Live imaging of neolymphangiogenesis identifies acute antimetastatic roles of dsRNA mimics (2021)
Figure 1

Figure 1. Identification of anti-lymphangiogenic compounds in Vegfr3Luc genetically engineered mouse models (GEMM).

Schematic representation of the Vegfr3Luc GEMM (MetAlert) mice to assess melanomas driven by melanocytic-specific induction of oncogenic BrafV600E in a Pten deficient background (1), as well as to monitor xenografts of human cells (2) and patient-derived specimens (PDX, 3).

Luciferase-based imaging of drug response in Vegfr3Luc;Tyr:CreERT2;BrafV600E; Ptenflox/flox mice. Panels labeled as "basal" and "induced" correspond to the bioluminescence of animals prior and 5 weeks after administration of 4OH-tamoxifen (5 mM, topical administration, 3 consecutive days) for the induction of melanomas. (Right panels) Treatment with anti-PD-L1 antibody (αPD-L1; clone 10F.9G2, 3 weeks) or the corresponding control IgG (200 ug/dose, twice per week, 3 weeks); vemurafenib (Vem, 50 mg/Kg, oral once per day, 3 weeks) or BO-110 (BO, 0.8 mg/kg, twice per week, 3 weeks). Scale: p/s/cm2/sr (x106).

Growth curves of Vegfr3Luc;Tyr:CreERT2;BrafV600E; Ptenflox/flox melanomas treated with αPD-L1 or IgG (200ug/dose, 2 doses/week; left panel), or with vemurafenib (Vem, 50 mg/Kg, daily dose), BO-110 (BO, 0.8 mg/kg, 2 doses/week) or vehicle control (V, daily dose) as indicated. Data correspond to the average tumor size ± SD at the indicated time points. Red arrows mark the initiation of treatment (n= min 5 mice per condition). Statistics Two-Way-ANNOVA. p=0.0012 (αPD-L1), p=0.0007 (BO-110) and p=0.0353 (Vemurafenib).

Histological visualization of lymphatic vessel density (dual Lyve11 Prox1 staining) in representative sections of tumors of Vegfr3Luc;Tyr::CreERT2;BrafV600E;Ptenflox/flox melanomas in mice treated with vehicle (V) or 4 doses of BO-110 (BO, 0.8 mg/kg). Double positive (Lyve1, Prox1) vessels were pseudocolored to green to ease the visualization. See also images for lung lymphatic vessels in Fig. EV1C. Red arrowheads indicate Lyve1-Prox1 positive lymphatic vessels.

Quantification of lymphatic and blood vessels density in tumors and lungs of Vegfr3Luc; Tyr::CreERT2;BrafV600E;Ptenflox/flox melanomas after treatment as indicated in B,C. Data correspond to the quantification of four fields per tumor, performed in biological triplicates. Statistical significance was determined by Mann Whitney t-test.

Treatment with BO-110 of human patient-derived xenografts (PDX) implanted in Vegfr3Luc nu/nu. 42 days after implantation (when systemic luciferase was detected), animals were randomized for treatment with vehicle (V) or with 0.8 mg/kg BO-110 (BO, twice per week), and luciferase emission was acquired at the indicated times. Scale, p/s/cm2/sr (x106).

Quantification of the inhibitory effect of BO-110 (BO, 0.8 mg/kg, 2 doses/week I.P. administration, 11 weeks) on the growth of melanoma PDXs. Red arrows mark the initiation of treatment. Shown are mean tumor size in mm3 ± SD in biological triplicates. Statistical significance was determined by Two-Way-ANOVA. P = 0.0009.

Representative sentinel, axillary and brachial lymph nodes (SLN, ALN and BLN, respectively) of mCherry-SK-Mel-147-driven xenografts in Vegfr3Luc nu/nu mice treated with vehicle (V) or 4 doses of BO-110 (BO, 0.8 mg/kg) and imaged for mCherry fluorescence to assess metastatic potential as a function of treatment. Numbers in parenthesis correspond to mice with positive metastases in at least one LN (lymph node) with respect to the total animals analyzed per condition. Scale, p/s/cm2/sr (x108). See also Fig. EV1D,E for images and quantification of lymphatic vessels in lymph nodes.