Wilber Quispe-Tintaya

STING Ligand c-di-GMP Improves Cancer Vaccination against Metastatic Breast Cancer

Chandra, Quispe-Tintaya, and colleagues show that stimulator of IFN genes (STING) ligand c-di-GMP activated caspase-3, stimulated T cells, and nearly completely eliminated all metastases in mouse breast cancer model 4T1, when combined with Listeria monocytogenes–based Mage-b vaccine in a therapeutic setting. Cancer vaccination may be our best and most benign option for preventing or treating metastatic cancer. However, breakthroughs are hampered by immune suppression in the tumor microenvironment. In this study, we analyzed whether cyclic diguanylate (c-di-GMP), a ligand for stimulator of interferon genes (STING), could overcome immune suppression and improve vaccination against metastatic breast cancer. Mice with metastatic breast cancer (4T1 model) were therapeutically immunized with an attenuated Listeria monocytogenes (LM)–based vaccine, expressing tumor-associated antigen Mage-b (LM-Mb), followed by multiple low doses of c-di-GMP (0.2 μmol/L). This treatment resulted in a striking and near elimination of all metastases. Experiments revealed that c-di-GMP targets myeloid-derived suppressor cells (MDSC) and tumor cells. Low doses of c-di-GMP significantly increased the production of IL12 by MDSCs, in correlation with improved T-cell responses to Mage-b, whereas a high dose of c-di-GMP (range, 0.3–3 mmol/L) activated caspase-3 in the 4T1 tumor cells and killed the tumor cells directly. On the basis of these results, we tested one administration of high-dose c-di-GMP (3 mmol/L) followed by repeated administrations of low-dose c-di-GMP (0.2 μmol/L) in the 4T1 model, and found equal efficacy compared with the combination of LM-Mb and c-di-GMP. This finding correlated with a mechanism of improved CD8 T-cell responses to tumor-associated antigens (TAA) Mage-b and Survivin, most likely through cross-presentation of these TAAs from c-di-GMP–killed 4T1 tumor cells, and through c-di-GMP–activated TAA-specific T cells. Our results demonstrate that activation of STING-dependent pathways by c-di-GMP is highly attractive for cancer immunotherapy. Cancer Immunol Res; 2(9); 901–10. ©2014 AACR.

Nontoxic radioactive Listeriaat is a highly effective therapy against metastatic pancreatic cancer

No significant improvement in therapy of pancreatic cancer has been reported over the last 25 y, underscoring the urgent need for new alternative therapies. Here, we coupled a radioisotope, 188Rhenium, to an attenuated (at) live Listeria monocytogenes (Listeriaat) using Listeria-binding antibodies, thus creating a unique radioactive Listeriaat (RL). We then demonstrated in a highly metastatic pancreatic mouse tumor model (Panc-02) that RL delivered radioactivity to the metastases and less abundantly to primary tumors in vivo, without harming normal cells. This result was possible because Listeriaat was efficiently cleared by the immune system in normal tissues but not in the heavily immune-suppressed microenvironment of metastases and primary tumor. Multiple treatments with low doses of the RL resulted in a dramatic decrease in the number of metastases (∼90%) compared with control groups in the Panc-02 model. This is the first report of using live attenuated bacteria delivering a highly radioactive payload to the metastases, resulting in killing tumor cells in vivo without harming normal cells. The nontoxic RL treatment is attractive for clinical development as a therapy to prevent pancreatic cancer recurrence and metastases.

Curcumin improves the therapeutic efficacy of Listeriaat-Mage-b vaccine in correlation with improved T-cell responses in blood of a triple-negative breast cancer model 4T1

Success of cancer vaccination is strongly hampered by immune suppression in the tumor microenvironment (TME). Interleukin (IL)‐6 is particularly and highly produced by triple‐negative breast cancer (TNBC) cells, and has been considered as an important contributor to immune suppression in the TME. Therefore, we hypothesized that IL‐6 reduction may improve efficacy of vaccination against TNBC cancer through improved T‐cell responses. To prove this hypothesis, we investigated the effect of curcumin, an inhibitor of IL‐6 production, on vaccination of a highly attenuated Listeria monocytogenes (Listeriaat), encoding tumor‐associated antigens (TAA) Mage‐b in a TNBC model 4T1. Two therapeutic vaccination strategies with Listeriaat‐Mage‐b and curcumin were tested. The first immunization strategy involved all Listeriaat‐Mage‐b vaccinations and curcumin after tumor development. As curcumin has been consumed all over the world, the second immunization strategy involved curcumin before and all therapeutic vaccinations with Listeriaat‐Mage‐b after tumor development. Here, we demonstrate that curcumin significantly improves therapeutic efficacy of Listeriaat‐Mage‐b with both immunization strategies particularly against metastases in a TNBC model (4T1). The combination therapy was slightly but significantly more effective against the metastases when curcumin was administered before compared to after tumor development. With curcumin before tumor development in the combination therapy, the production of IL‐6 was significantly decreased and IL‐12 increased by myeloid‐derived suppressor cells (MDSC), in correlation with improved CD4 and CD8 T‐cell responses in blood. Our study suggests that curcumin improves the efficacy of Listeriaat‐Mage‐b vaccine against metastases in TNBC model 4T1 through reversal of tumor‐induced immune suppression.

Identification of Old World Leishmania spp. by specific polymerase chain reaction amplification of cysteine proteinase B genes and rapid dipstick detection

We used the cysteine proteinase B (cpb) gene family of the trypanosomatid genus Leishmania as a target to develop rapid, specific, and easy-to-use polymerase chain reaction (PCR) tests to discriminate Leishmania infantum, Leishmania donovani, Leishmania tropica, Leishmania aethiopica, and Leishmania major. Identification of all 5 Old World species and validation of intraspecies variability are features lacking in other species-specific PCRs. Amplicon analysis was done on agarose gels and was further simplified by using an oligochromatography dipstick to detect L. infantum and L. donovani products. Because the analytical sensitivity is lower than that of certain other species- and genus-specific PCRs, our assays are especially valuable for use on cultured isolates or directly on cryostabilates. As such, they can be implemented by research and health centers having access to culturing, DNA isolation, and PCR.

DNA damage in normally and prematurely aged mice.

Steady‐state levels of spontaneous DNA damage, the by‐product of normal metabolism and environmental exposure, are controlled by DNA repair pathways. Incomplete repair or an age‐related increase in damage production and/or decline in repair could lead to an accumulation of DNA damage, increasing mutation rate, affecting transcription, and/or activating programmed cell death or senescence. These consequences of DNA damage metabolism are highly conserved, and the accumulation of lesions in the DNA of the genome could therefore provide a universal cause of aging. An important corollary of this hypothesis is that defects in DNA repair cause both premature aging and accelerated DNA damage accumulation. While the former has been well‐documented, the reliable quantification of the various lesions thought to accumulate in DNA during aging has been a challenge. Here, we quantified inhibition of long‐distance PCR as a measure of DNA damage in liver and brain of both normal and prematurely aging, DNA repair defective mice. The results indicate a marginal, but statistically significant, increase in spontaneous DNA damage with age in normal mouse liver but not in brain. Increased levels of DNA damage were not observed in the DNA repair defective mice. We also show that oxidative lesions do not increase with age. These results indicate that neither normal nor premature aging is accompanied by a dramatic increase in DNA damage. This suggests that factors other than DNA damage per se, for example, cellular responses to DNA damage, are responsible for the aging phenotype in mice.

Fast mitochondrial DNA isolation from mammalian cells for next-generation sequencing

Standard methods for mitochondrial DNA (mtDNA) extraction do not provide the level of enrichment for mtDNA sufficient for direct sequencing and must be followed by long-range-PCR amplification, which can bias the sequencing results. Here, we describe a fast, cost-effective, and reliable method for preparation of mtDNA enriched samples from eukaryotic cells ready for direct sequencing. Our protocol utilizes a conventional miniprep kit, paramagnetic bead-based purification, and an optional, limited PCR amplification of mtDNA. The first two steps alone provide more than 2000-fold enrichment for mtDNA when compared with total cellular DNA (~200-fold in comparison with current commercially available kits) as demonstrated by real-time PCR. The percentage of sequencing reads aligned to mtDNA was about 22% for non-amplified samples and greater than 99% for samples subjected to 10 cycles of long-range-PCR with mtDNA specific primers.

Application of PCR-RFLP for the exploration of the molecular diversity of Leishmania infantum in Algeria.

In recent years, new methods have been developed for the molecular typing of Leishmania that need to be extensively validated by studies of clinical isolates in a well defined epidemiological context. The present study is a contribution to this effort. Using PCR-RFLP of gp63 and cpb genes, we analysed 59 isolates of L. (L.) infantum obtained from different regions of Algeria and originating from different clinical forms, hosts and zymodemes. PCR-RFLP identified 15 different genotypes among the four zymodemes analysed, thereby demonstrating a higher discriminatory power than multilocus enzyme electrophoresis. We did not see any significant relationships between PCR-RFLP patterns and host origin. However, cpb polymorphism showed two interesting trends: a possible relationship with the cutaneous origin of the isolates and an association with a West-East cline. We verified the proof of evidence of the direct applicability of gp63 and cpb PCR-RFLP in blood samples from dogs. Further work is needed to compare the sensitivity of pattern detection with cpb and gp63 PCR-RFLP but our results pave the way to future multilocus PCR-RFLP studies of L. (L.) infantum populations.

High-throughput sequencing in mutation detection: A new generation of genotoxicity tests?

The advent of next generation sequencing (NGS) technology has provided the means to directly analyze the genetic material in primary cells or tissues of any species in a high throughput manner for mutagenic effects of potential genotoxic agents. In principle, direct, genome-wide sequencing of human primary cells and/or tissue biopsies would open up opportunities to identify individuals possibly exposed to mutagenic agents, thereby replacing current risk assessment procedures based on surrogate markers and extrapolations from animal studies. NGS-based tests can also precisely characterize the mutation spectra induced by genotoxic agents, improving our knowledge of their mechanism of action. Thus far, NGS has not been widely employed in genetic toxicology due to the difficulties in measuring low-abundant somatic mutations. Here, we review different strategies to employ NGS for the detection of somatic mutations in a cost-effective manner and discuss the potential applicability of these methods in testing the mutagenicity of genotoxic agents.

Improved transposon-based library preparation for the Ion Torrent platform

A transposon-based approach for the construction of sequencing libraries is an efficient way of preparing samples for processing on both Illumina and Ion Torrent platforms. However, PCR-mediated incorporation of adaptors in tagged DNA fragments leaves behind self-complementary regions flanking the DNA fragment. These regions are capable of forming hairpin structures and, together with adaptors, create conditions for the potential formation of template heteroduplexes. These negatively affect the sequencing process on the Ion Torrent platform and can lead to a more than 3-fold decline in output data compared with sequencing of conventional libraries. To address this problem, we have developed MuPlus, a transposon-based protocol for barcoded library preparation for Ion Torrent, in which one adaptor is integrated by PCR and the second is integrated by ligation as a single-stranded oligonucleotide after enzymatic cleavage of a complementary part on one strand of the tag. The resulting library does not contain self-complementary, hairpin-forming regions, is free of heteroduplexes, and can be analyzed on the Ion Torrent platform with the same efficiency as a library created with a ligation-based protocol.

Development and validation of a targeted next generation DNA sequencing panel outperforming whole exome sequencing for the identification of clinically relevant genetic variants

Next generation sequencing (NGS) technologies have revolutionized our approach to genomic research. The use of whole genome sequencing (WGS), whole exome sequencing (WES), transcriptome profiling, and targeted DNA sequencing has exponentially improved our understanding of the human genome and the genetic complexities underlying malignancy. Yet, WGS and WES clinical applications remain limited due to high costs and the large volume of data generated. When utilized to address biological questions in basic science studies, targeted sequencing panels have proven extremely valuable due to reduced costs and higher sequencing depth. However, the routine application of targeted sequencing to the clinical setting is limited to a few cancer subtypes. Some highly aggressive tumor types, like type 2 endometrial cancer (EC), could greatly benefit from routine genomic analysis using targeted sequencing. To explore the potential utility of a mid size panel (~150 genes) in the clinical setting, we developed and validated a custom panel against WGS, WES, and another commercially available targeted panel. Our results indicate that a mid size custom designed panel is as efficient as WGS and WES in mapping variants of biological and clinical relevance, rendering higher coverage, at a lower cost, with fewer variants of uncertain significance. Because of the much higher sequencing depth that could be achieved, our results demonstrate that targeted sequencing outperformed WGS and WES in the mapping of pathogenic variants in a breast cancer case, as well as a case of mixed serous and high-grade endometrioid EC, the most aggressive EC subtype.