FACS isolation and analysis of human circulating and tumor neutrophils.

The role of neutrophils in tumor growth and metastasis is still controversial. Studies in clinically relevant models of cancer have shown that neutrophils can promote tumor growth and development and metastasis, or inhibit it. Thus, further analysis is required to fully elucidate the role of neutrophils in cancer. A number of different methods are available for neutrophil isolation and characterization. However, Fluorescence-activated cell sorting (FACS) is particularly effective for isolating neutrophils and assessing their phenotype as it allows for the simultaneous use of multiple cell surface markers, can be used for isolation of both blood and tumor neutrophils and features a high purity and high yields.

Myeloid Cells in Metastasis.

Metastatic disease is the leading cause of death in patients with solid cancers. The progression to metastasis is a multistep process that involves detachment of tumor cells from their constraining basement membrane at the primary site, migration and intravasation into the circulation, survival in the circulation, extravasation into the secondary organ, and survival and growth at the secondary site. During these steps, tumor and immune cells interact and influence each other both within the tumor microenvironment and systemically. In particular, myeloid cells such as monocytes, macrophages, neutrophils, and myeloid-derived suppressor cells (myeloid regulatory cells) have been shown to play important roles in the metastatic process. These interactions open new avenues for targeting cancer metastasis, especially given the increasing interest in development of cancer immunotherapies. In this review, we describe the currently reported pathways and mechanisms involved in myeloid cell enhancement of the metastatic cascade.

Human Tumor-Associated Macrophage and Monocyte Transcriptional Landscapes Reveal Cancer-Specific Reprogramming, Biomarkers, and Therapeutic Targets.

The roles of tumor-associated macrophages (TAMs) and circulating monocytes in human cancer are poorly understood. Here, we show that monocyte subpopulation distribution and transcriptomes are significantly altered by the presence of endometrial and breast cancer. Furthermore, TAMs from endometrial and breast cancers are transcriptionally distinct from monocytes and their respective tissue-resident macrophages. We identified a breast TAM signature that is highly enriched in aggressive breast cancer subtypes and associated with shorter disease-specific survival. We also identified an auto-regulatory loop between TAMs and cancer cells driven by tumor necrosis factor alpha involving SIGLEC1 and CCL8, which is self-reinforcing through the production of CSF1. Together these data provide direct evidence that monocyte and macrophage transcriptional landscapes are perturbed by cancer, reflecting patient outcomes.

Synchrotron microbeam radiotherapy evokes a different early tumor immunomodulatory response to conventional radiotherapy in EMT6.5 mammary tumors.

BACKGROUND: Synchrotron microbeam radiation therapy (MRT) is a new, evolving form of radiotherapy that has potential for clinical application. Several studies have shown in preclinical models that synchrotron MRT achieves equivalent tumor control to conventional radiotherapy (CRT) but with significantly reduced normal tissue damage. METHODS: To explore differences between these two modalities, we assessed the immune cell infiltrate into EMT6.5 mammary tumors after CRT and MRT. RESULTS: CRT induced marked increases in tumor-associated macrophages and neutrophils while there were no increases in these populations following MRT. In contrast, there were higher numbers of T cells in the MRT treated tumors. There were also increased levels of CCL2 by immunohistochemistry in tumors subjected to CRT, but not to MRT. Conversely, we found that MRT induced higher levels of pro-inflammatory genes in tumors than CRT. CONCLUSION: Our data are the first to demonstrate substantial differences in macrophage, neutrophil and T cell numbers in tumors following MRT versus CRT, providing support for the concept that MRT evokes a different immunomodulatory response in tumors compared to CRT.

Isolation of Mouse and Human Tumor-Associated Macrophages.

The tumor microenvironment is a complex network of cells that support tumor progression and malignancy. It has been demonstrated that tumor cells can educate the immune system to promote a tumor-friendly environment. Among all these immune cells, tumor-associated macrophages (TAMs) are well represented and their presence in mouse models has been shown to promote tumor progression and metastasis. These effects are through the stimulation of angiogenesis, enhancement of tumor cell invasion and intravasation, immunosuppression, and at the metastatic site tumor cell extravasation and growth. However, the precise mechanisms are not fully understood. Furthermore there is limited information on TAMs derived from human cancers. For this reason it is important to be able to extract TAMs from tumors in order to compare their phenotypes, functions, and transcriptomes with normal resident tissue macrophages. Isolation of these cells is challenging due to the lack of markers and standardized protocols. Here we show an optimized protocol for the efficient isolation and extraction of resident macrophages and TAMs from human and mouse tissues by using multicolor flow cytometry. These protocols allow for the extraction of thousands of macrophages in less than 5 h from tissues as small as half a gram. The isolated macrophages can then be used for both "omics" and in vitro studies.

Neutrophils: important contributors to tumor progression and metastasis.

The presence of neutrophils in tumors has traditionally been considered to be indicative of a failed immune response against cancers. However, there is now evidence showing that neutrophils can promote tumor growth, and increasingly, the data support an active role for neutrophils in tumor progression to distant metastasis. Neutrophils have been implicated in promoting metastasis in cancer patients, where neutrophil numbers and neutrophil-related factors and functions have been associated with progressive disease. Nevertheless, the role of neutrophils in tumors, both at the primary and secondary sites, remains controversial, with some studies reporting their anti-tumor functions. This review will focus on the data demonstrating a role for neutrophils in both tumor growth and metastasis and will attempt to clarify the discrepancies in the literature.

The promotion of breast cancer metastasis caused by inhibition of CSF-1R/CSF-1 signaling is blocked by targeting the G-CSF receptor.

Treatment options are limited for patients with breast cancer presenting with metastatic disease. Targeting of tumor-associated macrophages through the inhibition of colony-stimulating factor-1 receptor (CSF-1R), a key macrophage signaling pathway, has been reported to reduce tumor growth and metastasis, and these treatments are now in clinical trials. Here, we report that, surprisingly, treatment with neutralizing anti-CSF-1R and anti-CSF-1 antibodies, or with two different small-molecule inhibitors of CSF-1R, could actually increase spontaneous metastasis without altering primary tumor growth in mice bearing two independently derived mammary tumors. The blockade of CSF-1R or CSF-1 led to increased levels of serum G-CSF, increased frequency of neutrophils in the primary tumor and in the metastasis-associated lung, as well as increased numbers of neutrophils and Ly6C(hi) monocytes in the peripheral blood. Neutralizing antibody against the G-CSF receptor, which regulates neutrophil development and function, reduced the enhanced metastasis and neutrophil numbers that resulted from CSF-1R blockade. These results indicate that the role of the CSF-1R/CSF-1 system in breast cancer is far more complex than originally proposed, and requires further investigation as a therapeutic target.

Mobilization of viable tumor cells into the circulation during radiation therapy.

PURPOSE: To determine whether radiation therapy (RT) could mobilize viable tumor cells into the circulation of non-small cell lung cancer (NSCLC) patients. METHODS AND MATERIALS: We enumerated circulating tumor cells (CTCs) by fluorescence microscopy of blood samples immunostained with conventional CTC markers. We measured their DNA damage levels using γ-H2AX, a biomarker for radiation-induced DNA double-strand breaks, either by fluorescence-activated cell sorting or by immunofluorescence microscopy. RESULTS: Twenty-seven RT-treated NSCLC patients had blood samples analyzed by 1 or more methods. We identified increased CTC numbers after commencement of RT in 7 of 9 patients treated with palliative RT, and in 4 of 8 patients treated with curative-intent RT. Circulating tumor cells were also identified, singly and in clumps in large numbers, during RT by cytopathologic examination (in all 5 cases studied). Elevated γ-H2AX signal in post-RT blood samples signified the presence of CTCs derived from irradiated tumors. Blood taken after the commencement of RT contained tumor cells that proliferated extensively in vitro (in all 6 cases studied). Circulating tumor cells formed γ-H2AX foci in response to ex vivo irradiation, providing further evidence of their viability. CONCLUSIONS: Our findings provide a rationale for the development of strategies to reduce the concentration of viable CTCs by modulating RT fractionation or by coadministering systemic therapies.

Did transmission of Helicobacter pylori from humans cause a disease outbreak in a colony of Stripe-faced Dunnarts (Sminthopsis macroura)?

Since the discovery that Helicobacter pylori causes a range of pathologies in the stomachs of infected humans, it has become apparent that Helicobacters are found in a diverse range of animal species where they are frequently associated with disease. In 2003 and 2004, there were two outbreaks of increased mortality associated with gastric bleeding and weight-loss in a captive colony of the Australian marsupial, the Stripe-faced Dunnart (Sminthopsis macroura). The presence of gastric pathology led to an investigation of potential Helicobacter pathogenesis in these animals. Histological examination revealed the presence of gastritis, and PCR analysis confirmed the presence of Helicobacter infection in the stomachs of these marsupials. Surprisingly, sequencing of 16S rRNA from these bacteria identified the species as H. pylori and PCR confirmed the strain to be positive for the important pathogenesis factor, cagA. We therefore describe, for the first time, an apparent reverse zoonotic infection of Stripe-faced Dunnarts with H. pylori. Already prone to pathological effects of stress (as experienced during breeding season), concomitant H. pylori infection appears to be a possible essential but not sufficient co-factor in prototypic gastric bleeding and weight loss in these marsupials. The Stripe-faced Dunnart could represent a new model for investigating Helicobacter-driven gastric pathology. Infections from their human handlers, specifically of H. pylori, may be a potential risk to captive colonies of marsupials.

Muc1 mucin limits both Helicobacter pylori colonization of the murine gastric mucosa and associated gastritis.

BACKGROUND & AIMS: The MUC1 mucin is expressed on the cell surface of epithelial cells lining the gastric mucosa. Epidemiologic studies suggest that functional allelic variations in the MUC1 gene may play a role in human susceptibility to Helicobacter pylori-associated pathologies, including gastric adenocarcinoma. We have evaluated the impact of Muc1 expression on the colonization and pathogenesis of gastric Helicobacter infections. METHODS: Wild-type and Muc1-deficient mice were infected with H pylori and colonization and gastritis levels determined. Primary gastric cells were used to examine the impact of Muc1 expression on bacterial adherence. RESULTS: Mice lacking Muc1 were colonized by 5-fold more H pylori within 1 day of infection, and this difference was maintained for at least 2 months postinfection. Mice heterozygous for the null Muc1 allele developed intermediate bacterial colonization. Although wild-type mice developed only a mild gastritis when infected for 2 months with H pylori, Muc1(-/-) mice developed an atrophic gastritis marked by loss of parietal cells. We demonstrate H pylori adhesion to purified MUC1 and significantly increased adhesion to cultured murine Muc1 null gastric epithelial cells, suggesting that Muc1 acts as a decoy limiting binding to the cell surface. CONCLUSIONS: Muc1 provides a protective barrier, which limits both acute and chronic colonization by H pylori, as well as playing a major role in limiting the inflammation induced by Helicobacter infection. We propose that Muc1 restricts access of H pylori to the epithelial surface, hence reducing exposure of the host to proinflammatory bacterial products.

Effectiveness of vaccination with recombinant HpaA from Helicobacter pylori is influenced by host genetic background.

Several studies have explored the production and immunogenicity of HpaA as a potential protective antigen against Helicobacter pylori but little is known regarding its protective capabilities. We therefore evaluated the protective efficacy of recombinant HpaA (rHpaA) as a candidate vaccine antigen against H. pylori. To explore the impact of genetic diversity, inbred and outbred mice were prophylactically and therapeutically immunized with rHpaA adjuvanted with cholera toxin (CT). Prophylactic immunization induced a reduction in bacterial colonization in BALB/c and QS mice, but was ineffective in C57BL/6 mice, despite induction of antigen-specific antibodies. By contrast, therapeutic immunization was effective in all three strains of mice. Prophylactic immunization with CT-adjuvanted rHpaA was more effective when delivered via the nasal route than following intragastric delivery in BALB/c mice. However, HpaA-mediated protection was inferior to that induced by bacterial lysate. Hence, protective efficacy is inducible with vaccines containing HpaA, most relevantly shown in an outbred population of mice. The effectiveness of protection induced by HpaA antigen was influenced by host genetics and was less effective than lysate. HpaA therefore has potential for the development of effective immunization against H. pylori but this would probably entail the antigen to be one component of a multiantigenic vaccine.