Leishmania braziliensis enhances monocyte responses to promote anti-tumor activity.

Innate immune cells can undergo long-term functional reprogramming after certain infections, a process called trained immunity (TI). Here, we focus on antigens of Leishmania braziliensis, which induced anti-tumor effects via trained immunity in human monocytes. We reveal that monocytes exposed to promastigote antigens of L. braziliensis develop an enhanced response to subsequent exposure to Toll-like receptor (TLR)2 or TLR4 ligands. Mechanistically, the induction of TI in monocytes by L. braziliensis is mediated by multiple pattern recognition receptors, changes in metabolism, and increased deposition of H3K4me3 at the promoter regions of immune genes. The administration of L. braziliensis exerts potent anti-tumor capabilities by delaying tumor growth and prolonging survival of mice with non-Hodgkin lymphoma. Our work reveals mechanisms of TI induced by L. braziliensis in vitro and identifies its potential for cancer immunotherapy.

Salmonella-induced immune response reduces recurrence and tumor dissemination in preclinical melanoma model.

Localized melanoma is easy to remove by surgery, resulting in a high five-year relative survival rate. However, when disseminated the disease management is challenging. The use of immunotherapies, such as anti-checkpoint monoclonal antibodies, has improved treatment options but still only a small percentage of patients responds to these expensive treatments. In this work, we apply a bacteria-based immunotherapy using LVR01, an attenuated Salmonella enterica serovar Typhimurium, as neoadjuvant therapy one week before surgery in a preclinical disseminated murine melanoma model. LVR01 administration resulted in tumor growth retardation prior to tumor resection, due to a rapid upregulation of inflammatory genes in the tumor microenvironment. As a consequence, cell infiltration increased, particularly neutrophils, macrophages and NK cells, being the latter involved in Salmonella anti-tumor activity. Besides, tumor-draining lymph node infiltration is characterized by reinvigorated CD4+ and CD8+ lymphocytes. Induced immune response could account for the prevention or delay of tumor recurrence and appearance of metastasis, resulting in a prolonged overall survival after surgery. Furthermore, upon rechallenge mice show partial protection, suggesting the existence of specific memory against melanoma. We propose that neoadjuvant LVR01 treatment could represent an interesting inexpensive alternative that may ease tumor resection, while preventing tumor recurrence in patients with melanoma.

Inflammasome activation, NLRP3 engagement and macrophage recruitment to tumor microenvironment are all required for Salmonella antitumor effect.

Salmonella-based cancer therapies show great potential in preclinical models, but for most cases the observed antitumor effect is transient. Understanding the basis of the antitumor efficacy might guide the design of improved strains that elicit long-lasting effects, paving the wave for clinical use.  Here, we deepened into the role of macrophages and inflammasome activation in the context of Salmonella anti-melanoma effect. We showed inflammasome activation in melanoma cells upon infection, which correlated with cell surface exposure of gasdermin-D (GSDM-D) and calreticulin (CRT) and High mobility group box 1 protein (HMGB-1) release, suggesting immunogenic cell death, particularly pyroptosis. Salmonella infection upregulated levels of Caspase-11 (Casp11) mRNA, but not Nlrp3 or Nlrc4 mRNA, the only described inflammasome receptors engaged by Salmonella, suggesting that non-canonical inflammasome activation could be occurring in melanoma cells. Intratumoral administration of Salmonella to melanoma-bearing mice elicited local inflammasome activation and interleukin-1ß (IL-1ß) production together with tumor growth retardation and extended survival in wild type but not Caspase-1/11 (Casp1/11) knockout mice despite similar levels of intratumoral IL-1ß in the later. Salmonella antitumor activity was also suppressed in melanoma bearing Nlrp3 knockout mice. Salmonella induced macrophage recruitment to the tumor site and infiltrating cells exhibited inflammasome activation. Depletion experiments confirmed that macrophages are also essential for Salmonella anti-melanoma effect. Intratumoral macrophages showed a marked M2/M1 shift soon after treatment but this inflammatory profile is then lost, which could explain the transient effect of therapy.  All in all, our results highlight CASP-1/11 axis and macrophages as essential players in Salmonella-based cancer immunotherapy and suggest a possible target for future interventions.

Preclinical Evaluation of LVR01 Attenuated Salmonella as Neoadjuvant Intralesional Therapy in Combination with Chemotherapy for Melanoma Treatment.

Treatment of malignant melanoma has improved in the last few years owing to early detection and new therapeutic options. Still, management of advanced disease remains a challenge because it requires systemic treatment. In such cases, dacarbazine-based chemotherapy has been widely used, despite low efficacy. Neoadjuvant therapies emerge as alternative options that could help chemotherapy to achieve increased benefit. In this work, we evaluate LVR01, an attenuated Salmonella enterica serovar typhimurium, as neoadjuvant intralesional therapy in combination with dacarbazine in a preclinical melanoma model. B16F1 melanoma‒bearing mice received intraperitoneal administration of dacarbazine for 3 consecutive days. LVR01 treatment, consisting of one single intratumoral injection, was applied 1 day before chemotherapy began. This therapeutic approach retarded tumor growth and prolonged overall survival, revealing a strong synergistic antitumor effect. Dacarbazine induced a drastic reduction of secondary lymphoid organ cellularity, which was partially restored by Salmonella, particularly potentiating activated cytotoxic cell compartments. Systemic immune reactivation could be a consequence of the intense inflammatory tumor microenvironment induced by LVR01. We propose that the use of LVR01 as neoadjuvant intralesional therapy could be considered as an interesting strategy with close clinical application to boost chemotherapy effect in patients with melanoma.