Stromal cells support the survival of human primary chronic lymphocytic leukemia (CLL) cells through Lyn-driven extracellular vesicles.

Introduction: In chronic lymphocytic leukemia (CLL), the tumor cells receive survival support from stromal cells through direct cell contact, soluble factors and extracellular vesicles (EVs). The protein tyrosine kinase Lyn is aberrantly expressed in the malignant and stromal cells in CLL tissue. We studied the role of Lyn in the EV-based communication and tumor support. Methods: We compared the Lyn-dependent EV release, uptake and functionality using Lyn-proficient (wild-type) and -deficient stromal cells and primary CLL cells. Results: Lyn-proficient cells caused a significantly higher EV release and EV uptake as compared to Lyn-deficient cells and also conferred stronger support of primary CLL cells. Proteomic comparison of the EVs from Lyn-proficient and -deficient stromal cells revealed 70 significantly differentially expressed proteins. Gene ontology studies categorized many of which to organization of the extracellular matrix, such as collagen, fibronectin, fibrillin, Lysyl oxidase like 2, integrins and endosialin (CD248). In terms of function, a knockdown of CD248 in Lyn+ HS-5 cells resulted in a diminished B-CLL cell feeding capacity compared to wildtype or scrambled control cells. CD248 is a marker of certain tumors and cancer-associated fibroblast (CAF) and crosslinks fibronectin and collagen in a membrane-associated context. Conclusion: Our data provide preclinical evidence that the tyrosine kinase Lyn crucially influences the EV-based communication between stromal and primary B-CLL cells by raising EV release and altering the concentration of functional molecules of the extracellular matrix.

CD30-Positive Extracellular Vesicles Enable the Targeting of CD30-Negative DLBCL Cells by the CD30 Antibody-Drug Conjugate Brentuximab Vedotin.

CD30, a member of the TNF receptor superfamily, is selectively expressed on a subset of activated lymphocytes and on malignant cells of certain lymphomas, such as classical Hodgkin Lymphoma (cHL), where it activates critical bystander cells in the tumor microenvironment. Therefore, it is not surprising that the CD30 antibody-drug conjugate Brentuximab Vedotin (BV) represents a powerful, FDA-approved treatment option for CD30+ hematological malignancies. However, BV also exerts a strong anti-cancer efficacy in many cases of diffuse large B cell lymphoma (DLBCL) with poor CD30 expression, even when lacking detectable CD30+ tumor cells. The mechanism remains enigmatic. Because CD30 is released on extracellular vesicles (EVs) from both, malignant and activated lymphocytes, we studied whether EV-associated CD30 might end up in CD30- tumor cells to provide binding sites for BV. Notably, CD30+ EVs bind to various DLBCL cell lines as well as to the FITC-labeled variant of the antibody-drug conjugate BV, thus potentially conferring the BV binding also to CD30- cells. Confocal microscopy and imaging cytometry studies revealed that BV binding and uptake depend on CD30+ EVs. Since BV is only toxic toward CD30- DLBCL cells when CD30+ EVs support its uptake, we conclude that EVs not only communicate within the tumor microenvironment but also influence cancer treatment. Ultimately, the CD30-based BV not only targets CD30+ tumor cell but also CD30- DLBCL cells in the presence of CD30+ EVs. Our study thus provides a feasible explanation for the clinical impact of BV in CD30- DLBCL and warrants confirming studies in animal models.

Intracellular Targeting of Poly Lactic-Co-Glycolic Acid Nanoparticles by Surface Functionalization with Peptides.

Nanoparticles (NPs) are a promising strategy for delivering drugs to specific sites because of their tunable size and surface chemistry variety. Among the availablematerials, NPs prepared with biopolymers are of particular interest because of their biocompatibility and controlled release of encapsulated drugs. Poly lactic-co-glycolic acid (PLGA) is one of the most widely used biopolymers in biomedical applications. In addition to material choice modulation of the interaction between NPs and biological systems is essential for the safety and effective use of NPs. Therefore, this work focused on evaluating different surface functionalization strategies to promote cancer cell uptake and intracellular targeting of PLGA NPs. Herein, cell-penetrating peptides (CPPs) were shown to successfully drive PLGA NPs to the mitochondria and nuclei. Furthermore, the functionalization of PLGA NPs with peptide AC-1001 H3 (GQYGNLWFAY) was proven to be useful for targeting actin filaments. The PLGA NPs cell internalization mechanism by B16F10-Nex2 cells was identified as caveolae-mediated endocytosis, which could be inhibited by the presence of methyl-ß-cyclodextrin. Notably, when peptide C (CVNHPAFAC) was used to functionalize PLGA NPs, none of the tested inhibitors could avoid cell internalization of PLGA NPs. Therefore, we suggest this peptide as a promising surface modification agent for enhancing drug delivery to cancer cells. Finally, PLGA NPs showed slow release kinetics and low cytotoxic profile, which, combined with the surface functionalization strategies addressed in this study, highlight the potential of PLGA NPs as a drug delivery platform for improving cancer therapy.

Inhibition of melanoma metastasis by dual-peptide PLGA NPS.

Despite the positive results observed in vitro and in vivo, clinical trials with bioactive peptides are generally hampered by their fast degradation in the biological system. Two bioactive peptides, P20 (CSSRTMHHC) and the combined peptide C (CVNHPAFACGYGHTMYYHHYQHHL) have been identified as anticancer therapeutics. Combined peptide C consists of peptide C (CVNHPAFAC), a tumor-homing peptide, conjugated to the antiangiogenic peptide HTMYYHHYQHHL with a GYG. In this work, PLGA NPs with peptide C were applied as a dual-peptide carrier for application in cancer therapy. Peptide P20 was loaded into the NPs and combined peptide C was conjugated to the NPs surface. These NPs were evaluated as a therapeutic system to treat metastatic melanoma. In vivo assays showed that P20 encapsulation in PLGA NPs enhanced its antitumor activity. The inhibitory activity of P20-PLGANPs was similar to the activity of non-encapsulated P20 in a dose fivefold higher. The inhibitory activity was even higher when P20PLGA NPs were functionalized with combined peptide C. P20PLGAPepC NPs reduced in 28% the number of lung nodules in a syngeneic model of metastatic melanoma as compared to untreated animals. Additionally to the better tumor targeting and the in situ release of P20, it is expected that the therapeutic efficiency of the dual-peptide PLGA NPs was further enhanced by a synergistic effect between P20 and combined peptide C. Our encouraging results showed that by enabling the co-delivery of two peptides and promoting tumor targeting, PLGA NPs coupled with peptide C is a promising platform for peptide-based cancer therapy.