Extracellular vesicles originating from melanoma cells promote dysregulation in haematopoiesis as a component of cancer immunoediting.

Haematopoiesis dysregulation with the presence of immature myeloid and erythroid immunosuppressive cells are key characteristics of the immune escape phase of tumour development. Here, the role of in vitro generated B16F10 tumour cell-derived extracellular vesicles (tEVs) as indirect cellular communicators, participating in tumour-induced dysregulation of haematopoiesis, was explored. The isolated tEVs displayed features of small EVs with a size range of 100-200 nm, expressed the common EV markers CD63, CD9, and Alix, and had a spherical shape with a lipid bilayer membrane. Proteomic profiling revealed significant levels of angiogenic factors, particularly vascular endothelial growth factor (VEGF), osteopontin, and tissue factor, associated with the tEVs. Systemic administration of these tEVs in syngeneic mice induced splenomegaly and disrupted haematopoiesis, leading to extramedullary haematopoiesis, expansion of splenic immature erythroid progenitors, reduced bone marrow cellularity, medullary expansion of granulocytic myeloid suppressor cells, and the development of anaemia. These effects closely mirrored those observed in tumour-bearing mice and were not seen after heat inactivating the tEVs. In vitro studies demonstrated that tEVs independently induced the expansion of bone marrow granulocytic myeloid suppressor cells and B cells while reducing the frequency of cells in the erythropoietic lineage. These effects of tEVs were significantly abrogated by the blockade of VEGF or heat inactivation. Our findings underscore the important role of tEVs in dysregulating haematopoiesis during the immune escape phase of cancer immunoediting, suggesting their potential as targets for addressing immune evasion and reinstating normal hematopoietic processes.

Tumor cell derived osteopontin and prostaglandin E2 synergistically promote the expansion of myeloid derived suppressor cells during the tumor immune escape phase.

The immune escape stage in cancer immunoediting is a pivotal feature, transitioning immune-controlled tumor dormancy to progression, and augmenting invasion and metastasis. Tumors employ diverse mechanisms for immune escape, with generating immunosuppressive cells from skewed hematopoiesis being a crucial mechanism. This led us to suggest that tumor cells with immune escape properties produce factors that induce dysregulations in hematopoiesis. In support of this suggestion, this study found that mice bearing advanced-stage tumors exhibited dysregulated hematopoiesis characterized by the development of splenomegaly, anemia, extramedullary hematopoiesis, production of immunosuppressive mediators, and expanded medullary myelopoiesis. Further ex vivo studies exhibited that conditioned medium derived from EL4lu2 cells could mediate the expansion of myeloid derived suppressor cells (MDSCs) in bone marrow cell cultures. The protein array profiling results revealed the presence of elevated levels of osteopontin (OPN), prostaglandin E2 (PGE2) and interleukin 17 (IL-17) in the culture medium derived from EL4luc2 cells. Accordingly, substantial levels of these factors were also detected in the sera of mice bearing EL4luc2 tumors. Among these factors, only PGE2 alone could increase the number of MDSCs in the BM cell cultures. This effect of PGE2 was significantly potentiated by the presence of OPN but not IL-17. Finally, in vitro treatment of EL4luc2 cells with pioglitazone, a modulator of OPN and cyclooxygenase 2 (COX-2) resulted in a significant reduction in cell proliferation in EL4luc2 cells. Our findings highlight the significant role played by tumor cell-derived OPN and PGE2 in fostering the expansion of medullary MDSCs and in promoting tumor cell proliferation. Furthermore, these intertwined cancer processes could be key targets for pioglitazone intervention.

Novel Orthogonally Hydrocarbon-Modified Cell-Penetrating Peptide Nanoparticles Mediate Efficient Delivery of Splice-Switching Antisense Oligonucleotides In Vitro and In Vivo.

Splice-switching therapy with splice-switching oligonucleotides (SSOs) has recently proven to be a clinically applicable strategy for the treatment of several mis-splice disorders. Despite this, wider application of SSOs is severely limited by the inherently poor bioavailability of SSO-based therapeutic compounds. Cell-penetrating peptides (CPPs) are a class of drug delivery systems (DDSs) that have recently gained considerable attention for improving the uptake of various oligonucleotide (ON)-based compounds, including SSOs. One strategy that has been successfully applied to develop effective CPP vectors is the introduction of various lipid modifications into the peptide. Here, we repurpose hydrocarbon-modified amino acids used in peptide stapling for the orthogonal introduction of hydrophobic modifications into the CPP structure during peptide synthesis. Our data show that α,α-disubstituted alkenyl-alanines can be successfully utilized to introduce hydrophobic modifications into CPPs to improve their ability to formulate SSOs into nanoparticles (NPs), and to mediate high delivery efficacy and tolerability both in vitro and in vivo. Conclusively, our results offer a new flexible approach for the sequence-specific introduction of hydrophobicity into the structure of CPPs and for improving their delivery properties.

Lipophilic Peptide Dendrimers for Delivery of Splice-Switching Oligonucleotides.

Non-viral transfection reagents are continuously being developed in attempt to replace viral vectors. Among those non-viral vectors, dendrimers have gained increasing interest due to their unique molecular structure and multivalency. However, more improvements are still needed to achieve higher efficacy and lower toxicity. In this study, we have examined 18 peptide dendrimers conjugated to lipophilic moieties, such as fatty acids or hydrophobic amino acids, that were previously explored for siRNA. Reporter cells were employed to investigate the transfection of single strand splice-switching oligonucleotides (ONs) using these peptide dendrimers. Luciferase level changes reflecting efficiency varied with amino acid composition, stereochemistry, and complexation media used. 3rd generation peptide dendrimers with D-amino acid configuration were superior to L-form. Lead formulations with 3rd generation, D-amino acid peptide dendrimers increased the correction level of the delivered ON up to 93-fold over untreated HeLa Luc/705 cells with minimal toxicity. To stabilize the formed complexes, Polyvinyl alcohol 18 (PVA18) polymer was added. Although PVA18 addition increased activity, toxicity when using our best candidates G 2,3KL-(Leu)4 (D) and G 2,3KL-diPalmitamide (D) was observed. Our findings demonstrate the potential of lipid-conjugated, D-amino acid-containing peptide dendrimers to be utilized as an effective and safe delivery vector for splice-switching ONs.