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Intercellular communication between different cell types in solid tumors contributes to tumor growth and metastatic dissemination. The secretome of cancer-associated fibroblasts (CAFs) plays major roles in these processes. Using human mammary CAFs, we showed that CAFs with a myofibroblast phenotype released extracellular vesicles that transferred proteins to endothelial cells (ECs) that affected their interaction with immune cells. Mass spectrometry–based proteomics identified proteins transferred from CAFs to ECs, which included plasma membrane receptors. Using THY1 as an example of a transferred plasma membrane–bound protein, we showed that CAF-derived proteins increased the adhesion of a monocyte cell line to ECs. CAFs produced high amounts of matrix-bound EVs, which were the primary vehicles of protein transfer. Hence, our work paves the way for future studies that investigate how CAF-derived matrix-bound EVs influence tumor pathology by regulating the function of neighboring cancer, stromal, and immune cells. Editor's summary: Cancer-associated fibroblasts promote tumor growth, in part, by releasing extracellular vesicles, which can carry proteins to cells in the tumor microenvironment. Santi et al. investigated intercellular communication between endothelial cells in blood vessels and cancer-associated fibroblasts isolated from patients with breast cancer. Endothelial cells in vitro and in vivo took up proteins from extracellular vesicles, specifically matrix-bound vesicles, released by cancer-associated fibroblasts. Uptake of the membrane glycoprotein THY1 from cancer-associated fibroblasts increased the adhesion of monocytes to endothelial cells. Cancer-associated fibroblasts that released the most matrix-bound vesicles resembled myofibroblasts. Thus, identifying the proteins released by myofibroblast-like cancer-associated fibroblasts that alter endothelial cell function could yield potential targets for disrupting this intercellular communication. —Wei Wong [ABSTRACT FROM AUTHOR]

Gene therapy holds great promise for treating prostate cancer unresponsive to conventional therapies. However, the lack of delivery systems that can transport therapeutic DNA and drugs while targeting tumors without harming healthy tissues presents a significant challenge. This study aimed to explore the potential of novel hybrid lipid nanoparticles, composed of biocompatible zein and conjugated to the cancer-targeting ligand transferrin. These nanoparticles were designed to entrap the anti-cancer drug docetaxel and carry plasmid DNA, with the objective of improving the delivery of therapeutic payloads to prostate cancer cells, thereby enhancing their anti-proliferative efficacy and gene expression levels. These transferrin-bearing, zein-based hybrid lipid nanoparticles efficiently entrapped docetaxel, leading to increased uptake by PC-3 and LNCaP cancer cells and significantly enhancing anti-proliferative efficacy at docetaxel concentrations exceeding 1 µg/mL. Furthermore, they demonstrated proficient DNA condensation, exceeding 80% at polymer–DNA weight ratios of 1500:1 and 2000:1. This resulted in increased gene expression across all tested cell lines, with the highest transfection levels up to 11-fold higher than those observed with controls, in LNCaP cells. These novel transferrin-bearing, zein-based hybrid lipid nanoparticles therefore exhibit promising potential as drug and gene delivery systems for prostate cancer therapy. [ABSTRACT FROM AUTHOR]

Simple Summary: The efficacy of some cancer chemotherapies relies on their ability to induce immunogenic cell death (ICD) to activate the immune system. In addition, it has been suggested that treatments that provoke ICD could be used in combination with chemotherapies to improve their anti-cancer actions. Having determined that mutating the caspase cleavage site in ROCK1 promoted necrosis-like inflammatory cell death during acute chemically induced liver damage and in hepatocellular carcinomas, we examined whether similar effects would be induced by pharmacological ROCK inhibition. Although mice expressing the caspase-resistant non-cleavable ROCK1 (ROCK1nc) had higher levels of neutrophils and CD8+ T cells in hepatocellular carcinoma tumours than in ROCK1 wild-type mice, indicative of greater ICD, two independent pharmacological ROCK inhibitors did not induce similar patterns. As a result, there is unlikely to be a clinical benefit derived from using pharmacological ROCK inhibitors to provoke ICD with standard of care therapies to increase their therapeutic efficacy. The morphological changes during apoptosis help facilitate "immunologically silent" cell death. Caspase cleavage of the ROCK1 kinase results in its activation, which drives the forceful contraction of apoptotic cells. We previously showed that when ROCK1 was mutated to render it caspase-resistant, there was greater liver damage and neutrophil recruitment after treatment with the hepatotoxin diethylnitrosamine (DEN). We now show that acute DEN-induced liver damage induced higher levels of pro-inflammatory cytokines/chemokines, indicative of immunogenic cell death (ICD), in mice expressing non-cleavable ROCK1 (ROCK1nc). Hepatocellular carcinoma (HCC) tumours in ROCK1nc mice had more neutrophils and CD8+ T cells relative to mice expressing wild-type ROCK1, indicating that spontaneous tumour cell death also was more immunogenic. Since ICD induction has been proposed to be tumour-suppressive, the effects of two distinct ROCK inhibitors on HCC tumours was examined. Both fasudil and AT13148 significantly decreased tumour numbers, areas and volumes, but neither resulted in greater numbers of neutrophils or CD8+ T cells to be recruited. In the context of acute DEN-induced liver damage, AT13148 inhibited the recruitment of dendritic, natural killer and CD8+ T cells to livers. These observations indicate that there is an important role for ROCK1 cleavage to limit immunogenic cell death, which was not replicated by systemic ROCK inhibitor administration. As a result, concomitant administration of ROCK inhibitors with cancer therapeutics would be unlikely to result in therapeutic benefit by inducing ICD to increase anti-tumour immune responses. [ABSTRACT FROM AUTHOR]

Human small and large intestinal tissue was used to study the interaction of Campylobacter jejuni with its target tissue. The strain used for the study was 81-176 (+ pVir). Tissue was processed for scanning and transmission electron microscopy, and by immunohistochemistry for light microscopy. Organisms adhered to the apical surface of ileal tissues at all time points in large numbers, in areas where mucus was present and in distinct groups. Microcolony formation was evident at 1-2 h, with bacteria adhering to mucus on the tissue surface and to each other by flagellar interaction. At later time points (3-4 h), biofilm formation on ileal tissue was evident. Flagellar mutants did not form microcolonies or biofilms in tissue. Few organisms were observed in colonic tissue, with organisms present but not as abundant as in the ileal tissue. This study shows that C. jejuni 81-1 76 can form microcolonies and biofilms on human intestinal tissue and that this may be an essential step in its ability to cause diarrhoea in man. DOI 10.1099/rnic.0.039867-0

Originally developed for the treatment of inflammatory disorders, the non-steroidal anti-inflammatory drug aspirin was shown to have a preventive effect against cancer in the past decades. Most importantly, recent studies suggested that it might also provide a therapeutic benefit in the treatment of cancer in vitro. However, this drug failed to specifically reach tumors at a therapeutic concentration following intravenous administration, thus resulting in lack of efficacy on tumors. In this work, we demonstrated that aspirin could be formulated in transferrin-bearing vesicles and that this tumor-targeted formulation could lead to an increase in the anti-proliferative efficacy of the drug in three cancer cell lines in vitro. The in vitro therapeutic efficacy of aspirin was significantly improved when formulated in transferrin-bearing vesicles, by about 2-fold compared to that of drug solution. These results are promising and support the optimization of this delivery system to further improve its potential as a therapeutic tool in combination with other anti-cancer therapies. [ABSTRACT FROM AUTHOR]

Cancer cells are softer than the normal cells, and metastatic cells are even softer. These changes in biomechanical properties contribute to cancer progression by facilitating cell movement through physically constraining environments. To identify properties that enabled passage through physical constraints, cells that were more efficient at moving through narrow membrane micropores were selected from established cell lines. By examining micropore-selected human MDA MB 231 breast cancer and MDA MB 435 melanoma cancer cells, membrane fluidity and nuclear elasticity were excluded as primary contributors. Instead, reduced actin cytoskeleton anisotropy, focal adhesion density and cell stiffnesswere characteristics associated with efficient passage through constraints. By comparing transcriptomic profiles between the parental and selected populations, increased Ras/MAPK signalling was linked with cytoskeleton rearrangements and cell softening. MEK inhibitor treatment reversed the transcriptional, cytoskeleton, focal adhesion and elasticity changes. Conversely, expression of oncogenic KRas in parental MDA MB 231 cells, or oncogenic BRaf in parental MDA MB 435 cells, significantly reduced cell stiffness. These results reveal that MAPK signalling, in addition to tumour cell proliferation, has a significant role in regulating cell biomechanics. [ABSTRACT FROM AUTHOR]

The therapeutic potential of plumbagin, a naphthoquinone extracted from the officinal leadwort with anticancer properties, is hampered by its failure to specifically reach tumours at a therapeutic concentration after intravenous administration, without secondary effects on normal tissues. Its use in clinic is further limited by its poor aqueous solubility, its spontaneous sublimation, and its rapid elimination in vivo. We hypothesize that the entrapment of plumbagin within liposomes grafted with transferrin, whose receptors are overexpressed on many cancer cells, could result in a selective delivery to tumours after intravenous administration. The objectives of this study were therefore to prepare and characterize transferrin‐targeted liposomes entrapping plumbagin and to evaluate their therapeutic efficacy in vitro and in vivo. The entrapment of plumbagin in transferrin‐bearing liposomes led to an increase in plumbagin uptake by cancer cells and improved antiproliferative efficacy and apoptosis activity in B16‐F10, A431, and T98G cell lines compared with that observed with the drug solution. In vivo, the intravenous injection of transferrin‐bearing liposomes entrapping plumbagin led to tumour suppression for 10% of B16‐F10 tumours and tumour regression for a further 10% of the tumours. By contrast, all the tumours treated with plumbagin solution or left untreated were progressive. The animals did not show any signs of toxicity. Transferrin‐bearing liposomes entrapping plumbagin are therefore highly promising therapeutic systems that should be further optimized as a therapeutic tool for cancer treatment. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells are multipotent adult stem cells capable of generating bone, cartilage and fat, and are thus currently being exploited for regenerative medicine. When considering osteogenesis, developments have been made with regards to chemical induction (e.g. differentiation media) and physical induction (e.g. material stiffness, nanotopography), targeting established early transcription factors or regulators such as runx2 or bone morphogenic proteins and promoting increased numbers of cells committing to osteo-specific differentiation. Recent research highlighted the involvement of microRNAs in lineage commitment and terminal differentiation. Herein, gold nanoparticles that confer stability to short single stranded RNAs were used to deliver MiR-31 antagomiRs to both pre-osteoblastic cells and primary human MSCs in vitro. Results showed that blocking miR-31 led to an increase in osterix protein in both cell types at day 7, with an increase in osteocalcin at day 21, suggesting MSC osteogenesis. In addition, it was noted that antagomiR sequence direction was important, with the 5 prime reading direction proving more effective than the 3 prime. This study highlights the potential that miRNA antagomiR-tagged nanoparticles offer as novel therapeutics in regenerative medicine. [ABSTRACT FROM AUTHOR]

Multicellular spheroids are an established system for three-dimensional cell culture. Spheroids are typically generated using hanging drop or non-adherent culture; however, an emerging technique is to use magnetic levitation. Herein, mesenchymal stem cell spheroids were generated using magnetic nanoparticles and subsequently cultured within a type I collagen gel, with a view towards developing a bone marrow niche environment. Cells were loaded with magnetic nanoparticles, and suspended beneath an external magnet, inducing self-assembly of multicellular spheroids. Cells in spheroids were viable and compared to corresponding monolayer controls, maintained stem cell phenotype and were quiescent. Interestingly, core spheroid necrosis was not observed, even with increasing spheroid size, in contrast to other commonly used spheroid systems. This mesenchymal stem cell spheroid culture presents a potential platform for modelling in vitro bone marrow stem cell niches, elucidating interactions between cells, as well as a useful model for drug delivery studies. [ABSTRACT FROM AUTHOR]

Photoreceptors are highly specialized sensory neurons that possess a modified primary cilium called the outer segment. Photoreceptor outer segment formation and maintenance require highly active protein transport via a process known as intraflagellar transport. Anterograde transport in outer segments is powered by the heterotrimeric kinesin II and coordinated by intraflagellar transport proteins. Here, we describe a new zebrafish model carrying a nonsense mutation in the kinesin II family member 3A ( kif3a) gene. Kif3a mutant zebrafish exhibited curved body axes and kidney cysts. Outer segments were not formed in most parts of the mutant retina, and rhodopsin was mislocalized, suggesting KIF3A has a role in rhodopsin trafficking. Both rod and cone photoreceptors degenerated rapidly between 4 and 9 days post fertilization, and electroretinography response was not detected in 7 days post fertilization mutant larvae. Loss of KIF3A in zebrafish also resulted in an intracellular transport defect affecting anterograde but not retrograde transport of organelles. Our results indicate KIF3A plays a conserved role in photoreceptor outer segment formation and intracellular transport. [ABSTRACT FROM AUTHOR]

Aims: RNAi is a powerful tool for gene silencing that can be used to reduce undesirable overexpression of oncogenes as a novel form of cancer treatment. However, when using RNAi as a therapeutic tool there is potential for associated gene effects. This study aimed to utilize gold nanoparticles to deliver siRNA into HeLa cells. Results: Knockdown of the c-myc oncogene by RNAi, at the RNA, protein and cell proliferation level was achieved, while also identifying associated gene responses. Discussion: The gold nanoparticles used in this study present an excellent delivery platform for siRNA, but do note associated gene changes. Conclusion: The study highlights the need to more widely assess the cell physiological response to RNAi treatment, rather than focus on the immediate RNA levels. [ABSTRACT FROM AUTHOR]

Nanotechnology plays an increasingly important role in the biomedical arena. In particular, magnetic nanoparticles (mNPs) have become important tools in molecular diagnostics, in vivo imaging and improved treatment of disease, with the ultimate aim of producing a more theranostic approach. Due to their small sizes, the nanoparticles can cross most of the biological barriers such as the blood vessels and the blood brain barrier, thus providing ubiquitous access to most tissues. In all biomedical applications maximum nanoparticle uptake into cells is required. Two promising methods employed to this end include functionalization of mNPs with cell-penetrating peptides to promote efficient translocation of cargo into the cell and the use of external magnetic fields for enhanced delivery. This study aimed to compare the effect of both penetratin and a static magnetic field with regards to the cellular uptake of 200 nm magnetic NPs and determine the route of uptake by both methods. Results demonstrated that both techniques increased particle uptake, with penetratin proving more cell specific. Clathrinmedicated endocytosis appeared to be responsible for uptake as shown via PCR and western blot, with Pitstop 2 (known to selectively block clathrin formation) blocking particle uptake.Interestingly, it was further shown that a magnetic field was able to reverse or overcome the blocking, suggesting an alternative route of uptake. [ABSTRACT FROM AUTHOR]

The formation of a protein layer "corona" on the nanoparticle surface upon entry into a biological environment was shown to strongly influence the interactions with cells, especially affecting the uptake of nanomedicines. In this work, we present the impact of the protein corona on the uptake of PEGylated zein micelles by cancer cells, macrophages, and dendritic cells. Zein was successfully conjugated with poly(ethylene glycol) (PEG) of varying chain lengths (5K and 10K) and assembled into micelles. Our results demonstrate that PEGylation conferred stealth effects to the zein micelles. The presence of human plasma did not impact the uptake levels of the micelles by melanoma cancer cells, regardless of the PEG chain length used. In contrast, it decreased the uptake by macrophages and dendritic cells. These results therefore make PEGylated zein micelles promising as potential drug delivery systems for cancer therapy. [ABSTRACT FROM AUTHOR]

Multicellular spheroids are an established system for three-dimensional cell culture. Spheroids are typically generated using hanging drop or non-adherent culture; however, an emerging technique is to use magnetic levitation. Herein, mesenchymal stem cell spheroids were generated using magnetic nanoparticles and subsequently cultured within a type I collagen gel, with a view towards developing a bone marrow niche environment. Cells were loaded with magnetic nanoparticles, and suspended beneath an external magnet, inducing self-assembly of multicellular spheroids. Cells in spheroids were viable and compared to corresponding monolayer controls, maintained stem cell phenotype and were quiescent. Interestingly, core spheroid necrosis was not observed, even with increasing spheroid size, in contrast to other commonly used spheroid systems. This mesenchymal stem cell spheroid culture presents a potential platform for modelling in vitro bone marrow stem cell niches, elucidating interactions between cells, as well as a useful model for drug delivery studies. [ABSTRACT FROM AUTHOR]

Genome sequencing of Clostridium clostridioforme strain LM41 revealed the presence of an atypically high proportion of mobile genetic elements for this species, with a particularly high abundance of prophages. Bioinformatic analysis of prophage sequences sought to characterize these elements and identify prophage-linked genes contributing to enhanced fitness of the host bacteria in the dysbiotic gut. Using PHASTER, PhageScope and manual curation, this work has identified 15 prophages: 4 predicted to be intact, 2 predicted to be defective and 9 which are unclassified. Quantitative PCR (qPCR) analysis revealed spontaneous release of four of the LM41 prophages (φ1, φ2, φ4 and φ10) into the culture supernatant, with virion-like particles visualized using transmission electron microscopy. The majority (12/14) of these particles had morphology akin to podoviruses, which is consistent with morphology predictions for φ1 and φ4. We observed diversity in the lysogeny mechanisms utilized by the prophages, with examples of the classical λ-like CI/Cro system, the ICE Bs 1 ImmR/ImmA-like system and the Mu-like C/Ner system. Classical morons, such as toxins or immune evasion factors, were not observed. We did, however, identify a variety of genes with roles in mediating restriction modification and genetic diversity, as well as some candidate genes with potential roles in host adaptation. Despite being the most abundant entities in the intestine, there is a dearth of information about phages associated with members of the microbiome. This work begins to shed light on the contribution of these elements to the lifestyle of C. clostridioforme LM41.

We present an autonomous chemical synthesis robot for the exploration, discovery, and optimization of nanostructures driven by real-time spectroscopic feedback, theory, and machine learning algorithms that control the reaction conditions and allow the selective templating of reactions. This approach allows the transfer of materials as seeds between cycles of exploration, opening the search space like gene transfer in biology. The open-ended exploration of the seed-mediated multistep synthesis of gold nanoparticles (AuNPs) via in-line ultraviolet-visible characterization led to the discovery of five categories of nanoparticles by only performing ca. 1000 experiments in three hierarchically linked chemical spaces. The platform optimized nanostructures with desired optical properties by combining experiments and extinction spectrum simulations to achieve a yield of up to 95%. The synthetic procedure is outputted in a universal format using the chemical description language (χDL) with analytical data to produce a unique digital signature to enable the reproducibility of the synthesis.