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]

The chemokine receptor CXCR2 and its ligands CXCL1, CXCL2 and CXCL5 play an important role in homing of tumor-associated neutrophils (TANs) into developing tumors. TANs are known to support the development of blood vessels in growing solid tumors, hence contributing to tumor growth. Here, we show that the migration of neutrophils is influenced by endogenous interferon-beta (IFN-b) via regulation of such chemokines and their receptor. We could demonstrate that CXCL1 and CXCL2 gradients are formed in tumor-bearing mice, i.e., low chemokine level in bone marrow (BM) and high level in the tumor. This supports migration of neutrophils into the tumor. Moreover, expression of CXCR2 was highest on neutrophils from BM and lowest in TANs. Importantly, although IFN-b appears to have only a minor influence on the expression of CXCR2, it strongly regulates the CXCR2 ligands. In the absence of endogenous IFN-b, they were expressed significantly higher in tumor-infiltrating neutrophils. Treatment of such neutrophils from tumor-bearing Ifnb1 2/2 mice with recombinant IFN-b downregulated CXCR2 ligand expression to wild-type levels. This explains the reduced migration of neutrophils into tumors and the diminished tumor angiogenesis in IFN-b-sufficient mice. Our results add a novel functional aspect of the type I IFN system as effector molecules of natural cancer surveillance and open interesting possibilities for antineutrophil therapies against cancer.

Cancer has become the second ranking cause of death in the industrialized world. Conventional anti-cancer therapies such as surgery, radiotherapy, and chemotherapy are effective in the treatment of solid tumors only to some extent. Furthermore, they are often associated with severe side effects. Use of bacteria as alternative cancer therapeutics has sporadically been followed over more than a century. The potential to target and colonize solid tumors could be shown for many different bacteria in the meantime. Such bacteria are either obligate anaerobic bacteria like Clostridium or Bifidobacterium or facultative anaerobic like Escherichia coli or Salmonella. Here we describe bacterial strains that were successfully applied mostly in animals bearing model tumors, although first clinical trials have been reported as well. Our review mainly concentrates on Salmonella enterica serovar Typhimurium (S. Typhimurium) since these bacteria were studied most intensively thus far. Importantly, S. Typhimurium were shown not only to colonize large established tumors but also exhibit the property to invade and affect metastases. We report on a potential mechanism by which such bacteria can invade solid tumors. Furthermore, we describe several successful attempts in which the bacteria have been used as carriers for recombinant therapeutic molecules that render bacteria more powerful in eradication of the established tumor. Such attempts should be considered starting points on the way to an effective and safe tumor therapy with the help of bacteria.