Oncotarget. 2015 Jul 10;6(19):17648-60.

Attenuated mutant strain of Salmonella Typhimurium lacking the ZnuABC transporter contrasts tumor growth promoting anti-cancer immune response.

 

Chirullo B1, Ammendola S2, Leonardi L3, Falcini R4, Petrucci P1, Pistoia C1, Vendetti S5, Battistoni A2, Pasquali P1.

  • 1Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome 00161, Italy.
  • 2Department of Biology, University of Rome Tor Vergata, Rome 00133, Italy.
  • 3Università degli Studi di Perugia, Department of Veterinary Medicine, Perugia 06126, Italy.
  • 4Veterinary Clinic, Rieti 02043, Italy.
  • 5Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome 00161, Italy.

 

Abstract

Salmonella Typhimurium has been shown to be highly effective as antitumor agent. The aim of this study was to investigate the tumor targeting efficacy and the mechanism of action of a specific attenuated mutant strain of Salmonella Typhimurium (STM) devoid of the whole operon coding for the high-affinity zinc transporter ZnuABC, which is required for bacterial growth in environments poor in zinc and for conferring full virulence to different Gram-negative pathogens. We showed that STM is able to penetrate and replicate into tumor cells in in vitro and in vivo models. The subcutaneous administration of STM in mammary adenocarcinoma mouse model led to both reduction of tumor growth and increase in life expectancy of STM treated mice. Moreover, investigating the potential mechanism behind the favorable clinical outcomes, we provide evidence that STM stimulates a potent inflammatory response and a specific immune pattern, recruiting a large number of innate and adaptive immune cells capable to contrast the immunosuppressive environment generated by tumors.

KEYWORDS: Antitumor efficacy; attenuated-Salmonella; bacterial therapy; cancer therapy; immune response

PMID: 26158862

 

SUPPLEMENT:

An important chapter in current anti-cancer therapies is the development of viable alternative therapies able to overcome their well-known intrinsic limitations and reduce their considerable costs. In this context, the employment of attenuated bacteria as anti-cancer therapy is a promising approach, already known for more than a century, but with a recent renewed interest due to the ability of the bacteria to show selective replication and preferential accumulation in the tumor zone [1-2].

In this study, we investigated the anti-cancer potential of an attenuated mutant strain of Salmonella enterica serovar Typhimurium devoid of the operon znuABC (STM), coding for the high-affinity zinc transporter, which is important for the bacteria growth in environments poor in zinc and for its virulence [3].

 

Figure 1

Figure 1. STM induces a significant reduction of the tumor masses (A), a significant increase of average life expectancy (B) and decreases of the incidence of metastatic nodules in lung (C) of STM-treated tumor-bearing mice.

 

The results indicated that STM is able to penetrate and replicate into the tumor zone in tumor bearing Balb/c mice, injected subcutaneously with 4T1 murine mammary adenocarcinoma tumor cells, which results in a final mass 9 fold smaller when compared to the control group (fig. 1A), and a statistically significant increase in the average life expectancy of STM-treated mice (Fig. 1B). Moreover, since the 4T1 tumor cells are known to have highly metastatic properties so as to disseminate and develop metastases, we showed that STM is able to remarkably decrease the incidence of lung metastatic nodules in STM-treated tumor bearing mice compared with the untreated control group (fig. 1C).

Next we showed that the STM-treatment induces an important accumulation of bacteria in tumor sites, which induces a potent inflammatory response, recruiting a large number of innate and adaptive immune cells in both spleens and tumor masses. In particular, the STM treatment led to increase the absolute number of cells in spleen by monocytes/macrophages, neutrophils, CD8+ T cells, B220+ cells, and the CD3+ γδ+ T cells. The modifications of the immune population were also confirmed by histopathological analysis on primary and metastatic tumors sampled at 30 dpi, which showed a marked inflammatory response that varied from lymphohistiocytic to neutrophilic/eosinophilic populations in the STM-treated group compared to the predominant lymphocytic population of the untreated group (Fig. 2).

 

Figure 2

Figure 2. Histopathological analysis from primary and metastatic tumors of untreated and STM-treated bearing-tumor mice. Panels A-D: Untreated bearing-tumor group. A) Primary adenocarcinoma. Hematoxylin-eosin, magnification 20x. B) Primary adenocarcinoma. Hematoxylin-eosin, magnification 40x. C) Adenocarcinoma, lung metastasis. Hematoxylin-eosin, magnification 20x. D) Adenocarcinoma, liver metastasis. Hematoxylin-eosin, magnification 10x. Panels E-G: STM treated bearing-tumor group. E) Primary adenocarcinoma. Hematoxylin-eosin, magnification 20x. F) Primary adenocarcinoma. Hematoxylin-eosin, magnification 10x. G) Adenocarcinoma, lung metastasis. Hematoxylin-eosin, magnification 20x. H) Adenocarcinoma, liver metastasis. Hematoxylin-eosin, magnification 20x.

 

Then, looking for the potential mechanism behind the favorable clinical outcomes, we found significantly increased level, per mg of tumor masses, of the Calreticulin (CRT), a protein involved in the Immunogenic Cell Death pathway [4], in STM-treated tumor bearing mice compared with the untreated group. Moreover, a significant increase of IFN-gamma and IL-1 beta production, per mg of tumor masses and spleen, for IFN-gamma, and only in tumor masses for the IL-1 beta, was found in the STM-treated group (Fig. 3). These results together suggest that the anti-tumor effect of STM is the result of a combination of different mechanisms involving a pro-inflammatory environment that can trigger antitumor immunity by facilitating the activation of specific anti-tumor T cell response, resulting in a significant reduction of the tumor masses growth and in an increase of the life expectancy.

 

 

Figure 3

Figure 3. STM treatment induces a release of Calreticulin (CRT) in mice and promotes IFN-γ and IL-1β production in the tumor masses and spleens, 30 days after tumor implantation.

 

References

  1. Coley, W.B. The treatment of malignant tumors by repeated inoculations of erysipelas. With a report of ten original cases (1893). Clin Orthop Relat Res 1991; 262: 3-11.
  2. Hoffman RM. Tumor-seeking Salmonella amino acid auxotrophs. Curr Opin Biotechnol 2011; 22:917-23.
  3. Ammendola S, Pasquali P, Pistoia C, Petrucci P, Petrarca P, Rotilio G, Battistoni A. High-affinity Zn2+ uptake system ZnuABC is required for bacterial zinc homeostasis in intracellular environments and contributes to the virulence of Salmonella enterica. Infect Immun 2007; 75:5867-76.
  4. Kroemer G, Galluzzi L, Kepp O, Zitvogel L. Immunogenic cell death in cancer therapy. Annu Rev Immunol 2013; 31:51-72.

 

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