Bioimaging approaches for quantification of individual cell behavior during cell fate decisions.

Tracking individual cells has allowed a new understanding of cellular behavior in human health and disease by adding a dynamic component to the already complex heterogeneity of single cells. Technically, despite countless advances, numerous experimental variables can affect data collection and interpretation and need to be considered. In this review, we discuss the main technical aspects and biological findings in the analysis of the behavior of individual cells. We discuss the most relevant contributions provided by these approaches in clinically relevant human conditions like embryo development, stem cells biology, inflammation, cancer and microbiology, along with the cellular mechanisms and molecular pathways underlying these conditions. We also discuss the key technical aspects to be considered when planning and performing experiments involving the analysis of individual cells over long periods. Despite the challenges in automatic detection, features extraction and long-term tracking that need to be tackled, the potential impact of single-cell bioimaging is enormous in understanding the pathogenesis and development of new therapies in human pathophysiology.

Cancer Cell Fitness Is Dynamic.

Several phenotypes that impact the capacity of cancer cells to survive and proliferate are dynamic. Here we used the number of cells in colonies as an assessment of fitness and devised a novel method called Dynamic Fitness Analysis (DynaFit) to measure the dynamics in fitness over the course of colony formation. DynaFit is based on the variance in growth rate of a population of founder cells compared with the variance in growth rate of colonies with different sizes. DynaFit revealed that cell fitness in cancer cell lines, primary cancer cells, and fibroblasts under unhindered growth conditions is dynamic. Key cellular mechanisms such as ERK signaling and cell-cycle synchronization differed significantly among cells in colonies after 2 to 4 generations and became indistinguishable from randomly sampled cells regarding these features. In the presence of cytotoxic agents, colonies reduced their variance in growth rate when compared with their founder cell, indicating a dynamic nature in the capacity to survive and proliferate in the presence of a drug. This finding was supported by measurable differences in DNA damage and induction of senescence among cells of colonies. The presence of epigenetic modulators during the formation of colonies stabilized their fitness for at least four generations. Collectively, these results support the understanding that cancer cell fitness is dynamic and its modulation is a fundamental aspect to be considered in comprehending cancer cell biology and its response to therapeutic interventions. SIGNIFICANCE: Cancer cell fitness is dynamic over the course of the formation of colonies. This dynamic behavior is mediated by asymmetric mitosis, ERK activity, cell-cycle duration, and DNA repair capacity in the absence or presence of a drug.

Dipyridamole impairs autophagic flux and exerts antiproliferative activity on prostate cancer cells.

Autophagy is a cellular bulk degradation process used as an alternative source of energy and metabolites and implicated in various diseases. Inefficient autophagy in nutrient-deprived cancer cells would be beneficial for cancer therapy making its modulation valuable as a therapeutic strategy for cancer treatment, especially in combination with chemotherapy. Dipyridamole (DIP) is a vasodilator and antithrombotic drug. Its major effects involve the block of nucleoside uptake and phosphodiestesase inhibition, leading to increased levels of intracellular cAMP. Here we report that DIP increases autophagic markers due to autophagic flux blockage, resembling autophagosome maturation and/or closure impairment. Treatment with DIP results in an increased number of autophagosomes and autolysosomes and impairs degradation of SQSTM1/p62. As blockage of autophagic flux decreases the recycling of cellular components, DIP reduced the intracellular ATP levels in cancer cells. Autophagic flux blockage was neither through inhibition of lysosome function nor blockage of nucleoside uptake, but could be prevented by treatment with a PKA inhibitor, suggesting that autophagic flux failure mediated by DIP results from increased intracellular levels of cAMP. Treatment with DIP presented antiproliferative effects in vitro alone and in combination with chemotherapy drugs. Collectively, these data demonstrate that DIP can impair autophagic degradation, by preventing the normal autophagosome maturation, and might be useful in combination anticancer therapy.

Os ramos colaterais da aorta abdominal em jaguatirica (Leopardus pardalis) / The collateral branches from abdominal aorta in ocelot (Leopardus pardalis)

A jaguatirica (Leopardus pardalis) é uma das espécies de felino silvestre que pouco foi investigada quanto a sua morfologia. Assim, o estudo objetivou detalhar a origem e distribuição dos ramos colaterais da aorta abdominal deste animal. [...] A aorta abdominal do L. pardalis teve origem entre T12 e L1, sendo a artéria celíaca o primeiro ramo visceral no sentido crânio-caudal, resultando nas artérias hepática, gástrica esquerda e esplênica. A artéria mesentérica cranial surgiu como segundo ramo da aorta abdominal, originando as artérias jejunais. Na sequência localizamos artéria pancreáticoduodenal caudal, artérias ileais, artérias ileocólicas, artérias renais direita e esquerda, artérias adrenais direita e esquerda e artérias ováricas ou testiculares direita e esquerda. Parietalmente, a aorta abdominal originou em média seis ramos lombares, bem como a artéria frenicoabdominal, as artérias circunflexas ilíacas profundas e artérias ilíacas externa e interna. A aorta abdominal gerou ainda a artéria mesentérica caudal, a qual dividiu-se em artérias cólica esquerda e retal cranial. A artéria cólica esquerda seguiu cranialmente paralela ao cólon descendente irrigando-o, originando em média 18 ramos, e anastomosando-se com a artéria cólica média. A artéria retal cranial seguiu em direção caudal distribuindo oito ramos à porção final do cólon descendente e ao reto, e uniu-se com a artéria retal média. Por fim, a aorta abdominal emitiu como ramo terminal a artéria sacral mediana. A vascularização arterial abdominal desta espécie é bastante semelhante ao descrito em felinos domésticos e demais mamíferos, com diferenças quanto ao número de artérias jejunais e origem das artérias renais.

The ocelot (Leopardus pardalis) is a species of wild cat that little has been investigated by their morphology. Thus, the study aimed to detail the origin and distribution of collateral branches from abdominal aorta this animal. [...]The abdominal aorta of L. pardalis originated between T12 and L1, and the celiac artery was first visceral branch in the craniocaudal direction, resulting in the hepatic a., gastric left a. and splenic a.. The caudal pancreaticoduodenal a., ileal aa., ileocolic aa., right and left renal aa., right and left adrenal aa., and. right and left ovarian aa. or testicular aa. were founded after. Parietally the abdominal aorta yielded an average of six lumbar branches, as well as frenicoabdominal aa., deep circumflex iliac aa. and external and internal iliac aa.. The abdominal aorta still originated the caudal mesenteric a., which is divided into the left colic a. and the cranial rectal a.. The left colic artery followed cranially parallel to the descending colon irrigating it, originating an average of 18 branches, and anastomosing with middle colic a.. The cranial rectal artery followed caudally emerging into eight branches uniting with the final portion of the descending colon and the rectum, together with the rectal middle a.. Finally, the abdominal aorta emerged as the terminal branch, the median sacral. The abdominal arterial vascularization of the ocelot is quite similar to that described in domestic cats, with differences in the number of jejunal arteries and origin of the renal arteries. The abdominal arterial vasculature of this species is quite similar to that described in domestic cats and other mammals, differing in number of the jejunal arteries and origin of the renal arteries.

Morphology of sympathetic chain in Saguinus niger.

Saguinus niger popularly known as Sauim, is a Brazilian North primate. Sympathetic chain investigation would support traumatic and/or cancer diagnosis which are little described in wild animals. The aim of this study was to describe the morphology and distribution of sympathetic chain in order to supply knowledge for neurocomparative research. Three female young animals that came death by natural causes were investigated. Animals were fixed in formaldehyde 10% and dissected along the sympathetic chain in neck, thorax and abdomen. Cranial cervical ganglion was located at the level of carotid bifurcation, related to carotid internal artery. In neck basis the vagosympathetic trunk divides into the sympathetic trunk and the parasympathetic vagal nerve. Sympathetic trunk ran in dorsal position and originated the stellate ganglia, formed by the fusion of caudal cervical and first thoracic ganglia. Vagal trunk laid ventrally to heart and formed the cardiac plexus. In abdomen, on the right side, were found the celiac ganglion and cranial mesenteric ganglion; in the left side these ganglia were fusioned into the celiac-mesenteric ganglion displaced closely to the celiac artery. In both sides, the caudal mesenteric ganglion was located near to the caudal mesenteric artery.