Transient nuclear lamin A/C accretion aids in recovery from vapor nanobubble-induced permeabilisation of the plasma membrane

Vapor nanobubble (VNB) photoporation is a physical method for intracellular delivery that has gained significant interest in the past decade. It has successfully been used to introduce molecular cargo of diverse nature into different cell types with high throughput and minimal cytotoxicity. For translational purposes, it is important to understand whether and how photoporation affects cell homeostasis. To obtain a comprehensive view on the transcriptional rewiring that takes place after VNB photoporation, we performed a longitudinal shotgun RNA-sequencing experiment. Six hours after photoporation, we found a marked upregulation of LMNA transcripts as well as their protein products, the A-type lamins. At the same time point, we observed a significant increase in several heterochromatin marks, suggesting a global stiffening of the nucleus. These molecular features vanished 24 h after photoporation. Since VNB-induced chromatin condensation was prolonged in LMNA knockout cells, A-type lamins may be required for restoring the nucleus to its original state. Selective depletion of A-type lamins reduced cell viability after VNB photoporation, while pharmacological stimulation of LMNA transcription increased the percentage of successfully transfected cells that survived after photoporation. Therefore, our results suggest that cells respond to VNB photoporation by temporary upregulation of A-type lamins to facilitate their recovery.

How pancreatic adenocarcinoma might cause diabetes? The role of TGF- β

Pancreatic ductal adenocarcinoma (PDAC) is a deadly sickness that stays incurable due to past due diagnosis, which renders any healing intervention challenging. Most PDAC sufferers expand de novo diabetes, which exacerbates their morbidity and mortality. How PDAC triggers diabetes continues to be unfolding. Using a mouse version of KrasG12D-pushed PDAC, which faithfully recapitulates the development of the human sickness, we determined a large and selective depletion of β-cells, taking place very early on the degrees of preneoplastic lesions. Mechanistically, it turned into observed that accelerated TGF beta (TGF-β) signaling throughout PDAC development induced erosion of β-mobileular mass thru apoptosis. Suppressing TGF-β signaling, both pharmacologically thru TGF-β immunoneutralization or genetically thru deletion of Smad4 or TGF-β kind II receptor (TβRII), afforded size able safety in opposition to PDAC-pushed β-mobileular depletion. From a translational perspective, each activation of TGF-β signaling and depletion of β-cells often arise in human PDAC, imparting a mechanistic cause of the pathogenesis of diabetes in PDAC sufferers, and similarly implicating new- onset diabetes as a capability early prognostic marker for PDAC. In this mini review we try to analyze the principle relationships between pancreatic cancer and diabetes and vice versa in addition to the implication of TGF-β signaling as a likely goal for attenuating diabetes in pancreatic most cancers patients.

Formulation of Boron Encapsulated Smart Nanocapsules for Targeted Drug Delivery to the Brain

Drug delivery through the Blood–Brain Barrier (BBB) represents a significant challenge. Despite the current strategies to circumvent the BBB, nanotechnology offers unprecedented opportunities for combining selective delivery, improved bioavailability, drug protection, and enhanced pharmacokinetics profiles. Chitosan nanocarriers allow for a more efficacious strategy at the cellular and sub-cellular levels. Boron Neutron Capture Therapy (BNCT) is a targeted chemo-radiotherapeutic technique that allows the selective depletion of cancer cells by means of selective tagging of cancer cells with 10B, followed by irradiation with low-energy neutrons. Consequently, the combination of a polymer-based nanodelivery system enclosing an effective BNCT pharmacophore can potentially lead to the selective delivery of the load to cancer cells beyond the BBB. In this work, synthesized novel boronated agents based on carborane-functionalized Delocalized Lipophilic Cations (DLCs) are assessed for safety and selective targeting of tumour cells. The compounds are then encapsulated in nanocarriers constituted by chitosan to promote permeability through the BBB. Additionally, chitosan was used in combination with polypyrrole to form a smart composite nanocapsule, which is expected to release its drug load with variations in pH. Results indicate the achievement of more selective boron delivery to cells via carboranyl DLCs. Finally, preliminary cell studies indicate no toxicity was detected in chitosan nanocapsules, further enhancing its viability as a potential delivery vehicle in the BNCT of brain tumours.

NIMG-48. TLR7/8-AGONIST-LOADED NANOPARTICLES REPROGRAM TUMOR-ASSOCIATED MYELOID CELLS FOR EFFECTIVE IMMUNOTHERAPY OF EXPERIMENTAL GLIOMA AND MRI-BASED TREATMENT MONITORING

Drivers of glioblastoma progression include the immunosuppressive tumor microenvironment (TME), dominated by tumor-associated myeloid cells. Therefore, we investigated a new approach targeting the myeloid compartment to reprogram myeloid cells in the TME using a β-cyclodextrin nanoparticle (CDNP) formulation encapsulating the toll-like receptor 7 and 8 (TLR7/8) agonist R848. Biodistribution confirmed specific targeting of CDNP-R848 to tumor-associated macrophages (TAMs) (labeling efficiency: 34.0% ± 22.2%), whereas tumor microglia (5.4% ± 4.4%) and splenic macrophages (13.2% ± 0.7%) revealed less uptake. Interestingly, intravenous application of CDNP-R848 induced strong tumor regression with an overall response rate of 80% (2.5% complete response, 52.5% partial response and 25% stable disease, n=40 mice) in Gl261 syngeneic experimental gliomas, while CDNP vehicle treated animals showed exponential tumor growth (100% progressive disease, n=12 mice). As advanced imaging is essential to monitor intracranial disease and possibly predict response and resistance, we performed high resolution magnetic resonance imaging using ultrasmall iron oxide nanoparticles (USPIO) for macrophage tracking. Increased levels of USPIO uptake in vehicle treated animals compared to CDNP-R848 treated animals were found as an early marker of responding mice (ΔT2*: -11.7 ± 4.2 vs -4.0 ± 2.8 ms, p=0.01). This correlated with an increased influx of myeloid cells into the TME of vehicle treated animals and showed a strong correlation of macrophage recruitment and USPIO uptake (R2: 0.78, p=0.004). Mechanistically, phenotyping of macrophages (CD45high/CD11b+) indicated a pro-inflammatory shift of TAMs with an increased infiltration of pro-inflammatory F4/80+/MHCII+ macrophages during CDNP-R848 treatment. Surprisingly, the anti-tumor effect of CDNP-R848 was independent of CD8+ T cells, CD4+ T cells or NK cells during selective depletion experiments. In summary, this work demonstrates the ability of myeloid-targeted therapies to re-shape the tumor microenvironment for an effective immunotherapy of glioma.

Neutrophil Extracellular Traps-DNase Balance and Autoimmunity

Neutrophil extracellular traps (NETs) are macromolecular structures programmed to trap circulating bacteria and viruses. The accumulation of NETs in the circulation correlates with the formation of anti-double-stranded (ds) DNA antibodies and is considered a causative factor for systemic lupus erythematosus (SLE). The digestion of DNA by DNase1 and DNases1L3 is the rate- limiting factor for NET accumulation. Mutations occurring in one of these two DNASE genes determine anti-DNA formation and are associated with severe Lupus-like syndromes and lupus nephritis (LN). A second mechanism that may lead to DNase functional impairment is the presence of circulating DNase inhibitors in patients with low DNase activity, or the generation of anti-DNase antibodies. This phenomenon has been described in a relevant number of patients with SLE and may represent an important mechanism determining autoimmunity flares. On the basis of the reviewed studies, it is tempting to suppose that the blockade or selective depletion of anti-DNase autoantibodies could represent a potential novel therapeutic approach to prevent or halt SLE and LN. In general, strategies aimed at reducing NET formation might have a similar impact on the progression of SLE and LN.

Selective depletion of a CD64-expressing phagocyte subset mediates protection against toxic kidney injury and failure

Dendritic cells (DC), macrophages, and monocytes, collectively known as mononuclear phagocytes (MPs), critically control tissue homeostasis and immune defense. However, there is a paucity of models allowing to selectively manipulate subsets of these cells in specific tissues. The steady-state adult kidney contains four MP subsets with Clec9a-expression history that include the main conventional DC1 (cDC1) and cDC2 subtypes as well as two subsets marked by CD64 but varying levels of F4/80. How each of these MP subsets contributes to the different phases of acute kidney injury and repair is unknown. We created a mouse model with a Cre-inducible lox-STOP-lox-diphtheria toxin receptor cassette under control of the endogenous CD64 locus that allows for diphtheria toxin–mediated depletion of CD64-expressing MPs without affecting cDC1, cDC2, or other leukocytes in the kidney. Combined with specific depletion of cDC1 and cDC2, we revisited the role of MPs in cisplatin-induced kidney injury. We found that the intrinsic potency reported for CD11c+ cells to limit cisplatin toxicity is specifically attributed to CD64+ MPs, while cDC1 and cDC2 were dispensable. Thus, we report a mouse model allowing for selective depletion of a specific subset of renal MPs. Our findings in cisplatin-induced injury underscore the value of dissecting the functions of individual MP subsets in kidney disease, which may enable therapeutic targeting of specific immune components in the absence of general immunosuppression.

Oncometabolite R-2 hydroxyglutarate aids the inflammatory transformation of peritumoral astrocytes

Reactive transformation of astrocytes in IDH wild-type glioma contribute to anti-tumor immunity and support pro-oncogenic signaling. The role of intra- and peritumoral astrocytes in IDH1/2 mutated glioma, a prognostically beneficial subgroup with oncogene-driven enrichment of R-2-hydroxygluterate (2-HG) remains sparsely explored. Here, we characterized the transcriptomic signature of IDH1/2-mutant glioma associated astrocytes and determined a unique inflammatory transformation, profoundly different to astrocytes in IDH wildtype glioma patients. IDH-mutated glioma inoculation into human neocortical sections or treatment with R-2-HG resulted in an oncometabolite-mediated transcriptional shift towards inflammation in astrocytes. This transcriptional shift was found to be mediated by myeloid cell polarization through R-2-HG in the tumor microenvironment, which was further confirmed by selective depletion of myeloid cells. Integrative analysis of bulk RNA-sequencing of purified microglia, single-cell transcriptomics and spatially resolved transcriptomics confirmed this microglia-mediated inflammatory activation of astrocytes. This inflammatory activation is accompanied by loss of neurotransmitter homeostasis. Further, treatment of cortical sections with RH2G results in increased neuronal activity and LFP activity, pointing to the excitotoxic nature of RH2G in the neural microenvironment, with a significant loss of neurons with chronic treatment. The presented findings provide insights into the role that R-2HG plays in the reactive transformation of astrocytes within the IDH-mutated tumor environment and is fundamentally different that seen in IDH wildtype glioma.