Chemotherapy-resistant osteosarcoma is highly susceptible to IL-15-activated allogeneic and autologous NK cells

High-grade osteosarcoma occurs predominantly in adolescents and young adults and has an overall survival rate of about 60%, despite chemotherapy and surgery. Therefore, novel treatment modalities are needed to prevent or treat recurrent disease. Natural killer (NK) cells are lymphocytes with cytotoxic activity toward virus-infected or malignant cells. We explored the feasibility of autologous and allogeneic NK cell–mediated therapies for chemotherapy-resistant and chemotherapy-sensitive high-grade osteosarcoma. The expression by osteosarcoma cells of ligands for activating NK cell receptors was studied in vitro and in vivo, and their contribution to NK cell–mediated cytolysis was studied by specific antibody blockade. Chromium release cytotoxicity assays revealed chemotherapy-sensitive and chemotherapy-resistant osteosarcoma cell lines and osteosarcoma primary cultures to be sensitive to NK cell–mediated cytolysis. Cytolytic activity was strongly enhanced by IL-15 activation and was dependent on DNAM-1 and NKG2D pathways. Autologous and allogeneic activated NK cells lysed osteosarcoma primary cultures equally well. Osteosarcoma patient–derived NK cells were functionally and phenotypically unimpaired. In conclusion, osteosarcoma cells, including chemoresistant variants, are highly susceptible to lysis by IL-15-induced NK cells from both allogeneic and autologous origin. Our data support the exploitation of NK cells or NK cell–activating agents in patients with high-grade osteosarcoma

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Marmots from space: assessing population size and habitat use of a burrowing mammal using publicly available satellite images

Social, burrowing mammals such as prairie dogs, ground squirrels or marmots are keystone species in grassland ecosystems. Grasslands have been converted into cropland or pastures globally, yet it remains virtually unknown how this has affected the biogeography of burrowing mammals, as efficient, broad-scale survey methods are lacking. We aimed to test whether structures created by burrowing rodents can be reliably detected on publicly available, very-high-resolution satellite images, in order to assess rodent distribution and abundance. We identified burrows of Bobak marmot (Marmota bobak), a keystone burrowing steppe rodent, on 1300 randomly selected plots of 1 km diameter (78.53 ha) across the species’ range (~950 000 km²) in Kazakhstan and southern Russia using Google Earth and Bing images. We then used burrow occurrences and species distribution models to map marmot distribution. We assessed how marmot occurrence and density vary across land-use types. We also combined satellite-based burrow densities and ground-survey data to derive a new population estimate for the species across Kazakhstan. We mapped a total of 7425 burrows from the satellite imagery. Field visits at a subsample of burrows suggested that burrow occurrence was detected reliably. Broad-scale marmot distribution was mainly determined by summer rainfall, land use and elevation. Occurrence probability was highest on arable croplands, followed by abandoned croplands and grazed steppe. The current Bobak marmot population size for Kazakhstan was estimated at 6.1 (±2.4) million individuals. Our results demonstrate that publicly available, very-high-resolution images can be used to reliably map the distribution of burrowing mammals across large geographic scales. The observed and predicted distributions indicate that the Bobak's range has remained almost unchanged in Kazakhstan since the 1950s, despite several drastic episode of land-use change. This suggests that burrowing mammals can be remarkably resilient to land-use pressure, questioning prevailing narratives of population collapse in these species following agricultural expansion

Tumor Selective Hyperthermia Induced by Short-Wave Capacitively-Coupled RF Electric-Fields

<div><p>There is a renewed interest in developing high-intensity short wave capacitively-coupled radiofrequency (RF) electric-fields for nanoparticle-mediated tumor-targeted hyperthermia. However, the direct thermal effects of such high-intensity electric-fields (13.56 MHZ, 600 W) on normal and tumor tissues are not completely understood. In this study, we investigate the heating behavior and dielectric properties of normal mouse tissues and orthotopically-implanted human hepatocellular and pancreatic carcinoma xenografts. We note tumor-selective hyperthermia (relative to normal mouse tissues) in implanted xenografts that can be explained on the basis of differential dielectric properties. Furthermore, we demonstrate that repeated RF exposure of tumor-bearing mice can result in significant anti-tumor effects compared to control groups without detectable harm to normal mouse tissues.</p></div

Dielectric properties of tumor tissues.

<p><i>Ex-vivo</i> dielectric spectroscopy of tumors (Hep3B and MDA PATC-3 xenografts) and normal tissues (liver and pancreas) was performed. Permittivity (ε’) and imaginary permittivity (ε”) are shown for each tissue. <i>(n = 4–15, Solid line represents mean, dashed line represents standard deviation, and vertical dotted line represents frequency of 13.56 MHz). To compare permittivity values of normal and tumor tissues, two-tailed unpaired Student’s t-test was performed. p<0.01 for all datasets.</i></p

Dielectric properties of normal mouse tissues.

<p><i>Panel A, B. Ex-vivo</i> dielectric spectroscopy of normal mouse tissues was performed. Permittivity (ε’) and imaginary permittivity (ε”) are shown for each tissue. <i>(n = 3–15, Solid line represents mean, dashed line represents standard deviation, and vertical dotted line represents frequency of 13.56 MHz)). Panel C.</i> Variation of tumor dielectric properties with respect to tumor mass. <i>The</i> permittivity (ε’) and imaginary permittivity (ε”) values of MDA PATC-3 xenografts with variable mass are shown at 13.56 MHz. <i>(n = 3–6, data points represent mean, and error bars represent standard deviation). Panel D.</i> Areas of spontaneous necrosis were seen in untreated orthotopic xenografts, which were not seen in adjacent normal liver or pancreas. A representative figure of a HepG2 xenograft is shown at the tumor margin. Similar observations were noted for Hep3B, Panc1 and MDA PATC-3 xenografts <i>(not shown)</i>.</p

Thermal dose quantification in Hep3B and MDA PATC-3 xenografts under RF field exposure (13.56 MHz, 600 W).

<p><i>Panel A.</i> Hep3B xenograft and normal mouse liver temperatures were measured in real-time using fiber optic thermography while abdominal surface/skin temperatures were measured using infrared thermography. RF exposure was started at a tumor temperature of 35°C. <i>(n = 6). Panel B.</i> MDA PATC-3 xenograft and intra-peritoneal temperatures were measured in real-time using fiber optic thermography while abdominal surface/skin temperatures were measured using infrared thermography. RF exposure was started at a tumor temperature of 37°C. <i>(n = 9). (Solid line represents mean, dashed line represents standard deviation, and vertical dotted line represents frequency of 13.56 MHz).</i></p