To supplement and modify the diagnosis and clinical research of B-cell Chronic Lymphocytic Leukemia (B-CLL), a new method based on cell imaging and image processing was developed and applied to the B-CLL patient samples. The fluorophore-labelled leukemia cells were clearly visualized, reflecting the positive/negative expression of the corresponding surface markers and their distribution. Computer algorithms were devised and used to analyze a large number of images. The fluorescence intensity of the labelled antibodies on a given cell directly reflects the expression of the corresponding surface markers. The morphology and size of leukemia cells were not identical even in the same patient’s sample and the size variation does not correlate with the number of surface markers. The amount of each surface marker was approximately fixed for each patient, but there were some relationships, for instance, the number of CD19 and CD38 markers were correlated to each other. The heterogeneous expression of surface markers confirmed an assumption that surface markers have their preferred membrane positions. One of the most important results is that the cell imaging and our image processing method has provided an alternative and reliable way to diagnose B-CLL and new insights in the prognosis of subtype of B-CLL.

<p>It has been long known that many types of cancers have high metabolic requirements and use reprogrammed metabolism to support cellular activities. The first identified metabolic alteration in cancer cells was elevated glucose uptake, glycolysis activity and lactate production even in the presence of oxygen. This metabolic program, termed aerobic glycolysis or the Warburg effect, provides cells with energy as well as biosynthetic substrates to sustain cell survival and rapid cell proliferation. Cancer metabolism is closely linked to genetic mutations and oncogenic signaling pathways, such as PI3K/Akt, cMyc and HIF pathways. These oncogenic signals can direct metabolic reprogramming while changes in metabolic status can regulate activities of these signaling pathways in turn. In addition to glucose, later studies also found utilization of alternate nutrients in cancer cells, including glutamine and lipids. Glutamine is the second major metabolic fuel and can be converted to various substrates to support cell bioenergetics needs and biosynthetic reactions. Usage of metabolic fuels in cancer cells, however, is variable. While certain cancers display addiction to one type of nutrient, others are capable of using multiple nutrients. </p><p>The unique metabolic features of cancer cells raise the possibility of targeting metabolism as a novel therapeutic approach for cancer treatment. Using pharmacological inhibitors, previous research has provided corroborating evidence that metabolic stress can impact survival and growth of proliferative cancer cells by regulating cell apoptotic machinery and cell cycle checkpoints. Due to lack of genetic tools and side effects from these inhibitors, however, mechanistic understanding of cell response to metabolic inhibition was limited in these studies. More importantly, how metabolic stress affects cancer progression in a physiological condition has not yet been well investigated. Lastly, current research has not examined metabolic program in indolent cancers and the metabolic requirements and activities in less proliferative cells also remain to be understood.</p><p>This work examines nutrients utilization in B cell derived acute and chronic leukemia (B-ALL and B-CLL). B-ALL is an aggressive form of leukemia. Using cell lines and primary patient samples, we found B-ALL cells primarily used glucose through aerobic glycolysis, similar to other proliferative cancer cells. B-ALL cells were also more sensitive to inhibition of glycolysis than normal B cells. Employing an untargeted metabolomics profiling in combination with isotope labeled glucose tracing approach, we show in a B-ALL model that genetic ablation of glucose transporter Glut1 partially reduced glucose uptake, sufficiently hindered anabolic pathways and promoted catabolic metabolism. This metabolic shift led to sharply curtailed B-ALL proliferation in vitro and reduced leukemic burden in vivo. Furthermore, this partial inhibition of glucose metabolism sensitized B-ALL cells to apoptotic stimuli and non-cytotoxic metabolic inhibition significantly enhanced efficacy of a tyrosine kinase inhibitor to eliminate B-ALL cells in vitro and in vivo. Thus, partial inhibition of glucose metabolism can provide a plausible adjuvant therapy to treat cancers that depend on glycolysis for survival and proliferation. </p><p>In contrast to B-ALL, B-CLL is an indolent form of cancer. Most B-CLL cells exhibited low glucose metabolic activities that were comparable with normal B cells at resting stage. Similar to chronically stimulated and anergic B cells, these B-CLL cells also failed to upregulate glucose metabolism in response to IgM stimulation. We also observed an altered amino acid and acyl-carnitine profile and increased glutaminase mRNA in B-CLL relative to normal B cells, suggesting the capability of using alternate nutrients such as glutamine in these cells. Finally, we explored the possibility of suppressing mitochondria metabolism to induce B-CLL cell death through inhibition of the nuclear hormone receptor and metabolic regulator ERRalpha. ERRalpha is known to regulate mitochondrial metabolism and was expressed higher in B-CLL than normal B cells. ERRalpha inhibition decreased viability of oncogene transformed pro-B cells, suggesting ERRalpha as a potential target for B-CLL treatment.</p><p> Collectively, this work investigates metabolic phenotype in two forms of leukemia derived from B cells. It reveals different metabolic requirements and activities in aggressive and indolent leukemia and explores different approaches to suppress metabolism in these cancers. Findings of this work shed light on how to potentially design metabolic approach to improve cancer treatment.</p> / Dissertation

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Adequate cell production for adoptive cell transfer therapies such as Chimeric Antigen Receptor (CAR)-T cell therapy remains a critical barrier to treatment for indications that fail to achieve clinical success. One such disease is Chronic Lymphocytic Leukemia (CLL), a B-cell lymphoma with their characteristically exhausted T cells, marked by a progressive loss of the ability to secrete cytokines and proliferate, as well as an increase in the expression of checkpoint inhibitor molecules such as PD-1. The goal of this thesis is to characterize the functional differences or specific biomarkers within the CLL patient population that is indicative of the proliferation outcomes. Conventional clinical markers such as Rai stage or PD-1 expression alone were inadequate to describe the complex variability among patients. In order to better characterize exhaustion using microscopy-based cell function assays, we developed a sample sparing microscopy chamber that requires as little as 1000 cells per sample. The microscopy chambers were mass produced via injection molding, and made compatible with the antibody microcontact printing technique developed in the Kam lab. The chambers typically reduced cell usage per experiment by 20-fold. This reduction allowed us to measure IL-2 secretion, T cell arrest response to activating antibody patterns (pattern alignment), and motility of scarce human samples simultaneously from a single experiment. Results from these functional readouts along with other clinical markers were used as inputs for a multifactor exploratory analysis to cluster patients according to their functional similarities from the combination of responses in an unbiased manner. The resulting clusters based on the combination of the top 3 parameters IL-2, pattern alignment, and PD-1 resulted in better separation of patient groups and provided a basis for predicting max doubling outcomes from these inputs. We further used motility measurements as a way to understand initial T cell response to activation before the stop response, which was measured as pattern alignment previously. The time it takes for cells to come to a stop at the signal was most informative for translating T cell activation response to a stop response, and eventually to downstream effector functions of cytokine secretion and proliferation. The results of this work provide a powerful framework to describe different donors, and can be applied to cells from additional donors to guide future cell expansion studies.

Phosphatidylinositol 3-kinase δ (PIK3δ) is a tyrosine kinase essential for B cell survival, making it an important target in the treatment of chronic lymphocytic leukemia. Idelalisib is an inhibitor of PIK3δ demonstrating initial success in disease response, but is now shown to have a decreased overall survival and life-threatening serious adverse events. The following is an unfortunate case of a grade III adverse skin reaction secondary to idelalisib with the likely complication of methicillin-resistant Staphylococcus aureus bacteremia.

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