Direccionament de fàrmacs per a l'eliminació selectiva de cèl·lules canceroses cxcr4+ en models de càncer colorrectal metastàtic

• Autores: Rita Sala Faig
• Directores de la Tesis: Ramon Mangues Bafalluy (dir. tes.), María Virtudes Céspedes Navarro (codir. tes.)
• Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2020
• Idioma: catalán
• ISBN: 9788449098253
• Tribunal Calificador de la Tesis: Diego Arango del Corro (presid.), Pilar Vinardell Martínez-Hidalgo (secret.), Jan Paul Medema (voc.)
• Programa de doctorado: Programa de Doctorado en Bioquímica, Biología Molecular y Biomedicina por la Universidad Autónoma de Barcelona
• Materias:
  • Ciencias de la vida
    • Biología celular
• Enlaces
  • Tesis en acceso abierto en: TDX
• Resumen

  • Colorectal cancer (CRC) remains the third cause of cancer-related mortality in Western countries, being metastases the main cause of death. Despite progress in prevention strategies that decreased CRC incidence and mortality, still nearly a quarter of patients are diagnosed at an advanced metastatic stage, with only a 15% five-year survival rate. Thus, inhibition of metastasis development by targeting cancer stem cells, which are associated with cancer dissemination, will significantly increase the benefits of current cancer therapies. In collaboration with the Nanobiotechnology group from de UAB, we developed self-assembling protein-based nanoparticles targeting the CXCR4 receptor whose overexpression correlates with tumor dissemination, poor survival, and recurrence in CRC patients. Preclinical evaluation of the efficacy and toxicity of the T22-GFP-H6 nanocarrier and its therapeutic derivatives required the use of adequate in vivo disseminated CRC models. For that purpose, we generated subcutaneous and highly metastatic models of CRC that overexpress CXCR4, derived from the SW1417 CRC cell line or the SP5 patient sample. In order to increase the metastatic efficiency of previous CXCR4+ CRC models, we orthotopically implanted luciferase expressing SW1417 cells in the cecum of severe immunodeficient mice. NOD/SCID mice (deficient in T and B cells) presented a low metastatic rate. In contrast, orthotopic microinjection in NSG mice (deficient in T, B and NK cells) replicated the dissemination pattern observed in patients, causing mice death and resulting in a higher number and size of hepatic and pulmonary metastases as compared to NOD/SCID mice.

    In the assessment of nanoparticles’ biodistribution, T22-GFP-H6 achieved a highly selective tumor uptake in a CXCR4+ CRC subcutaneous model, as detected by fluorescent emission (around 70% of the total), while displaying only transient accumulation in non-tumor organs. We demonstrated that the nanocarrier tumor accumulation was CXCR4-dependent because pre-treatment with AMD3100, a CXCR4 antagonist, reduced tumor uptake. Furthermore, tumor accumulation was increased by the functionalization of the nanocarrier with the fusogenic HA2 peptide, which promotes endosomal escape. On the one hand, we observed that the therapeutic nanoconjugate T22-GFP-H6-Aur maintained the nanocarrier’s biodistribution but its antitumoral effect was surprisingly poor. T22-GFP-H6-Aur inhibited only tanscelomic metastasis in a highly metastatic CRC model, while activating a lethal immunogenic response when repeatedly administered in low immunosuppressed mice. As an alternative therapeutic option, we replaced the GFP protein in the nanoparticle by the de-immunized PE24 toxin, to reduce its immunogenicity while promoting a potent and intrinsic cytotoxic activity. The administration of low doses of the T22-PE24-H6 nanotoxin prevented the development of lymphatic and hematogenous metastasis in the highly metastatic CRC model without toxicity. We demonstrated that the T22-PE24-H6 nanotoxin induced cancer cell death through the non-apoptotic pathway, pyroptosis. In conclusion, the use of the T22-PE24-H6 nanotoxin could be a promising strategy to selectively eliminate CXCR4+ CRC stem cells in the absence of systemic toxicity, applicable to chemotherapy-resistant and disseminated CRC associated with the upregulation of CXCR4 and antiapoptotic mechanisms.