Optimizing high-intensity focused ultrasound-induced immunogenic cell-death using passive cavitation mapping as a monitoring tool.

Over the past decade, ultrasound (US) has gathered significant attention and research focus in the realm of medical treatments, particularly within the domain of anti-cancer therapies. This growing interest can be attributed to its non-invasive nature, precision in delivery, availability, and safety. While the conventional objective of US-based treatments to treat breast, prostate, and liver cancer is the ablation of target tissues, the introduction of the concept of immunogenic cell death (ICD) has made clear that inducing cell death can take different non-binary pathways through the activation of the patient's anti-tumor immunity. Here, we investigate high-intensity focused ultrasound (HIFU) to induce ICD by unraveling the underlying physical phenomena and resulting biological effects associated with HIFU therapy using an automated and fully controlled experimental setup. Our in-vitro approach enables the treatment of adherent cancer cells (B16F10 and CT26), analysis for ICD hallmarks and allows to monitor and characterize in real time the US-induced cavitation activity through passive cavitation detection (PCD). We demonstrate HIFU-induced cell death, CRT exposure, HMGB1 secretion and antigen release. This approach holds great promise in advancing our understanding of the therapeutic potential of HIFU for anti-cancer strategies.

Alpha-galactosylceramide improves the potency of mRNA LNP vaccines against cancer and intracellular bacteria.

Although various types of mRNA-based vaccines have been explored, the optimal conditions for induction of both humoral and cellular immunity remain rather unknown. In this study, mRNA vaccines of nucleoside-modified mRNA in lipoplexes (LPXs) or lipid nanoparticles (LNPs) were evaluated after administration in mice through different routes, assessing mRNA delivery, tolerability and immunogenicity. In addition, we investigated whether mRNA vaccines could benefit from the inclusion of the adjuvant alpha-galactosylceramide (αGC), an invariant Natural Killer T (iNKT) cell ligand. Intramuscular (IM) vaccination with ovalbumin (OVA)-encoding mRNA encapsulated in LNPs adjuvanted with αGC showed the highest antibody- and CD8+ T cell responses. Furthermore, we observed that addition of signal peptides and endocytic sorting signals of either LAMP1 or HLA-B7 in the OVA-encoding mRNA sequence further enhanced CD8+ T cell activation although reducing the induction of IgG antibody responses. Moreover, mRNA LNPs with the ionizable lipidoid C12-200 exhibited higher pro-inflammatory- and reactogenic activity compared to mRNA LNPs with SM-102, correlating with increased T cell activation and antitumor potential. We also observed that αGC could further enhance the cellular immunity of clinically relevant mRNA LNP vaccines, thereby promoting therapeutic antitumor potential. Finally, a Listeria monocytogenes mRNA LNP vaccine supplemented with αGC showed synergistic protective effects against listeriosis, highlighting a key advantage of co-activating iNKT cells in antibacterial mRNA vaccines. Taken together, our study offers multiple insights for optimizing the design of mRNA vaccines for disease applications, such as cancer and intracellular bacterial infections.

Nanomedicine to aid immunogenic cell death (ICD)-based anticancer therapy.

Immunogenic cell death (ICD) is emerging as a key component of antitumor therapy that harnesses the immune system of the patient to combat cancer. In recent years, several efforts were made to improve the ICD-based therapies. Here, we discuss how nanomaterial-based strategies increase the efficacy of ICD and highlight their benefits and challenges.

Laser-induced vapor nanobubbles for B16-F10 melanoma cell killing and intracellular delivery of chemotherapeutics.

The most lethal form of skin cancer is cutaneous melanoma, a tumor that develops in the melanocytes, which are found in the epidermis. The treatment strategy of melanoma is dependent on the stage of the disease and often requires combined local and systemic treatment. Over the years, systemic treatment of melanoma has been revolutionized and shifted toward immunotherapeutic approaches. Phototherapies like photothermal therapy (PTT) have gained considerable attention in the field, mainly because of their straightforward applicability in melanoma skin cancer, combined with the fact that these strategies are able to induce immunogenic cell death (ICD), linked with a specific antitumor immune response. However, PTT comes with the risk of uncontrolled heating of the surrounding healthy tissue due to heat dissipation. Here, we used pulsed laser irradiation of endogenous melanin-containing melanosomes to induce cell killing of B16-F10 murine melanoma cells in a non-thermal manner. Pulsed laser irradiation of the B16-F10 cells resulted in the formation of water vapor nanobubbles (VNBs) around endogenous melanin-containing melanosomes, causing mechanical cell damage. We demonstrated that laser-induced VNBs are able to kill B16-F10 cells with high spatial resolution. When looking more deeply into the cell death mechanism, we found that a large part of the B16-F10 cells succumbed rapidly after pulsed laser irradiation, reaching maximum cell death already after 4 h. Practically all necrotic cells demonstrated exposure of phosphatidylserine on the plasma membrane and caspase-3/7 activity, indicative of regulated cell death. Furthermore, calreticulin, adenosine triphosphate (ATP) and high-mobility group box 1 (HMGB1), three key damage-associated molecular patterns (DAMPs) in ICD, were found to be exposed from B16-F10 cells upon pulsed laser irradiation to an extent that exceeded or was comparable to the bona fide ICD-inducer, doxorubicin. Finally, we could demonstrate that VNB formation from melanosomes induced plasma membrane permeabilization. This allowed for enhanced intracellular delivery of bleomycin, an ICD-inducing chemotherapeutic, which further boosted cell death with the potential to improve the systemic antitumor immune response.