Tumor-Secreted Extracellular Vesicles Counteract Therapy Response by Triggering Inflammatory Mesenchymal Stem Cell Development.

PURPOSE: Therapy resistance is a major clinical hurdle in bone cancer treatment and seems to be largely driven by poorly understood microenvironmental factors. Recent evidence suggests a critical role for a unique subpopulation of mesenchymal stem cells with inflammatory features (iMSC), though their origin and function remained unexplored. We demonstrate that cancer-secreted extracellular vesicles (EV) trigger the development of iMSCs, which hinder therapy response in vivo, and set out to identify strategies to counteract their function. EXPERIMENTAL DESIGN: The role of iMSCs in therapy resistance was evaluated in an orthotopic xenograft mouse model of osteosarcoma. EV-induced alterations of the MSC transcriptome were analyzed and compared with single-cell RNA sequencing data of biopsies from patients with osteosarcoma and multiple myeloma. Functional assays identified EV components driving iMSC development. We assessed the efficacy of clinical drugs in blocking iMSC-induced resistance in vivo. RESULTS: We found that iMSCs are induced by interaction with cancer EVs and completely abrogate the antimetastatic effect of TGFß signaling inhibition. Importantly, EV-induced iMSCs faithfully recapitulate the inflammatory single-cell RNA signature of stromal cells enriched in biopsies from patients with multiple myeloma and osteosarcoma. Mechanistically, cancer EVs act through two distinct mechanisms. EV-associated TGFß induces IL6 production, whereas the EV-RNA cargo enhances TLR3-mediated chemokine production. We reveal that simultaneous blockade of downstream EV-activated pathways with ladarixin and tocilizumab disrupts metastasis formation and overcomes iMSC-induced resistance. CONCLUSIONS: Our observations establish iMSCs as major contributors to drug resistance, reveal EVs as triggers of iMSC development, and highlight a promising combination strategy to improve therapy response in patients with bone cancer.

Exosomal MicroRNAs: Comprehensive Methods from Exosome Isolation to miRNA Extraction and Purity Analysis.

Exosomes are extracellular vesicles secreted by cells with a key role in a wide range of biological processes including cancer. These vesicles are involved in intercellular communication and deliver diverse cargo molecules, including miRNAs (exo-miRNAs), to recipient cells affecting their physiology. Exo-miRNAs have a role in promoting tumor, progression, metastatization, and remodeling of tumor microenvironment, therefore making them interesting biomarkers to study.Here we provide a detailed technical protocol for exosome isolation (which can be applied to cell culture as well as physiological fluids), validation of their vesicular identity, miRNA extraction, and quantitative and qualitative analysis to evaluate the sample purity and concentration.

Harnessing EV communication to restore antitumor immunity.

Restoring effective anti-tumor immune responses to cure cancer is a promising strategy, but challenging to achieve due to the intricate crosstalk between tumor and immune cells. While it is established that tumor cells acquire traits to escape immune recognition, the involvement of extracellular vesicles (EVs) in curbing immune cell activation is rapidly emerging. By assisting cancer cells in spreading immunomodulatory signals in the form of (glyco)proteins, lipids, nucleic acids and metabolic regulators, EVs recently emerged as versatile mediators of immune suppression. Blocking their action might reactivate immune cell function and natural antitumor immune responses. Alternatively, EV communication may be exploited to boost anti-tumor immunity. Indeed, novel insights into EV biology paved the way for efficient ex vivo production of 'rationally engineered' EVs that function as potent antitumor vaccines or carry out specific functional tasks. In this review we discuss the latest findings on immune regulation by cancer EVs and explore how EV-mediated communication can be either targeted or harnessed to restore immunity as a means for cancer therapy.

Extracellular Vesicle-Based Nucleic Acid Delivery: Current Advances and Future Perspectives in Cancer Therapeutic Strategies.

Extracellular vesicles (EVs) are sophisticated and sensitive messengers released by cells to communicate with and influence distant and neighboring cells via selective transfer of bioactive content, including protein lipids and nucleic acids. EVs have therefore attracted broad interest as new and refined potential therapeutic systems in many diseases, including cancer, due to their low immunogenicity, non-toxicity, and elevated bioavailability. They might serve as safe and effective vehicles for the transport of therapeutic molecules to specific tissues and cells. In this review, we focus on EVs as a vehicle for gene therapy in cancer. We describe recent developments in EV engineering to achieve efficient intracellular delivery of cancer therapeutics and avoid off-target effects, to provide an overview of the potential applications of EV-mediated gene therapy and the most promising biomedical advances.

MicroRNA-Assisted Hormone Cell Signaling in Colorectal Cancer Resistance.

Despite substantial progress in cancer therapy, colorectal cancer (CRC) is still the third leading cause of cancer death worldwide, mainly due to the acquisition of resistance and disease recurrence in patients. Growing evidence indicates that deregulation of hormone signaling pathways and their cross-talk with other signaling cascades inside CRC cells may have an impact on therapy resistance. MicroRNAs (miRNAs) are small conserved non-coding RNAs thatfunction as negative regulators in many gene expression processes. Key studies have identified miRNA alterations in cancer progression and drug resistance. In this review, we provide a comprehensive overview and assessment of miRNAs role in hormone signaling pathways in CRC drug resistance and their potential as future targets for overcoming resistance to treatment.

Trifolium Repens Blocks Proliferation in Chronic Myelogenous Leukemia via the BCR-ABL/STAT5 Pathway.

Some species of clover are reported to have beneficial effects in human diseases. However, little is known about the activity of the forage plant Trifolium repens, or white clover, which has been recently found to exert a hepatoprotective action. Scientific interest is increasingly focused on identifying new drugs, especially natural products and their derivatives, to treat human diseases including cancer. We analyzed the anticancer effects of T. repens in several cancer cell lines. The phytochemical components of T. repens were first extracted in a methanol solution and then separated into four fractions by ultra-high-performance liquid chromatography. The effects of the total extract and each fraction on cancer cell proliferation were analyzed by MTT assay and Western blotting. T. repens and, more robustly, its isoflavonoid-rich fraction showed high cytotoxic effects in chronic myelogenous leukemia (CML) K562 cells, with IC50 values of 1.67 and 0.092 mg/mL, respectively. The block of cell growth was associated with a total inhibition of BCR-ABL/STAT5 and activation of the p38 signaling pathways. In contrast, these strongly cytotoxic effects did not occur in normal cells. Our findings suggest that the development of novel compounds derived from phytochemical molecules contained in Trifolium might lead to the identification of new therapeutic agents active against CML.