Thierry Marysael

A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death

Surface‐exposed calreticulin (ecto‐CRT) and secreted ATP are crucial damage‐associated molecular patterns (DAMPs) for immunogenic apoptosis. Inducers of immunogenic apoptosis rely on an endoplasmic reticulum (ER)‐based (reactive oxygen species (ROS)‐regulated) pathway for ecto‐CRT induction, but the ATP secretion pathway is unknown. We found that after photodynamic therapy (PDT), which generates ROS‐mediated ER stress, dying cancer cells undergo immunogenic apoptosis characterized by phenotypic maturation (CD80high, CD83high, CD86high, MHC‐IIhigh) and functional stimulation (NOhigh, IL‐10absent, IL‐1βhigh) of dendritic cells as well as induction of a protective antitumour immune response. Intriguingly, early after PDT the cancer cells displayed ecto‐CRT and secreted ATP before exhibiting biochemical signatures of apoptosis, through overlapping PERK‐orchestrated pathways that require a functional secretory pathway and phosphoinositide 3‐kinase (PI3K)‐mediated plasma membrane/extracellular trafficking. Interestingly, eIF2α phosphorylation and caspase‐8 signalling are dispensable for this ecto‐CRT exposure. We also identified LRP1/CD91 as the surface docking site for ecto‐CRT and found that depletion of PERK, PI3K p110α and LRP1 but not caspase‐8 reduced the immunogenicity of the cancer cells. These results unravel a novel PERK‐dependent subroutine for the early and simultaneous emission of two critical DAMPs following ROS‐mediated ER stress.

A Dual-targeting Anticancer Approach: Soil and Seed Principle

PURPOSE To test the hypothesis that targeting the microenvironment (soil) may effectively kill cancer cells (seeds) through a small-molecular weight sequential dual-targeting theragnostic strategy, or dual-targeting approach. MATERIALS AND METHODS With approval from the institutional animal care and use committee, 24 rats were implanted with 48 liver rhabdomyosarcomas (R1). First, the vascular-disrupting agent combretastatin A4 phosphate (CA4P) was injected at a dose of 10 mg/kg to cause tumor necrosis, which became a secondary target. Then, the necrosis-avid agent hypericin was radiolabeled with iodine 131 to form (131)I-hypericin, which was injected at 300 MBq/kg 24 hours after injection of CA4P. Both molecules have small molecular weight, are naturally or synthetically derivable, are intravenously injectable, and are of unique targetablities. The tumor response in the dual-targeting group was compared with that in vehicle-control and single-targeting (CA4P or (131)I-hypericin) groups with in vivo magnetic resonance imaging and scintigrams and ex vivo gamma counting, autoradiography, and histologic analysis. Tumor volumes, tumor doubling time (TDT), and radiobiodistribution were analyzed with statistical software. P values below .05 were considered to indicate a significant difference. RESULTS Eight days after treatment, the tumor volume of rhabdomyosarcoma in the vehicle-control group was double that in both single-targeting groups (P < .001) and was five times that in the dual-targeting group (P < .0001), without treatment-related animal death. The TDT was significantly longer in the dual-targeting group (P < .0001). Necrosis appeared as hot spots on scintigrams, corresponding to 3.13% of the injected dose of (131)I-hypericin per gram of tissue (interquartile range, 2.92%-3.97%) and a target-to-liver ratio of 20. The dose was estimated to be 100 times the cumulative dose of 50 Gy needed for radiotherapeutic response. Thus, accumulated (131)I-hypericin from CA4P-induced necrosis killed residual cancer cells with ionizing radiation and inhibited tumor regrowth. CONCLUSION This dual-targeting approach may be a simple and workable solution for cancer treatment and deserves further exploitation.

Radiolabeled iodohypericin as tumor necrosis avid tracer: diagnostic and therapeutic potential.

It is estimated that 30–80% of solid tumor mass represents necrotic tissue that consists out of a significant number of dead and dying cells. The fact that these necrotic zones are restricted to dysplastic and malignant tissue and are rarely present in normal tissue makes necrosis an interesting target both for cancer diagnosis and therapy. In this study, the avidity of hypericin, [123I]iodohypericin and [131I]iodohypericin to tumor necrosis was explored for both diagnosis and therapy of experimental malignancies. The intratumoral distribution in RIF‐1 tumors was investigated by means of fluorescence microscopy (hypericin) and autoradiography ([123I]iodohypericin). Results show high uptake of the tracers in necrosis at 24 hr, lasting for up to 72 hr p.i. Ratios of activity of [123I]iodohypericin in necrotic tissue over viable tumor reached up to 19.63 ± 4.66, correlating with 9.20% ID/g in necrosis. Nude mice bearing RIF‐1 tumors that received three injections of 300 μCi over a 3‐week treatment period showed stabilization in tumor growth for 5 days, as measured by caliper and micro‐positron emission tomography using [18F]fluorodeoxyglucose. Based on these results, we suggest the potentials of radiolabeled hypericin (1) in diagnostic aspects including prognosis or staging assessment of bulky necrotic cancers, monitoring of treatments and therapeutic follow‐up; and (2) in cancer treatment based on tumor necrosis. In conclusion, we showed that hypericin radiolabeled with iodine is a necrosis avid tracer that can be used both as a tumor diagnostic and therapeutic.