Neoadjuvant administration of Semliki Forest virus expressing interleukin-12 combined with attenuated Salmonella eradicates breast cancer metastasis and achieves long-term survival in immunocompetent mice.
BACKGROUND
Metastatic breast cancer is a major cause of death among women worldwide; therefore efficient therapeutic strategies are extremely needed. In this work we have developed a gene therapy- and bacteria-based combined neoadjuvant approach and evaluated its antitumor effect in a clinically relevant animal model of metastatic breast cancer.


METHODS
2×10(8) particles of a Semliki Forest virus vector expressing interleukin-12 (SFV-IL-12) and/or 2×10(7) units of an aroC (-) Samonella Typhimurium strain (LVR01) were injected into 4T1 tumor nodules orthotopically implanted in mice. Tumors were surgically resected and long-term survival was determined. IL-12 and interferon-γ were quantified by Enzyme-Linked ImmunoSorbent Assay, bacteria was visualized by inmunohistochemistry and the number of lung metastasis was calculated with a clonogenic assay.


RESULTS
SFV-IL-12 and LVR01 timely inoculated and followed by surgical resection of tumors succeeded in complete inhibition of lethal lung metastasis and long-term survival in 90% of treated mice. The combined therapy was markedly synergistic compared to each treatment alone, since SFV-IL-12 monotherapy showed a potent antiangiogenic effect, being able to inhibit tumor growth and extend survival, but could not prevent establishment of distant metastasis and death of tumor-excised animals. On the other hand, LVR01 alone also showed a significant, although limited, antitumor potential, despite its ability to invade breast cancer cells and induce granulocyte recruitment. The efficacy of the combined therapy depended on the order in which both factors were administered; inasmuch the therapeutic effect was only observed when SFV-IL-12 was administered previous to LVR01, whereas administration of LVR01 before SFV-IL-12 had negligible antitumor activity. Moreover, pre-treatment with LVR01 seemed to suppress SFV-IL-12 antiangiogenic effects associated to lower IL-12 expression in this group. Re-challenged mice were unable to reject a second 4T1 tumor; however 100% of them could be totally cured by applying the same neoadjuvant combined regimen. To our knowledge, these are the most encouraging results obtained to date in a post-operatory setting using the highly aggressive 4T1 animal model.


CONCLUSIONS
SFV-IL-12-based gene therapy combined with Salmonella LVR01 neoadjuvant administration has a synergic antitumor effect and may be a promising therapeutic option to prevent and/or eradicate pre-operatory metastasis in locally advanced breast cancer.
Department of Biotechnology, Instituto de Higiene, Facultad de Medicina, Universidad de la República, (UdelaR), Av. A. Navarro 3051, 11600, Montevideo, Uruguay. mgkramer@higiene.edu.uy.
M Gabriela
MG
Kramer
Department of Biotechnology, Instituto de Higiene, Facultad de Medicina, Universidad de la República, (UdelaR), Av. A. Navarro 3051, 11600, Montevideo, Uruguay. mmasner@gmail.com.
Martín
M
Masner
Division Gene Therapy, Center for Applied Medical Research, University of Navarra, Av. Pio XII 55, 31008, Pamplona, Spain. erkudenc@unav.es.|IdiSNA, Navarra Institute for Health Research, c/Irunlarrea 3, 31008, Pamplona, Spain. erkudenc@unav.es.
Erkuden
E
Casales
Department of Biotechnology, Instituto de Higiene, Facultad de Medicina, Universidad de la República, (UdelaR), Av. A. Navarro 3051, 11600, Montevideo, Uruguay. mmoreno@higiene.edu.uy.
María
M
Moreno
Division Gene Therapy, Center for Applied Medical Research, University of Navarra, Av. Pio XII 55, 31008, Pamplona, Spain. csmerdou@unav.es.|IdiSNA, Navarra Institute for Health Research, c/Irunlarrea 3, 31008, Pamplona, Spain. csmerdou@unav.es.
Cristian
C
Smerdou
Department of Biotechnology, Instituto de Higiene, Facultad de Medicina, Universidad de la República, (UdelaR), Av. A. Navarro 3051, 11600, Montevideo, Uruguay. jachabal@higiene.edu.uy.
José A
JA
Chabalgoity
D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D045744:Cell Line, Tumor; D003131:Combined Modality Therapy; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology*; D015316:Genetic Therapy; D005822:Genetic Vectors; D007121:Immunocompetence; D007150:Immunohistochemistry; D007371:Interferon-gamma / Q000097:blood; D018664:Interleukin-12 / Q000097:blood / Q000235:genetics*; D008175:Lung Neoplasms / Q000235:genetics / Q000556:secondary / Q000628:therapy*; D051379:Mice; D008807:Mice, Inbred BALB C; D020360:Neoadjuvant Therapy; D009368:Neoplasm Transplantation; D010446:Peptide Fragments / Q000097:blood; D012486:Salmonella typhimurium / Q000235:genetics*; D012672:Semliki forest virus / Q000235:genetics*; D015996:Survival Rate
D010446:Peptide Fragments; C069261:interferon gamma (1-39); D018664:Interleukin-12; D007371:Interferon-gamma