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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

Combined neoadjuvant antitumor effect of SFV-IL-12 and LVR01. One dose of SFV-IL-12 or SFV-LacZ (2×108 vp in 50 μl PBS), or LVR01 (2×107 cfu in 50 μl PBS), or 50 μl PBS was i.t. injected at days 10 and/or 13 after 4 T1 cells inoculation. In most groups, treated tumors were surgically removed at day 16 (S-16). a Schematic representation of the therapeutic protocol for the combinations relevant to this study. b Kaplan–Meier plot shows the survival rate of the indicated groups of mice. The total number of animals included in the SFV-IL-12 + LVR01 + S-16 group was 31 (consecutive experiments) and the 28 survivor mice were employed in the re-challenge study presented in Fig. 8. For the rest of the groups n = 8-10. c Tumor size was measured before surgery (day 16) and mean + SD tumor volumes were calculated for each group. d Representative images of treated tumors excised 16 days after 4 T1 cells inoculation. p< 0.05 (*); p< 0.01 (**); p< 0.001 (***)
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Fig6: Combined neoadjuvant antitumor effect of SFV-IL-12 and LVR01. One dose of SFV-IL-12 or SFV-LacZ (2×108 vp in 50 μl PBS), or LVR01 (2×107 cfu in 50 μl PBS), or 50 μl PBS was i.t. injected at days 10 and/or 13 after 4 T1 cells inoculation. In most groups, treated tumors were surgically removed at day 16 (S-16). a Schematic representation of the therapeutic protocol for the combinations relevant to this study. b Kaplan–Meier plot shows the survival rate of the indicated groups of mice. The total number of animals included in the SFV-IL-12 + LVR01 + S-16 group was 31 (consecutive experiments) and the 28 survivor mice were employed in the re-challenge study presented in Fig. 8. For the rest of the groups n = 8-10. c Tumor size was measured before surgery (day 16) and mean + SD tumor volumes were calculated for each group. d Representative images of treated tumors excised 16 days after 4 T1 cells inoculation. p< 0.05 (*); p< 0.01 (**); p< 0.001 (***)

Mentions: On the other hand, when SFV-IL-12 (day 10) was combined with LVR01 administration (day 13) followed by surgical removal of the treated tumor (day 16, Fig. 6a), a clear synergy in the antitumor action was observed, evidenced by 90 % long-term survival without metastasis. However, if treated tumors were not resected, all mice died, indicating that this combined therapy only works in neoadjuvancy (Fig. 6b). Interestingly, we found that the order of the injected factors dramatically affected the antitumor response, since the administration of LVR01 prior to SFV-IL-12 did not show any synergy and all animals died at similar rate than control groups (Fig. 6a and b). It is worth to note, that at the time of surgery, tumors receiving only LVR01 or the ineffective LVR01 + SFV-IL-12 combination showed a bigger size compared to SFV-IL-12 alone or SFV-IL-12 + LVR01 treated animals (Fig. 6c). In addition, inhibition of vasculature was observed with effective SFV-IL-12 + LVR01, but not with the LVR01 + SFV-IL-12 combination (Fig. 6d). Moreover, we found that IL-12 expression was significantly reduced in mice receiving LVR01 prior to SFV-IL-12 (day 14, Fig. 7a), while a higher amount of IFN-γ (a main mediator of IL-12 activity) was induced earlier in the effective combination (day 11, Fig. 7b), indicating that the right levels and dynamics of both cytokines expression may be associated with the curative effect of the combined therapy. In this regard, we also observed a significant increase of CD8+ and CD4+ T cells in draining lymph nodes isolated from the effective LVR01 + SFV-IL-12 combined group (see Additional file 2). Overall, these data demonstrate that the synergic neoadjuvant action of SFV-IL-12 and Salmonella LVR01 prevent and eradicate breast cancer metastasis that could be disseminated in the organism before surgical removal of the tumors. Our observations point out to the importance of reducing angiogenesis and inducing an efficient immune response at early stages of the disease.Fig. 6


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)

Combined neoadjuvant antitumor effect of SFV-IL-12 and LVR01. One dose of SFV-IL-12 or SFV-LacZ (2×108 vp in 50 μl PBS), or LVR01 (2×107 cfu in 50 μl PBS), or 50 μl PBS was i.t. injected at days 10 and/or 13 after 4 T1 cells inoculation. In most groups, treated tumors were surgically removed at day 16 (S-16). a Schematic representation of the therapeutic protocol for the combinations relevant to this study. b Kaplan–Meier plot shows the survival rate of the indicated groups of mice. The total number of animals included in the SFV-IL-12 + LVR01 + S-16 group was 31 (consecutive experiments) and the 28 survivor mice were employed in the re-challenge study presented in Fig. 8. For the rest of the groups n = 8-10. c Tumor size was measured before surgery (day 16) and mean + SD tumor volumes were calculated for each group. d Representative images of treated tumors excised 16 days after 4 T1 cells inoculation. p< 0.05 (*); p< 0.01 (**); p< 0.001 (***)
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Related In: Results  -  Collection

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Fig6: Combined neoadjuvant antitumor effect of SFV-IL-12 and LVR01. One dose of SFV-IL-12 or SFV-LacZ (2×108 vp in 50 μl PBS), or LVR01 (2×107 cfu in 50 μl PBS), or 50 μl PBS was i.t. injected at days 10 and/or 13 after 4 T1 cells inoculation. In most groups, treated tumors were surgically removed at day 16 (S-16). a Schematic representation of the therapeutic protocol for the combinations relevant to this study. b Kaplan–Meier plot shows the survival rate of the indicated groups of mice. The total number of animals included in the SFV-IL-12 + LVR01 + S-16 group was 31 (consecutive experiments) and the 28 survivor mice were employed in the re-challenge study presented in Fig. 8. For the rest of the groups n = 8-10. c Tumor size was measured before surgery (day 16) and mean + SD tumor volumes were calculated for each group. d Representative images of treated tumors excised 16 days after 4 T1 cells inoculation. p< 0.05 (*); p< 0.01 (**); p< 0.001 (***)
Mentions: On the other hand, when SFV-IL-12 (day 10) was combined with LVR01 administration (day 13) followed by surgical removal of the treated tumor (day 16, Fig. 6a), a clear synergy in the antitumor action was observed, evidenced by 90 % long-term survival without metastasis. However, if treated tumors were not resected, all mice died, indicating that this combined therapy only works in neoadjuvancy (Fig. 6b). Interestingly, we found that the order of the injected factors dramatically affected the antitumor response, since the administration of LVR01 prior to SFV-IL-12 did not show any synergy and all animals died at similar rate than control groups (Fig. 6a and b). It is worth to note, that at the time of surgery, tumors receiving only LVR01 or the ineffective LVR01 + SFV-IL-12 combination showed a bigger size compared to SFV-IL-12 alone or SFV-IL-12 + LVR01 treated animals (Fig. 6c). In addition, inhibition of vasculature was observed with effective SFV-IL-12 + LVR01, but not with the LVR01 + SFV-IL-12 combination (Fig. 6d). Moreover, we found that IL-12 expression was significantly reduced in mice receiving LVR01 prior to SFV-IL-12 (day 14, Fig. 7a), while a higher amount of IFN-γ (a main mediator of IL-12 activity) was induced earlier in the effective combination (day 11, Fig. 7b), indicating that the right levels and dynamics of both cytokines expression may be associated with the curative effect of the combined therapy. In this regard, we also observed a significant increase of CD8+ and CD4+ T cells in draining lymph nodes isolated from the effective LVR01 + SFV-IL-12 combined group (see Additional file 2). Overall, these data demonstrate that the synergic neoadjuvant action of SFV-IL-12 and Salmonella LVR01 prevent and eradicate breast cancer metastasis that could be disseminated in the organism before surgical removal of the tumors. Our observations point out to the importance of reducing angiogenesis and inducing an efficient immune response at early stages of the disease.Fig. 6

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