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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.

Kramer MG, Masner M, Casales E, Moreno M, Smerdou C, Chabalgoity JA - BMC Cancer (2015)

Bottom Line: Moreover, pre-treatment with LVR01 seemed to suppress SFV-IL-12 antiangiogenic effects associated to lower IL-12 expression in this group.To our knowledge, these are the most encouraging results obtained to date in a post-operatory setting using the highly aggressive 4T1 animal model.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

Antitumor effect of LVR01 in 4T1 tumor bearing and tumor-excised animals. a Schematic representation of the therapeutic protocol performed in this study. Two doses of LVR01 (2×107 cfu in 50 μl PBS) or 50 μl PBS were i.t. injected at days 10 and 13 after 4T1 cells inoculation. In the indicated groups, the treated tumors were surgically removed at day 16 (S-16). b Kaplan–Meier plot shows the survival rate of treated and control mice (n = 5-9). Non-significant (n.s); p< 0.001 (***)
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Fig4: Antitumor effect of LVR01 in 4T1 tumor bearing and tumor-excised animals. a Schematic representation of the therapeutic protocol performed in this study. Two doses of LVR01 (2×107 cfu in 50 μl PBS) or 50 μl PBS were i.t. injected at days 10 and 13 after 4T1 cells inoculation. In the indicated groups, the treated tumors were surgically removed at day 16 (S-16). b Kaplan–Meier plot shows the survival rate of treated and control mice (n = 5-9). Non-significant (n.s); p< 0.001 (***)

Mentions: We hypothesized that live attenuated Salmonella could be a good complement for IL-12-based antitumor therapy due to the intrinsic immune-stimulatory properties and the better antitumor responses observed when administering bacteria carrying plasmids that express pro-inflammatory cytokines (reviewed in [41]). Additionally, a recent related study of our group has shown that i.t. administrations of Salmonella LVR01 generate a considerable dose-dependent antitumor effect in a mouse model of B-cell lymphoma [42]. Despite this result, when two doses of LVR01 were inoculated into primary 4T1 tumors, no survival improvement was seen in tumor-bearing animals (Fig. 4). Surgical removal of the tumor after LVR01 inoculations led to a significant death delay, although no animals survived for the long-term (Fig. 4). In this case, all mice died with large macroscopic lung and disseminated metastasis, indicating that LVR01 alone was insufficient to control the 4T1 metastatic process. Nevertheless, the inoculated bacteria were able to invade tumor cells and induce local microscopic tissue distortion and granulocyte infiltration (Fig. 5), persisting inside the tumor mass for several days (see Additional file 1).Fig. 4


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.

Kramer MG, Masner M, Casales E, Moreno M, Smerdou C, Chabalgoity JA - BMC Cancer (2015)

Antitumor effect of LVR01 in 4T1 tumor bearing and tumor-excised animals. a Schematic representation of the therapeutic protocol performed in this study. Two doses of LVR01 (2×107 cfu in 50 μl PBS) or 50 μl PBS were i.t. injected at days 10 and 13 after 4T1 cells inoculation. In the indicated groups, the treated tumors were surgically removed at day 16 (S-16). b Kaplan–Meier plot shows the survival rate of treated and control mice (n = 5-9). Non-significant (n.s); p< 0.001 (***)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4562361&req=5

Fig4: Antitumor effect of LVR01 in 4T1 tumor bearing and tumor-excised animals. a Schematic representation of the therapeutic protocol performed in this study. Two doses of LVR01 (2×107 cfu in 50 μl PBS) or 50 μl PBS were i.t. injected at days 10 and 13 after 4T1 cells inoculation. In the indicated groups, the treated tumors were surgically removed at day 16 (S-16). b Kaplan–Meier plot shows the survival rate of treated and control mice (n = 5-9). Non-significant (n.s); p< 0.001 (***)
Mentions: We hypothesized that live attenuated Salmonella could be a good complement for IL-12-based antitumor therapy due to the intrinsic immune-stimulatory properties and the better antitumor responses observed when administering bacteria carrying plasmids that express pro-inflammatory cytokines (reviewed in [41]). Additionally, a recent related study of our group has shown that i.t. administrations of Salmonella LVR01 generate a considerable dose-dependent antitumor effect in a mouse model of B-cell lymphoma [42]. Despite this result, when two doses of LVR01 were inoculated into primary 4T1 tumors, no survival improvement was seen in tumor-bearing animals (Fig. 4). Surgical removal of the tumor after LVR01 inoculations led to a significant death delay, although no animals survived for the long-term (Fig. 4). In this case, all mice died with large macroscopic lung and disseminated metastasis, indicating that LVR01 alone was insufficient to control the 4T1 metastatic process. Nevertheless, the inoculated bacteria were able to invade tumor cells and induce local microscopic tissue distortion and granulocyte infiltration (Fig. 5), persisting inside the tumor mass for several days (see Additional file 1).Fig. 4

Bottom Line: Moreover, pre-treatment with LVR01 seemed to suppress SFV-IL-12 antiangiogenic effects associated to lower IL-12 expression in this group.To our knowledge, these are the most encouraging results obtained to date in a post-operatory setting using the highly aggressive 4T1 animal model.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus