High-content microscopy reveals a morphological signature of bortezomib resistance.

Drug resistance is a challenge in anticancer therapy, particularly with targeted therapeutics and cytotoxic compounds. In many cases, cancers can be resistant to the drug prior to exposure, i.e., possess intrinsic drug resistance. However, we lack target-independent methods to anticipate resistance in cancer cell lines or characterize intrinsic drug resistance without a priori knowledge of its cause. We hypothesized that cell morphology could provide an unbiased readout of drug sensitivity prior to treatment. We therefore isolated clonal cell lines that were either sensitive or resistant to bortezomib, a well-characterized proteasome inhibitor and anticancer drug to which many cancer cells possess intrinsic resistance. We then measured high-dimensional single-cell morphology profiles using Cell Painting, a high-content microscopy assay. Our imaging- and computation-based profiling pipeline identified morphological features typically different between resistant and sensitive clones. These features were compiled to generate a morphological signature of bortezomib resistance, which correctly predicted the bortezomib treatment response in seven of ten cell lines not included in the training dataset. This signature of resistance was specific to bortezomib over other drugs targeting the ubiquitin-proteasome system. Our results provide evidence that intrinsic morphological features of drug resistance exist and establish a framework for their identification.

Pipeline for illumination correction of images for high-throughput microscopy.

The presence of systematic noise in images in high-throughput microscopy experiments can significantly impact the accuracy of downstream results. Among the most common sources of systematic noise is non-homogeneous illumination across the image field. This often adds an unacceptable level of noise, obscures true quantitative differences and precludes biological experiments that rely on accurate fluorescence intensity measurements. In this paper, we seek to quantify the improvement in the quality of high-content screen readouts due to software-based illumination correction. We present a straightforward illumination correction pipeline that has been used by our group across many experiments. We test the pipeline on real-world high-throughput image sets and evaluate the performance of the pipeline at two levels: (a) Z'-factor to evaluate the effect of the image correction on a univariate readout, representative of a typical high-content screen, and (b) classification accuracy on phenotypic signatures derived from the images, representative of an experiment involving more complex data mining. We find that applying the proposed post-hoc correction method improves performance in both experiments, even when illumination correction has already been applied using software associated with the instrument. To facilitate the ready application and future development of illumination correction methods, we have made our complete test data sets as well as open-source image analysis pipelines publicly available. This software-based solution has the potential to improve outcomes for a wide-variety of image-based HTS experiments.

Morphology-guided graph search for untangling objects: C. elegans analysis.

We present a novel approach for extracting cluttered objects based on their morphological properties. Specifically, we address the problem of untangling Caenorhabditis elegans clusters in high-throughput screening experiments. We represent the skeleton of each worm cluster by a sparse directed graph whose vertices and edges correspond to worm segments and their adjacencies, respectively. We then search for paths in the graph that are most likely to represent worms while minimizing overlap. The worm likelihood measure is defined on a low-dimensional feature space that captures different worm poses, obtained from a training set of isolated worms. We test the algorithm on 236 microscopy images, each containing 15 C. elegans worms, and demonstrate successful cluster untangling and high worm detection accuracy.

Death by edible mushroom: first report of Volvariella volvacea as an etiologic agent of invasive disease in a patient following double umbilical cord blood transplantation.

We describe a case of invasive fungal infection caused by Volvariella volvacea following double umbilical cord blood transplantation (UCBT). Although infections caused by several mushroom species have been documented, we believe this to be the first published report of invasive infection with Volvariella volvacea, an edible mushroom belonging to Agaricales.

Small cell carcinoma of the uterine cervix.

From 1962 to 1985, 2201 patients with invasive cervical cancer were staged, evaluated, and treated at the University of Kentucky Medical Center. After a thorough evaluation, 25 cases (1.1%) fulfilled the histologic criteria for small cell cancer defined by Reagan and coworkers. These patients were computer-matched for age, disease stage, and lesion size to 25 patients with large cell nonkeratinizing cancer and 25 patients with keratinizing squamous cell cancer. Morphometric analyses of nuclear size and maximum nuclear diameter were performed on all cases without knowledge of cell type. Small cell cancers were characterized by a nuclear area of 160 mu 2 or less and a maximum nuclear diameter of 16.2 mu, which was significantly lower than that for large cell tumors. Thirty-three percent of the small cell carcinomas stained positively for the neuroendocrine markers (neuron-specific enolase [NSE] and chromogranin [CGR]), whereas the remainder contained only epithelial markers such as cytokeratin (CYK) and epithelial membrane antigen (EMA). Small cell cancers were associated with a high frequency of lymph-vascular space invasion and a diminished lymphoplasmacytic response. Patients with small cell cancer had a significantly higher recurrence rate, particularly to extrapelvic sites, than the matched patients with large cell cancers, and their survival was lower. Clinical trials to determine the efficacy of adjuvant chemotherapy in the treatment of small cell cervical cancer are needed.