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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

Effect of 4T1 breast cancer metastases in animals that underwent tumor surgery. Tumor cells were orthotopically implanted in mice mammary fat pads (day 0). Primary tumors were left without surgery or removed by surgical excision, 10 or sixteen (S-16) days later. a Kaplan–Meier plot shows the survival rate of the indicated groups (n = 8). b Additional tumor-bearing or tumor excised mice were sacrificed at 10, 16 or 35 days after 4T1 cells inoculation, lungs were extracted and processed and the number of lung metastases were quantified as described in Methods. 35 (S-16) refers to a group of mice that underwent surgery at day 16 and were checked for lung metastases 19 days later (i.e. 35 days after 4T1 tumor implantation). c Lungs extracted from a healthy control mouse (Control) and from moribund mice showing numerous metastatic nodules (Metastatic lungs). Non-significant (n.s.)
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Fig1: Effect of 4T1 breast cancer metastases in animals that underwent tumor surgery. Tumor cells were orthotopically implanted in mice mammary fat pads (day 0). Primary tumors were left without surgery or removed by surgical excision, 10 or sixteen (S-16) days later. a Kaplan–Meier plot shows the survival rate of the indicated groups (n = 8). b Additional tumor-bearing or tumor excised mice were sacrificed at 10, 16 or 35 days after 4T1 cells inoculation, lungs were extracted and processed and the number of lung metastases were quantified as described in Methods. 35 (S-16) refers to a group of mice that underwent surgery at day 16 and were checked for lung metastases 19 days later (i.e. 35 days after 4T1 tumor implantation). c Lungs extracted from a healthy control mouse (Control) and from moribund mice showing numerous metastatic nodules (Metastatic lungs). Non-significant (n.s.)

Mentions: Breast cancer aggressiveness and poor prognosis is commonly associated with metastasis occurrence to distant organs, such as lungs, liver and bones. In this regard, it is well known that early resection of primary tumors contributes to control metastasis; however clinical outcome depends on many factors, including growth dynamics and metastatic characteristics of these tumors. In order to correlate the 4T1 breast cancer model with clinical cases of LABC in respect to its metastatic progression, we evaluated the therapeutic impact of primary tumor resection at different time points. Small tumors (4-5 mm diameter) could be homogeneously detected 10 days after orthotopic implantation of 7×104 4T1 cells and by day 16 tumors reached 6–7 mm diameter, still a feasible surgically size. Thus, we defined days 10 and 16 post-4T1 implantation as early and middle-early time points for tumor resection, respectively. Control tumor-bearing mice have a life span of 44 ± 5 days after 4T1 cells implantation (Figs. 1 and 8b). Animals whose tumors were surgically resected at day 10 showed an improved overall survival (with 40 % mice surviving more than 6 months), while 100 % animals operated at day 16 died at the same rate as non-operated mice (Fig. 1a). In agreement with these data, lung micro-metastatic cells were present at day 10 in 40 % of the animals, while the rest were metastasis-free; however, 6 days later (day 16), 100 % animals presented a variable number (10-1300) of lung micro-metastasis (Fig. 1b). Middle-early excised tumor mice and non-operated controls showed comparable life span and a similar amount of established lung metastasis (in the order of 105 4T1 tumor cells/lungs) 35 days after tumor implantation (Fig. 1b) suggesting that both facts are associated. The autopsy of moribund mice revealed in all cases the presence of numerous macroscopic metastatic foci and a massive distortion of both lungs (Fig. 1c), accompanied by severe breathing difficulties at the time of death. These data confirmed the determinant role of the metastatic disease in the survival outcome of the 4T1 model of breast cancer [6]. In addition, non-survivor animals occasionally presented visible metastatic tumor nodules in liver, lymph nodes, spleen and/or heart (not shown). It is important to note, that just a small number of 4T1 metastatic cells (around 10–20, Fig. 1b) are sufficient to progress to a severe lung metastatic disease in the absence of primary tumors, indicating that these tumor cells may be particularly resistant or indifferent to the host immune system in order to persist. Due to the early metastatic dissemination and their growing characteristics, we conclude that the 4T1-tumor model could reflect most aggressive clinical cases of LABC. Moreover, middle-early tumor-excised mice would represent an excellent model of minimal residual disease (MRD) for therapeutic intervention against metastatic disease, since 100 % of animals develop lung metastasis and do not survive in the long-term. Our data also point out to a temporal window between days 10 and 16 (initiation - establishment of the metastatic process) for evaluation of novel neoadjuvant strategies (prior to surgical removal of primary tumors) aimed at preventing and treating lethal MRD.Fig. 1


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)

Effect of 4T1 breast cancer metastases in animals that underwent tumor surgery. Tumor cells were orthotopically implanted in mice mammary fat pads (day 0). Primary tumors were left without surgery or removed by surgical excision, 10 or sixteen (S-16) days later. a Kaplan–Meier plot shows the survival rate of the indicated groups (n = 8). b Additional tumor-bearing or tumor excised mice were sacrificed at 10, 16 or 35 days after 4T1 cells inoculation, lungs were extracted and processed and the number of lung metastases were quantified as described in Methods. 35 (S-16) refers to a group of mice that underwent surgery at day 16 and were checked for lung metastases 19 days later (i.e. 35 days after 4T1 tumor implantation). c Lungs extracted from a healthy control mouse (Control) and from moribund mice showing numerous metastatic nodules (Metastatic lungs). Non-significant (n.s.)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Effect of 4T1 breast cancer metastases in animals that underwent tumor surgery. Tumor cells were orthotopically implanted in mice mammary fat pads (day 0). Primary tumors were left without surgery or removed by surgical excision, 10 or sixteen (S-16) days later. a Kaplan–Meier plot shows the survival rate of the indicated groups (n = 8). b Additional tumor-bearing or tumor excised mice were sacrificed at 10, 16 or 35 days after 4T1 cells inoculation, lungs were extracted and processed and the number of lung metastases were quantified as described in Methods. 35 (S-16) refers to a group of mice that underwent surgery at day 16 and were checked for lung metastases 19 days later (i.e. 35 days after 4T1 tumor implantation). c Lungs extracted from a healthy control mouse (Control) and from moribund mice showing numerous metastatic nodules (Metastatic lungs). Non-significant (n.s.)
Mentions: Breast cancer aggressiveness and poor prognosis is commonly associated with metastasis occurrence to distant organs, such as lungs, liver and bones. In this regard, it is well known that early resection of primary tumors contributes to control metastasis; however clinical outcome depends on many factors, including growth dynamics and metastatic characteristics of these tumors. In order to correlate the 4T1 breast cancer model with clinical cases of LABC in respect to its metastatic progression, we evaluated the therapeutic impact of primary tumor resection at different time points. Small tumors (4-5 mm diameter) could be homogeneously detected 10 days after orthotopic implantation of 7×104 4T1 cells and by day 16 tumors reached 6–7 mm diameter, still a feasible surgically size. Thus, we defined days 10 and 16 post-4T1 implantation as early and middle-early time points for tumor resection, respectively. Control tumor-bearing mice have a life span of 44 ± 5 days after 4T1 cells implantation (Figs. 1 and 8b). Animals whose tumors were surgically resected at day 10 showed an improved overall survival (with 40 % mice surviving more than 6 months), while 100 % animals operated at day 16 died at the same rate as non-operated mice (Fig. 1a). In agreement with these data, lung micro-metastatic cells were present at day 10 in 40 % of the animals, while the rest were metastasis-free; however, 6 days later (day 16), 100 % animals presented a variable number (10-1300) of lung micro-metastasis (Fig. 1b). Middle-early excised tumor mice and non-operated controls showed comparable life span and a similar amount of established lung metastasis (in the order of 105 4T1 tumor cells/lungs) 35 days after tumor implantation (Fig. 1b) suggesting that both facts are associated. The autopsy of moribund mice revealed in all cases the presence of numerous macroscopic metastatic foci and a massive distortion of both lungs (Fig. 1c), accompanied by severe breathing difficulties at the time of death. These data confirmed the determinant role of the metastatic disease in the survival outcome of the 4T1 model of breast cancer [6]. In addition, non-survivor animals occasionally presented visible metastatic tumor nodules in liver, lymph nodes, spleen and/or heart (not shown). It is important to note, that just a small number of 4T1 metastatic cells (around 10–20, Fig. 1b) are sufficient to progress to a severe lung metastatic disease in the absence of primary tumors, indicating that these tumor cells may be particularly resistant or indifferent to the host immune system in order to persist. Due to the early metastatic dissemination and their growing characteristics, we conclude that the 4T1-tumor model could reflect most aggressive clinical cases of LABC. Moreover, middle-early tumor-excised mice would represent an excellent model of minimal residual disease (MRD) for therapeutic intervention against metastatic disease, since 100 % of animals develop lung metastasis and do not survive in the long-term. Our data also point out to a temporal window between days 10 and 16 (initiation - establishment of the metastatic process) for evaluation of novel neoadjuvant strategies (prior to surgical removal of primary tumors) aimed at preventing and treating lethal MRD.Fig. 1

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