclonal antibodies (NmAbs) elicited against the receptor-binding domain (RBD). METHODS: In the present study, recombinant RBD (rRBD) protein was produced in E. coli, followed by immunizing mice with purified rRBD. ELISA was applied to screen the hybridomas for positive reactivity with rRBD protein. The linear and conformational epitopes of the mAbs were subsequently identified using western blot. Finally, the reactivity, affinity, and neutralization activity of the purified mAbs were evaluated using ELISA. RESULTS: All mAbs exhibited similar reactivity trends towards both eukaryotic RBD and prokaryotic rRBD in ELISA. Among them, 2E7-D2 and 2B4-G8 mAbs demonstrated higher reactivity than other mAbs. Additionally, in western blot assays, these two mAbs could detect reducing and non-reducing rRBD, indicating recognition of linear epitopes. Notably, five mAbs effectively blocked rRBD- angiotensin-converting enzyme 2 (ACE2) interaction, while two high-affinity mAbs exhibited potent neutralizing activity against eukaryotic RBD. CONCLUSION: In the current study, we generated and characterized new RBD-specific mAbs using the hybridoma technique that recognized linear and conformational epitopes in RBD with neutralization potency. Our mAbs are novel candidates for diagnosing and treating SARS-CoV-2.

Higher expression of SALL4-A isoform is correlated with worse outcomes and progression of the disease in subtype of testicular germ cell tumours.

BACKGROUND: The transcription factor SALL4 is associated with embryonic pluripotency and has proposed as a novel immunohistochemistry (IHC) marker for diagnosing germ cell tumours. SALL4 comprises three isoforms, and SALL4-A being the full-length isoform. Studying its isoforms could revolutionize testicular cancer prognosis and subtype differentiation. METHODS: The expression and clinical significance of isoform 'A' of SALL4 was evaluated in 124 testicular germ cell tumours (TGCTs) subtypes, adjacent 67 normal tissues and 22 benign tumours, using immunohistochemistry on tissue microarrays (TMA). RESULTS: A statistically significant higher expression of nuclear and cytoplasmic SALL4-A was detected in TGCTs histological subtypes and benign tumours compared to the normal tissues. Seminoma and yolk sac tumours had the highest nuclear and cytoplasmic expression of SALL4-A. A significant correlation was detected between the higher nuclear expression of SALL4-A and increased pT stages (P = 0.026) in seminomas. Whereas in embryonal carcinomas, cytoplasmic expression of SALL4-A was associated with the tumour recurrence (P = 0.04) and invasion of the epididymis (P = 0.011). CONCLUSIONS: SALL4-A isoform expression in the cytoplasm and nucleus of TGCTs may be associated with histological differentiation. In the seminoma subtype of TGCTs, higher expression of SALL4-A may be used as a predictive indicator of poorer outcomes and prognosis.

Tumor matrix stiffness provides fertile soil for cancer stem cells.

Matrix stiffness is a mechanical characteristic of the extracellular matrix (ECM) that increases from the tumor core to the tumor periphery in a gradient pattern in a variety of solid tumors and can promote proliferation, invasion, metastasis, drug resistance, and recurrence. Cancer stem cells (CSCs) are a rare subpopulation of tumor cells with self-renewal, asymmetric cell division, and differentiation capabilities. CSCs are thought to be responsible for metastasis, tumor recurrence, chemotherapy resistance, and consequently poor clinical outcomes. Evidence suggests that matrix stiffness can activate receptors and mechanosensor/mechanoregulator proteins such as integrin, FAK, and YAP, modulating the characteristics of tumor cells as well as CSCs through different molecular signaling pathways. A deeper understanding of the effect of matrix stiffness on CSCs characteristics could lead to development of innovative cancer therapies. In this review, we discuss how the stiffness of the ECM is sensed by the cells and how the cells respond to this environmental change as well as the effect of matrix stiffness on CSCs characteristics and also the key malignant processes such as proliferation and EMT. Then, we specifically focus on how increased matrix stiffness affects CSCs in breast, lung, liver, pancreatic, and colorectal cancers. We also discuss how the molecules responsible for increased matrix stiffness and the signaling pathways activated by the enhanced stiffness can be manipulated as a therapeutic strategy for cancer.

Circulating exosomal microRNAs as potential prognostic biomarkers in gastrointestinal cancers: a systematic review and meta-analysis.

BACKGROUND: Recent reports suggested that circulating exosomal microRNAs (exomiRs) may serve as non-invasive prediction biomarkers in gastrointestinal (GI) cancers, yet their clinicopathological and prognostic values need to be more clarified. Hence, the present meta-analysis was aimed to quantitatively assess the evidence regarding the association between circulating exomiRs and prognosis in GI cancer patients. METHODS: A comprehensive search was carried out in prominent literature databases, including PubMed, ISI Web of Science, Scopus, and Embase. Odds ratios (ORs) or hazard ratios (HRs) with 95% confidence intervals (CIs) were gathered to evaluate the strength of the association. The quality assessment was investigated through the Newcastle-Ottawa Scale (NOS) and publication bias via Eggers' test and funnel plots. RESULTS: A total of 47 studies, comprising of 4881 patients, were considered eligible for this meta-analysis. Both up-regulated and down-regulated circulating exomiRs are significantly associated with differentiation (HR = 1.353, P = 0.015; HR = 1.504, P = 0.016), TNM stage (HR = 2.058, P < 0.001; HR = 2.745, P < 0.001), lymph node metastasis (HR = 1.527, P = 0.004; HR = 2.009, P = 0.002), distant metastasis (HR = 2.006, P < 0.001; HR = 2.799, P = 0.002), worse overall survival (OS) (HR = 2.053, P < 0.001; HR = 1.789, P = 0.001) and poorer disease/relapse/progression-free survival (DFS/RFS/PFS) (HR = 2.086, P < 0.001; HR = 1.607, P = 0.001) in GI cancer patients, respectively. In addition, subgroup analyses based on seven subcategories indicated the robustness of the association. The majority of findings were lack of publication bias except for the association between up-regulated exomiRs and OS or DFS/RFS/PFS and for the down-regulated exomiRs and TNM stage. CONCLUSION: This study supports that up- and down-regulated circulating exomiRs are associated with poorer survival outcomes and could be served as potential prognostic biomarkers in GI cancers. Given the limitations of the current findings, such as significant heterogeneity, more investigations are needed to fully clarify the exomiRs prognostic role.

High expression of Talin-1 is associated with tumor progression and recurrence in melanoma skin cancer patients.

BACKGROUND: Talin-1 as a component of multi-protein adhesion complexes plays a role in tumor formation and migration in various malignancies. This study investigated Talin-1 in protein levels as a potential prognosis biomarker in skin tumors. METHODS: Talin-1 was evaluated in 106 skin cancer (33 melanomas and 73 non-melanomas skin cancer (NMSC)) and 11 normal skin formalin-fixed paraffin-embedded (FFPE) tissue samples using immunohistochemical technique on tissue microarrays (TMAs). The association between the expression of Talin-1 and clinicopathological parameters, as well as survival outcomes, were assessed. RESULTS: Our findings from data minings through bioinformatics tools indicated dysregulation of Talin-1 in mRNA levels for skin cancer samples. In addition, there was a statistically significant difference in Talin-1 expression in terms of intensity of staining, percentage of positive tumor cells, and H-score in melanoma tissues compared to NMSC (P = 0.001, P < 0.001, and P < 0.001, respectively). Moreover, high cytoplasmic expression of Talin-1 was found to be associated with significantly advanced stages (P = 0.024), lymphovascular invasion (P = 0.023), and recurrence (P = 0.006) in melanoma cancer tissues. Our results on NMSC showed a statistically significant association between high intensity of staining and the poor differentiation (P = 0.044). No significant associations were observed between Talin-1 expression levels and survival outcomes of melanoma and NMSC patients. CONCLUSION: Our observations showed that higher expression of Talin1 in protein level may be significantly associated with more aggressive tumor behavior and advanced disease in patients with skin cancer. However, further studies are required to find the mechanism of action of Talin-1 in skin cancers.

RNA-sequencing for transcriptional profiling of whole blood in early stage and metastatic pancreatic cancer patients.

We investigated the transcriptional profile of whole blood in early and metastatic stages of pancreatic cancer (PaC) patients to identify potential diagnostic factors for early diagnosis. Blood samples from 18 participants (6 healthy individuals, 6 patients in early stage (I/II) PaC, and 6 patients in metastatic PaC) were analyzed by RNA-sequencing. The expression levels of identified genes were subsequently compared with their expression in pancreatic tumor tissues based on TCGA data reported in UALCAN and GEPIA2 databases. Overall, 331 and 724 genes were identified as differentially expressed genes in early and metastatic stages, respectively. Of these, 146 genes were shared by early and metastatic stages. Upregulation of PTCD3 and UBA52 genes and downregulation of A2M and ARID1B genes in PaC patients were observed from early stage to metastasis. TCGA database showed increasing trend in expression levels of these genes from stage I to IV in pancreatic tumor tissue. Finally, we found that low expression of PTCD3, A2M, and ARID1B genes and high expression of UBA52 gene were positively correlated with PaC patients survival. We identified a four-gene set (PTCD3, UBA52, A2M, and ARID1B) expressed in peripheral blood of early stage and metastatic PaC patients that may be useful for PaC early diagnosis.

Heterotypic tumor spheroids: a platform for nanomedicine evaluation.

Nanomedicine has emerged as a promising therapeutic approach, but its translation to the clinic has been hindered by the lack of cellular models to anticipate how tumor cells will respond to therapy. Three-dimensional (3D) cell culture models are thought to more accurately recapitulate key features of primary tumors than two-dimensional (2D) cultures. Heterotypic 3D tumor spheroids, composed of multiple cell types, have become more popular than homotypic spheroids, which consist of a single cell type, as a superior model for mimicking in vivo tumor heterogeneity and physiology. The stromal interactions demonstrated in heterotypic 3D tumor spheroids can affect various aspects, including response to therapy, cancer progression, nanomedicine penetration, and drug resistance. Accordingly, to design more effective anticancer nanomedicinal therapeutics, not only tumor cells but also stromal cells (e.g., fibroblasts and immune cells) should be considered to create a more physiologically relevant in vivo microenvironment. This review aims to demonstrate current knowledge of heterotypic 3D tumor spheroids in cancer research, to illustrate current advances in utilizing these tumor models as a novel and versatile platform for in vitro evaluation of nanomedicine-based therapeutics in cancer research, and to discuss challenges, guidelines, and future directions in this field.

Quantitative measurement of CA 15-3 cancer biomarker using an electrochemical aptasensor based on the electrodeposition of Au thin film on cauliflower-like rGO-MoS2 nanocomposite.

The present research was conducted to design and construct an electrochemical aptasensor for evaluating carbohydrate antigen 15-3 (CA15-3) as a biomarker for breast cancer. The aptasensor has been fabricated by a gold thin film (AuTF) electrodeposited on a cauliflower-like reduced graphene oxide-molybdenum sulfide nanocomposite (rGO-MoS2). The modified electrode's surface was used to immobilize the thiolated aptamer, which was subsequently treated with CA 15-3 antigen. The aptasensor fabrication process was assessed using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). This research also applied EIS to the quantitative measurement of CA 15-3 antigen by the proposed aptasensor. The interfacial charge transfer resistance (Rct) alteration before and after incubation of CA 15-3 by the immobilized aptamer was considered a signal for the quantitative measurement of CA 15-3. A linear concentration ranging from 5.0 to 200.0 U mL-1 with a detection limit of 3.0 × 10-1 U mL-1 was obtained for CA 15-3 using the EIS method. This designed aptasensor indicates satisfactory repeatability and stability, good selectivity, and high sensitivity. Moreover, clinical samples were assayed by the prepared aptasensor and compared with the ELISA method, yielding acceptable results. The recovery and relative standard deviation (RSD) of CA 15-3 in human serum samples were in the range 95.0 to 107.0% and 3.5 to 7.5%, respectively.

Oncogenic functions and clinical significances of DCLK1 isoforms in colorectal cancer: a systematic review and meta-analysis.

BACKGROUND: The oncogenic role of doublecortin-like kinase 1 (DCLK1) as a putative cancer stem cell (CSC) marker has been clarified in colorectal cancer (CRC). Isoform-specific functions of DCLK1 have shed new light on different functions of DCLK1 short (DCLK1-S) and DCLK1 long (DCLK1-L) isoforms in tumor initiation, growth, and metastasis. Therefore, the current systematic review and meta-analysis aimed to review the available in vitro, in vivo, and clinical evidence on the oncogenic roles and clinical significance of DCLK1 isoforms in colorectal cancer. METHODS: The literature databases of PubMed, Scopus, ISI Web of Science, and Embase were searched to identify eligible articles. The description characteristics of in vitro and pre-clinical studies were extracted from identified reports. In addition, hazard ratios (HRs) or odds ratios (ORs) with 95% confidence intervals (CIs) were recorded to determine the relationships between DCLK1-L and DCLK1-S expression and prognostic outcomes in patients with CRC. RESULTS: Both in vitro and in vivo evidence have emphasized the potential oncogenic functions of DCLK1 in tumor initiation, self-renewal ability, tumor invasion, epithelial-mesenchymal transition (EMT), and metastasis. However, the anti-DCLK1 antibodies generally utilized in these studies could detect sequence homology epitopes of both isoforms. Recent limited isoform-specific evidence has strongly supported the significant positive expression and rather oncogenic efficacy of DCLK1-S in tumorigenesis, EMT, and invasion compared with DCLK1-L in human CRC cell lines. Our meta-analysis findings of limited clinical studies indicated that only overexpression of DCLK1-S is associated with worse overall survival (OS) (HR = 7.930, 95% CI 2.252-27.924, p = 0.001). Increased expression of both DCLK1-S (HR = 1.610, 95% CI 1.020-2.541, p = 0.041) and DCLK1-L (HR = 5.890, 95% CI 1.219-28.453, p = 0.027) isoforms was closely associated with worse DSS/CSS in CRC patients. Furthermore, the high expression of DCLK1-S was found to be associated with poor DFS/RFS/PFS (HR = 1.913, 95% CI 1.230-2.973, p = 0.004). CONCLUSIONS: The current findings strongly supported that the DCLK1-S isoform may play a crucial role in the invasion, aggressive tumor behavior, and worsened survival outcomes of CRC patients. However, further critical investigations related to the potential preclinical and clinical utilities of DCLK1-S as a specific CRC-CSC marker are warranted.

Periostin: biology and function in cancer.

Periostin (POSTN), a member of the matricellular protein family, is a secreted adhesion-related protein produced in the periosteum and periodontal ligaments. Matricellular proteins are a nonstructural family of extracellular matrix (ECM) proteins that regulate a wide range of biological processes in both normal and pathological conditions. Recent studies have demonstrated the key roles of these ECM proteins in the tumor microenvironment. Furthermore, periostin is an essential regulator of bone and tooth formation and maintenance, as well as cardiac development. Also, periostin interacts with multiple cell-surface receptors, especially integrins, and triggers signals that promote tumor growth. According to recent studies, these signals are implicated in cancer cell survival, epithelial-mesenchymal transition (EMT), invasion, and metastasis. In this review, we will summarize the most current data regarding periostin, its structure and isoforms, expressions, functions, and regulation in normal and cancerous tissues. Emphasis is placed on its association with cancer progression, and also future potential for periostin-targeted therapeutic approaches will be explored.

Introduction of an efficient method for placenta decellularization with high potential to preserve ultrastructure and support cell attachment.

The placenta, as a large discarded tissue and rich in extracellular matrix (ECM), is an excellent candidate for biological scaffolds in reconstructive medicine. Considering the importance of ECM structure in cell fate, the aim of this study was to achieve human placenta decellularization protocol that preserve the structure of scaffolds. Thus, human placenta was decellularized by four protocols and decellularization efficacy was compared by hematoxylin and eosin (H&E), 4',6-diamidino-2-phenylindole (DAPI) staining, and DNA measurement. Decellularized placenta structure preservation was assessed by Masson's trichrome staining, scanning electron microscopy (SEM), and immunofluorescence (IF) for collagen I, IV, and fibronectin. Finally, liquid displacement measured scaffolds' porosity. After culturing menstrual blood-derived stem cells (MenSCs) on placenta scaffolds, cell adhesion was investigated by SEM imaging, and cell viability and proliferation were assessed by MTT assay. According to H&E and DAPI staining, only protocols 1 and 3 could completely remove cells from the scaffolds. DNA measurements confirmed a significant reduction in the genetic material of decellularized scaffolds compared to native placenta. According to Masson's trichrome, IF, and SEM imaging, scaffold structure is better preserved in P3 than P1 protocol. Liquid displacement showed higher porosity of P3 scaffold than P1. SEM imaging confirmed cells adhesion to the decellularized placenta, and the attached cells showed good viability and maintained their proliferative capacity, indicating the suitability of the scaffolds for cell growth. Results introduced an optimized protocol for placenta decellularization that preserves the scaffold structure and supports cell adhesion and proliferation.

Bioengineering of fibroblast-conditioned polycaprolactone/gelatin electrospun scaffold for skin tissue engineering.

BACKGROUND: Synthetic tissue engineering scaffolds has poor biocompatiblity with very low angiogenic properties. Conditioning the scaffolds with functional groups, coating with biological components, especially extracellular matrix (ECM), is an excellent strategy for improving their biomechanical and biological properties. METHODS: In the current study, a composite of polycaprolactone and gelatin (PCL/Gel) was electrospun in the ratio of 70/30 and surface modified with 1% gelatin-coating (G-PCL/Gel) or plasma treatment (P-PCL/Gel). The surface modification was determined by SEM and ATR-FTIR spectroscopy, respectively. The scaffolds were cultured with fibroblast 3T3, then decellularized during freeze-thawing process to fabricate a fibroblast ECM-conditioned PCL/Gel scaffold (FC-PCL/Gel). The swelling and degaradtion as well as in vitro and in vivo biocompatibility and angiogenic properties of the scaffolds were evaluated. RESULTS: The structure of the surface-modified G-PCL/Gel and P-PCL/Gel were unique and not changed compared with the PCL/Gel scaffolds. ATR-FTIR analysis admitted the formation of oxygen-containing groups, hydroxyl and carboxyl, on the surface of the P-PCL/Gel scaffold. The SEM micrographs and DAPI staining confirmed the cell attachment and the ECM deposition on the platform and successful removal of the cells after decellularization. P-PCL/Gel showed better cell attachment, ECM secretion and deposition after decellularization compared with G-PCL/Gel. The FC-PCL/Gel was considered as an optimized scaffold for further assays in this study. The FC-PCL/Gel showed increased hydrophilic behavior and cytobiocompatibility compared with P-PCL/Gel. The ECM on the FC-PCL/Gel scaffold showed a gradual degradation during 30 days of degradation time, as a small amount of ECM remained over the FC-PCL/Gel scaffold at day 30. The FC-PCL/Gel showed significant biocompatibility and improved angiogenic property compared with P-PCL/Gel when subcutaneously implanted in a mouse animal model for 7 and 28 days. CONCLUSIONS: Our findings suggest FC-PCL/Gel as an excellent biomimetic construct with high angiogenic properties. This bioengineered construct can serve as a possible application in our future pre-clinical and clinical studies for skin regeneration.

Significant co-expression of putative cancer stem cell markers, EpCAM and CD166, correlates with tumor stage and invasive behavior in colorectal cancer.

BACKGROUND: The crucial oncogenic role of cancer stem cells (CSCs) in tumor maintenance, progression, drug resistance, and relapse has been clarified in different cancers, particularly in colorectal cancer (CRC). The current study was conducted to evaluate the co-expression pattern and clinical significance of epithelial cell adhesion molecules (EpCAM) and activated leukocyte cell adhesion (CD166 or ALCAM) in CRC patients. METHODS: This study was carried out on 458 paraffin-embedded CRC specimens by immunohistochemistry on tissue microarray (TMA) slides. RESULTS: Elevated expression of EpCAM and CD166 was observed in 61.5% (246/427) and 40.5% (164/405) of CRC cases. Our analysis showed a significant positive association of EpCAM expression with tumor size (P = 0.02), tumor stage (P = 0.007), tumor differentiate (P = 0.005), vascular (P = 0.01), neural (P = 0.01), and lymph node (P = 0.001) invasion. There were no significant differences between CD166 expression and clinicopathological parameters. Moreover, the combined analysis demonstrated a reciprocal significant correlation between EpCAM and CD166 expression (P = 0.02). Interestingly, there was a significant positive correlation between EpCAM/CD166 phenotypes expression and tumor stage (P = 0.03), tumor differentiation (P = 0.05), neural, and lymph node invasion (P =0.01). CONCLUSIONS: The significant correlation of EpCAM and CD166 expression and their association with tumor progression and aggressive behavior is the reason for the suggestion of these two CSC markers as promising targets to promote novel effective targeted-therapy strategies for cancer treatment in the present study.

Production and characterization of a new specific monoclonal antibody against A-isoform of SALL4: A novel emerging testicular cancer marker.

SALL4 transcription factor plays an important role to maintain the pluripotent and self-renewal of embryonic stem cells. It contributes to the growth of many cancers and embryonic development. With the exception of spermatogonia, SALL4 expression is silenced in most adult tissues after birth; nevertheless, it is re-expressed in a subset of different solid malignancies. SALL4 is a new, precise biomarker for testicular germ cell cancers that was just introduced. The whole isoform of SALL4 is called SALL4-A. Regarding the lack of antibody against human SALL4 isoforms, the pattern of expression, the role of each isoform remain unknown. Furthermore, in isoform specific evaluations, we aimed, for the first time, to produce and characterize mAb against human SALL4-A. Immunization of mice were performed with a selected 33-mer synthetic peptide of SALL4-A conjugated with KLH. Hybridoma cells were screened by ELISA for positive reactivity with SALL4-A peptide. From the ascites fluid of mice that had been injected with hybridoma cells, anti-SALL4-A mAbs were isolated using a protein G column. Reactivity of the mAbs was evaluated using the peptide and SALL4-A recombinant protein by ELISA and IHC on testicular cancer tissue as positive control, and normal kidney, stomach and prostate tissues as negative control. The produced mAb could well detect SALL4-A in testicular cancer tissues using IHC, while the reactivity was negative in normal kidney, stomach and prostate tissues. Using ELISA, the mAb affinity for the peptide and SALL4-A recombinant protein was assessed, and it was shown to be reasonably high. The mAb detected SALL4-A in nucleus and cytoplasm of several cancer cells and spermatogonia in testicular cancer tissue. In addition, it could recognize SALL4-A recombinant protein. Our produced monoclonal antibody against isoform-A of human SALL4 can specifically recognize SALL4-A using either IHC or ELISA. We hope that this mAb could help researchers in isoform-specific study of human SALL4.

Dendritic cells loaded with exosomes derived from cancer stem cell-enriched spheroids as a potential immunotherapeutic option.

Cancer stem cells (CSCs) are responsible for therapeutic resistance and recurrence in colorectal cancer. Despite advances in immunotherapy, the inability to specifically eradicate CSCs has led to treatment failure. Hence, identification of appropriate antigen sources is a major challenge in designing dendritic cell (DC)-based therapeutic strategies against CSCs. Here, in an in vitro model using the HT-29 colon cancer cell line, we explored the efficacy of DCs loaded with exosomes derived from CSC-enriched colonospheres (CSCenr -EXOs) as an antigen source in activating CSC-specific T-cell responses. HT-29 lysate, HT-29-EXOs and CSCenr lysate were independently assessed as separate antigen sources. Having confirmed CSCs enrichment in spheroids, CSCenr -EXOs were purified and characterized, and their impact on DC maturation was investigated. Finally, the impact of the antigen-pulsed DCs on the proliferation rate and also spheroid destructive capacity of autologous T cells was assessed. CSCenr -EXOs similar to other antigen groups had no suppressive/negative impacts on phenotypic maturation of DCs as judged by the expression level of costimulatory molecules. Notably, similar to CSCenr lysate, CSCenr -EXOs significantly increased the IL-12/IL-10 ratio in supernatants of mature DCs. CSCenr -EXO-loaded DCs effectively promoted T-cell proliferation. Importantly, T cells stimulated with CSCenr -EXOs disrupted spheroids' structure. Thus, CSCenr -EXOs present a novel and promising antigen source that in combination with conventional tumour bulk-derived antigens should be further explored in pre-clinical immunotherapeutic settings for the efficacy in hampering recurrence and metastatic spread.

Highly Photoluminescent Nitrogen- and Zinc-Doped Carbon Dots for Efficient Delivery of CRISPR/Cas9 and mRNA.

Safe and efficient delivery of CRISPR/Cas9 systems is still a challenge. Here we report the development of fluorescent nitrogen- and zinc-doped carbon dots (N-Zn-doped CDs) using one-step microwave-aided pyrolysis based on citric acid, branched PEI25k, and different zinc salts. These versatile nanovectors with a quantum yield of around 60% could not only transfect large CRISPR plasmids (∼9 kb) with higher efficiency (80%) compared to PEI25k and lipofectamine 2000 (Lipo 2K), but they also delivered mRNA into HEK 293T cells with the efficiency 20 times greater than and equal to that of PEI25k and Lipo 2K, respectively. Unlike PEI25k, N-Zn-doped CDs exhibited good transfection efficiency even at low plasmid doses and in the presence of 10% fetal bovine serum (FBS). Moreover, these nanovectors demonstrated excellent efficiency in GFP gene disruption by transferring plasmid encoding Cas9 and sgRNA targeting GFP as well as Cas9/sgRNA ribonucleoproteins into HEK 293T-GFP cells. Hence, N-Zn-doped CDs with remarkable photoluminescence properties and high transfection efficiency in the delivery of both CRISPR complexes and mRNA provide a promising platform for developing safe, efficient, and traceable delivery systems for biological research.

Overexpression of DDIT4 and TPTEP1 are associated with metastasis and advanced stages in colorectal cancer patients: a study utilizing bioinformatics prediction and experimental validation.

BACKGROUND: Various diagnostic and prognostic tools exist in colorectal cancer (CRC) due to multiple genetic and epigenetic alterations causing the disease. Today, the expression of RNAs is being used as prognostic markers for cancer. METHODS: In the current study, various dysregulated RNAs in CRC were identified via bioinformatics prediction. Expression of several of these RNAs were measured by RT-qPCR in 48 tissues from CRC patients as well as in colorectal cancer stem cell-enriched spheroids derived from the HT-29 cell line. The relationships between the expression levels of these RNAs and clinicopathological features were analyzed. RESULTS: Our bioinformatics analysis determined 11 key mRNAs, 9 hub miRNAs, and 18 lncRNAs which among them 2 coding RNA genes including DDIT4 and SULF1 as well as 3 non-coding RNA genes including TPTEP1, miR-181d-5p, and miR-148b-3p were selected for the further investigations. Expression of DDIT4, TPTEP1, and miR-181d-5p showed significantly increased levels while SULF1 and miR-148b-3p showed decreased levels in CRC tissues compared to the adjacent normal tissues. Positive relationships between DDIT4, SULF1, and TPTEP1 expression and metastasis and advanced stages of CRC were observed. Additionally, our results showed significant correlations between expression of TPTEP1 with DDIT4 and SULF1. CONCLUSIONS: Our findings demonstrated increased expression levels of DDIT4 and TPTEP1 in CRC were associated with more aggressive tumor behavior and more advanced stages of the disease. The positive correlations between TPTEP1 as non-coding RNA and both DDIT4 and SULF1 suggest a regulatory effect of TPTEP1 on these genes.

Clinical and prognostic significances of cancer stem cell markers in gastric cancer patients: a systematic review and meta-analysis.

BACKGROUND: Gastric cancer (GC) is considered one of the most lethal malignancies worldwide, which is accompanied by a poor prognosis. Although reports regarding the importance of cancer stem cell (CSC) markers in gastric cancer progression have rapidly developed over the last few decades, their clinicopathological and prognostic values in gastric cancer still remain inconclusive. Therefore, the current meta-analysis aimed to quantitatively re-evaluate the association of CSC markers expression, overall and individually, with GC patients' clinical and survival outcomes. METHODS: Literature databases including PubMed, Scopus, ISI Web of Science, and Embase were searched to identify the eligible articles. Hazard ratios (HRs) or odds ratios (ORs) with 95% confidence intervals (CIs) were recorded or calculated to determine the relationships between CSC markers expression positivity and overall survival (OS), disease-free survival (DFS)/relapse-free survival (RFS), disease-specific survival (DSS)/ cancer-specific survival (CSS), and clinicopathological features. RESULTS: We initially retrieved 4,425 articles, of which a total of 66 articles with 89 studies were considered as eligible for this meta-analysis, comprising of 11,274 GC patients. Overall data analyses indicated that the overexpression of CSC markers is associated with TNM stage (OR = 2.19, 95% CI 1.84-2.61, P = 0.013), lymph node metastasis (OR = 1.76, 95% CI 1.54-2.02, P < 0.001), worse OS (HR = 1.65, 95% CI 1.54-1.77, P < 0.001), poor CSS/DSS (HR = 1.69, 95% CI 1.33-2.15, P < 0.001), and unfavorable DFS/RFS (HR = 2.35, 95% CI 1.90-2.89, P < 0.001) in GC patients. However, CSC markers expression was found to be slightly linked to tumor differentiation (OR = 1.25, 95% CI 1.01-1.55, P = 0.035). Sub-analysis demonstrated a significant positive relationship between most of the individual markers, specially Gli-1, Oct-4, CD44, CD44V6, and CD133, and clinical outcomes as well as the reduced survival, whereas overexpression of Lgr-5, Nanog, and sonic hedgehog (Shh) was not found to be related to the majority of clinical outcomes in GC patients. CONCLUSION: The expression of CSC markers is mostly associated with worse outcomes in patients with GC, both overall and individual. The detection of a combined panel of CSC markers might be appropriate as a prognostic stratification marker to predict tumor aggressiveness and poor prognosis in patients with GC, which probably results in identifying novel potential targets for therapeutic approaches.

Morphological and molecular characteristics of spheroid formation in HT-29 and Caco-2 colorectal cancer cell lines.

BACKGROUND: Relapse and metastasis in colorectal cancer (CRC) are often attributed to cancer stem-like cells (CSCs), as small sub-population of tumor cells with ability of drug resistance. Accordingly, development of appropriate models to investigate CSCs biology and establishment of effective therapeutic strategies is warranted. Hence, we aimed to assess the capability of two widely used and important colorectal cancer cell lines, HT-29 and Caco-2, in generating spheroids and their detailed morphological and molecular characteristics. METHODS: CRC spheroids were developed using hanging drop and forced floating in serum-free and non-attachment conditions and their morphological features were evaluated by scanning electron microscopy (SEM). Then, the potential of CSCs enrichment in spheroids was compared to their adherent counterparts by analysis of serial sphere formation capacity, real-time PCR of key stemness genes (KLF4, OCT4, SOX2, NANOG, C-MYC) and the expression of potential CRC-CSCs surface markers (CD166, CD44, and CD133) by flow cytometry. Finally, the expression level of some EMT-related (Vimentin, SNAIL1, TWIST1, N-cadherin, E-cadherin, ZEB1) and multi-drug resistant (ABCB1, ABCC1, ABCG2) genes was evaluated. RESULTS: Although with different morphological features, both cell lines were formed CSCs-enriched spheroids, indicated by ability to serial sphere formation, significant up-regulation of stemness genes, SOX2, C-MYC, NANOG and OCT4 in HT-29 and SOX2, C-MYC and KLF4 in Caco-2 spheroids (p-value < 0.05) and increased expression of CRC-CSC markers compared to parental cells (p-value < 0.05). Additionally, HT-29 spheroids exhibited a significant higher expression of both ABCB1 and ABCG2 (p-value = 0.02). The significant up-regulation of promoting EMT genes, ZEB1, TWIST1, E-cadherin and SNAIL1 in HT-29 spheroids (p-value = 0.03), SNAIL1 and Vimentin in Caco-2 spheroids (p-value < 0.05) and N-cadherin down-regulation in both spheroids were observed. CONCLUSION: Enrichment of CSC-related features in HT-29 and Caco-2 (for the first time without applying special scaffold/biochemical) spheroids, suggests spheroid culture as robust, reproducible, simple and cost-effective model to imitate the complexity of in vivo tumors including self-renewal, drug resistance and invasion for in vitro research of CRC-CSCs.