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.

Background/Objectives: Cancer vaccines represent the next frontier in immunotherapy, aiming to elicit long-lasting protective anti-tumor immune responses. Human epidermal growth factor receptor 2 (HER2) is a well-established therapeutic target in breast cancer. Active immunization with HER2-displaying M13 bacteriophages can induce a therapeutic immune response against HER2-positive breast cancer, offering a promising alternative to trastuzumab. However, the duration of anticancer immune protection triggered by anti-HER2 phage-based vaccines is limited by tumor-immune suppressive mechanisms. Methods: In this study, two vaccination cycles with ECTM phages displaying the extracellular (EC) and transmembrane (TM) domains of human HER2 were combined with palbociclib, a CDK4/6 inhibitor, to enhance antitumor immunity in the clinically relevant Δ16HER2 transgenic preclinical model of breast cancer. Results: The proposed combination treatment resulted in a better and long-lasting control of tumor growth rate and multiplicity than either palbociclib or phage vaccination alone, correlating with a significantly stronger anti-HER2 humoral response (IgG2a isotype). Analysis of the tumor immune infiltrate revealed an increased presence of CD8+ T cells concomitant with a reduction in FoxP3+ regulatory T cells (Tregs) in tumors explanted from mice receiving the combination therapy. Conclusions: These preclinical results provide a rationale for the clinical translation of CDK4/6 inhibitors combined with anti-HER2 active immunotherapies in breast cancer, as they may yield sustained antitumor responses by reverting the immunosuppressive tumor environment.

Nucleolin (NCL), an RNA-binding protein which regulates critical cellular processes, is frequently dysregulated in human cancers, including breast cancer, making it an attractive therapeutic target. However, molecular details of the RNA-NCL interaction have not been investigated yet. A tRNA fragment named tRF3E, displaying tumor suppressor roles in breast cancer, was found to bind NCL with high affinity displacing NCL-controlled transcripts. Here, we investigated the determinants and cooperativity of tRF3E-NCL interaction by Electrophoretic Mobility Shift Assays and in silico docking analysis, using wild-type or mutated tRF3E. We found that NCL, through its RNA-binding domains (RBD1-2 and RBD3-4), binds simultaneously two tRF3E molecules, giving rise to an energetically favored complex. Instead, a mutant form of tRF3E (M19-24), in which the NCL recognition element in position 19-24 has been disrupted, contacts NCL exclusively at RBD3-4, causing the loss of cooperativity among RBDs. Importantly, when expressed in MCF7 breast cancer cells, tRF3E significantly reduced cell proliferation and colony formation, confirming its role as tumor suppressor, but tRF3E functional properties were lost when the 19-24 motif was mutated, suggesting that cooperativity among multiple domains is required for the NCL-mediated tRF3E antitumor function. This study sheds light on the dynamic of RNA-NCL interaction and lays the foundations for using tRF3E as a promising NCL-targeted biodrug candidate.

Inonotus obliquus (Chaga) is a medicinal mushroom with several pharmacological properties that is used as a tea in traditional Chinese medicine. In this study, Chaga water extract was digested in vitro to mimic the natural processing and absorption of its biocomponents when it is consumed as functional beverage, and its anticancer activities were evaluated in breast cancer (BC) cell lines, representing HER2-positive and triple-negative subtypes. After chemical characterization by liquid chromatography/mass spectrometry (HR-QTOF) analysis, the effect of Chaga biocomponents on cell viability and cell cycle progression was assessed by MTT assay, FACS analysis, and Western blot. Dihydrofolate reductase (DHFR) activity was measured by an enzymatic assay. Four highly bioactive triterpenoids (inotodiol, trametenolic acid, 3-hydroxy-lanosta-8,24-dien-21-al, and betulin) were identified as the main components, able to decrease BC cell viability and block the cell cycle in G0/G1 by inducing the downregulation of cyclin D1, CDK4, cyclin E, and phosphorylated retinoblastoma protein. DHFR was identified as their crucial target. Moreover, bioactive Chaga components exerted a synergistic action with cisplatin and with trastuzumab in SK-BR-3 cells by inhibiting both HER2 and HER1 activation and displayed an immunomodulatory effect. Thus, Inonotus obliquus represents a source of triterpenoids that are effective against aggressive BC subtypes and display properties of targeted drugs.

Pollution from micro- and nanoplastics (MNPs) has long been a topic of concern due to its potential impact on human health. MNPs can circulate through human blood and, thus far, have been found in the lungs, spleen, stomach, liver, kidneys and even in the brain, placenta, and breast milk. While data are already available on the adverse biological effects of pristine MNPs (e.g. oxidative stress, inflammation, cytotoxicity, and even cancer induction), no report thus far clarified whether the same effects are modulated by the formation of a protein corona around MNPs. To this end, here we use pristine and human-plasma pre-coated polystyrene (PS) nanoparticles (NPs) and investigate them in cultured breast cancer cells both in terms of internalization and cell biochemical response to the exposure. It is found that pristine NPs tend to stick to the cell membrane and inhibit HER-2-driven signaling pathways, including phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways, which are associated with cancer cell survival and growth. By contrast, the formation of a protein corona around the same NPs can promote their uptake by endocytic vesicles and final sequestration within lysosomes. Of note is that such intracellular fate of PS-NPs is associated with mitigation of the biochemical alterations of the phosphorylated AKT (pAKT)/AKT and phosphorylated ERK (pERK)/ERK levels. These findings provide the distribution of NPs in human breast cancer cells, may broaden our understanding of the interactions between NPs and breast cancer cells and underscore the crucial role of the protein corona in modulating the impact of MNPs on human health. Formation and characterization of the protein corona on polystyrene nanoplastics and its impact on cytotoxicity, cellular uptake, and signaling pathways in breast cancer cells.

Currently available vaccines against COVID-19 showed high efficacy against the original strain of SARS-CoV-2 but progressively lower efficacy against new variants. In response to emerging SARS-CoV-2 strains, we propose chimeric DNA vaccines encoding the spike antigen, including a combination of selected key mutations from different variants of concern. We developed two DNA vaccines, pVAX-S1-TM-D614G and pVAX-S1-TM-INDUK (INDUK), encoding the SARS-CoV-2 S1 spike subunit in fusion with the transmembrane region that allows protein trimerization as predicted by in silico analysis. pVAX-S1-TM-D614G included the dominant D614G substitution, while the chimeric vaccine INDUK contained additional selected mutations from the Delta (E484Q and L452R) and Alpha (N501Y and A570D) variants. Considering that aging is a risk factor for severe disease and that suboptimal vaccine responses were observed in older individuals, the immunogenicity of pVAX-S1-TM-D614G and INDUK was tested in both young and aged C57BL/6 mice. Two vaccine doses were able to trigger significant anti-SARS-CoV-2 antibody production, showing neutralizing activity. ELISA tests confirmed that antibodies induced by pVAX-S1-TM-D614G and INDUK were able to recognize both Wuhan Spike and Delta variant Spike as trimers, while neutralizing antibodies were detected by an ACE2:SARS-CoV-2 Spike S1 inhibitor screening assay, designed to assess the capacity of antibodies to block the interaction between the viral spike S1 protein and the ACE2 receptor. Although antibody titer declined within six months, a third booster dose significantly increased the magnitude of humoral response, even in aged individuals, suggesting that immune recall can improve antibody response durability. The analysis of cellular responses demonstrated that vaccination with INDUK elicited an increase in the percentage of SARS-CoV-2-specific IFN-γ producing T lymphocytes in immunized young mice and TNF-α-producing T lymphocytes in both young and aged mice. These findings not only hold immediate promise for addressing evolving challenges in SARS-CoV-2 vaccination but also open avenues to refine strategies and elevate the effectiveness of next-generation vaccines.

Supplementary Figures 1-2 from Combining Human and Rat Sequences in Her-2 DNA Vaccines Blunts Immune Tolerance and Drives Antitumor Immunity

Supplementary Information includes Supplementary Materials and Methods, Supplementary Figures 1-7, with Legends and Supplementary References. Supplementary Figure S1: Loss of stathmin impairs mouse mammary gland development. Supplementary Figure S2: Loss of stathmin decreases the growth of mammary epithelial cells. Supplementary Figure S3: Loss of stathmin alters the mammary gland organization. Supplementary Figure S4: mRNA levels of STAT5 target genes are differently modulated when stathmin is silenced. Supplementary Figure S5: Stathmin expression correlates with poor prognosis in breast cancer patients. Supplementary Figure S6: Î"16HER2-driven mammary neoplastic lesions display luminal features. Supplementary Figure S7: Absence of stathmin in pre-neoplastic mouse mammary glands (13 weeks of age) weakens the activation of Î"16HER2 transformation-related pathways

Supplementary Figure 5 from A Better Immune Reaction to Erbb-2 Tumors Is Elicited in Mice by DNA Vaccines Encoding Rat/Human Chimeric Proteins

<p>HER2-positive BC patient pathobiological and clinical characteristics.</p>

Supplementary Figure 7 from A Better Immune Reaction to Erbb-2 Tumors Is Elicited in Mice by DNA Vaccines Encoding Rat/Human Chimeric Proteins

Flow cytometry analysis of live transgenically expressed MI6 and WTHER2 tumor cell lines.

Enrichment plots of significantly enriched gene sets for the comparison between active d16HER2 tumors and inactive or negative d16HER2 tumors.

List of cases of the GSE 55348.

Characterization of IgGs yielded from DNA vaccination by means of cellular ELISA

Anti-tumor effect triggered by phage-based vaccines in Î"16HER2 mice

<p>Table S1: Clinical Data of BC samples collected at Centro di Riferimento Oncologico di Aviano (CRO), IRCCS, National Cancer Institute, Aviano, Italy. Table S2: Clinical Data of BC samples collected in University "Sapienza" of Rome, Santo Andrea Hospital, Rome, Italy</p>

Supplementary Figure Legends 1-4, Tables Legends 1-8 from A Better Immune Reaction to Erbb-2 Tumors Is Elicited in Mice by DNA Vaccines Encoding Rat/Human Chimeric Proteins

Supplementary Figure 1 from Dual Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Blockade Is an Effective Radiosensitizing Strategy for the Treatment of Non–Small Cell Lung Cancer Harboring K-RAS Mutations