Angiogenesis is a hallmark of malignant neoplasias, as the formation of new blood vessels is required for tumors to acquire oxygen and nutrients essential for their continued growth and metastasis. However, the signaling pathways leading to tumor vascularization are not fully understood. Here, using a transplantable mouse tumor model, we have demonstrated that endogenous IFN-β inhibits tumor angiogenesis through repression of genes encoding proangiogenic and homing factors in tumor-infiltrating neutrophils. We determined that IFN-β-deficient mice injected with B16F10 melanoma or MCA205 fibrosarcoma cells developed faster-growing tumors with better-developed blood vessels than did syngeneic control mice. These tumors displayed enhanced infiltration by CD11b + Gr1 + neutrophils expressing elevated levels of the genes encoding the proangiogenic factors VEGF and MMP9 and the homing receptor CXCR4. They also expressed higher levels of the transcription factors c-myc and STAT3, known regulators of VEGF, MMP9, and CXCR4. In vitro, treatment of these tumor-infiltrating neutrophils with low levels of IFN-β restored expression of proangiogenic factors to control levels. Moreover, depletion of these neutrophils inhibited tumor growth in both control and IFN-β-deficient mice. We therefore suggest that constitutively produced endogenous IFN-β is an important mediator of innate tumor surveillance. Further, we believe our data help to explain the therapeutic effect of IFN treatment during the early stages of cancer development.

BackgroundSeveral facultative anaerobic bacteria with potential therapeutic abilities are known to preferentially colonize solid tumors after systemic administration. How they efficiently find and invade the tumors is still unclear. However, this is an important issue to be clarified when bacteria should be tailored for application in cancer therapy.Methodology/Principal FindingsWe describe the initial events of colonization of an ectopic transplantable tumor by Salmonella enterica serovar Typhimurium. Initially, after intravenous administration, bacteria were found in blood, spleen, and liver. Low numbers were also detected in tumors associated with blood vessels as could be observed by immunohistochemistry. A rapid increase of TNF-α in blood was observed at that time, in addition to other pro-inflammatory cytokines. This induced a tremendous influx of blood into the tumors by vascular disruption that could be visualized in H&E stainings and quantified by hemoglobin measurements of tumor homogenate. Most likely, together with the blood, bacteria were flushed into the tumor. In addition, blood influx was followed by necrosis formation, bacterial growth, and infiltration of neutrophilic granulocytes. Depletion of TNF-α retarded blood influx and delayed bacterial tumor-colonization.ConclusionOur findings emphasize similarities between Gram-negative tumor-colonizing bacteria and tumor vascular disrupting agents and show the involvement of TNF-α in the initial phase of tumor-colonization by bacteria.

Administration of facultative anaerobic bacteria like Salmonella typhimurium, Shigella flexneri, and Escherichia coli to tumor-bearing mice leads to a preferential accumulation and proliferation of the microorganisms within the solid tumor. Until now, all known tumor-targeting bacteria have shown poor dissemination inside the tumors. They accumulate almost exclusively in large necrotic areas and spare a rim of viable tumor cells. Interestingly, the bacteria-containing necrotic region is separated from viable tumor cells by a barrier of host neutrophils that have immigrated into the tumor. We here report that depletion of host neutrophils results in a noticeably higher total number of bacteria in the tumor and that bacteria were now also able to migrate into vital tumor tissue. Most remarkably, an increase in the size of the necrosis was observed, and complete eradication of established tumors could be observed under these conditions. Thus, bacteria-mediated tumor therapy can be amplified by depletion of host neutrophils. [Cancer Res 2008;68(8):2952–60]

Cholesterol-dependent cytolysins (CDCs) represent a large family of conserved pore-forming toxins produced by several Gram-positive bacteria such as Listeria monocytogenes, Streptococcus pyrogenes and Bacillus anthracis. These toxins trigger a broad range of cellular responses that greatly influence pathogenesis. Using mast cells, we demonstrate that listeriolysin O (LLO), a prototype of CDCs produced by L. monocytogenes, triggers cellular responses such as degranulation and cytokine synthesis in a Ca(2+)-dependent manner. Ca(2+) signalling by LLO is due to Ca(2+) influx from extracellular milieu and release of from intracellular stores. We show that LLO-induced release of Ca(2+) from intracellular stores occurs via at least two mechanisms: (i) activation of intracellular Ca(2+) channels and (ii) a Ca(2+) channels independent mechanism. The former involves PLC-IP(3)R operated Ca(2+) channels activated via G-proteins and protein tyrosine kinases. For the latter, we propose a novel mechanism of intracellular Ca(2+) release involving injury of intracellular Ca(2+) stores such as the endoplasmic reticulum. In addition to Ca(2+) signalling, the discovery that LLO causes damage to an intracellular organelle provides a new perspective in our understanding of how CDCs affect target cells during infection by the respective bacterial pathogens.

SummaryWe have used Salmonella enterica serovar Typhimurium (S. typhimurium) which are able to colonize tumours besides spleen and liver. Bacteria were equipped with constructs encoding green fluorescent protein or luciferase as reporters under control of the promoter PBAD that is inducible with L-arabinose. Reporter genes could be induced in culture but also when the bacteria resided within the mouse macrophages J774A.1. More important, strong expression of reporters by the bacteria could be detected in mice after administration of L-arabinose. This was especially pronounced in bacteria colonizing tumours. Histology demonstrated that the bacteria had accumulated in and close to necrotic areas of tumours. Bacterial gene induction was observed in both regions. PBAD is tightly controlled also in vivo because gene E of bacteriophage FX174 could be introduced as inducible suicide gene. The possibility to deliberately induce genes in bacterial carriers within the host should render them extremely powerful tools for tumour therapy.