Limits...
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 SFV-IL-12 in 4T1 tumor bearing mice. Two doses of SFV-LacZ or SFV-IL-12 (2×108 vp in 50 μl PBS) were i.t. injected at days 10 and 13 after 4T1 cells inoculation. An equivalent volume of PBS was administered to control animals a Tumor size progression was measured every 3-6 days and mean tumor volumes were calculated for each group (n = 6). b Kaplan–Meier plot shows the survival rate of the same mice. c Pictures of representative tumors 21 days after 4T1 cells inoculation showing the external aspect (upper panel) and interior tumor region (lower panel). A reduced volume and vasculature was observed in treated mice compared to control mice. d An additional group of treated and control animals were sacrificed 35 days after tumor cells inoculation and 4T1 lung metastases were quantified as described in Methods. Non-significant (n.s), p< 0.01 (**); p< 0.001 (***)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Antitumor effect of SFV-IL-12 in 4T1 tumor bearing mice. Two doses of SFV-LacZ or SFV-IL-12 (2×108 vp in 50 μl PBS) were i.t. injected at days 10 and 13 after 4T1 cells inoculation. An equivalent volume of PBS was administered to control animals a Tumor size progression was measured every 3-6 days and mean tumor volumes were calculated for each group (n = 6). b Kaplan–Meier plot shows the survival rate of the same mice. c Pictures of representative tumors 21 days after 4T1 cells inoculation showing the external aspect (upper panel) and interior tumor region (lower panel). A reduced volume and vasculature was observed in treated mice compared to control mice. d An additional group of treated and control animals were sacrificed 35 days after tumor cells inoculation and 4T1 lung metastases were quantified as described in Methods. Non-significant (n.s), p< 0.01 (**); p< 0.001 (***)

Mentions: We and other groups have shown that the amount of IL-12 expressed at the tumor site using different viral vectors is critical for tumor regression in a number of cancer models [14–16, 18, 28, 32]. In order to evaluate the therapeutic potential of SFV-IL-12 against breast cancer, we took into consideration all these previous experiences, and injected a relatively high dose of SFV-IL-12 (2×108 vp) at two early stages of the disease: the first dose was given at day 10 and the second dose was given at day 13 after 4T1 implantation (Fig. 2). Tumor growth was periodically measured and animals were monitored for survival outcome. Similarly, control groups received two i.t. doses of an SFV vector expressing β-galactosidase (SFV-LacZ) or PBS. As shown in Fig. 2a, treatment with SFV-IL-12 caused a significant inhibition of tumor growth prolonged until days 20-25. At this stage, control animals showed large ulcerated and irrigated tumors, whereas SFV-IL-12 treated tumors were small and whitish, due to a visible reduction of vasculature (Fig. 2c). These data are consistent with the described antiangiogenic effect of IL-12 in 4T1 tumors and in other IL-12-treated tumor models [18, 32–35] indicating the in vivo activity of this cytokine. The survival of SFV-IL-12 injected animals was significantly extended compared to the SFV-LacZ and PBS control groups, although no animals showed long-term survival (Fig. 2c). Moreover, a significant lower number of lung metastasis was observed at day 35 in SFV-IL-12 treated mice (Fig. 2d), suggesting that their extended survival rate may be a consequence of the reduced metastatic burden at a time point when untreated animals start to die. Together, these results show that SFV-IL-12 induce a marked local antiangiogenic effect in implanted 4T1 tumors, which may contribute to inhibit primary tumor growth for a limited period of time, as well as to reduce dissemination of metastasis in the organism, prolonging consequently animal survival. However, SFV-IL-12 monotherapy, at least at the employed dose regimen, was insufficient to achieve complete tumor remission and to prevent the spread of 4T1 lung metastasis.Fig. 2


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 SFV-IL-12 in 4T1 tumor bearing mice. Two doses of SFV-LacZ or SFV-IL-12 (2×108 vp in 50 μl PBS) were i.t. injected at days 10 and 13 after 4T1 cells inoculation. An equivalent volume of PBS was administered to control animals a Tumor size progression was measured every 3-6 days and mean tumor volumes were calculated for each group (n = 6). b Kaplan–Meier plot shows the survival rate of the same mice. c Pictures of representative tumors 21 days after 4T1 cells inoculation showing the external aspect (upper panel) and interior tumor region (lower panel). A reduced volume and vasculature was observed in treated mice compared to control mice. d An additional group of treated and control animals were sacrificed 35 days after tumor cells inoculation and 4T1 lung metastases were quantified as described in Methods. Non-significant (n.s), p< 0.01 (**); p< 0.001 (***)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Antitumor effect of SFV-IL-12 in 4T1 tumor bearing mice. Two doses of SFV-LacZ or SFV-IL-12 (2×108 vp in 50 μl PBS) were i.t. injected at days 10 and 13 after 4T1 cells inoculation. An equivalent volume of PBS was administered to control animals a Tumor size progression was measured every 3-6 days and mean tumor volumes were calculated for each group (n = 6). b Kaplan–Meier plot shows the survival rate of the same mice. c Pictures of representative tumors 21 days after 4T1 cells inoculation showing the external aspect (upper panel) and interior tumor region (lower panel). A reduced volume and vasculature was observed in treated mice compared to control mice. d An additional group of treated and control animals were sacrificed 35 days after tumor cells inoculation and 4T1 lung metastases were quantified as described in Methods. Non-significant (n.s), p< 0.01 (**); p< 0.001 (***)
Mentions: We and other groups have shown that the amount of IL-12 expressed at the tumor site using different viral vectors is critical for tumor regression in a number of cancer models [14–16, 18, 28, 32]. In order to evaluate the therapeutic potential of SFV-IL-12 against breast cancer, we took into consideration all these previous experiences, and injected a relatively high dose of SFV-IL-12 (2×108 vp) at two early stages of the disease: the first dose was given at day 10 and the second dose was given at day 13 after 4T1 implantation (Fig. 2). Tumor growth was periodically measured and animals were monitored for survival outcome. Similarly, control groups received two i.t. doses of an SFV vector expressing β-galactosidase (SFV-LacZ) or PBS. As shown in Fig. 2a, treatment with SFV-IL-12 caused a significant inhibition of tumor growth prolonged until days 20-25. At this stage, control animals showed large ulcerated and irrigated tumors, whereas SFV-IL-12 treated tumors were small and whitish, due to a visible reduction of vasculature (Fig. 2c). These data are consistent with the described antiangiogenic effect of IL-12 in 4T1 tumors and in other IL-12-treated tumor models [18, 32–35] indicating the in vivo activity of this cytokine. The survival of SFV-IL-12 injected animals was significantly extended compared to the SFV-LacZ and PBS control groups, although no animals showed long-term survival (Fig. 2c). Moreover, a significant lower number of lung metastasis was observed at day 35 in SFV-IL-12 treated mice (Fig. 2d), suggesting that their extended survival rate may be a consequence of the reduced metastatic burden at a time point when untreated animals start to die. Together, these results show that SFV-IL-12 induce a marked local antiangiogenic effect in implanted 4T1 tumors, which may contribute to inhibit primary tumor growth for a limited period of time, as well as to reduce dissemination of metastasis in the organism, prolonging consequently animal survival. However, SFV-IL-12 monotherapy, at least at the employed dose regimen, was insufficient to achieve complete tumor remission and to prevent the spread of 4T1 lung metastasis.Fig. 2

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