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

Abstract

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.

Keywords: Melanoma; Memory immune response; Minimal residual disease; Neoadjuvant therapies; Salmonella.

Graphical abstract

Fig. 1

Tumor size, free of disease and overall survival of B16F1 melanoma bearing mice treated with LVR01 prior to surgery. (a) Schematic representation of treatment schedule. Mice were treated with 1 × 10⁶ cfu LVR01 i.t. as described in M&M. (b) Tumor size at day 18, prior to surgery. Volume was calculated as (LxWxD)xπ/6 and results are shown as median and quartiles. **p < 0.01 Student's T test. (c) Reemergence of the disease is plotted as disease-free survival (DFS) curve. Then tumor size was measured every 2–3 days. (d) Animal survival is plotted as overall survival (OS) curve. Survival was followed up for 100 days, ***p < 0.001, Log-rank Test (Control group n = 12, LVR01 group n = 30).

Fig. 2

Tumor setting at days 3 or 8 induced by LVR01 neoadjuvant intralesional administration in mice prior to surgery. (a) Cytokine/chemokine profile in LVR01-treated mice, normalized against control untreated mice, expressed as mean Log2 (n = 5–6) (b) Tumor immune cell phenotypification. Tumor homogenates were stained for detecting different tumor infiltrating populations of granulocytes and lymphocytes. Gating strategies are shown in Suppl. Fig. 1. All results are shown as mean ± SD (n = 5–11). Student's T test, *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig. 3

Immune profile induced by LVR01 in secondary lymphoid organs at day 16 after Salmonella treatment. TDLN homogenates were stained for detecting different lymphocyte subpopulations. (a) Counts of lymphocytes are expressed as absolute numbers. (b) CTLA-4 and PD-1 expression on CD4⁺ T and CD8⁺ T cells. (c) Effector and (d) effector memory CD4⁺ and CD8⁺ T cell counts. (e) Secreted IFNγ levels by B16F1 re-stimulated splenocytes, measured by ELISA. All gating strategies are available in Suppl Fig. 1. Results in (a), (c) and (d) are shown as box and whiskers (min to max), while (b) and (e) are shown as mean ± SD (n = 5–6). Student's T test, *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig. 4

Relevance of different cell types in tumor control induced by LVR01. Tumor growth after Salmonella-treated melanoma-bearing mice depleted of (a) CD8⁺ T cells, (b) Neutrophils, and (c) NK cells. Gating strategies and depletion followup are shown in Suppl. Fig. 3. All results are shown as mean ± SD (n = 11–12). Student's T test, *p < 0.05.

Fig. 5

Tumor size and overall survival of B16F1 melanoma bearing mice treated with neoadjuvant LVR01, after rechallenge. (a) Schematic representation of treatment schedule. Mice were treated with 1 × 10⁶ cfu LVR01 i.t., tumors were excised a week later and 76 days later surviving mice were rechallenged subcutaneously in the opposite flank with 1 × 10⁵ B16F1 cells. (b) Tumor growth curve, expressed as days post primary tumor implantation. Tumor size was measured every 2–3 days and volume was calculated as (LxWxD)xπ/6. Results are shown as mean ± SD (n = 12–16) ***p < 0.001, Student's T test. (c) Survival was followed up for 160 days ***p < 0.001, Log-rank Test.

Fig. 6

Tumor size and overall survival of B16F1 melanoma bearing mice treated with LVR01 post surgery. (a) Schematic representation of treatment schedule. 18 days post tumor implantation, tumors were excised and starting 9 days later mice were treated weakly with 1 × 10⁶ cfu LVR01 i.t., or s.c. when tumors were not palpable. (b) Tumor growth curve, expressed as days post primary tumor removal. Tumor size was measured every 2–3 days and volume was calculated as (LxWxD)xπ/6. Results are shown as mean ± SD (n = 12) ***p < 0.001, two-way ANOVA. (c) Survival was followed up for 60 days after primary tumor removal ***p < 0.001, Log-rank Test.