Identification of immunogenic tumor antigens that are efficiently processed and delivered by dendritic cells to prime the immune system and to induce an appropriate immune response is a research hotspot in the field of cancer vaccine development. High biosafety is an additional demand. Tumor-derived exosomes (TEXs) are nanosized lipid bilayer encapsulated vesicles that shuttle bioactive information to the tumor microenvironment facilitating tumor progression. However, accumulating evidence points toward the capacity of TEXs to efficiently stimulate immune responses against tumors provided they are appropriately administered. After briefly describing the function of exosomes in cancer biology and their communication with immune cells, we summarize in this review in vitro and preclinical studies eliciting the potency of TEXs in inducing effective anti-tumor responses and recently modified strategies further improving TEX-vaccination efficacy. We interpret the available data as TEXs becoming a lead in cancer vaccination based on tumor antigen-selective high immunogenicity.
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.
Carbon dots (CDs) have become the focus of many studies due to their outstanding optical properties and good biocompatibility. We investigated their potential application to produce a smart and highly efficient yet nontoxic nanovector for gene delivery. This was achieved by conjugating PEI1.8k-functionalized CDs (synthesized by one-step microwave-assisted pyrolysis) with arginine-disulfide linkers to produce CD-PEI1.8k-Arg nanoparticles. This nanovector could deliver p-CRISPR (9.3 kb) into different types of cell lines with higher efficiency compared to native PEI1.8k or PEI25k. CD-PEI1.8k-Arg also maintained its outstanding transfection efficiency at a high serum concentration and low p-CRISPR dose, compared to PEI25k, which was ineffective under those conditions. Additionally, CD-PEI1.8k-Arg could knock out the GFP gene with great efficiency by delivering the required components of CRISPR/Cas9, including a plasmid encoding Cas9, sgRNA targeting GFP, and Cas9/sgRNA ribonucleoproteins (RNPs) into the HEK 293T-GFP cells. Moreover, the nanoparticles showed potential for the local delivery of p-CRISPR into brain tissue. The remarkable properties of CD-PEI1.8k-Arg could enable the development of a safe, highly efficient gene-delivery nanovector for the treatment of various diseases in the near future.
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.
Diabetes is a major risk factor for cataract, the leading cause of blindness worldwide.There is an unmet need for a realistic model of diabetic cataract for mechanistic and longitudinal studies, as existing models do not reflect key aspects of the complex human disease. Here, we introduce and characterize diabetic cataract in the Nile grass rat (NGR, Arvicanthis niloticus), an established model of metabolic syndrome and type 2 diabetes (T2D). We conducted a longitudinal study of cataract in over 88 NGRs in their non-diabetic, pre-diabetic, and diabetic stages of metabolism. Oral glucose tolerance test (OGTT) results distinguished the metabolic stages. Diverse cataract types were observed in the course of diabetes, including cortical, posterior subcapsular (PSC), and anterior subcapsular (ASC), all of which succeeded a characteristic dotted ring stage in all animals. The onset ages of diabetes and cataract were 44 ± 3 vs 29 ± 1 (P < .001) and 66 ± 5 vs 58 ± 6 (not significant) weeks in females and males, respectively. Histological analysis revealed fiber disorganization, vacuolar structures, and cellular proliferation and migration in cataractous lenses. The lens epithelial cells (LECs) in non-diabetic young NGRs expressed the stress marker GRP78, as did LECs and migrated cells in the lenses of diabetic animals. Elucidating mechanisms underlying LEC proliferation and migration will be clinically valuable in prevention and treatment of posterior capsule opacification, a dreaded complication of cataract surgery. Marked changes in N-cadherin expression emphasized a role for LEC integrity in cataractogenesis. Apoptotic cells were dispersed in the equatorial areas in early cataractogenesis. Our study reveals diverse cataract types that spontaneously develop in the diabetic NGR, and which uniquely mirror the cataract and its chronic course of development in individuals with diabetes. We provide mechanistic insights into early stages of diabetic cataract. These unique characteristics make NGR highly suited for mechanistic studies, especially in the context of metabolism, diabetes, and aging.
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.
To explore the proper prognostic markers for the likelihood of metastasis in CRC patients. Seventy-seven fresh CRC samples were collected to evaluate the mRNA level of the selected marker using Real-time PCR. Moreover, 648 formalin-fixed paraffin-embedded CRC tissues were gathered to evaluate protein expression by immunohistochemistry (IHC) on tissue microarrays. The results of Real-Time PCR showed that low expression of Talin1 was significantly associated with advanced TNM stage (p = 0.034) as well as gender (p = 0.029) in mRNA levels. Similarly, IHC results indicated that a low level of cytoplasmic expression of Talin1 was significantly associated with advanced TNM stage (p = 0.028) as well as gender (p = 0.009) in CRC patients. Moreover, decreased expression of cytoplasmic Talin1 protein was found to be a significant predictor of worse disease-specific survival (DSS) (p = 0.011) in the univariate analysis. In addition, a significant difference was achieved (p = 0.039) in 5-year survival rates of DSS: 65% for low, 72% for moderate, and 88% for high Talin1 protein expression. Observations showed that lower expression of Talin1 at both the gene and protein level may drive the disparity of CRC patients’ outcomes via worse DSS and provide new insights into the development of progression indicators because of its correlation with increased tumor aggressiveness.
Retinal degenerative diseases, due to the lack of regeneration systems and self-renewable cells, often lead to visual impairment. Pax6 is a pleiotropic transcription factor and its expression level determines self-renewal status or differentiation of retinal cells. Here, we investigated the fate of simultaneous induction of retinal ganglion cell death and Pax6 overexpression in retro-differentiation of retinal cells and their commitment to re-enter into the cell cycle. Induction of acute retinal ganglion cell death and generation of mouse experimental model was performed by N-methyl D-aspartic acid (NMDA) injection. Recombinant AAV2 virus harboring PAX6 cDNA and reporter gene was injected into untreated and model mouse eyes. Histological analyses, including IHC and retinal flatmounts immunostaining were performed. The number of Ki67+ cells was clearly increased in model mice, presumably due to NMDA treatment and regardless of Pax6 over-expression. Unlike previous studies, Ki67+ cells were found in GCL layer and interestingly ONL cells expressed Sox2 stemness marker after NMDA cytotoxicity. The potential of retinal cells for robust Ki67 expression, after injury, and expression of Sox2, confirmed their intrinsic plasticity and made a vivid prospect for retinal regenerative medicine.
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