Cell Biology 2016 ASCB Annual Meeting

An integrated platform real-time dynamic culturing and analysis of hypoxia with single cell resolution

Maintaining tissue homeostasis is dependent upon the complex interaction between cells and the surrounding milieu. One key factor impacting this dynamic network is the adequate supply of oxygen. Hypoxia is a hallmark of primary tumors, arising due to impaired vascularization and increased respiratory demand of rapidly proliferating cancer cells. Clinically, hypoxia-driven RNA signatures correlate with aggressive tumor behavior and adverse prognosis of many tumors. At the cell level, hypoxic responses are mediated by HIF (Hypoxia-inducible factor) transcription factors which regulate expression of genes that drive adaptation of resident cells. In fact, hypoxia has a major impact on critical aspects of metastatic disease including cell proliferation, metabolic capacity, immune response, invasion/metastasis, and resistance to chemotherapeutic intervention.

Unraveling the mechanisms underlying hypoxic responses is essential to developing therapeutics targeting tumor progression. Cell culture platforms capable of precise regulation and maintenance of microenvironments, including gas exchange, are essential for oncology research. Standard cell culture has two major limitations for studying effects of steady-state oxygen levels and hypoxia. First, media overlying cells presents substantial resistance to oxygen diffusion resulting in a significant lag in cell-level gas changes (relative to the gas phase). Second, and more significantly, due to convective mixing, diffusion resistance varies unpredictably making precise control of oxygen pressure, at the single cell level, difficult to achieve. Moreover, current platforms are not strictly designed for microscope-based visualization and thus cannot be employed for live imaging analysis of single cells.

Here we present a combined cell culture/imaging platform for analysis of hypoxic responses in real time. The culturing unit consists of a microfluidic plate and environmental control system. Micro-volume cultures enable more precise chemio-temporal and spatial control, including rapid changing of conditions. Plates are constructed with gas-permeable materials and aeration channels to minimize diffusive effects. The controller regulates pressurized cell loading, programmable media perfusion, temperature, and gas levels. Due it to small size (96-well plate) and high optical clarity, the plate can be paired with a digital inverted microscope enabling automated dynamic analysis. Fluorescent visualization further permits selective discrimination of unique cell types and changes in expression of labeled proteins. In summary, the synergistic interplay between micro-culture and real-time imaging provides an automated framework for studying the onset of hypoxia as well as the action of potential inhibitory therapeutics.

Understanding the complexity of budding yeast colonies can give insight into metabolite exchange interactions, growth parameters and cell lifespan. Yet, it is difficult to monitor yeast colonies due to their ability to escape the focal planes as well as imaging issues associated with rapid growth and overcrowding. To address these challenges, we have developed a microfluidic device that mechanically immobilizes cell communities under an array of 72 x 72 x 0.65 µm (L x W x H) soft trap areas, positioned within a 3mm x 3mm x 10 µm (L x W x H) chamber. This trap design enables focused monolayer cell proliferation under the elastic ceiling and continuous removal of excess cells from the trap site to prevent cell congestion. The platform works in tandem with a micro-environmental system equipped with programmable and automated control of media perfusion (up to 6 reagents), gas, and temperature levels. In this 72 hour experiment, we used high magnification (60x) time-lapse imaging to observe single budding yeast cells develop into multiple colonies in a controlled microenvironment. After reaching steady state growth, colonies were exposed to a nutrient gradient and cell division was quantitatively measured using image analysis tools. Software-driven perturbation provided precise media flow to the thriving colony which was verified by two-color fluorescence imaging to measure viability. In summary, we demonstrate the use of a highly sophisticated microfluidic cell culture platform enabling the analysis of dynamic colony growth and response in real time to automated, user-defined changes in micro-environment.

Protein translational alterations can increase the overall rate of protein synthesis as well as activate regulatory mechanisms leading to the translation of specific messenger RNAs for proteins that promote cancer progression and survival. Several components of translation initiation complex have been found to be mutated, post-translationally modified, or differentially expressed, and have been shown to act as oncogenes. We have developed a Luminex® bead based multiplex immunoassay that allows for the simultaneous detection of multiple components of the translation machinery in a single well, including phosphorylated eIF2a, eIF-4B, eIF4E, eIF4G and 4E-BP1 as well as total 4E-BP1. Using the Protein Translation 6-plex kit, we analyzed the levels of phosphorylated and total proteins in human cancer cell lines as well as breast and colon tumor extracts. We observed significant phosphorylation of all the analytes in both HeLa and Jurkat cell lines. Anisomycin, a protein translation inhibitor, led to a decrease in phosphorylation of eIF2a, eIF-4B, eIF4E and eIF4G, but not phospho- nor total 4E-BP1, in a time-dependent manner. Furthermore, the phosphorylation of eIF2a, eIF-4B, eIF4E and eIF4G by 2 hours of anisomycin treatment correlated with the time of activation of Caspase-3, a marker of apoptosis. In order to determine if these analytes in the Protein Translation 6-plex kit are hyperphosphorylated in tumors, we ran matched normal and tumor tissue samples from the same patients for both breast and colon cancer. Hyperphosphorylation was observed for four of the analytes (eIF2a, eIF-4B, eIF4E and eIF4G) in some tumors, with hyperphosphorylated eIF4E showing the highest frequency. In addition, increased expression levels of total 4E-BP1 were observed in all tumors tested. Interestingly, the phospho-4E-BP1 levels when normalized to total 4E-BP1 levels decreased by nearly 2-fold in all tumor samples tested, even though total 4E-BP1 levels increased.

We have developed a Luminex® bead based multiplex immunoassay that allows for the simultaneous detection of multiple proteins in the protein translation initiation complex in a single well, including phosphorylated eIF2a, eIF-4B, eIF4E, eIF4G and 4E-BP1, and total protein level of 4E-BP1. These studies demonstrate the validity of using the Protein Translation 6-plex kit to examine the mechanism of protein translation.

Solid tumors grow in a three-dimensional (3D) spatial conformation, resulting in a heterogeneous exposure to oxygen and nutrients as well as to other physical and chemical stresses. To mimic the 3D spatial conformation, 3D in vitro culture models have been used in cancer research since the diffusion-limited distribution of oxygen (hypoxia), nutrients, metabolites, and signaling molecules is not mimicked in conventional two-dimensional (2D) monolayer cultures. One of the 3D in vitro culture models, the tumorsphere culture is an emerging model for studying and expanding the cancer stem cell (CSC) population. E006AA cell line is a spontaneously immortalized cell line derived from a Gleason 6 localized prostate cancer in a hormone-naive prostate cancer patient of African American descent. This cell line expresses androgen receptor and was repeatedly reported to be non-tumorigenic in nude mice. However this cell line forms continuously growing tumor in NOG-SCID triple-deficient mice (i.e., NOG-SCID mice not having NK, B, and T-cells) and an establishment of highly tumorgenic subline E006AA-hT was reported suggesting this cell line has a potential for CSC proliferation by 3D sphere cultures. Here we report tumorsphere cultures of E006AA cells by a new 3D sphere culture media system. The cells showed continuous proliferation supported during serial passage of 3D tumorsphere cultures. In addition to the stable proliferation, increases of Aldehyde Dehydrogenase (ALDH) expressing CSC population were observed with the increase of passages of 3D tumorsphere cultures.

Protein kinase B (PKB/AKT) proteins, including three isoforms Akt1, Akt2, and Akt3, are downstream of PI3-kinase signals from receptor tyrosine kinases (RTKs) and influence cell growth, proliferation, and apoptosis. Knock-out animals and isoform-specific inhibitors have been developed to illustrate the contribution of each Akt isoform and its effects on regulation of metabolism and cancer development/progression. Using Luminex technology, we have developed multiplex Akt isoform-specific immunoassays for simultaneous quantification of Akt1/pAkt1, Akt2/pAkt2, and Akt3/pAkt3 proteins for human and mouse. Isoform specificity was validated using recombinant Akt proteins for each isoform. For each multiplex assay, these proteins can be used to generate standard curves for the quantification of total and phospho-Akt isoforms in tissue extracts and cell lysates. Here we report the methods and applications of these bead-based multiplex isoform-specific Akt immunoassays in dissecting the S473 phosphorylated Akt and total isoform-specific Akt levels in mouse and human in vivo/in vitro models.

In the human models, we have characterized the Akt isoforms (Akt1, Akt2, Akt3) and their phospho-isoforms (pAkt1, pAkt2, pAkt3) in human breast and colon cancer tissue as well as the matched adjacent normal tissue. We have also generated the Akt isoform profiles showing differential expression levels of Akt1, pAkt1, Akt2, pAkt2, Akt3, and pAkt3 in a panel of the tumor cell lines (including MCF7, MDA-MB-231, HeLa, Jurkat, HepG2, U87, A549 tumor cells, +/- stimuli) and cultured stroma cells (including HUVEC, HFF1, NIH 3T3, bEND.3). In the Streptozotocin (STZ)-induced diabetes mouse model, we investigated the total and phosphorylated Akt isoforms in liver, pancreas, pituitary gland, prostate, skeletal muscle, and brain tissues. Expression of Akt1 and Akt2 was detected in all tissue extracts. Akt3 expression was enriched in the brain compared to the other examined tissues, where Akt3 was detected at low levels. STZ treatment increased expression of all three Akt isoforms in the brain, particularly in the cerebral cortex. While the preliminary studies indicated expression of all three Akt isoforms in the brain, only Akt3 exhibited detectable levels of phosphorylation in the brain extracts (hippocampus, cortex, and the rest of the brain). Altogether, the results of this study demonstrate the utility of these human/mouse isoform-specific Akt multiplex panels as useful tools for studying the tissue protein biomarker profiles of Akt isoforms in cancer and metabolism research.