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Dynamic and adaptive cancer stem cell population admixture in colorectal neoplasia.
Intestinal homeostasis is underpinned by LGR5+ve crypt-base columnar stem cells (CBCs), but following injury, dedifferentiation results in the emergence of LGR5-ve regenerative stem cell populations (RSCs), characterized by fetal transcriptional profiles. Neoplasia hijacks regenerative signaling, so we assessed the distribution of CBCs and RSCs in mouse and human intestinal tumors. Using combined molecular-morphological analysis, we demonstrate variable expression of stem cell markers across a range of lesions. The degree of CBC-RSC admixture was associated with both epithelial mutation and microenvironmental signaling disruption and could be mapped across disease molecular subtypes. The CBC-RSC equilibrium was adaptive, with a dynamic response to acute selective pressure, and adaptability was associated with chemoresistance. We propose a fitness landscape model where individual tumors have equilibrated stem cell population distributions along a CBC-RSC phenotypic axis. Cellular plasticity is represented by position shift along this axis and is influenced by cell-intrinsic, extrinsic, and therapeutic selective pressures.
Holding space for spatial biology
Spatial biology is revolutionising our understanding of cellular organisation and disease by preserving the interactions between cells within tissues. Traditional methods often disrupted these delicate structures, making it challenging to study cells in their natural environments. This article explores key technologies, such as immunohistochemistry (IHC) and in situ hybridisation, which enable precise molecular analysis while maintaining spatial context. IHC remains essential for identifying protein markers in pathology, while multiplex imaging systems significantly enhance biomarker detection and high-throughput spatial profiling. Additionally, new computational tools like MuSpAn are advancing our understanding of spatial cell organisation. Despite challenges posed by data complexity, spatial biology opens exciting new possibilities for precision medicine, facilitating targeted therapies and advancing personalised treatment strategies. As the field rapidly evolves, it continues to drive groundbreaking breakthroughs in disease research and therapeutic development.
The International Natural Product Sciences Taskforce (INPST) and the power of Twitter networking exemplified through #INPST hashtag analysis.
BackgroundThe development of digital technologies and the evolution of open innovation approaches have enabled the creation of diverse virtual organizations and enterprises coordinating their activities primarily online. The open innovation platform titled "International Natural Product Sciences Taskforce" (INPST) was established in 2018, to bring together in collaborative environment individuals and organizations interested in natural product scientific research, and to empower their interactions by using digital communication tools.MethodsIn this work, we present a general overview of INPST activities and showcase the specific use of Twitter as a powerful networking tool that was used to host a one-week "2021 INPST Twitter Networking Event" (spanning from 31st May 2021 to 6th June 2021) based on the application of the Twitter hashtag #INPST.Results and conclusionThe use of this hashtag during the networking event period was analyzed with Symplur Signals (https://www.symplur.com/), revealing a total of 6,036 tweets, shared by 686 users, which generated a total of 65,004,773 impressions (views of the respective tweets). This networking event's achieved high visibility and participation rate showcases a convincing example of how this social media platform can be used as a highly effective tool to host virtual Twitter-based international biomedical research events.
Design of a novel electrospun PVA platform for gene therapy applications using the CHAT peptide.
The electrospinning of polymers has previously shown excellent potential for localised gene therapy. Thus, it was proposed that for the first time, the cell-penetrating CHAT peptide could be utilised to deliver DNA via electrospun nanofibres for localised gene therapy treatment. CHAT is an effective delivery system that encapsulates pDNA to form nanoparticles with the physicochemical characteristics for cellular uptake and protein generation. In this study, the production of smooth, bead-free PVA nanofibres by electrospinning was optimised through a Design of Experiments approach. Bead-free PVA nanofibres were consistently produced using the optimised parameters as follows: applied voltage (8 kV); collector-emitter distance (8 cm); polymer flow rate (4 µL/min); polymer concentration (9 wt% polymer); PVA MW (146-180 kDa). PVA nanofibres were subsequently crosslinked in 1 vol% glutaraldehyde in methanol to confer stability under aqueous conditions with minimal change to morphology, and no compromise to biocompatibility. Nanoparticles of CHAT/pDNA were synthesised and incorporated into the crosslinked nanofibres via soak-loading. Evaluation studies indicated that 100% of the loaded cargo was released within 48 h from the nanofibres. Furthermore, the released pDNA retained structural integrity and functionality as confirmed by gel electrophoresis and transfection studies in NCTC-929 fibroblast cells. Taken together, this data demonstrates that delivery of CHAT/pDNA nanoparticles from electrospun PVA nanofibres represents a solution for localised gene therapy.
Rational design and characterisation of a linear cell penetrating peptide for non-viral gene delivery.
The design of a non-viral gene delivery system that can release a functional nucleic acid at the intracellular destination site is an exciting but also challenging proposition. The ideal gene delivery vector must be non-toxic, non-immunogenic, overcome extra- and intra-cellular barriers, protect the nucleic acid cargo from degradation with stability over a range of temperatures. A new 15 amino acid linear peptide termed CHAT was designed in this study with the goal of delivering DNA with high efficiency into cells in vitro and tissues in vivo. Rational design involved incorporation of key amino acids including arginine for nucleic acid complexation and cellular uptake, tryptophan to enhance hydrophobic interaction with cell membranes, histidine to facilitate endosomal escape and cysteine for stability and controlled cargo release. Six linear peptides were synthesised with strategic sequences and amino acid substitutions. Data demonstrated that all six peptides complexed pDNA to produce cationic nanoparticles less than 200 nm in diameter, but not all peptides resulted in successful transfection; indicating the influence of peptide design for endosomal escape. Peptide 4, now termed CHAT, was non-cytotoxic, traversed the plasma membrane of breast and prostate cancer cell lines, and elicited reporter-gene expression following intra-tumoural and intravenous delivery in vivo. CHAT presents an exciting new peptide for the delivery of nucleic acid therapeutics.
Improving the Intercellular Uptake and Osteogenic Potency of Calcium Phosphate via Nanocomplexation with the RALA Peptide.
Calcium phosphate-base materials (e.g., alpha tri-calcium phosphate (α-TCP)) have been shown to promote osteogenic differentiation of stem/progenitor cells, enhance osteoblast osteogenic activity and mediate in vivo bone tissue formation. However, variable particle size and hydrophilicity of the calcium phosphate result in an extremely low bioavailability. Therefore, an effective delivery system is required that can encapsulate the calcium phosphate, improve cellular entry and, consequently, elicit a potent osteogenic response in osteoblasts. In this study, collagenous matrix deposition and extracellular matrix mineralization of osteoblast lineage cells were assessed to investigate osteogenesis following intracellular delivery of α-TCP nanoparticles. The nanoparticles were formed via condensation with a novel, cationic 30 mer amphipathic peptide (RALA). Nanoparticles prepared at a mass ratio of 5:1 demonstrated an average particle size of 43 nm with a zeta potential of +26 mV. The average particle size and zeta potential remained stable for up to 28 days at room temperature and across a range of temperatures (4-37 °C). Cell viability decreased 24 h post-transfection following RALA/α-TCP nanoparticle treatment; however, recovery ensued by Day 7. Immunocytochemistry staining for Type I collagen up to Day 21 post-transfection with RALA/α-TCP nanoparticles (NPs) in MG-63 cells exhibited a significant enhancement in collagen expression and deposition compared to an untreated control. Furthermore, in porcine mesenchymal stem cells (pMSCs), there was enhanced mineralization compared to α-TCP alone. Taken together these data demonstrate that internalization of RALA/α-TCP NPs elicits a potent osteogenic response in both MG-63 and pMSCs.
Exploring the Potential of MicroRNA Let-7c as a Therapeutic for Prostate Cancer.
Prostate cancer (PCa) is one of the leading causes of mortality worldwide and often presents with aberrant microRNA (miRNA) expression. Identifying and understanding the unique expression profiles could aid in the detection and treatment of this disease. This review aims to identify miRNAs as potential therapeutic targets for PCa. Three bio-informatic searches were conducted to identify miRNAs that are reportedly implicated in the pathogenesis of PCa. Only hsa-Lethal-7 (let-7c), recognized for its role in PCa pathogenesis, was common to all three databases. Three further database searches were conducted to identify known targets of hsa-let-7c. Four targets were identified, HMGA2, c-Myc (MYC), TRAIL, and CASP3. An extensive review of the literature was undertaken to assess the role of hsa-let-7c in the progression of other malignancies and to evaluate its potential as a therapeutic target for PCa. The heterogeneous nature of cancer makes it logical to develop mechanisms by which the treatment of malignancies is tailored to an individual, harnessing specific knowledge of the underlying biology of the disease. Resetting cellular miRNA levels is an exciting prospect that will allow this ambition to be realized.
MicroRNAs in Pancreatic Cancer: biomarkers, prognostic, and therapeutic modulators
AbstractA severe lack of early diagnosis coupled with resistance to most available therapeutic options renders pancreatic cancer as a major clinical concern. The limited efficacy of current treatments necessitates the development of novel therapeutic strategies that are based on an understanding of the molecular mechanisms involved in pancreatic cancer progression. MicroRNAs (miRNAs) are non-coding small RNAs that regulate the expression of multiple proteins in the post-translation process and thus have promise as biomarkers, prognostic agents, and as advanced pancreatic therapies.Profiling of deregulated miRNAs in pancreatic cancer can correlate to diagnosis, indicate optimal treatment and predict response to therapy. Furthermore, understanding the main effector genes in pancreatic cancer along with downstream pathways can identify possible miRNAs as therapeutic candidates. Additionally, obstacles to the translation of miRNAs into the clinic are also considered.Distinct miRNA expression profiles can correlate to stages of malignant pancreatic disease, and hold potential as biomarkers, prognostic markers and clinical targets. However, a limited understanding and validation of the specific role of such miRNAs stunts clinical application. Target prediction using algorithms provides a wide range of possible targets, but these miRNAs still require validation through pre-clinical studies to determine the knock-on genetic effects.Graphical abstract
Delivery of RALA/siFKBPL nanoparticles via electrospun bilayer nanofibres: An innovative angiogenic therapy for wound repair.
FK506-binding protein-like (FKBPL) has previously been shown to inhibit angiogenesis viain vitro and in vivo experimentation. Thus, it was proposed that the delivery of a siRNA targeting FKBPL could hold great potential in promoting angiogenesis for advanced wound healing applications. An effective delivery system has been utilised to encapsulate the siFKBPL to form nanoparticles, thereby improving cellular entry and eliciting a potent angiogenic response. In this study, nanoparticles were formed via condensation of siFKBPL with RALA; a novel, cationic 30 mer amphipathic peptide. Nanoparticles prepared at a N:P ratio of 6 demonstrated an average particle size of 76.6nm with a zeta potential of +16.5mV. Treatment of HMEC-1 cells at N:P 6 resulted in a transfection efficiency of 33.7%, negligible cytotoxicity, and significant knockdown of endogenous FKBPL expression. Functionally, treatment with RALA/siFKBPL resulted in significant improvements in cell migration and endothelial tubule formation in vitro. The process of electrospinning was employed to fabricate a nanofibrous wound patch to facilitate the controlled delivery of the RALA/siFKBPL nanoparticles. Alginate/poly-(vinyl alcohol) was electrospun following electrospinning of Chitosan/poly-(vinyl alcohol) to form a bilayered wound patch. Subsequently, the nanofibres were crosslinked to improve stability, before nanoparticle incorporation via soak loading. In vivo wound healing studies using C57BL/6J mice demonstrated a significant increase in angiogenesis when the RALA/siFKBPL nanoparticles were delivered from the bilayered wound patch; a 326% increase in blood vessel density was observed compared to untreated wounds. Taken together, this data demonstrates that delivery of RALA/siFKBPL nanoparticles from the bilayered wound patch represents an innovative wound healing therapy.
DNA vaccination via RALA nanoparticles in a microneedle delivery system induces a potent immune response against the endogenous prostate cancer stem cell antigen.
Castrate resistant prostate cancer (CRPC) remains a major challenge for healthcare professionals. Immunotherapeutic approaches, including DNA vaccination, hold the potential to harness the host's own immune system to mount a cell-mediated, anti-tumour response, capable of clearing disseminated tumour deposits. These anti-cancer vaccines represent a promising strategy for patients with advanced disease, however, to date DNA vaccines have demonstrated limited efficacy in clinical trials, owing to the lack of a suitable DNA delivery system. This study was designed to evaluate the efficacy of a two-tier delivery system incorporating cationic RALA/pDNA nanoparticles (NPs) into a dissolvable microneedle (MN) patch for the purposes of DNA vaccination against prostate cancer. Application of NP-loaded MN patches successfully resulted in endogenous production of the encoded Prostate Stem Cell Antigen (PSCA). Furthermore, immunisation with RALA/pPSCA loaded MNs elicited a tumour-specific immune response against TRAMP-C1 tumours ex vivo. Finally, vaccination with RALA/pPSCA loaded MNs demonstrated anti-tumour activity in both prophylactic and therapeutic prostate cancer models in vivo. This is further evidence that this two-tier MN delivery system is a robust platform for prostate cancer DNA vaccination. STATEMENT OF SIGNIFICANCE: This research describes the development and utilisation of our unique microneedle (MN) DNA delivery system, which enables penetration through the stratum corneum and deposition of the DNA within the highly immunogenic skin layers via a dissolvable MN matrix, and facilitates cellular uptake via complexation of pDNA cargo into nanoparticles (NPs) with the RALA delivery peptide. We report for the first time on using the NP-MN platform to immunise mice with encoded Prostate Stem Cell Antigen (mPSCA) for prostate cancer DNA vaccination. Application of the NP-MN system resulted in local mPSCA expression in vivo. Furthermore, immunisation with the NP-MN system induced a tumour-specific cellular immune response, and inhibited the growth of TRAMP-C1 prostate tumours in both prophylactic and therapeutic challenge models in vivo.
MicroRNA as Therapeutic Targets for Chronic Wound Healing.
Wound healing is a highly complex biological process composed of three overlapping phases: inflammation, proliferation, and remodeling. Impairments at any one or more of these stages can lead to compromised healing. MicroRNAs (miRs) are non-coding RNAs that act as post-transcriptional regulators of multiple proteins and associated pathways. Thus, identification of the appropriate miR involved in the different phases of wound healing could reveal an effective third-generation genetic therapy in chronic wound care. Several miRs have been shown to be upregulated or downregulated during the wound healing process. This article examines the biological processes involved in wound healing, the miR involved at each stage, and how expression levels are modulated in the chronic wound environment. Key miRs are highlighted as possible therapeutic targets, either through underexpression or overexpression, and the healing benefits are interrogated. These are prime miR candidates that could be considered as a gene therapy option for patients suffering from chronic wounds. The success of miR as a gene therapy, however, is reliant on the development of an appropriate delivery system that must be designed to overcome both extracellular and intracellular barriers.
Epithelial GREMLIN1 disrupts intestinal epithelial-mesenchymal crosstalk to induce a wnt-dependent ectopic stem cell niche through stromal remodelling
Abstract In homeostasis, counterbalanced morphogen signalling gradients along the vertical axis of the intestinal mucosa regulate the fate and function of epithelial and stromal cell compartments. Here, we use a disease-positioned mouse and human tissue to explore the consequences of pathological BMP signalling dysregulation on epithelial-mesenchymal interaction. Aberrant pan-epithelial expression of the secreted BMP antagonist Grem1 results in ectopic crypt formation, with lineage tracing demonstrating the presence of Lgr5(−) stem/progenitor cells. Isolated epithelial cell Grem1 expression has no effect on individual cell fate, indicating an intercompartmental impact of mucosal-wide BMP antagonism. Treatment with an anti-Grem1 antibody abrogates the polyposis phenotype, and triangulation of specific pathway inhibitors defines a pathological sequence of events, with Wnt-ligand-dependent ectopic stem cell niches forming through stromal remodelling following BMP disruption. These data support an emerging co-evolutionary model of intestinal cell compartmentalisation based on bidirectional regulation of epithelial-mesenchymal cell fate and function.