Methods of generating populations of tumour-infiltrating T cells
A tumor-infiltrating, tumor-cell technique for the generation of T-cell populations of scientific, diagnostic or therapeutic relevance
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Embodiment 1
[0214] LTX-315 disintegrates the plasma membrane of osteosarcoma cells
[0215] 1 Materials and methods
[0216] Plasma membrane disintegration was performed as described in FORVEILLE S. et al., Cell Cycle 2015, 14:3506-12.
[0217] 1.1 Chemicals and cell culture
[0218] Media and supplements for cell culture were obtained from Gibco-Life Technologies (Carlsbad, CA, USA), except from Lytix Biopharma ( Norway) provided chemicals other than LTX-315 (K-K-W-W-K-K-W-Dip-K-NH2) from Sigma-Aldrich (St. Louis, MO, USA); plastics were from Greiner Bio-One (Monroe, CA, USA).
[0219] Human osteosarcoma U2OS cells were cultured in Glutamax-containing DMEM medium supplemented with 10% fetal calf serum (FCS) and 10MM HEPES buffer. At 37 °C in a humidified incubator at 5% CO 2 Cells were grown under atmosphere.
[0220] 1.2 Transmission electron microscopy
[0221] For ultramicrostudies, human osteosarcoma U2OS cells were fixed in 1.6% glutaraldehyde (v / v, dissolved in 0.1M ...
Embodiment 2
[0225] LTX-315 internalizes and interacts with mitochondria
[0226] In this study, we investigated the tumoricidal effect of LTX-315 on human melanoma cells. The peptide is internalized and shown to associate with mitochondria, ultimately leading to lytic cell death. Intratumoral injection of LTX-315 peptide to treat solid tumors through the following two-stage mode of action: the first stage is the collapse of the tumor itself, and the second node is the release of damage-associated molecular pattern molecules (DAMPs) by dying tumor cells , which is capable of inducing subsequent immune protection against relapse and metastasis.
[0227] 1 Materials and methods
[0228] Studies were performed as described in FORVEILLE S. et al., Cell Cycle 2015, 6:34910-23.
[0229] 1.1 Reagents
[0230] LTX-315 and LTX-328 (K-A-Q-Dip-Q-K-Q-A-W-NH 2 ). Respectively from Innovagen (Lund, Sweden) and ( Norway) purchased LTX-315Pacific Blue and LTX-328Pacific Blue.
[0231] 1.2...
Embodiment 3
[0271] LTX-401 induces DAMP release from melanoma cells
[0272] In this study, we examined the ability of the amino acid derivative LTX-401 to induce cell death in cancer cell lines, and to induce regression in a murine melanoma model. In vitro mode of action studies revealed lytic cell death and release of hazard-associated molecular pattern molecules, preceded by massive cytoplasmic vacuolization and destruction of lysosomes in treated cells. Using a murine melanoma model, most animals treated with intratumoral injections of LTX-401 exhibited complete and durable remissions. Taken together, these results suggest the potential of LTX-401 as an immunotherapeutic agent for the treatment of solid tumors.
[0273] 1 Materials and methods
[0274] 1.1 Reagents
[0275] LTX-401 (Mw net = 367.53). The chemical structure is shown in Figure 11 middle.
[0276] 1.2 Cell culture
[0277] JM1, rat hepatocellular carcinoma, HEPG2 and BEL7402, both human hepatocellular ca...
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