LTX-401 induces the release of danger signals.

<p>(A) Release of HMGB1 into the supernatant of LTX-401-treated cells as determined with Western blot. Translocation of the nuclear protein HMGB1 from the cell lysate (L) to the culture supernatant (S) was evident after 30 minutes of treatment with LTX-401 (108 μM), and the translocation was absolute after 90 minutes of incubation. Control cells showed no translocation after 240 minutes. Experiment was conducted thrice, and this figure is a digital image of a representative blot; (B) B16F1 cells were treated with LTX-401 (108 μM) for different time points (30, 60, 90, 120 and 240 minutes) before determining the amount of cytochrome <i>c</i> in supernatants using an ELISA assay. The results are presented as mean +/- SEM; (C) B16F1 cells were treated with LTX-401 (54 μM) for designated time points (10, 30, 60, 90 and 120 min). The quantification of ATP level was performed by luciferase bioluminescence, and the results are presented as mean +/- SEM.</p

LTX-401 treatment induced ultrastructural changes with vacuolization.

<p>Representative TEM micrographs of B16F1 cells treated with LTX-401 (108 μM). Untreated control cells (A and B) were kept in a serum-free RPMI 1640 only until the experimental endpoint (60 minutes), and compared with cells treated for both 5 min (C and D) and 60 min (E and F); scale bars = 10 μm for A, C, E, 5 μm for B, D, F.</p

LTX-401 treatment induced loss of lysosomal integrity.

<p>(A) B16F1 cells were treated with LTX-401 (27 μM) for 60 minutes before determining the signal strength from LysoTracker (DND-26) using flow cytometry. The figure is representative for one of three conducted experiments; (B) B16F1 cells were treated with LTX-401 (27 μM) for 60 minutes, and the signal from LysoTracker was imaged using confocal microscopy (z-stack, maximum intensity projection).</p

Tumor-treatment schedule.

<p>Tumor-treatment schedule.</p

Intratumoral treatment induced complete regression in B16F1 melanomas.

<p>(A) The intratumoral treatment of murine melanoma induced a rapid and complete regression in 9/11 animals, and offered long-term protection against a rechallenge in the majority of the cured mice. Palpable B16F1 tumors were injected with either a sterile 0.9% NaCl (vehicle controls) or 5 mg/ml LTX-401 once a day for three consecutive days. P<0.0001 (Mantel-Cox test); (B) Infiltrating CD3+ lymphocytes were detected in tumors following one single injection of LTX-401 (7 days post-injection) in a murine experimental animal model.</p