A common observation in drug delivery studies is that preincubation of nanoparticles (NPs) with high concentrations of human plasma (HP) markedly reduces in vitro transfection efficiency (TE). This effect is commonly attributed to the formation of a protein corona (PC), which is assumed to impair cellular uptake or intracellular trafficking of gene delivery systems such as lipid nanoparticles (LNPs). However, the evidence presented here suggests an alternative mechanism underlying this phenomenon. To reproduce conditions in which LNPs encounter an excess of circulating proteins, NPs were incubated in HP prior to exposure to cells in standard calcium-containing culture medium. Under these conditions, plasma undergoes gelation, leading to the formation of a clot-like network. A combination of complementary approaches, including dynamic light scattering (DLS), fluorescence-activated cell sorting (FACS), confocal fluorescence microscopy, raster image correlation spectroscopy (RICS), and functional assays evaluating TE and cell viability, demonstrates that this gel-like matrix restricts LNP diffusion in the extracellular environment. In contrast, coronated LNPs that reach the intracellular space display comparable trafficking behavior, indicating that the PC does not compromise intracellular processing. These findings highlight the importance of extracellular factors when evaluating the impact of the PC and extrapolating in vitro results to in vivo settings.

IntroductionNatural Killer (NK) cells are cytotoxic innate lymphocytes able to detect transformed cells through the balanced action of inhibitory and activating receptors. NKG2D is one of the main activating receptors involved in tumor surveillance thanks to its ability to recognize stress-induced ligands. Of note, the prolonged exposure to NKG2D ligands promotes receptor down-modulation that results in defective activation of NKG2D and other unrelated activating receptors, including DNAM-1 that is also involved in tumor clearance. However, further investigations are necessary to characterize how the NKG2D/DNAM-1 interplay affects NK cell anti-tumor function.MethodsPrimary cultured human NK cells were stimulated with the natural ligand MICA or an anti-NKG2D agonist antibody. The expression of activating and inhibitory receptors as well as DNAM-1-triggered signaling events and cytotoxicity were evaluated by flow cytometry. DNAM-1-mediated granule polarization was evaluated by confocal microscopy.ResultsWe showed that NKG2D crosslinking mediated by the natural ligand MICA or an agonist antibody had different consequences on primary cultured human NK cells. Indeed, MICA stimulation increases the expression of the checkpoint receptor TIGIT that is able to counteract DNAM-1 activation. Stimulation with the agonist antibody, without altering TIGIT expression, directly inhibits DNAM-1-mediated signal transduction and cytotoxic function with a mechanism that required NKG2D endocytosis.DiscussionOur findings contribute to shed light on the functional consequences of NKG2D engagement, demonstrating that a direct impact on DNAM-1-mediated signal transduction occurs independently from the modality of NKG2D crosslinking. Understanding the molecular mechanisms responsible for suppression of NK cell activation may help the development of therapeutic anti-cancer strategies aimed to prevent NK cell dysfunction or to reinvigorate an impaired cytotoxic activity.

Understanding interactions between nanomaterials and biomolecules is essential for advancing biomedical nanotechnologies. This study investigates how double-stranded DNA of varying sizes affects the protein corona (PC) surrounding Graphene Oxide (GO) nanosheets in DNA-supplemented human plasma. The findings reveal that DNA plays a pivotal role in modulating the PC composition through dynamic competition governed by factors like surface charge, affinity, and DNA fragment size. At lower DNA concentrations, competition between DNA and proteins for binding sites on GO leads to a corona predominantly composed of proteins, with some DNA molecules also bound. However, as the DNA concentration increases beyond a threshold, a shift occurs. DNA increasingly outcompetes proteins for binding sites, resulting in a two-component corona enriched with both DNA and proteins. Notably, the proportion of DNA within the corona progressively increases with rising DNA concentration, while the protein content decreases. This dynamic interplay between DNA and proteins has significant biological implications. A monotonic increase in Toll-like receptor 9 (TLR9) activation is observed as the DNA content within the corona increases. As the corona composition and its influence on cellular responses are crucial, this study emphasizes the relevance of exploring competition at the bio-nano interface for the advancement of these applications.

ADAR1 edits double-stranded RNAs (dsRNAs) by deaminating adenosines into inosines, preventing aberrant activation of innate immunity by endogenous dsRNAs, which may resemble viral structures. Several tumors exploit ADAR1 to evade immune surveillance; indeed, its deletion reduces tumor viability and reshapes infiltrating leukocytes. Here we investigated the role of ADAR1 in immune evasion mechanisms during cervical cancer (CC) progression. Patients’ biopsy samples showed higher ADAR1 expression already in premalignant lesions (squamous intraepithelial lesions [SIL]) and a substantially reduced percentage of infiltrating CD7+ innate cells in in situ and invasive carcinomas compared with normal mucosa, with CD56+ NK cells showing phenotypic alterations that may have affected their functional responses. In CC-derived cell lines (SiHa, CaSki), ADAR1 silencing reduced cell proliferation, an effect further enhanced by exogenous IFN-β administration. It also induced proinflammatory gene expression, as demonstrated by RNA-Seq analysis, and conditioned supernatants collected from these cells activated several NK cell effector functions. NK cell infiltration and activation were also confirmed in organotypic 3D tissue models of SiHa cells knocked out for ADAR1. In conclusion, ADAR1 expression increased with CC progression and was accompanied by alterations in tumor-infiltrating NK cells, but its silencing in CC-derived cell lines potentiated antitumor NK cell activities. Thus, ADAR1 inhibition may represent a therapeutic perspective for CC and possibly other malignancies.

Multiple myeloma (MM) remains an incurable disease despite therapeutic advancements extending survival. Relapses driven by drug resistance and minimal residual disease underscore the need for novel treatment strategies. Natural Killer (NK) cells play a key role in MM immunity, yet their function is suppressed by inhibitory cytokines and metabolites from the tumor microenvironment. Developing anticancer drugs with immunomodulatory properties, such as enhancing tumor sensitivity to NK cell recognition, remains a critical challenge. MM cells exhibit high protein synthesis rates, making them vulnerable to proteostasis disruption. Dysregulated ribosome function and aberrant mRNA translation contribute to proteasome inhibitor resistance. RNA Polymerase I (RNA Pol I)-mediated rDNA transcription, the rate-limiting step in ribosome biogenesis (RiBi), is significantly upregulated in MM. Targeting rDNA transcription and inducing nucleolar stress response (NSR) presents a promising therapeutic approach, though its immunomodulatory role is not well understood. Our study examined two "first-in-class" RNA Pol I inhibitors, CX-5461 and BMH-21, which differentially regulate NK cell-activating and inhibitory ligand expression in MM. BMH-21 enhanced NK cell degranulation and increased IFN-γ and TNF-α secretion, demonstrating stronger immunostimulatory effects than CX-5461. Conversely, CX-5461 induced a significant DNA damage response (DDR) and senescence, leading to HLA-E upregulation and suppressing NK cell activity. Mechanistic analyses revealed that HLA-E presentation is governed by ATR/AKT/mTORC1/S6K signaling and Pioneer Round of Translation (PRT), linking its regulation to DDR. This effect was modulated by Lenalidomide and Panobinostat. Moreover, RNA Pol I inhibition enhanced Daratumumab-mediated antibody-dependent cellular cytotoxicity (ADCC) of NK cells against MM, uncovering novel immuno-mediated antitumor mechanisms.

The advent of chimeric antigen receptor (CAR) T-cell therapy has introduced a novel and personalized approach to cancer treatment. Despite its promise, the challenge of developing a system that bypasses the need for viral vectors remains significant, particularly in terms of achieving a clinical efficacy and sustained durability. To address these challenges, lipid nanoparticles (LNPs) produced through advanced microfluidic technology have been recently utilized to encapsulate plasmid DNA (pDNA) encoding CAR receptors. However, the intrinsic challenges associated with pDNA encapsulation, along with the critical requirement for efficient expression, remain substantial obstacles. Here, we show that incorporating DNA-condensing agents into the microfluidic manufacturing of LNPs effectively overcomes these limitations. Briefly, we conducted a preliminary investigation to characterize LNPs with and without the commercial condensing agent P3000-Reagent (PR), focusing on their physicochemical properties and scrutinizing the biological outcomes primarily in the HEK-293 cell line. Our results demonstrated that precondensation of the pDNA with PR differentially increased the transfection efficiency of the tested formulations, whereas confocal microscopy indicated reduced lysosomal colocalization and major nuclear localization. Finally, PR was found to enhance LNP efficiency upon multiple administrations to the immortalized T-lymphocyte Jurkat cell line, enabling the delivery of both a luciferase reporter gene and a functional CAR-encoding plasmid. Overall, these findings underscore the great potential of introducing DNA-condensing agents into the LNP preparation process, especially for systems designed for challenging delivery applications, such as multiadministration transfection protocols.

Cancer progression is continuously controlled by the immune system which can identify and destroy nascent tumor cells or inhibit metastatic spreading. However, the immune system and its deregulated activity in the tumor microenvironment can also promote tumor progression favoring the outgrowth of cancers capable of escaping immune control, in a process termed cancer immunoediting. This process, which has been classified into three phases, i.e. “elimination”, “equilibrium” and “escape”, is influenced by several cancer- and microenvironment-dependent factors. Senescence is a cellular program primed by cells in response to different pathophysiological stimuli, which is based on long-lasting cell cycle arrest and the secretion of numerous bioactive and inflammatory molecules. Because of this, cellular senescence is a potent immunomodulatory factor promptly recruiting immune cells and actively promoting tissue remodeling. In the context of cancer, these functions can lead to both cancer immunosurveillance and immunosuppression. In this review, the authors will discuss the role of senescence in cancer immunoediting, highlighting its context- and timing-dependent effects on the different three phases, describing how senescent cells promote immune cell recruitment for cancer cell elimination or sustain tumor microenvironment inflammation for immune escape. A potential contribution of senescent cells in cancer dormancy, as a mechanism of therapy resistance and cancer relapse, will be discussed with the final objective to unravel the immunotherapeutic implications of senescence modulation in cancer.

Lipid nanoparticles (LNPs) play a crucial role in addressing genetic disorders, and cancer, and combating pandemics such as COVID-19 and its variants. Yet, the ability of LNPs to effectively encapsulate large-size DNA molecules remains elusive. This is a significant limitation, as the successful delivery of large-size DNA holds immense potential for gene therapy. To address this gap, the present study focuses on the design of PEGylated LNPs, incorporating large-sized DNA, departing from traditional RNA and ionizable lipids. The resultant LNPs demonstrate a unique particle morphology. These particles were further engineered with a DNA coating and plasma proteins. This multicomponent bionanoconstruct exhibits enhanced transfection efficiency and safety in controlled laboratory settings and improved immune system evasion in in vivo tests. These findings provide valuable insights for the design and development of bionanoarchitectures for large-size DNA delivery, opening new avenues for transformative gene therapies. Encapsulation of large-size DNA molecules into lipid nanoparticles (LNPs) remains challenging. Here, the authors engineer PEGylated LNPs with DNA and plasma proteins to improve the delivery of large DNA molecules, enhancing the gene therapy potential.

2'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) is the endogenous agonist of STING; as such, cGAMP has powerful immunostimulatory activity, due to its capacity to stimulate type I interferon-mediated immunity. Recent evidence indicates that cancer cells, under certain conditions, can release cGAMP extracellularly, a phenomenon currently considered important for therapeutic responses and tumor rejection. Nonetheless, the mechanisms that regulate cGAMP activity in the extracellular environment are still largely unexplored. In this work, we collected evidence demonstrating that CD38 glycohydrolase can inhibit extracellular cGAMP activity through its direct binding. We firstly used different cell lines and clinical samples to demonstrate a link between CD38 and extracellular cGAMP activity; we then performed extensive in silico molecular modeling and cell-free biochemical assays to show a direct interaction between the catalytic pocket of CD38 and cGAMP. Altogether, our findings expand the current knowledge about the regulation of cGAMP activity.

The existing manufacturing protocols for CAR-T cell therapies pose notable challenges, particularly in attaining a transient transfection that endures for a significant duration. To address this gap, this study aims to formulate a transfection protocol utilizing multiple lipid-based nanoparticles (LNPs) administrations to enhance transfection efficiency (TE) to clinically relevant levels. By systematically fine-tuning and optimizing our transfection protocol through a series of iterative refinements, we have accomplished a remarkable one-order-of-magnitude augmentation in TE within the immortalized T-lymphocyte Jurkat cell line. This enhancement has been consistently observed over 2 weeks, and importantly, it has been achieved without any detrimental impact on cell viability. In the subsequent phase of our study, we aimed to optimize the gene delivery system by evaluating three lipid-based formulations tailored for DNA encapsulation using our refined protocol. These formulations encompassed two LNPs constructed from ionizable lipids and featuring systematic variations in lipid composition (iLNPs) and a cationic lipoplex (cLNP). Our findings showcased a notable standout among the three formulations, with cLNP emerging as a frontrunner for further refinement and integration into the production pipeline of CAR-T therapies. Consequently, cLNP was scrutinized for its potential to deliver CAR-encoding plasmid DNA to the HEK-293 cell line. Confocal microscopy experiments demonstrated its efficiency, revealing substantial internalization compared to iLNPs. By employing a recently developed confocal image analysis method, we substantiated that cellular entry of cLNP predominantly occurs through macropinocytosis. This mechanism leads to heightened intracellular endosomal escape and mitigates lysosomal accumulation. The successful expression of anti-CD19-CD28-CD3z, a CAR engineered to target CD19, a protein often expressed on the surface of B cells, was confirmed using a fluorescence-based assay. Overall, our results indicated the effectiveness of cLNP in gene delivery and suggested the potential of multiple administration transfection as a practical approach for refining T-cell engineering protocols in CAR therapies. Future investigations may focus on refining outcomes by adjusting transfection parameters like nucleic acid concentration, lipid-to-DNA ratio, and incubation time to achieve improved TE and increased gene expression levels.

Supplementary Figure S5. Proposed model: MM alteration of chemokine/chemokine receptor axes in BM shapes NK cell effector function by reducing the number of functional KLRG1- subset

Supplementary Table S2. Expression of chemokine genes by BM cell populations

The success of senescence-based anticancer therapies relies on their anti-proliferative power and on their ability to trigger anti-tumor immune responses. Indeed, genotoxic drug-induced senescence increases the expression of NK cell-activating ligands on multiple myeloma (MM) cells, boosting NK cell recognition and effector functions. Senescent cells undergo morphological change and context-dependent functional diversification, acquiring the ability to secrete a vast pool of molecules termed the senescence-associated secretory phenotype (SASP), which affects neighboring cells. Recently, exosomes have been recognized as SASP factors, contributing to modulating a variety of cell functions. In particular, evidence suggests a key role for exosomal microRNAs in influencing many hallmarks of cancer. Herein, we demonstrate that doxorubicin treatment of MM cells leads to the enrichment of miR-433 into exosomes, which in turn induces bystander senescence. Our analysis reveals that the establishment of the senescent phenotype on neighboring MM cells is p53- and p21-independent and is related to CDK-6 down-regulation. Notably, miR-433-dependent senescence does not induce the up-regulation of activating ligands on MM cells. Altogether, our findings highlight the possibility of miR-433-enriched exosomes to reinforce doxorubicin-mediated cellular senescence.

Supplemental Table 2. Patient characteristics. Analysis of patient characteristics used in the set of experiments in Figure 6. Patients were classified according to the state of the disease. PC=plasma cell.

Supplementary Figure S1. Chemokine expression in serum and spleen of healthy control or MM-bearing mice and 5TGM1 culture supernatants

Natural Killer (NK) cells act as important regulators in the development and progression of hematological malignancies and their suppressor activity against Multiple Myeloma (MM) cells has been confirmed in many studies. Significant changes in the distribution of NK cell subsets and dysfunctions of NK cell effector activities were described in MM patients and correlated with disease staging. Thus, restoring or enhancing the functionality of these effectors for the treatment of MM represents a critical need. Neddylation is a post-translational modification that adds a ubiquitin-like molecule, NEDD8, to the substrate protein. One of the outcomes is the activation of the Cullin Ring Ligases (CRLs), a class of ubiquitin-ligases that controls the degradation of about 20% of proteasome-regulated proteins. Overactivation of CRLs has been described in cancer and can lead to tumor growth and progression. Thus, targeting neddylation represents an attractive approach for cancer treatment. Our group has recently described how pharmacologic inhibition of neddylation increases the expression of the NKG2D activating receptor ligands, MICA and MICB, in MM cells, making these cells more susceptible to NK cell degranulation and killing. Here, we extended our investigation to the direct role of neddylation on NK cell effector functions exerted against MM. We observed that inhibition of neddylation enhanced NK cell-mediated degranulation and killing against MM cells and improved Daratumumab/Elotuzumab-mediated response. Mechanistically, inhibition of neddylation increased the expression of Rac1 and RhoA GTPases in NK cells, critical mediators for an efficient degranulation at the immunological synapse of cytotoxic lymphocytes, and augmented the levels of F-actin and perforin polarization in NK cells contacting target cells. Moreover, inhibition of neddylation partially abrogated TGFβ-mediated repression of NK cell effector activity. This study describes the role of neddylation on NK cell effector functions and highlights the positive immunomodulatory effects achieved by the inhibition of this pathway in MM.

Figure S3. MEL-treated MM cells induce NK cells entry in the S phase of the cell cycle.

Fig S5. MEL-treated SKO-007(J3) and ARK exosomes display a similar IL-15RA expression.