Stepwise-targeting and hypoxia-responsive liposome AMVY@NPs carrying siYAP and verteporfin for glioblastoma therapy.

BACKGROUND: The Hippo pathway is a conserved tumour suppressor signalling pathway, and its dysregulation is often associated with abnormal cell growth and tumorigenesis. We previously revealed that the transcriptional coactivator Yes-associated protein (YAP), the key effector of the Hippo pathway, is a molecular target for glioblastoma (GBM), the most common malignant brain tumour. Inhibiting YAP with small interfering RNA (siYAP) or the specific inhibitor verteporfin (VP) can diminish GBM growth to a certain degree. RESULTS: In this study, to enhance the anti-GBM effect of siYAP and VP, we designed stepwise-targeting and hypoxia-responsive liposomes (AMVY@NPs), which encapsulate hypoxia-responsive polymetronidazole-coated VP and DOTAP adsorbed siYAP, with angiopep-2 (A2) modification on the surface. AMVY@NPs exhibited excellent blood‒brain barrier crossing, GBM targeting, and hypoxia-responsive and efficient siYAP and VP release properties. By inhibiting the expression and function of YAP, AMVY@NPs synergistically inhibited both the growth and stemness of GBM in vitro. Moreover, AMVY@NPs strongly inhibited the growth of orthotopic U87 xenografts and improved the survival of tumour-bearing mice without adverse effects. CONCLUSION: Specific targeting of YAP with stepwise-targeting and hypoxia-responsive liposome AMVY@NPs carrying siYAP and VP efficiently inhibited GBM progression. This study provides a valuable drug delivery platform and creative insights for molecular targeted treatment of GBM in the future.

Intravascular innate immune cells reprogrammed via intravenous nanoparticles to promote functional recovery after spinal cord injury.

Traumatic primary spinal cord injury (SCI) results in paralysis below the level of injury and is associated with infiltration of hematogenous innate immune cells into the injured cord. Methylprednisolone has been applied to reduce inflammation following SCI, yet was discontinued due to an unfavorable risk-benefit ratio associated with off-target effects. In this study, i.v. administered poly(lactide-coglycolide) nanoparticles were internalized by circulating monocytes and neutrophils, reprogramming these cells based on their physicochemical properties and not by an active pharmaceutical ingredient, to exhibit altered biodistribution, gene expression, and function. Approximately 80% of nanoparticle-positive immune cells were observed within the injury, and, additionally, the overall accumulation of innate immune cells at the injury was reduced 4-fold, coinciding with down-regulated expression of proinflammatory factors and increased expression of antiinflammatory and proregenerative genes. Furthermore, nanoparticle administration induced macrophage polarization toward proregenerative phenotypes at the injury and markedly reduced both fibrotic and gliotic scarring 3-fold. Moreover, nanoparticle administration with the implanted multichannel bridge led to increased numbers of regenerating axons, increased myelination with about 40% of axons myelinated, and an enhanced locomotor function (score of 6 versus 3 for control group). These data demonstrate that nanoparticles provide a platform that limits acute inflammation and tissue destruction, at a favorable risk-benefit ratio, leading to a proregenerative microenvironment that supports regeneration and functional recovery. These particles may have applications to trauma and potentially other inflammatory diseases.

Microporous scaffolds loaded with immunomodulatory lentivirus to study the contribution of immune cell populations to tumor cell recruitment in vivo.

Metastases are preceded by stochastic formation of a hospitable microenvironment known as the premetastatic niche, which has been difficult to study. Herein, we employ implantable polycaprolactone scaffolds as an engineered premetastatic niche to independently investigate the role of interleukin-10 (IL10), CXCL12, and CCL2 in recruiting immune and tumor cells and impacting breast cancer cell phenotype via lentiviral overexpression. Lentivirus delivered from scaffolds in vivo achieved sustained transgene expression for 56 days. IL10 lentiviral expression, but not CXCL12 or CCL2, significantly decreased tumor cell recruitment to scaffolds in vivo. Delivery of CXCL12 enhanced CD45+ immune cell recruitment to scaffolds while delivery of IL10 reduced immune cell recruitment. CCL2 did not alter immune cell recruitment. Tumor cell phenotype was investigated using conditioned media from immunomodulated scaffolds, with CXCL12 microenvironments reducing proliferation, and IL10 microenvironments enhancing proliferation. Migration was enhanced with CCL2 and reduced with IL10-driven microenvironments. Multiple linear regression identified populations of immune cells associated with tumor cell abundance. CD45+ immune and CD8+ T cells were associated with reduced tumor cell abundance, while CD11b+Gr1+ neutrophils and CD4+ T cells were associated with enhanced tumor cell abundance. Collectively, biomaterial scaffolds provide a tool to probe the formation and function of the premetastatic niche.

Mn-Based Artificial Mitochondrial Complex "VI" Acts as an Electron and Free Radical Conversion Factory to Suppress Macrophage Inflammatory Response.

Disturbance in the mitochondrial electron transport chain (ETC) is a key factor in the emerging discovery of immune cell activation in inflammatory diseases, yet specific regulation of ETC homeostasis is extremely challenging. In this paper, a mitochondrial complex biomimetic nanozyme (MCBN), which plays the role of an artificial "VI" complex and acts as an electron and free radical conversion factory to regulate ETC homeostasis is creatively developed. MCBN is composed of bovine serum albumin (BSA), polyethylene glycol (PEG), and triphenylphosphine (TPP) hierarchically encapsulating MnO2 polycrystalline particles. It has nanoscale size and biological properties like natural complexes. In vivo and in vitro experiments confirm that MCBN can target the mitochondrial complexes of inflammatory macrophages, absorb excess electrons in ETC, and convert the electrons to decompose H2O2. By reducing the ROS and ATP bursts and converting existing free radicals, inhibiting NLRP3 inflammatory vesicle activation and NF-κB signaling pathway, MCBN effectively suppresses macrophage M1 activation and inflammatory factor secretion. It also demonstrates good inflammation control and significantly alleviates alveolar bone loss in a mouse model of ligation-induced periodontitis. This is the first nanozyme that mimics the mitochondrial complex and regulates ETC, demonstrating the potential application of MCBN in immune diseases.

A synthetic metastatic niche reveals antitumor neutrophils drive breast cancer metastatic dormancy in the lungs.

Biomaterial scaffolds mimicking the environment in metastatic organs can deconstruct complex signals and facilitate the study of cancer progression and metastasis. Here we report that a subcutaneous scaffold implant in mouse models of metastatic breast cancer in female mice recruits lung-tropic circulating tumor cells yet suppresses their growth through potent in situ antitumor immunity. In contrast, the lung, the endogenous metastatic organ for these models, develops lethal metastases in aggressive breast cancer, with less aggressive tumor models developing dormant lungs suppressing tumor growth. Our study reveals multifaceted roles of neutrophils in regulating metastasis. Breast cancer-educated neutrophils infiltrate the scaffold implants and lungs, secreting the same signal to attract lung-tropic circulating tumor cells. Second, antitumor and pro-tumor neutrophils are selectively recruited to the dormant scaffolds and lungs, respectively, responding to distinct groups of chemoattractants to establish activated or suppressive immune environments that direct different fates of cancer cells.

The combined effects of microplastics and the heavy metal cadmium on the marine periphytic ciliate Euplotes vannus.

Microplastics could be grazed by marine organisms and possibly transferred to higher trophic levels along the microbial loop. Due to their size and capacity to concentrate heavy metals that trigger joint toxic effects, microplastics (MPs) have already become a severe threat to marine organisms. The detrimental effects of MPs on large marine organisms have been studied, but the combined toxicity of MPs and cadmium (Cd) on protozoan ciliates remains unclear. In the present study, we selected different diameters and concentrations of polystyrene microspheres (PS-MPs) and Cd2+ as model MPs and heavy metals to evaluate their single and combined effects on the periphytic marine ciliate Euplotes vannus in relation to carbon biomass and oxidative stress. The MPs were indeed ingested by Euplotes vannus and significantly reduced the abundance and carbon biomass of ciliate populations. Combined exposure to MPs and Cd2+ not only increased the bioaccumulation of Cd2+ in ciliates but also exacerbated the decrease in ciliate biomass by increasing oxidative stress and membrane damage. In comparison, the effects of nano-sized plastics (0.22 µm) were more harmful than those of micro-sized plastics (1.07 µm, 2.14 µm and 5.00 µm). A smaller size represents a higher potential for penetrating biological members and a stronger adsorption capacity for cadmium. These results provide new insight into the combined toxicity of microplastics and heavy metals on ciliated protozoa and lay a foundation for higher trophic levels and ecosystems.

High Frequency Spectral Ultrasound Imaging to Detect Metastasis in Implanted Biomaterial Scaffolds.

For most cancers, metastasis is the point at which disease is no longer curable. Earlier detection of metastasis, when it is undetectable by current clinical methods, may enable better outcomes. We have developed a biomaterial implant that recruits metastatic cancer cells in mouse models of breast cancer. Here, we investigate spectral ultrasound imaging (SUSI) as a non-invasive strategy for detecting metastasis to the implanted biomaterial scaffolds. Our results show that SUSI, which detects parameters related to tissue composition and structure, identified changes at an early time point when tumor cells were recruited to scaffolds in orthotopic breast cancer mouse models. These changes were not associated with acellular components in the scaffolds but were reflected in the cellular composition in the scaffold microenvironment, including an increase in CD31 + CD45-endothelial cell number in tumor bearing mice. In addition, we built a classification model based on changes in SUSI parameters from scaffold measurements to stratify tumor free and tumor bearing status. Combination of a linear discriminant analysis and bagged decision trees model resulted in an area under the curve of 0.92 for receiver operating characteristics analysis. With the potential for early non-invasive detection, SUSI could facilitate clinical translation of the scaffolds for monitoring metastatic disease.

Metastatic Conditioning of Myeloid Cells at a Subcutaneous Synthetic Niche Reflects Disease Progression and Predicts Therapeutic Outcomes.

Monitoring metastatic events in distal tissues is challenged by their sporadic occurrence in obscure and inaccessible locations within these vital organs. A synthetic biomaterial scaffold can function as a synthetic metastatic niche to reveal the nature of these distal sites. These implanted scaffolds promote tissue ingrowth, which upon cancer initiation is transformed into a metastatic niche that captures aggressive circulating tumor cells. We hypothesized that immune cell phenotypes at synthetic niches reflect the immunosuppressive conditioning within a host that contributes to metastatic cell recruitment and can identify disease progression and response to therapy. We analyzed the expression of 632 immune-centric genes in tissue biopsied from implants at weekly intervals following inoculation. Specific immune populations within implants were then analyzed by single-cell RNA-seq. Dynamic gene expression profiles in innate cells, such as myeloid-derived suppressor cells, macrophages, and dendritic cells, suggest the development of an immunosuppressive microenvironment. These dynamics in immune phenotypes at implants was analogous to that in the diseased lung and had distinct dynamics compared with blood leukocytes. Following a therapeutic excision of the primary tumor, longitudinal tracking of immune phenotypes at the implant in individual mice showed an initial response to therapy, which over time differentiated recurrence versus survival. Collectively, the microenvironment at the synthetic niche acts as a sentinel by reflecting both progression and regression of disease. SIGNIFICANCE: Immune dynamics at biomaterial implants, functioning as a synthetic metastatic niche, provides unique information that correlates with disease progression. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/3/602/F1.large.jpg.See related commentary by Wolf and Elisseeff, p. 377.

Biomaterial Scaffolds Recruit an Aggressive Population of Metastatic Tumor Cells In Vivo.

For most cancers, metastasis is the point at which clinical treatment shifts from curative intent to extending survival. Biomaterial implants acting as a synthetic premetastatic niche recruit metastatic cancer cells and provide a survival advantage, and their use as a diagnostic platform requires assessing their relevance to disease progression. Here, we showed that scaffold-captured tumor cells (SCAF) were 30 times more metastatic to the lung than primary tumor (PT) cells, similar to cells derived from lung micrometastases (LUNG). SCAF cells were more aggressive in vitro, demonstrated higher levels of migration, invasion, and mammosphere formation, and had a greater proportion of cancer stem cells than PT. SCAF cells were highly enriched for gene expression signatures associated with metastasis and had associated genomic structural changes, including globally enhanced entropy. Collectively, our findings demonstrate that SCAF cells are distinct from PT and more closely resemble LUNG, indicating that tumor cells retrieved from scaffolds are reflective of cells at metastatic sites. SIGNIFICANCE: These findings suggest that metastatic tumor cells captured by a biomaterial scaffold may serve as a diagnostic for molecular staging of metastasis.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/8/2042/F1.large.jpg.

Biomaterial Scaffolds as Pre-metastatic Niche Mimics Systemically Alter the Primary Tumor and Tumor Microenvironment.

Primary tumor (PT) immune cells and pre-metastatic niche (PMN) sites are critical to metastasis. Recently, synthetic biomaterial scaffolds used as PMN mimics are shown to capture both immune and metastatic tumor cells. Herein, studies are performed to investigate whether the scaffold-mediated redirection of immune and tumor cells would alter the primary tumor microenvironment (TME). Transcriptomic analysis of PT cells from scaffold-implanted and mock-surgery mice identifies differentially regulated pathways relevant to invasion and metastasis progression. Transcriptomic differences are hypothesized to result from scaffold-mediated modulations of immune cell trafficking and phenotype in the TME. Culturing tumor cells with conditioned media generated from PT immune cells of scaffold-implanted mice decrease invasion in vitro more than two-fold relative to mock surgery controls and reduce activity of invasion-promoting transcription factors. Secretomic characterization of the conditioned media delineates interactions between immune cells in the TME and tumor cells, showing an increase in the pan-metastasis inhibitor decorin and a concomitant decrease in invasion-promoting chemokine (C-C motif) ligand 2 (CCL2) in scaffold-implanted mice. Flow cytometric and transcriptomic profiling of PT immune cells identify phenotypically distinct tumor-associated macrophages (TAMs) in scaffold-implanted mice, which may contribute to an invasion-suppressive TME. Taken together, this study demonstrates biomaterial scaffolds systemically influence metastatic progression through manipulation of the TME.

Functional expression of secreted proteins from a bicistronic retroviral cassette based on foot-and-mouth disease virus 2A can be position dependent.

The expression of two or more genes from a single viral vector has been widely used to label or select for cells containing the transgenic element. Identification of the foot-and-mouth disease virus (FMDV) 2A cleavage peptide as a polycistronic linker capable of producing equivalent levels of transgene expression has greatly improved this approach in the field of gene therapy. However, as a consequence of 2A posttranslational cleavage the upstream protein is left with a residual 19 amino acids from the 2A sequence on its carboxy terminus, and the downstream protein is left with an additional 2 to 5 amino acids on its amino terminus. Here we have assessed the functional consequences of the FMDV 2A cleavage motif on two secreted proteins (interleukin [IL]-2 and transforming growth factor [TGF]-ß) when expressed from a retroviral bicistronic vector. Whereas IL-2 expression and function were found to be unaffected by the 2A motif in either orientation, functional expression of secreted TGF-ß was significantly abrogated when the transgene was expressed upstream of the 2A sequence. We believe this is a consequence of aberrant cleavage and intracellular trafficking of the TGF-ß polyprotein. These results highlight that to achieve functional expression of secreted proteins consideration must be taken of the transgenic protein's posttranslational modification and trafficking when using 2A-based bicistronic cassettes.