Évaluation in-vitro et in-vivo de la thérapie photodynamique associée à l’acide 5-Aminolevulinique sur le carcinome hépatocellulaire humain : impact sur la régulation du système immunitaire

In recent years, there has been a steady increase in patients with Hepatocellular Carcinoma (HCC) mainly in the north of France. Despite a large number of therapeutic options, HCC has a high cancer-related mortality. Hence there is a need to develop novel therapeutic strategies for HCC treatment which could augment the efficacy of existing options and at the same time provide longer tumor free survival for the patients.Photodynamic Therapy (PDT) is a local anti-tumoral modality relying on systemic administration of a non-toxic dye, called Photosensitizer (PS), and subsequent illumination by light of appropriate wavelength and energy to elicit tumor cell death. Initially used for treatment of dematological ailments, PDT has shown motivating results as a possible anti-tumoral therapy in last few decades.Using 5-Aminolevulinic acid (5-ALA), the US-FDA approved oral pro-drug of Protoporphyrin IX, the actual PS, we investigated the in-vitro impact of PDT over HCC cell lines, along with patient tumoral hepatocytes, healthy donor liver myofibroblasts and human immune system. At last, we validated our results on an in-vivo model of HCC. The overall aim is to establish 5-ALA PDT as an adjuvant to partial hepatectomy through intra-operative procedures.When the HCC cell lines were treated with varying 5-ALA concentration and light doses, we observed a dose-dependent decrease of cellular viability. We also observed varying sensitivity for 5-ALA PDT between the three HCC cell lines used (HuH7, Hep3B, HepG2), highlighting the role of p53 for underlying cell death mechanism. Thereafter, an IC50 dose for each of the cell line was obtained which induced decreased cellular proliferation and increased cell death with respect to non-treated control. These results confirm that 5-ALA PDT induce cell death to the HCC cell lines.Furthermore, in order to validate the ex vivo efficacy of 5- ALA-PDT we studied the impact of this therapy on tumor hepatocytes from four patients with HCC, showing that cell viability decreased continuously up to 4 times, from day 3 after illumination, compared to the untreated control. In addition, we have studied the safety of 5-ALA-PDT in liver myofibroblasts from healthy donors. Our results show that 5-ALA-PDT does not induce any change in terms of viability and proliferation of healthy myofibroblasts and that the levels of expression of the mRNA of the markers of fibrosis, namely collagen-1, HSP47, αSMA, TIMP1 and MMP2 are not modified. In addition, no modulation of the levels of collagen secretion was observed.Most interestingly, when we cultured human activated Peripheral Blood Mononuclear Cells (PBMCs), with conditioned media obtained from 5-ALA PDT treated HCC cell lines, we observed increased cellular proliferation with respect to non-treated control. This highlights clonogenic expansion and possible anti-tumor immune response activation by 5-ALA PDT. This conditioned media have further inhibit the proliferation of respective cancer cell lines, reflecting secretion of anti-proliferation factors by cells due to PDT.At last, our in-vivo results demonstrated that PpIX has higher accumulation in the tumor with respect to non-tumoral tissues and a decrease in the rate of tumor growth for humanized SCID mice treated by 5-ALA PDT, with respect to Non-Treated control group.To date, no other study has evaluated the impact of 5-ALA PDT on primary patient and healthy donor samples. We are also the first to assess the impact of 5-ALA PDT treated conditioned media on PBMCs and cancer cell line proliferation. Our study strengthens the notion of the application of intra-operative 5-ALA PDT for treatment of HCC patients. Further studies will aim to understand the role of p53 in the cell death mechanism, identify the immune population activated by PDT, evaluate the efficacy of the therapy in a humanized SCID mice model and establish a standard treatment protocol to be utilized in a clinical set-up.