Reevaluating the concept of treating experimental tumors with a mixed bacterial vaccine: Coley's Toxin.

Several decades after Coley's initial work, we here systematically analyzed tumoricidal as well as immunostimulatory effects of the historical preparation Coley's Toxin (CT), a safe vaccine made of heat-inactivated S. pyogenes and S. marcescens. First, by performing in vitro analysis, established human pancreatic carcinoma cell lines responded with dose- and time-dependent growth inhibition. Effects were attributed to necrotic as well as apoptotic cell death as determined by increased Caspase 3/7 levels, raised numbers of cells with sub-G1-DNA, and induced p21(waf) expression, indicative for cell cycle arrest. Besides, CT effectively stimulated human peripheral blood leukocytes (huPBL) from healthy volunteers. Quantitative gene expression analysis revealed upregulated mRNA levels of selected Toll-like receptors. Flow cytometric phenotyping of CT-stimulated huPBLs identified raised numbers of CD25(+)-activated leukocytes. In vivo, repetitive, local CT application was well tolerated by animals and induced considerable delay of Panc02 tumors. However, systemic treatment failed to affect tumor growth. Antitumoral effects following local therapy were primarily accompanied by stimulation of innate immune mechanisms. Data presented herein prove that the historical approach of using killed bacteria as active immunotherapeutic agents still holds promise, and further careful preclinical analyses may pave the way back into clinical applications.

Inflammation and immunity in the tumor environment.

The relationship between inflammation, innate immunity and cancer is widely accepted. Cancer-associated inflammation includes infiltrating leukocytes, cytokines, chemokines, growth factors, lipid messengers and matrix-degrading enzymes. Tumor-associated macrophages and lymphocyte subpopulations are major components of the leukocyte infiltrate in most tumors. However, the cytokine and chemokine expression profile of the tumor microenvironment may be more relevant than its specific immune cell content. Apart from inflammatory cells, tumor stroma consists of new blood vessels and connective tissue. Many factors produced by tumor cells promote tumor angiogenesis and generation of extracellular matrix. Investigations regarding the link between inflammation and cancer are vital for identifying cell or protein targets for cancer prevention and therapy. Based on the relation between inflammation and cancer, different forms of immunotherapy have been developed. In a mouse model, we investigated the potential of Streptococcus pyogenes to achieve a bacteria-related immune response against tumor cells followed by tumor regression. As a model of pancreatic carcinoma, the aggressively growing and poorly immunogenic Panc02 tumor model was chosen. Our findings showed that a local application of bacteria mediates complete tumor regression. Future investigations should focus on the optimization of immunotherapeutic approaches that incorporate live bacteria or bacterial components.

Pancreatic cancer regression by intratumoural injection of live Streptococcus pyogenes in a syngeneic mouse model.

BACKGROUND: This study addressed the potential of bacteriolytic therapy using Streptococcus pyogenes in a syngeneic pancreatic carcinoma mouse model. METHODS: Panc02 tumours were either infected with S pyogenes or were treated with the equivalent volume of vehicle. In addition to assessment of tumour histology and immunohistochemistry, isolated splenocytes were analysed by flow cytometry. Interferon (IFN) gamma secretion as a reaction of splenocytes against tumour cells was shown through the ELISpot technique. A cytotoxic effect of lymphocytes against tumour targets was detected by lactate dehydrogenase (LDH) release. Cytokine levels in serum were measured. RESULTS: A single application of live bacteria into established Panc02 tumours resulted in complete tumour regression. This antitumoral effect was accompanied by massive leucocyte infiltration into the tumours as well as a significant and sustained elevation of systemic levels of the proinflammatory cytokines IFN gamma, tumour necrosis factor alpha and interleukin 6. Lymphocytes obtained from treated mice specifically recognised syngeneic tumour cells in IFN gamma-ELISpot, and most importantly in cellular cytotoxicity assays, indicating a tumour-specific immune response. CONCLUSIONS: We provide data that both the direct lytic activity of S pyogenes towards tumour cells and the infection-driven infiltration of tumours by cells of the innate immune system lead to damage of tumour cells followed by a dissemination of tumour components. This last outcome allows for the activation of tumour-specific effector cells, most probably in draining lymph nodes, promoted by the proinflammatory context. Taken together, these data indicate that the application of live S pyogenes may be a promising new treatment strategy for advanced pancreatic cancer patients that warrants further investigation.