Amani A. Gillette

The secret languages of coevolved symbioses: Insights from the Euprymna scolopes-Vibrio fischeri symbiosis

Recent research on a wide variety of systems has demonstrated that animals generally coevolve with their microbial symbionts. Although such relationships are most often established anew each generation, the partners associate with fidelity, i.e., they form exclusive alliances within the context of rich communities of non-symbiotic environmental microbes. The mechanisms by which this exclusivity is achieved and maintained remain largely unknown. Studies of the model symbiosis between the Hawaiian squid Euprymna scolopes and the marine luminous bacterium Vibrio fischeri provide evidence that the interplay between evolutionarily conserved features of the innate immune system, most notably MAMP/PRR interactions, and a specific feature of this association, i.e., luminescence, are critical for development and maintenance of this association. As such, in this partnership and perhaps others, symbiotic exclusivity is mediated by the synergism between a general animal-microbe language and a secret language that is decipherable only by the specific partners involved.

The first engagement of partners in the Euprymna scolopes–Vibrio fischeri symbiosis is a two-step process initiated by a few environmental symbiont cells

We studied the Euprymna scolopes-Vibrio fischeri symbiosis to characterize, in vivo and in real time, the transition between the bacterial partners free-living and symbiotic life styles. Previous studies using high inocula demonstrated that environmental V. fischeri cells aggregate during a 3 h period in host-shed mucus along the light organs superficial ciliated epithelia. Under lower inoculum conditions, similar to the levels of symbiont cells in the environment, this interaction induces haemocyte trafficking into these tissues. Here, in experiments simulating natural conditions, microscopy revealed that at 3 h following first exposure, only ∼ 5 V. fischeri cells aggregated on the organ surface. These cells associated with host cilia and induced haemocyte trafficking. Symbiont viability was essential and mutants defective in symbiosis initiation and/or production of certain surface features, including the Mam7 protein, which is implicated in host cell attachment of V. cholerae, associated normally with host cilia. Studies with exopolysaccharide mutants, which are defective in aggregation, suggest a two-step process of V. fischeri cell engagement: association with host cilia followed by aggregation, i.e. host cell-symbiont interaction with subsequent symbiont-symbiont cell interaction. Taken together, these data provide a new model of early partner engagement, a complex model of host-symbiont interaction with exquisite sensitivity.

Shaping the microenvironment: evidence for the influence of a host galaxin on symbiont acquisition and maintenance in the squid-vibrio symbiosis

Most bacterial species make transitions between habitats, such as switching from free living to symbiotic niches. We provide evidence that a galaxin protein, EsGal1, of the squid Euprymna scolopes participates in both: (i) selection of the specific partner Vibrio fischeri from the bacterioplankton during symbiosis onset and, (ii) modulation of V.u2009fischeri growth in symbiotic maintenance. We identified two galaxins in transcriptomic databases and showed by quantitative reverse-transcriptase polymerase chain reaction that one (esgal1) was dominant in the light organ. Further, esgal1 expression was upregulated by symbiosis, a response that was partially achieved with exposure to symbiont cell-envelope molecules. Confocal immunocytochemistry of juvenile animals localized EsGal1 to the apical surfaces of light-organ epithelia and surrounding mucus, the environment in which V.u2009fischeri cells aggregate before migration into the organ. Growth assays revealed that one repeat of EsGal1 arrested growth of Gram-positive bacterial cells, which represent the cell type first winnowed during initial selection of the symbiont. The EsGal1-derived peptide also significantly decreased the growth rate of V.u2009fischeri in culture. Further, when animals were exposed to an anti-EsGal1 antibody, symbiont population growth was significantly increased. These data provide a window into how hosts select symbionts from a rich environment and govern their growth in symbiosis.

Protein-bound NAD(P)H Lifetime is Sensitive to Multiple Fates of Glucose Carbon

While NAD(P)H fluorescence lifetime imaging (FLIM) can detect changes in flux through the TCA cycle and electron transport chain (ETC), it remains unclear whether NAD(P)H FLIM is sensitive to other potential fates of glucose. Glucose carbon can be diverted from mitochondria by the pentose phosphate pathway (via glucose 6-phosphate dehydrogenase, G6PDH), lactate production (via lactate dehydrogenase, LDH), and rejection of carbon from the TCA cycle (via pyruvate dehydrogenase kinase, PDK), all of which can be upregulated in cancer cells. Here, we demonstrate that multiphoton NAD(P)H FLIM can be used to quantify the relative concentrations of recombinant LDH and malate dehydrogenase (MDH) in solution. In multiple epithelial cell lines, NAD(P)H FLIM was also sensitive to inhibition of LDH and PDK, as well as the directionality of LDH in cells forced to use pyruvate versus lactate as fuel sources. Among the parameters measurable by FLIM, only the lifetime of protein-bound NAD(P)H (τ2) was sensitive to these changes, in contrast to the optical redox ratio, mean NAD(P)H lifetime, free NAD(P)H lifetime, or the relative amount of free and protein-bound NAD(P)H. NAD(P)H τ2 offers the ability to non-invasively quantify diversions of carbon away from the TCA cycle/ETC, which may support mechanisms of drug resistance.

Organotypic microfluidic breast cancer model reveals starvation-induced spatial-temporal metabolic adaptations

Background Ductal carcinoma in situ (DCIS) is the earliest stage of breast cancer. During DCIS, tumor cells remain inside the mammary duct, growing under a microenvironment characterized by hypoxia, nutrient starvation, and waste product accumulation; this harsh microenvironment promotes genomic instability and eventually cell invasion. However, there is a lack of biomarkers to predict what patients will transition to a more invasive tumor or how DCIS cells manage to survive in this harsh microenvironment. Methods In this work, we have developed a microfluidic model that recapitulates the DCIS microenvironment. In the microdevice, a DCIS model cell line was grown inside a luminal mammary duct model, embedded in a 3D hydrogel with mammary fibroblasts. Cell behavior was monitored by confocal microscopy and optical metabolic imaging. Additionally, metabolite profile was studied by NMR whereas gene expression was analyzed by RT-qPCR. Findings DCIS cell metabolism led to hypoxia and nutrient starvation; revealing an altered metabolism focused on glycolysis and other hypoxia-associated pathways. In response to this starvation and hypoxia, DCIS cells modified the expression of multiple genes, and a gradient of different metabolic phenotypes was observed across the mammary duct model. These genetic changes observed in the model were in good agreement with patient genomic profiles; identifying multiple compounds targeting the affected pathways. In this context, the hypoxia-activated prodrug tirapazamine selectively destroyed hypoxic DCIS cells. Interpretation The results showed the capacity of the microfluidic model to mimic the DCIS structure, identifying multiple cellular adaptations to endure the hypoxia and nutrient starvation generated within the mammary duct. These findings may suggest new potential therapeutic directions to treat DCIS. In summary, given the lack of in vitro models to study DCIS, this microfluidic device holds great potential to find new DCIS predictors and therapies and translate them to the clinic.

Microfluidic models and optical imaging to monitor microenvironmental stimuli for breast cancer invasion (Conference Presentation)

Breast cancer is the most common cancer in women and usually originates from the epithelial cells of the mammary duct. During the earliest stage, the tumor cells remain trapped inside the duct, generating an indolent “Ductal Carcinoma In Situ” (DCIS). However, DCIS cells can break the wall of the mammary duct, invade the surrounding stromal tissue, and metastasize to other organs. Unfortunately, the mechanisms that trigger this invasion remain elusive. One hypothesis is that the harsh microenvironment (i.e., hypoxia, nutrient-starvation) of DCIS leads to epithelial cell invasion into the stroma. In this work, a microfluidic DCIS model was developed including DCIS and normal epithelial cells, fibroblasts, and blood vessel-like structures. Optical metabolic imaging (OMI) of the metabolic co-factors NAD(P)H and FAD was used to assess spatial gradients in metabolism within the microfluidic model. We observed that the epithelial cells hindered the penetration of nutrients inside the lumen, and led to severe hypoxia. This hypoxia exerted OMI-measured metabolic changes in both normal and DCIS cells. Nuclear magnetic resonance (NMR) metabolomics analysis showed DCIS-specific metabolic differences compared with normal cells, in agreement with OMI results. Metabolic changes included an increase in glycolysis products and production of cancer-associated metabolites. A hypoxia-activated prodrug (Tirapazamine) selectively destroyed the hypoxic tumor cells inside the lumen, without affecting cells at the lumen surface or the fibroblasts in the matrix. In the future, OMI of this microfluidic model will be used to test metabolic therapies that prevent the growth of DCIS into an invasive tumor.

Abstract 3312: A novel plate-based assay for screening autophagic activity in 2D and 3D cell culture models

The critical importance of autophagy in cell health and its proposed role in disease-relevant biology, including cancer, inflammation, and immunology, has increased the need for more effective assays to screen for agents that modulate autophagic activity. Here we utilize NanoLuc Binary Technology (NanoBiT) to develop a homogeneous plate-based assay to measure autophagic flux in cell culture models. In this approach, an exogenous LC3B (Atg8) fusion protein was tagged on its N-terminus with an 11 amino acid peptide (HiBiT) and stably expressed in mammalian cells, including U2OS and HEK293. After exposure to various treatment conditions, cellular levels of this novel autophagy reporter were determined by addition of a lytic detection reagent containing Large BiT (LgBiT). LgBiT rapidly associates with HiBiT in the cell lysate, producing a bright, luminescent enzyme in the presence of the furimazine substrate. The bright signal allows low levels of expression of the reporter, maximizing the assay response, and the signal is stable, allowing assay of multiple 96- or 384-well plates in the same experiment. In response to autophagic stimuli, including nutrient deprivation and various mTORC inhibitors (e.g., PP242 and rapamycin), autophagic degradation of expressed LC3 reporter was evident by reduced assay signal. In contrast, in response to both upstream (e.g., 3-MA and wortmannin) and downstream (e.g., bafilomycin A1 and chloroquine) inhibitors of the autophagy pathway, degradation of the autophagic reporter was effectively blocked and assay signal was consistently increased as predicted. Compound effects were time dependent and stratified according to expected potency and efficacy of the test agents employed. The use of a mutant reporter based on LC3G120A further demonstrated the specificity of the wild-type LC3 reporter for the detection of autophagic activity. When assayed in 384-well plates with automation, HEK293 autophagy reporter cells produced Z’ values of ~0.7 in response to autophagy induction with PP242, while subsequent blockade of autophagy with bafilomycin A1 resulted in Z’ values of ~0.8. This data, and subsequent LOPAC library screening, indicates the potential utility of this assay method for HTS applications. In addition, the HEK293 autophagy reporter cells can be induced to form 3D cell spheroids, thus allowing investigation of assay performance in this more complex model. Autophagy reporter levels increased with increasing spheroid size (up to 650 μm diameter tested) in a manner proportional to a surrogate measure of viable cell number. Importantly, both induction and inhibition of autophagic activity was easily detected following PP242 and bafilomycin A1 treatment, respectively. Using this novel plate-based assay system for the determination of autophagic flux, it is possible to screen test agents and quantitatively determine both the potency and efficacy of autophagy modulation. Citation Format: Dan F. Lazar, Amani A. Gillette, Braeden L. Butler, Christopher T. Eggers, Brock F. Binkowski, Gediminas Vidugiris, Michael R. Slater, Dongping Ma, James J. Cali. A novel plate-based assay for screening autophagic activity in 2D and 3D cell culture models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3312. doi:10.1158/1538-7445.AM2017-3312