Chemoresistance is mediated by ovarian cancer leader cells in vitro.

BACKGROUND: Leader cells are a subset of cancer cells that coordinate the complex cell-cell and cell-matrix interactions required for ovarian cancer migration, invasion, tumour deposition and are negatively associated with progression-free survival and response to therapy. Emerging evidence suggests leader cells may be enriched in response to chemotherapy, underlying disease recurrence following treatment. METHODS: CRISPR was used to insert a bicistronic T2A-GFP cassette under the native KRT14 (leader cell) promoter. 2D and 3D drug screens were completed in the presence of chemotherapies used in ovarian cancer management. Leader cell; proliferative (Ki67); and apoptotic status (Cleaved Caspase 3) were defined by live cell imaging and flow cytometry. Quantitative real-time PCR defined "stemness" profiles. Proliferation was assessed on the xCELLigence real time cell analyser. Statistical Analysis was performed using unpaired non-parametric t-tests or one-way ANOVA and Tukey's multiple comparison post hoc. RESULTS: Leader cells represent a transcriptionally plastic subpopulation of ovarian cancer cells that arise independently of cell division or DNA replication, and exhibit a "stemness" profile that does not correlate with epithelial-to-mesenchymal transition. Chemotherapeutics increased apoptosis-resistant leader cells in vitro, who retained motility and expressed known chemo-resistance markers including ALDH1, Twist and CD44v6. Functional impairment of leader cells restored chemosensitivity, with leader cell-deficient lines failing to recover following chemotherapeutic intervention. CONCLUSIONS: Our data demonstrate that ovarian cancer leader cells are resistant to a diverse array of chemotherapeutic agents, and are likely to play a critical role in the recurrence of chemo-resistant disease as drivers of poor treatment outcomes.

Using Proales similis (Rotifera) for toxicity assessment in marine waters.

There is a need to develop more animal species for assessing toxicity in marine environments. Cyst-based toxicity tests using invertebrates are especially fast, technically simple, cost-effective, and sensitive to a variety of toxicants. Over the past 30 years, a variety of toxicity endpoints have been measured using the marine rotifer Brachionus plicatilis hatched from cysts, including mortality, reproduction, ingestion, swimming, enzyme activity, and gene expression. A consensus has developed that the most ecologically relevant toxicity measurements should be made using more than one species. Furthermore, it has been noted that the rotifer species toxicant sensitivity distribution is much broader than which endpoint is measured. This implies that toxicity should be measured with the simplest, fastest, least expensive test available on as many species as feasible. If a battery of test species is to be used to estimate toxicity, diapause egg-based toxicity tests that do not require culturing of test animals will be key. In this paper, we describe how diapause eggs of a new marine rotifer, Proales similis, can be produced, stored and hatched under controlled conditions to produce animals for toxicity tests. Methods are described for quantifying the toxicity of copper, mercury and cadmium based on mortality, ingestion, reproduction, and diapause egg hatching endpoints. We found that reproduction and ingestion endpoints were generally more sensitive to the metals than mortality or diapause egg hatching. When the copper sensitivity of P. similis was compared to Brachionus manjavacas and B. plicatilis using an ingestion test, similar EC50s were observed. In contrast, the B. rotundiformis ingestion EC50 for copper was about 4× more sensitive. Although diapause egg hatching was not the most sensitive endpoint, it is the most ecologically relevant for assessing sediment toxicity. Our discovery of diapausing eggs in the P. similis life cycle has created a conundrum. We have not observed males or sex in P. similis populations, which is a direct contradiction to the orthodox view of the monogonont rotifer life cycle. Work is needed to clarify how diapause egg production is accomplished by P. similis and whether sexual reproduction is involved.

Repurposed FDA-approved drugs targeting genes influencing aging can extend lifespan and healthspan in rotifers.

Pharmaceutical interventions can slow aging in animals, and have advantages because their dose can be tightly regulated and the timing of the intervention can be closely controlled. They also may complement environmental interventions like caloric restriction by acting additively. A fertile source for therapies slowing aging is FDA approved drugs whose safety has been investigated. Because drugs bind to several protein targets, they cause multiple effects, many of which have not been characterized. It is possible that some of the side effects of drugs prescribed for one therapy may have benefits in retarding aging. We used computationally guided drug screening for prioritizing drug targets to produce a short list of candidate compounds for in vivo testing. We applied the virtual ligand screening approach FINDSITEcomb for screening potential anti-aging protein targets against FDA approved drugs listed in DrugBank. A short list of 31 promising compounds was screened using a multi-tiered approach with rotifers as an animal model of aging. Primary and secondary survival screens and cohort life table experiments identified four drugs capable of extending rotifer lifespan by 8-42%. Exposures to 1 µM erythromycin, 5 µM carglumic acid, 3 µM capecitabine, and 1 µM ivermectin, extended rotifer lifespan without significant effect on reproduction. Some drugs also extended healthspan, as estimated by mitochondria activity and mobility (swimming speed). Our most promising result is that rotifer lifespan was extended by 7-8.9% even when treatment was started in middle age.

Freshwater toxicity testing using rehydrated Philodina sp. (Rotifera) as test animals.

Rotifers have become widely used in aquatic toxicology as a rapid screening test for toxicity. The commercial availability of diapausing embryos (cysts) have facilitated their popularity because test animals can be obtained without having to master the details of culturing. Other rotifer species have life stages capable of surviving desiccation and also could be used in non-culture systems for toxicity assessment. In this article, we describe a system for toxicity testing in freshwater based on rehydrating desiccated bdelloid rotifers in the genus Philodina. These animals can remain in this anhydrobiotic state for more than one year and then rehydrate within hours to provide animals for toxicity tests. We describe three endpoints: a 1.5 h ingestion test, a 24 h mortality test, and a five day reproductive test. The latter test requires feeding and a method using a dried commercial product is explained. Using desiccated rotifers and dried food in toxicity tests make this system especially attractive because of its flexibility and low threshold of biological expertise required to execute the tests. The use of the Philodina toxicity test is illustrated with four metals: copper, lead, mercury and cadmium. Reproduction generally was the most sensitive endpoint, with EC50s of 0.33, 0.44, 0.60, and 0.12 mg/L, respectively. Ingestion was a close second with EC50s of 0.13, 1.64, 0.64, and 6.26 mg/L, respectively.