Development of Cyclic Peptides Targeting the Epidermal Growth Factor Receptor in Mesenchymal Triple-Negative Breast Cancer Subtype.

Triple-negative breast cancer (TNBC) is an aggressive malignancy characterized by the lack of expression of estrogen and progesterone receptors and amplification of human epidermal growth factor receptor 2 (HER2). Being the Epidermal Growth Factor Receptor (EGFR) highly expressed in mesenchymal TNBC and correlated with aggressive growth behavior, it represents an ideal target for anticancer drugs. Here, we have applied the phage display for selecting two highly specific peptide ligands for targeting the EGFR overexpressed in MDA-MB-231 cells, a human TNBC cell line. Molecular docking predicted the peptide-binding affinities and sites in the extracellular domain of EGFR. The binding of the FITC-conjugated peptides to human and murine TNBC cells was validated by flow cytometry. Confocal microscopy confirmed the peptide binding specificity to EGFR-positive MDA-MB-231 tumor xenograft tissues and their co-localization with the membrane EGFR. Further, the peptide stimulation did not affect the cell cycle of TNBC cells, which is of interest for their utility for tumor targeting. Our data indicate that these novel peptides are highly specific ligands for the EGFR overexpressed in TNBC cells, and thus they could be used in conjugation with nanoparticles for tumor-targeted delivery of anticancer drugs.

SARS CoV-2 spike protein-guided exosome isolation facilitates detection of potential miRNA biomarkers in COVID-19 infections.

OBJECTIVES: Nearly three years into the pandemic, SARS-CoV-2 infections are occurring in vaccinated and naturally infected populations. While humoral and cellular responses in COVID-19 are being characterized, novel immune biomarkers also being identified. Recently, an increase in angiotensin-converting enzyme 2 expressing (aka, ACE2 positive) circulating exosomes (ExoACE2) were identified in the plasma of COVID-19 patients (El-Shennawy et al.). In this pilot study, we describe a method to characterize the exosome-associated microRNA (exo-miRNA) signature in ACE2-positive and ACE2-negative exosomal populations (non-ExoACE2). METHODS: We performed a sorting protocol using the recombinant biotin-conjugated SARS CoV-2 spike protein containing the receptor binding domain (RBD) on plasma samples from six patients. Following purification, exo-miRNA were characterized for ACE2-positive and ACE2-negative exosome subpopulations by RT-PCR. RESULTS: We identified differential expression of several miRNA. Specifically let-7g-5p and hsa-miR-4454+miR-7975 were upregulated, while hsa-miR-208a-3p and has-miR-323-3p were downregulated in ExoACE2 vs. non-ExoACE2. CONCLUSIONS: The SARS CoV-2 spike-protein guided exosome isolation permits isolation of ExoACE2 exosomes. Such purification facilitates detailed characterization of potential biomarkers (e.g. exo-miRNA) for COVID-19 patients. This method could be used for future studies to further the understanding mechanisms of host response against SARS CoV-2.

Unmutated IGHV1-69 CLL Clone Displays a Distinct Gene Expression Profile by a Comparative qRT-PCR Assay.

Chronic Lymphocytic Leukemia (CLL) is a heterogeneous disease characterized by variable clinical courses among different patients. This notion was supported by the possible coexistence of two or more independent CLL clones within the same patients, identified by the characterization of the B cell receptor immunoglobulin (BcR IG) idiotypic sequence. By using the antigen-binding site of the BcR IG as bait, the identification and isolation of aggressive and drug-resistance leukemic B-cell clones could allow a deeper biological and molecular investigation. Indeed, by the screening of phage display libraries, we previously selected a peptide binder of the idiotypic region of CLL BCR IGs expressing the unmutated rearrangement IGHV1-69 and used it as a probe to perform a peptide-based cell sorting by flow cytometry in peripheral blood samples from patients with CLL. Since the IGHV1-69 clones persisted during the follow-up time in both patients, we explored the possibility of these clones having acquired an evolutive advantage compared to the other coexisting clones in terms of a higher expression of genes involved in the survival and apoptosis escape processes. To this end, we studied the expression patterns of a panel of genes involved in apoptosis regulation and in NF-kB-dependent pro-survival signals by comparative qRT-PCR assays. According to the results, IGHV1-69 clones showed a higher expression of pro-survival and anti-apoptotic genes as compared to the other CLL clones with different immunogenetic characteristics. Moreover, these IGHV1-69 clones did not carry any characteristic genetic lesions, indicating the relevance of our approach in performing a comprehensive molecular characterization of single tumor clones, as well as for designing new personalized therapeutic approaches for the most aggressive and persistent tumor clones.

IBtkα Activates the ß-Catenin-Dependent Transcription of MYC through Ubiquitylation and Proteasomal Degradation of GSK3ß in Cancerous B Cells.

The IBTK gene encodes the IBtkα protein that is a substrate receptor of E3 ubiquitin ligase, Cullin 3. We have previously reported the pro-tumorigenic activity of Ibtk in MYC-dependent B-lymphomagenesis observed in Eµ-myc transgenic mice. Here, we provide mechanistic evidence of the functional interplay between IBtkα and MYC. We show that IBtkα, albeit indirectly, activates the ß-catenin-dependent transcription of the MYC gene. Of course, IBtkα associates with GSK3ß and promotes its ubiquitylation, which is associated with proteasomal degradation. This event increases the protein level of ß-catenin, a substrate of GSK3ß, and results in the transcriptional activation of the MYC and CCND1 target genes of ß-catenin, which are involved in the control of cell division and apoptosis. In particular, we found that in Burkitt's lymphoma cells, IBtkα silencing triggered the downregulation of both MYC mRNA and protein expression, as well as a strong decrease of cell survival, mainly through the induction of apoptotic events, as assessed by using flow cytometry-based cell cycle and apoptosis analysis. Collectively, our results shed further light on the complex puzzle of IBtkα interactome and highlight IBtkα as a potential novel therapeutic target to be employed in the strategy for personalized therapy of B cell lymphoma.

A novel phage display based platform for exosome diversity characterization.

We present an innovative approach allowing the identification, isolation, and molecular characterization of disease-related exosomes based on their different antigenic reactivities. The designed strategy could be immediately translated into any disease in which exosomes are involved. The identification of specific markers and their subsequent association with exosome subtypes, together with the possibility to engineer target-guided exosome-like particles, could represent the key for the effective adoption of exosomes in clinical practice.

Metabolites Profiling of Melanoma Interstitial Fluids Reveals Uridine Diphosphate as Potent Immune Modulator Capable of Limiting Tumor Growth.

Tumor interstitial fluid (TIF) surrounds and perfuses tumors and collects ions, metabolites, proteins, and extracellular vesicles secreted by tumor and stromal cells. Specific metabolites, accumulated within the TIF, could induce metabolic alterations of immune cells and shape the tumor microenvironment. We deployed a metabolomic approach to analyze the composition of melanoma TIF and compared it to the plasma of C57BL6 mice, engrafted or not with B16-melanoma cells. Among the classes of metabolites analyzed, monophosphate and diphosphate nucleotides resulted enriched in TIF compared to plasma samples. The analysis of the effects exerted by guanosine diphosphate (GDP) and uridine diphosphate (UDP) on immune response revealed that GDP and UDP increased the percentage of CD4+CD25+FoxP3- and, on isolated CD4+ T-cells, induced the phosphorylation of ERK, STAT1, and STAT3; increased the activity of NF-κB subunits p65, p50, RelB, and p52; increased the expression of Th1/Th17 markers including IFNγ, IL17, T-bet, and RORγt; and reduced the expression of IL13, a Th2 marker. Finally, we observed that local administrations of UDP in B16-engrafted C57BL6 mice reduced tumor growth and necrotic areas. In addition, UDP-treated tumors showed a higher presence of MHCIIhi tumor-associated macrophage (TAM) and of CD3+CD8+ and CD3+CD4+ tumor-infiltrating T-lymphocytes (TILs), both markers of anti-tumor immune response. Consistent with this, intra-tumoral gene expression analysis revealed in UDP-treated tumors an increase in the expression of genes functionally linked to anti-tumor immune response. Our analysis revealed an important metabolite acting as mediator of immune response, which could potentially represent an additional tool to be used as an adjuvant in cancer immunotherapy.

Predominant VH1-69 IgBCR Clones Show Higher Expression of CD5 in Heterogeneous Chronic Lymphocytic Leukemia Populations.

The immunoglobulin B cell receptor (IgBCR) expressed by chronic lymphocytic leukemia (CLL) B cells plays a pivotal role in tumorigenesis, supporting neoplastic transformation, survival, and expansion of tumor clones. We demonstrated that in the same patient, two or more CLL clones could coexist, recognized by the expression of different variable regions of the heavy chain of IgBCR, composing the antigen-binding site. In this regard, phage display screening could be considered the easier and most advantageous methodology for the identification of small peptide molecules able to mimic the natural antigen of the tumor IgBCRs. These molecules, properly functionalized, could be used as a probe to specifically identify and isolate single CLL subpopulations, for a deeper analysis in terms of drug resistance, phenotype, and gene expression. Furthermore, CLL cells express another surface membrane receptor, the CD5, which is commonly expressed by normal T cells. Piece of evidence supports a possible contribution of CD5 to the selection and maintenance of autoreactivity in B cells and the constitutive expression of CD5 on CLL cells could induce pro-survival stimuli. In this brief research report, we describe a peptide-based single-cell sorting using as bait the IgBCR of tumor cells; in the next step, we performed a quantitative analysis of CD5 expression by qRT-PCR related to the expressed IgBCR. Our approach could open a new perspective for the identification, isolation, and investigation of all subsets of IgBCR-related CLL clones, with particular attention to the more aggressive clones.

Phage-Displayed Peptides for Targeting Tyrosine Kinase Membrane Receptors in Cancer Therapy.

Receptor tyrosine kinases (RTKs) regulate critical physiological processes, such as cell growth, survival, motility, and metabolism. Abnormal activation of RTKs and relative downstream signaling is implicated in cancer pathogenesis. Phage display allows the rapid selection of peptide ligands of membrane receptors. These peptides can target in vitro and in vivo tumor cells and represent a novel therapeutic approach for cancer therapy. Further, they are more convenient compared to antibodies, being less expensive and non-immunogenic. In this review, we describe the state-of-the-art of phage display for development of peptide ligands of tyrosine kinase membrane receptors and discuss their potential applications for tumor-targeted therapy.

Role of Chronic Lymphocytic Leukemia (CLL)-Derived Exosomes in Tumor Progression and Survival.

Chronic lymphocytic leukemia (CLL) is a B-lymphoproliferative disease, which consists of the abnormal proliferation of CD19/CD5/CD20/CD23 positive lymphocytes in blood and lymphoid organs, such as bone marrow, lymph nodes and spleen. The neoplastic transformation and expansion of tumor B cells are commonly recognized as antigen-driven processes, mediated by the interaction of antigens with the B cell receptor (BCR) expressed on the surface of B-lymphocytes. The survival and progression of CLL cells largely depend on the direct interaction of CLL cells with receptors of accessory cells of tumor microenvironment. Recently, much interest has been focused on the role of tumor release of small extracellular vesicles (EVs), named exosomes, which incorporate a wide range of biologically active molecules, particularly microRNAs and proteins, which sustain the tumor growth. Here, we will review the role of CLL-derived exosomes as diagnostic and prognostic biomarkers of the disease.

Uncovering the Exosomes Diversity: A Window of Opportunity for Tumor Progression Monitoring.

Cells can communicate through special "messages in the bottle", which are recorded in the bloodstream inside vesicles, namely exosomes. The exosomes are nanovesicles of 30-100 nm in diameter that carry functionally active biological material, such as proteins, messanger RNA (mRNAs), and micro RNA (miRNAs). Therefore, they are able to transfer specific signals from a parental cell of origin to the surrounding cells in the microenvironment and to distant organs through the circulatory and lymphatic stream. More and more interest is rising for the pathological role of exosomes produced by cancer cells and for their potential use in tumor monitoring and patient follow up. In particular, the exosomes could be an appropriate index of proliferation and cancer cell communication for monitoring the minimal residual disease, which cannot be easily detectable by common diagnostic and monitoring techniques. The lack of unequivocal markers for tumor-derived exosomes calls for new strategies for exosomes profile characterization aimed at the adoption of exosomes as an official tumor biomarker for tumor progression monitoring.

Insights about MYC and Apoptosis in B-Lymphomagenesis: An Update from Murine Models.

The balance between cell survival and cell death represents an essential part of human tissue homeostasis, while altered apoptosis contributes to several pathologies and can affect the treatment efficacy. Impaired apoptosis is one of the main cancer hallmarks and some types of lymphomas harbor mutations that directly affect key regulators of cell death (such as BCL-2 family members). The development of novel techniques in the field of immunology and new animal models has greatly accelerated our understanding of oncogenic mechanisms in MYC-associated lymphomas. Mouse models are a powerful tool to reveal multiple genes implicated in the genesis of lymphoma and are extensively used to clarify the molecular mechanism of lymphoma, validating the gene function. Key features of MYC-induced apoptosis will be discussed here along with more recent studies on MYC direct and indirect interactors, including their cooperative action in lymphomagenesis. We review our current knowledge about the role of MYC-induced apoptosis in B-cell malignancies, discussing the transcriptional regulation network of MYC and regulatory feedback action of miRs during MYC-driven lymphomagenesis. More importantly, the finding of new modulators of apoptosis now enabling researchers to translate the discoveries that have been made in the laboratory into clinical practice to positively impact human health.

Use of Complementary Natural Feed for Gastrointestinal Nematodes Control in Sheep: Effectiveness and Benefits for Animals.

The treatments of gastrointestinal nematodes (GIN) infection in sheep is almost exclusively based on the use of synthetic drugs. In some European regions the intensive use of antiparasitic drugs is leading to widespread development of anthelmintic resistance (AR). Currently in southern Italy AR is rare, but a constant monitoring of anthelmintic efficacy and the use of effective alternative therapies is strongly recommended. The aim of our study was to evaluate the effectiveness of a complementary natural feed (natural vegetable mixture), based on natural extracts, registered for GIN treatment in sheep, and its comparison with the drug ivermectin. The study was conducted in two sheep breeding farms in southern Italy and 75 sheep were divided in groups of 15 animals each (treated and untreated groups), homogeneous by GIN eggs per gram (EPG) of faeces, using the natural anthelmintic administered at full dose (10 g/sheep/orally) in the first breeding and at double dose (20 g/sheep/orally) in the second. In the latter we compared the effectiveness of mixture with ivermectin administered at full dose (200 µg/kg/BW). To determine the effectiveness, individual faecal samples were collected to evaluate the faecal eggs count (FEC) using FLOTAC technique and FEC reduction (FECR) on different days. The formula used FECR = 100 × (1 - (T2/C2)), based on the comparison of post-treatment EPG mean of the treated and untreated group (T2 and C2, respectively), is the one recommended by World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guidelines to monitor drug efficacy against GIN in livestock. The results reported that complementary natural feed, at two different dosages, was ineffective against GIN, while the drug, at conventional dosage, showed good anthelmintic efficacy, also confirming the importance of in vivo effectiveness studies.