Davin Means, bringing glioblastoma cells into focus using the Bruker Ultima Investigator inverted multiphoton microscope.

Author: Davin Means | Major: Biology

I began working with Dr. Rajaram during the spring semester of 2020, but it wasn’t until my first grant term (Spring 2021), that I began to focus on glioblastomas and the differentiation between IDH mutant and IDH wildtype cells. Glioblastomas are responsible for 80% of primary malignant central nervous system tumors in adults and have one of the poorest prognoses of all cancers. Invasion into the surrounding brain (the main contributor to patient death) is stem cell mediated, and diffuse gliomas have malignancy grades I-IV with diagnostic genotypes of IDH mutant and IDH wildtype. Around 80% of low-grade diffuse gliomas (grades I and II) are IDH mutant, and 90% of high-grade diffuse gliomas (grades III and IV) are IDH wildtype. While standard therapy for these tumors involves surgical resectioning followed by adjuvant chemoradiation, outcomes are poor, and relapse is inevitable. The stem cell characteristics within glioma cells are what enable them to evade chemotherapy and radiation treatment and leave residual cell populations that cause resurgence and relapse. While this outcome is unfavorable, the fact that IDH mutant gliomas have improved patient prognosis compared to IDH wildtype gliomas, provides hope and an avenue for better treatment outcomes. Thus, it is of great importance to be able to detect, monitor, and elucidate, IDHm and IDHwt cells and their response to stress in order to therapeutically target diffuse gliomas and develop precision medicine and new medications to improve health outcomes for DG patients.

In this regard, the primary aim of my research is to perform nondestructive, label-free, quantitative, and real time monitoring of cellular metabolism of invasive IDHwt and IDHm diffuse gliomas using optical metabolic imaging and determine optical metabolic imaging’s sensitivity to differences between IDHm and IDHwt cells. Concurrently, we determine if exposure to radiation or hypoxic stress elicits real-time metabolic changes that let us clearly distinguish between IDHm and IDHwt cells, thus allowing for real time sensing of the phenotype of glioma stem cells undergoing mesenchymal transition (GSCMT). Thus, under the guidance of Christine Shamblin (the imaging technologist within Dr. Quinn’s lab, I began the exciting, yet daunting task of learning to operate the Bruker Ultima Investigator inverted multiphoton microscope. Furthermore, under the instruction of Dr. Rajaram, Christine Shamblin, and Paola Rodriguez, I learned how to operate MATLAB and analyze my imaging data.

To acquire TPEF intensity and lifetime images from IDHm and IDHwt cells, I first 2D cultured IDHm and IDHwt U87 cells in monolayer and seeded onto glass bottom dishes using complete growth medium (Eagle’s Minimal Essential Medium containing a 10% concentration of fetal bovine serum). After 24 hours, I imaged the cells using a Bruker Ultima Investigator inverted multiphoton microscope. NADH fluorescence was spectrally isolated using 755 nm excitation and a non-descanned detector with a 460±20 nm filter. FAD fluorescence was isolated in a second detector with a 525±25 nm filter using 900 nm excitation. NADH and FAD fluorescence were normalized by laser power and detector gain to facilitate comparisons across different days, and an optical redox ratio of FAD/[NADH+FAD] fluorescence was computed. I acquired images from at least six fields of view (600 x 600 μm) on each cell plate. A total of two cell plates were imaged in each cell group (mutant and wild type). I collected NADH fluorescence lifetime imaging (FLIM) data at the same locations where NADH and FAD intensity images were collected. The NADH fluorescence time decay was then fit to a biexponential model to quantify the ratio of free to protein-bound NADH and the mean fluorescence lifetime. Acquisition of fluorescence lifetime in addition to the optical redox ratio enabled me to differentiate between NADH and NADPH. These studies established changes in the mean lifetime, free to protein bound NAD(P)H contributions, and the optical redox ratio in response to IDH mutations in a controlled 2D culture model.

Hypoxia induces invasion and differentiation in GSCs and causes differential metabolic shifts in IDHm versus IDHwt cells. Therefore, hypoxia can be used to highlight the differences in real time sensing of the metabolic phenotype of GSCMT among different DG groups. During my second funded grant term, I utilized hypoxia and radiation as inducible factors to study both GSC differentiation and metabolism in real time using optical imaging. One group of cells grown in glass bottom wells was exposed to hypoxia (1% O2) for at least six hours and imaged with the two-photon microscope during hypoxic exposure. A separate group was exposed to radiation therapy (dose 2 Gy) and imaged with the two-photon microscope before and after radiation exposure. The cells were imaged every 15 minutes to ascertain hypoxia or radiation induced changes within the GSCs.

The preliminary findings from my first round of experimentation indicate that optical metabolic imaging is sensitive to the differentiation of U87 IDHm and IDHwt glioma cells. IDHwt glioma cells yielded higher baseline optical redox ratios and NADH lifetimes and lower baseline NADH intensities. Optical metabolic imaging also was able to differentiate IDHm and IDHwt in response to hypoxia and radiation dose therapy, with IDHm cells having lower optical redox ratios, higher NADH intensities, and lower NADH lifetimes under hypoxic conditions and increased radiation sensitivity. Over the course of the next semester and in preparation for my Honors College Thesis, I will determine the validity of these preliminary findings through multiple experimental repeats. Furthermore, these findings were sourced from fifth generation glioblastoma cells cultured within our lab. To maintain clinical significance, I will utilize primary cell culture shipped directly from Dr. Analiz Rodriguez’s lab at the University of Arkansas for Medical Sciences during my experimental repeats. I will also incorporate other radiation therapy dosages (10Gy especially) and the use of temozolomide (a prominent chemotherapy for the treatment of glioblastoma), in my future explorations in the use of optical metabolic imaging in differentiating between IDHm and IDHwt glioblastoma and elucidating their response to treatment.

There were many challenges within the second grant term. It took months to learn how to correctly and efficiently utilize MATLAB and the Bruker Ultima Investigator inverted multiphoton microscope. Furthermore, due to the pandemic, there has been an extensive shortage on base supplies such as media, micropipettes, and cell culture dishes. Despite, or perhaps even because of, these challenges, my funded grant terms have been exciting and intense learning experiences. The Honors College Research Grant has transformed me into a more patient, detail-oriented person, and under the guidance of Dr. Rajaram, my critical thinking skills in regard to experimental design have greatly improved. In reviewing relevant scientific literature, analyzing the experiments of the other peers within my lab, and navigating my own research, I have in depth training in the formation of hypotheses and scientific questions that are clear, concise, and testable. I have learned methods in prioritizing experiments and sub hypotheses in a way that is conducive to maximum use of time and resources. I have gained ways to reduce technical and biological variation and have learned the importance of replication in reducing experimental noise. I have learned that carefully planned, methodically organized, extensively detailed, and consistently documented experimentation minimizes the risk of confounding variables, and I have come to understand the importance of scientific rigor and transparency in reducing experimental bias. I’ve gained crucial micro pipetting skills and learned how to operate the Seahorse XFp Extracellular Flux Analyzer, radiation dose therapy equipment, MATLAB, and the Bruker Ultima Investigator inverted multiphoton microscope. Furthermore, in working with Dr. Rajaram, Lisa Rebello, Christine Shamblin, Paola Rodriguez, and other peers in the lab, I’ve become acquainted with the synergy, communication, and cooperation necessary for the advancement of science and research. I am extremely thankful to the Honors College, Dr. Rajaram, and my lab peers for two semesters of phenomenal research experience which have honed my critical thinking skills and empowered me to thrive within any immersion into the realm of science and research.