Sample storage conditions induce post-collection biases in microbiome profiles

Abstract Background Here we investigated the influence of different stabilization and storage strategies on the quality and composition of the fecal microbial community. Namely, same-day isolated murine DNA was compared to samples stored for 1 month in air at ambient temperature, with or without preservative buffers (i.e. EDTA and lysis buffer), different temperatures (i.e. 4 °C, − 20 °C, and − 80 °C), and hypoxic conditions. Results Only storage in lysis buffer significantly reduced DNA content, yet without integrity loss. Storage in EDTA affected alpha diversity the most, which was also reflected in cluster separation. Distinct changes were also seen in the phyla and bacterial species abundance per storage strategy. Metabolic function analysis showed 22 pathways not significantly affected by storage conditions, whereas the tyrosine metabolism pathway was significantly changed in all strategies except by EDTA. Conclusion Each long-term storage strategy introduced a unique post-collection bias, which is important to take into account when interpreting data

Targeting Artificial Tumor Stromal Targets for Molecular Imaging of Tumor Vascular Hypoxia

<div><p>Developed and tested for many years, a variety of tumor hypoxia detection methods have been inconsistent in their ability to predict treatment outcomes or monitor treatment efficacy, limiting their present prognostic capability. These variable results might stem from the fact that these approaches are based on inherently wide-ranging global tumor oxygenation levels based on uncertain influences of necrotic regions present in most solid tumors. Here, we have developed a novel non-invasive and specific method for tumor vessel hypoxia detection, as hypoxemia (vascular hypoxia) has been implicated as a key driver of malignant progression, therapy resistance and metastasis. This method is based on high-frequency ultrasound imaging of α-pimonidazole targeted-microbubbles to the exogenously administered hypoxia marker pimonidazole. The degree of tumor vessel hypoxia was assessed in three mouse models of mammary gland carcinoma (4T1, SCK and MMTV-Wnt-1) and amassed up to 20% of the tumor vasculature. In the 4T1 mammary gland carcinoma model, the signal strength of α-pimonidazole targeted-microbubbles was on average 8-fold fold higher in tumors of pimonidazole-injected mice than in non-pimonidazole injected tumor bearing mice or non-targeted microbubbles in pimonidazole-injected tumor bearing mice. Overall, this provides proof of principle for generating and targeting artificial antigens able to be ‘created’ on-demand under tumor specific microenvironmental conditions, providing translational diagnostic, therapeutic and treatment planning potential in cancer and other hypoxia-associated diseases or conditions.</p></div

Enhancement of T-cell–Mediated Antitumor Response: Angiostatic Adjuvant to Immunotherapy against Cancer

Purpose: Tumor-released proangiogenic factors suppress endothelial adhesion molecule (EAM) expression and prevent leukocyte extravasation into the tumor. This is one reason why immunotherapy has met with limited success in the clinic. We hypothesized that overcoming EAM suppression with angiogenesis inhibitors would increase leukocyte extravasation and subsequently enhance the effectiveness of cellular immunotherapy. Experimental Design: Intravital microscopy, multiple color flow cytometry, immunohistochemistry, and various tumor mouse (normal and T-cell deficient) models were used to investigate the temporal dynamics of cellular and molecular events that occur in the tumor microenvironment during tumor progression and angiostatic intervention. Results: We report that while EAM levels and T-cell infiltration are highly attenuated early on in tumor growth, angiostatic therapy modulates these effects. In tumor models with normal and T-cell-deficient mice, we show the active involvement of the adaptive immune system in cancer and differentiate antiangiogenic effects from antiangiogenic mediated enhancement of immunoextravasation. Our results indicate that a compromised immune response in tumors can be obviated by the use of antiangiogenic agents. Finally, with adoptive transfer studies in mice, we show that a phased combination of angiostatic therapy and T-cell transfer significantly (P < 0.0013) improves tumor growth inhibition. Conclusions: This research contributes to understand the cellular mechanism of action of angiostatic agents and the immune response within the tumor microenvironment, in particular as a consequence of the temporal dynamics of EAM levels. Moreover, our results suggest that adjuvant therapy with angiogenesis inhibitors holds promise for cellular immunotherapy in the clinic. ©2011 AACR

High-frequency ultrasound imaging of targeted-microbubbles detects tumor vessel hypoxia.

<p>Representative image and quantified data of anti-pimonidazole labeled microbubbles (MBα-pimo) bound in perfused hypoxic tumor vasculature without pimonidazole injection <b>(A)</b>, and with pimonidazole injection <b>(B)</b> in 4T1 tumor bearing mice. Top image shows the signal before the burst sequence and the bottom image shows after the burst sequence <b>(A, B)</b>. <b>(C)</b> Quantified data of different experimental conditions using targeting and non-targeting microbubbles (as indicated). <b>D)</b> Summary of quantitated data statistically analyzed represented as mean ± SEM, ^#p < 0.05, versus non-targeting MB, MBα-pimo without pimonidazole injection, and MBα-pimo in muscle tissue (ANOVA post-hoc Holm-Sidak).</p

3D modeling of MBα-pimo distribution in mammary gland carcinoma.

<p><b>A)</b> Single slice images taken from a 3D imaging sequence in B-mode <i>(left)</i>. Single slice images taken from a 3D imaging sequence depicting the differential targeted expression (d.T.E) <i>(right)</i>. <b>B)</b> Three-dimensional contrast projection of 3D stack image data from hypoxia targeted, MBα-pimo, contrast signal collected in a rear-limb 4T1 tumor. Images (0.152mm/slice) generated using Visualsonics imaging system and post-processed using the Huygens essential software.</p

Schematic of anti-pimonidazole targeted-microbubbles (MBα-pimo) with ultrasound imaging for detection of vascular hypoxia.

<p><b>(A)</b> Illustration showing the differential distribution of MBα-pimo in well-oxygenated tumor endothelium (red) compared to hypoxic tumor endothelium (blue) during imaging and intervention by ultrasound. <b>(B)</b> Representative quantification graphic of MBα-pimo where the binding occurs over a 5 minute window after IV injection followed by a data collection period of contrast signal, a single ultrasound pulse to burst bound and free MBα-pimo, and a final data collection during the immediate reperfusion window. Subsequently, the difference in signal from the steady state prior to microbubble burst (‘pre’) and following burst (‘post’) can be calculated. This differential targeted expression (d.T.E.; linear, a.u.) represents the relative amount of bound MBα-pimo and indirectly indicates the location and amount of vascular hypoxia within the tumor (x-axis scale not linear).</p