Fig. 2

Lm^at-LLO inhibits the growth of primary melanoma tumors and their metastatization to lymph nodes. a–c Lm^at-LLO inhibits the growth of primary melanoma tumors. a Representative pictures of primary tumors (red line) developed by mice treated with saline solution (NaCl) or the indicated doses of Lm^at-LLO (one injection of 10⁷ CFU in the tumor area was followed by one IP injection per day of 10⁵ or 10⁶ CFU for 14 days). For each experimental group, pictures were taken at three time points (before treatment, after 1 week of treatment and after 2 weeks of treatment). b, c Volume (b) and weight (c) of primary tumors. The number of primary tumors (mice) analyzed are: 12 (volume) and 13 (weight) for the NaCl group, 7 (volume) and 8 (weight) for the Lm^at-LLO 10⁷–10⁵ group and 12 (volume and weight) for the Lm^at-LLO 10⁷–10^6.group. d Expression levels of undeleted Pten mRNA and IL-2 mRNA. Total RNA extracted from paraffin embedded primary tumor samples was analyzed by qRT-PCR. Left: Undeleted Pten mRNA levels were measured using a forward primer located on exon 3 and a reverse located on exon 4–5, as reported in [26]. The higher levels of undeleted Pten mRNA detected in mice treated with Lm^at-LLO compared to control mice are consistent with the smaller size of primary tumors. Right: The higher levels of IL-2 mRNA detected in Lm^at-LLO treated mice compared to control mice provide a molecular confirmation of the induction of the immune system by the vaccine. e Infection of tumor cells with Lm^at-LLO causes a significant increase in apoptotic cell death, as indicated by Cleaved Caspase-3 immunostaining. The number of primary tumors (mice) analyzed is five for each experimental group. Original magnification: 40× (scale bar: 25 μm). f, g Infection of tumor cells with Lm^at-LLO causes a significant increase in T-lymphocytes infiltration, as indicated by immunostaining of CD3+ (f) and CD8+ (g) cells. The number of primary tumors (mice) analyzed is five for each experimental group. Original magnification: 40× (scale bar: 20 μm). In b–g the mean ± SEM is reported. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. h, i Lm^at-LLO inhibits melanoma metastatization to lymph nodes. h Volume of inguinal lymph nodes in mice treated with saline solution (NaCl) or the indicated doses of Lm^at-LLO, as measured by ultrasound imaging. The volume of right and left inguinal lymph nodes was measured at three time points (before treatment, after 1 week and after 2 weeks of treatment). The total number of lymph nodes (mice) analyzed was eight (four) for each experimental group. In the graph, individual measurements are reported, as well as a line connecting mean values. **p < 0.01. i 3D ultra high-frequency ultrasound reconstruction of a representative inguinal lymph node per experimental group (with surface rendering and volume measurement reported as picture in picture). The upper images were acquired before treatment, while the lower images were acquired after 2 weeks of treatment. j Melanin content of axillary, brachial, and inguinal lymph nodes in mice treated with saline solution (NaCl) or the indicated doses of Lm^at-LLO. The total number of lymph nodes (mice) analyzed was the following: 59 (13) for the NaCl group, 44 (8) for the Lm^at-LLO 10⁷–10⁵ group, 59 (12) for the Lm^at-LLO 10⁷–10⁶ group. Black, high melanin content; white, no or low melanin content. *p < 0.05, **p < 0.01