Bacterial Therapy of Cancer: Promises, Limitations, and Insights for Future Directions

Abstract

Spontaneous tumors regression has been associated with microbial infection for 100s of years and inspired the use of bacteria for anticancer therapy. Dr. William B. Coley (1862-1936), a bone- sarcoma surgeon, was a pioneer in treating his patients with both live bacterial-based and mixture of heat-killed bacteria known as "Coley's toxins." Unfortunately, Coley was forced to stop his work which interrupted this field for about half a century. Currently, several species of bacteria are being developed against cancer. The bacterial species, their genetic background and their infectious behavior within the tumor microenvironment are thought to be relevant factors in determining their anti-tumor effectiveness in vivo. In this perspective article we will update the most promising results achieved using bacterial therapy (alone or combined with other strategies) in clinically-relevant animal models of cancer and critically discuss the impact of the bacterial variants, route of administration and mechanisms of bacteria-cancer-cell interaction. We will also discuss strategies to apply this information using modern mouse models, molecular biology, genetics and imaging for future bacterial therapy of cancer patients.

Keywords: Coley’s toxins; Salmonella enterica serovar Typhimurium (S. Typhimurium); animal models of cancer; antitumor effect; bacterial-based therapies; bactofection; combined therapies; immune response.

FIGURE 1

Diagram showing main antitumor mechanisms induced by S. Typhimurium (Salmonella). Links are established between direct cytotoxicity induced by bacteria and indirect tumor cell death triggered by the immune system. (a) Bacterial infection within the tumor microenvironment results in inhibition of tumor growth and cell death. (b) Detection of bacterial pathogen-associated molecular patterns (PAMP) by immune cells, trigger cytokine release and recruitment of leukocytes capable of initiating anti-tumor immune responses (Patyar et al., 2010). (c) Using their Type III secretion system, S. Typhimurium can introduce bacterial factors in cancer cells allowing its internalization and intra-cellular replication (Avogadri et al., 2005; Knodler et al., 2010). (cI) Invasive Salmonella induces cell stress responses through danger-associated molecular patterns (DAMP), which are interpreted as damage signals by the immune system. (cII) Simultaneously, this same process can lead to cytokine expression and the transfer of antigens from the bacteria to the cancer cell, enabling the adaptive immune system to recognize and target the invaded cancer cell as infected and bearer of exogenous antigens (Avogadri et al., 2005). Gap junctions are concomitantly induced in the invaded cell and enable cross presentation of antigens to antigen presenting cells (Saccheri et al., 2010). Both processes can give rise to antigen-dependent elimination of infected cancer cells. (cIII) Salmonella can lead to the death of the infected cell, by inducing apoptosis or pyroptosis. The later is a programmed inflammatory cell death, characterized by activation of caspase 1, activation of the inflamosome, and IL-1B and IL-18 secretion, as well as cell rounding and detachment, cytoskeleton reorganization, nucleus deformation and rupture of the cell membrane, resulting in the release of inflammatory signals (Fink and Cookson, 2005, 2007; Knodler et al., 2010; Wang et al., 2013). This mechanism can result in cancer-cell death and immune-cell activation. Pyroptosis was first described in macrophages, which die quickly as a result of this process, and is of particular interest in cancer immunotherapy, as tumor-associated macrophages have been shown to have immune-suppressive proprieties. Reducing their number could be another component of the S. Typhimurium anti-tumor effect. Cancer cell death leads to tumor-antigen liberation, and the released bacteria can infect surrounding cancer cells. (cIV) In the process of pyroptosis, pro-inflammatory cytokines IL1-B and IL-18 can trigger recruitment and activation of immune cells (Knodler et al., 2010; Zhao et al., 2012; Wang et al., 2013). (d) Various mechanisms enhance and converge to enable tumor-antigen recognition and activation of cytotoxic responses both in an antigen-dependent and -independent manner. S. Typhimurium proteins injected into the cancer cell cytosol are subject to proteasomal degradation, resulting in bacterial peptides that can be presented through MHC I to cytotoxic lymphocytes (Avogadri et al., 2005; Saccheri et al., 2010).

FIGURE 2

Direct and synergystic anti-tumor effects of attenuated S. Typhimurium integrating cellular and systemic immune responses. (A) Induction of cell death and granulocyte recruitment associated with intracellular replication of attenuated S. Typhimurium LVR01 (Salmonella), which previously showed a modest antitumor effect in the 4T1 metastatic breast cancer model (Kramer et al., 2015). (a) Confocal microscopy indicates bacteria invasion and replication in breast cancer cell lines: 4T1 (ATCC-CRL2539) (upper line) and NMU (ATCC-CRL1743) (lower line) in a time-course experiment. Cell cultures were grown in glass coverslips, infected with Salmonella expressing the GFP gene and sampled at 2, 12, 24, or 48 h to follow progression of intracellular replication. Specimens were fixed in paraformaldehyde 4%, washed in PBS and stained with DAPI and Phalloidin-Alexa555 (Invitrogen^TM). After the staining, the coverslips were washed with PBS, mounted using Pro Long Gold (Invitrogen^TM) and sealed with nail polish. This three color fluorescence pattern allowed the 3D analysis of the infected cultures, by simultaneously visualizing the bacteria, the nucleus and the F-actin cytoskeleton. Intracellular/extracellular determination of bacteria was possible due to the delimited borders of the actin cytoskeleton which are close to the cell membrane. Images were obtained with a LEICA^®TCS SP5 II spectral confocal microscope and processed with the software Leica^®LAS AF. As observed, bacterial invasion progresses, showing intracellular cytoplasmic hyperreplication over time. (b) Epifluorescence microscopy of 4T1 monolayers infected cells. Cancer cells were infected with Salmonella-GFP for 2 h and observed at different time points. Specimens were washed in PBS, fixed in paraformaldehyde 4%, and stained with DAPI (Invitrogen^TM). After 5 min staining, invaded cultures were washed and observed in a Nikon^®Ti-S epifluorescence inverted microscope. At 2 h few peri-nuclear bacteria could be seen (b.I) At 24 h (b.II) bacteria replicated in the cytoplasm and some infected cells appear rounded and extruded. At 48 h (b.III) densely-infected cells were similar, and eventually burst and release their cellular contents (b.IV). (c) Live infected cultures were observed either intact or in the presence of propidium iodide (500 nM) to assess intracellular bacterial mobility and cell viability, respectively. Monolayers of mammary cancer cells: 4T1 (c.I) and NMU (c.II), as well as macrophage cells J774.A (c.III) were infected with Salmonella-GFP. At 24 h post-infection, infected cells (green) die as indicated by propidium iodide staining (red). Macrophages died at earlier time points (2–16 h). Arrows point the extruded cells. (d) Flow cytometry of intratumor immune cells at 6 days after Salmonella inoculation of 4T1 tumors in vivo. As observed, the intra-tumor granulocyte/myeloid-derived-suppressor cell (Ly6G+CD11b+) levels increase and macrophage (F4/80+CD11b+) levels decreased after bacteria administration among total leukocytes (CD45+ cells). (f) X-gal agar plates were used to seed the untransformed bacteria (control) or bacteria transformed with a plasmid containing the lacZ gene under the control of the eukaryotic cytomegalovirus (CMV) promoter (pCMV-lacZ). As observed, the lacZ gene product β-galactosidase was detected, indicating that the CMV promoter was active in prokaryotic cell species. (B) In vivo effects of attenuated S. Typhimurium (Salmonella) in mice bearing metastatic cancer. This integrative diagram shows the anti-tumor effects of attenuated variants of Salmonella evaluated as mono-therapy. The bacteria inoculation by different routes (systemic or intratumoral) results in its biodistribution to most organs, but with a marked preference for tumors, including metastasic sites (Pawelek et al., 1997; Low et al., 1999; Forbes et al., 2003; Yu et al., 2004; Hoffman, 2016a). In tumors, bacterial infection is associated with tumor-tissue architecture deterioration, a rise in granulocytic cells and INF-γ induction and a decrease of intra-tumor macrophages (Avogadri et al., 2005; Westphal et al., 2008; Zheng et al., 2017). Late effects (10–20 days after bacteria administration) are characterized by a moderate decrease in tumor size, adaptive immune responses including INF-γ production, antibody recognition of tumor antigens, and cytotoxic immune activities (Avogadri et al., 2005; Kramer et al., 2015; Masner et al., unpublished results). Repeated administration of attenuated bacteria could result in a better targeting of metastases (Zhao et al., 2012), while stimulating immune responses that enhance cancer-cell elimination.