Controlling cancer-induced inflammation with a nucleic acid scavenger prevents lung metastasis in murine models of breast cancer.

Tumor cells release nucleic acid-containing proinflammatory complexes, termed nucleic acid-containing damage-associated molecular patterns (NA DAMPs), passively upon death and actively during stress. NA DAMPs activate pattern recognition receptors on cells in the tumor microenvironment leading to prolonged and intensified inflammation that potentiates metastasis. No strategy exists to control endogenous or therapy-induced inflammation in cancer patients. We discovered that the generation 3.0 polyamidoamine dendrimer (PAMAM-G3) scavenges NA DAMPs and mitigates their proinflammatory effects. In this study, we tested if the nucleic acid scavenger (NAS) PAMAM-G3 reduces lung metastasis in murine models of breast cancer. Our data indicate that PAMAM-G3 treatment decreases cell-free DNA levels and reduces lung metastasis in the experimental intravenous tumor-injection model and the postsurgical tumor-resection model of 4T1 breast cancer. Reduction in lung metastasis is associated with reduction in inflammatory immune cell subsets and proinflammatory cytokine levels in the tumor and the periphery. This study is the first example of NAS-mediated inhibition of metastasis to the lung. The study results provide a strong rationale for inclusion of NAS therapy in women with breast cancer undergoing standard-of-care surgery.

Degenerate PCR primers for assays to track steps of nitrogen metabolism by taxonomically diverse microorganisms in a variety of environments.

Steps in the global nitrogen cycle are mainly catalyzed by microorganisms. Accordingly, the activities of these microorganisms affect the health and productivity of ecosystems. Their activities are also used in wastewater treatment systems to remove reactive nitrogen compounds and prevent eutrophication events triggered by nutrient discharges. Therefore, tracking the activities of these microorganisms can provide insights into the functioning of these systems. The presence and abundance of genes encoding nitrogen-metabolizing enzymes can be traced via polymerase chain reaction (PCR); however, this requires primers that are sensitive to a heterogenous gene pool yet specific enough to the target biomarker. The ever-expanding diversity of sequences available from databases includes many sequences relevant to nitrogen metabolism that match poorly with primers previously designed to track their presence and/or abundance. This includes genes encoding ammonia monooxygenase (AMO) of ammonia oxidizing microorganisms, nitrite oxidoreductase (NXR) of nitrite oxidizing bacteria, and nitrous oxide reductase (NOS) of denitrifying bacteria. Some primers are also not designed to generate the short (~200 nucleotides) amplicons required for real-time quantitative PCR (qPCR) and reverse-transcriptase qPCR (qRT-PCR). In this study, genes collected from the Integrated Microbial Genomes database (IMG) were aligned to design PCR primers that could capture more sequence diversity than is possible using existing primers. Primers were designed to target three clades of AMO (Betaproteobacteria, Chrenarchaeota, and complete ammonia oxidizing Nitrospira), periplasmic NXR and two clades of NOS (Proteobacteria and Bacteroidetes/Firmicutes). These primers successfully amplified target sequences from two wastewater treatment plants with biological nitrogen removal (one with simultaneous nitrification/denitrification and one with distinct anoxic/oxic zones) and estuary sediment. Nucleotide sequences of the amplicons retrieved homologs when used to query GenBank by BLAST. While convincingly identified as target sequences for these primer pairs, these amplicons were divergent from each other, and quite divergent (as low as 73%) from those present in GenBank, suggesting these primers are capable of capturing a diverse range of sequences. A direct comparison showed that primers designed here are better suited to environmental samples, such as wastewater treatment facilities, by producing a greater number of amplicons from the same sample than primers currently established in literature.

Examining Peripheral and Tumor Cellular Immunome in Patients With Cancer.

Immunotherapies are rapidly being integrated into standard of care (SOC) therapy in conjunction with surgery, chemotherapy, and radiotherapy for many cancers and a large number of clinical studies continue to explore immunotherapy alone and as part of combination therapies in patients with cancer. It is evident that clinical effectiveness of immunotherapy is limited to a subset of patients and improving immunotherapy related outcomes remains a major scientific and clinical effort. Understanding the immune cell subset phenotype and activation/functional status (cellular immunome) prior to and post therapy is therefore critical to develop biomarkers that (1) will predict if a patient will respond to immunotherapy and (2) are a result of immunotherapy. In this study, we investigated local (tumor) and peripheral (blood) cellular immunome of patients with melanoma, breast cancer, and brain cancer using a rapid and reliable standardized, multiparameter flow cytometry assay. We used this approach to monitor changes in the peripheral cellular immunome in women with breast cancer undergoing SOC therapy. Our analysis is unique because it is conducted using matched fresh tumor tissue and blood from patients in real-time, within 2-3 h of sample acquisition, and provides insight into the innate and adaptive immune cell profile in blood and tumor. Specific to blood, this approach involves no manipulation and evaluates all immune subsets such as T cells, B cells, natural killer (NK) cells, monocytes, dendritic cells (DCs), neutrophils, eosinophils, and basophils using 0.5 ml of blood. Analysis of the corresponding tumor provides much needed insight into the phenotype and activation status of immune cells, especially T and B cells, in the tumor microenvironment vs. the periphery. This analysis will be used to assess baseline and therapy-mediated changes in local and peripheral cellular immunome in patients with glioblastoma, breast cancer, and melanoma in planned immunotherapy clinical studies.