Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor-associated macrophages (TAMs) secrete EVs, but their molecular features and functions are poorly characterized. Here, we report methodology for the enrichment, quantification, and proteomic and lipidomic analysis of EVs released from mouse TAMs (TAM-EVs). Compared to source TAMs, TAM-EVs present molecular profiles associated with a Th1/M1 polarization signature, enhanced inflammation and immune response, and a more favorable patient prognosis. Accordingly, enriched TAM-EV preparations promote T cell proliferation and activation ex vivo. TAM-EVs also contain bioactive lipids and biosynthetic enzymes, which may alter pro-inflammatory signaling in the cancer cells. Thus, whereas TAMs are largely immunosuppressive, their EVs may have the potential to stimulate, rather than limit, anti-tumor immunity.

Plitidepsin is an antitumoral compound safe for treating COVID-19 that targets the translation elongation factor eEF1A. Here we detect that plitidepsin decreases de novo cap-dependent translation of SARS-CoV-2 and non-viral RNAs but affects less than 13% of the host proteome, thus preserving cellular viability. In response to plitidepsin, cells upregulate EIF2AK3 and proteins that reduce translation, but also proteins that support proteostasis via ribosome synthesis and cap-independent translation by eIF4G2 and IGF2BP2. While plitidepsin inhibits cap- or internal ribosome entry sites (IRES)-mediated translation, its impact on N6-methyladenosine (m⁶A) translation is limited. In agreement, plitidepsin blocks members of Coronaviridae, Flaviviridae, Pneumoviridae and Herpesviridae families. Yet, it fails to inhibit retroviruses that exploit m⁶A synthesis routes and are blocked by drugs targeting IGF2BP2 m⁶A reader. By deciphering the molecular fingerprint of cells treated with therapies targeting translation we identify a rational approach to select broad-spectrum antivirals with potential to counteract future pandemic viruses.

Identifying animal species that are susceptible to the plethora of existing and emerging viruses is critical for predicting and containing disease outbreaks. Current efforts to assess viral tropism largely rely on experimental infection models, but such experiments are logistically and ethically infeasible for many wildlife species. To tackle this challenge, we developed a panel of airway organoids from ten taxonomically diverse wildlife and livestock species and evaluated their susceptibility to influenza viruses of mammalian (pH1N1) and avian (H5N1) origin. Our analyses revealed large host-specific differences in infection rate and cytopathogenicity that aligned with known in vivo data and field observations. Furthermore, we demonstrated that this organoid panel can serve as a powerful tool to elucidate receptor-binding mechanisms, viral dynamics and early host adaptation in poorly characterized animal hosts. In summary, this work provides a robust and ethically viable approach for evaluating viral tropism and adaptation in wildlife species, and may aid current pandemic preparedness, zoonotic disease surveillance and wildlife conservation efforts.

Despite the significant advances in cancer research made in recent years, this disease remains one of the leading causes of death worldwide. In part, this is due to the fact that after therapy, a subpopulation of self-renewing tumor cells can survive and promote cancer relapse, resistance to therapies and metastasis. Targeting these cancer stem cells (CSCs) is therefore essential to improve the clinical outcome of cancer patients. In this sense, multi-targeted drugs may be promising agents targeting CSC-associated multifocal effects. We have previously constructed different human pancreatic ribonuclease (RNase) variants that are cytotoxic for tumor cells due to a non-classical nuclear localization signal introduced in their sequence. These cytotoxic RNases affect the expression of multiple genes involved in deregulated metabolic and signaling pathways in cancer cells and are highly cytotoxic for multidrug-resistant tumor cell lines. Here, we show that these cytotoxic nuclear-directed RNases are highly selective for tumor cell lines grown in 3D, inhibit CSCs’ development and diminish the self-renewal capacity of the CSCs population. Moreover, these human RNase variants reduce the migration and invasiveness of highly invasive breast cancer cells and downregulate N-cadherin expression.

Antimicrobial resistance is an impactful One Health issue. One of its drivers is the extensive use of antibiotics in both human and animal production systems, and despite regulatory restrictions on antibiotic use in poultry farming, antimicrobial resistance remains a major challenge. Consequently, animals are at higher risk of harder-to-treat diseases and play a role as resistance reservoirs, highlighting the need for alternative antimicrobial strategies. Towards this end, antimicrobial peptides (AMPs) have emerged as promising candidates due to their broad-spectrum activity and lower propensity to induce resistance. However, the effectiveness of AMPs against poultry pathogens, and in particular multi drug-resistant strains, is largely unclear. To tackle this question, we evaluated the synthetic AMP SET-M33 against four species of clinically relevant pathogens in poultry, namely Escherichia coli, Salmonella enterica, Enterococcus faecalis and Enterococcus cecorum. Using a panel of 141 field isolates, we found that SET-M33 broadly inhibited bacterial growth at low micromolar concentrations (median MICs of 2.5 μM and 5 μM for Gram-negative and Gram-positive strains, respectively), including in multi drug-resistant isolates. To examine the potential impact of SET-M33 on the host, we established a new in vitro co-cultivation system using chicken intestinal organoids. We found that SET-M33 retains its antimicrobial activity in organoid-microbe co-cultures at concentrations that preserved host viability. These findings demonstrate the potential of SET-M33 as a new antimicrobial agent against pathogens in poultry.