To metabolomics and beyond: a technological portfolio to investigate cancer metabolism

Tumour cells have exquisite flexibility in reprogramming their metabolism in order to support tumour initiation, progression, metastasis and resistance to therapies. These reprogrammed activities include a complete rewiring of the bioenergetic, biosynthetic and redox status to sustain the increased energetic demand of the cells. Over the last decades, the cancer metabolism field has seen an explosion of new biochemical technologies giving more tools than ever before to navigate this complexity. Within a cell or a tissue, the metabolites constitute the direct signature of the molecular phenotype and thus their profiling has concrete clinical applications in oncology. Metabolomics and fluxomics, are key technological approaches that mainly revolutionized the field enabling researchers to have both a qualitative and mechanistic model of the biochemical activities in cancer. Furthermore, the upgrade from bulk to single-cell analysis technologies provided unprecedented opportunity to investigate cancer biology at cellular resolution allowing an in depth quantitative analysis of complex and heterogenous diseases. More recently, the advent of functional genomic screening allowed the identification of molecular pathways, cellular processes, biomarkers and novel therapeutic targets that in concert with other technologies allow patient stratification and identification of new treatment regimens. This review is intended to be a guide for researchers to cancer metabolism, highlighting current and emerging technologies, emphasizing advantages, disadvantages and applications with the potential of leading the development of innovative anti-cancer therapies

Hydrogel-Tissue Chemistry: Principles and Applications

Over the past five years, a rapidly developing experimental approach has enabled high-resolution and high-content information retrieval from intact multicellular animal (metazoan) systems. New chemical and physical forms are created in the hydrogel-tissue chemistry process, and the retention and retrieval of crucial phenotypic information regarding constituent cells and molecules (and their joint interrelationships) are thereby enabled. For example, rich data sets defining both single-cell-resolution gene expression and single-cell-resolution activity during behavior can now be collected while still preserving information on three-dimensional positioning and/or brain-wide wiring of those very same neurons—even within vertebrate brains. This new approach and its variants, as applied to neuroscience, are beginning to illuminate the fundamental cellular and chemical representations of sensation, cognition, and action. More generally, reimagining metazoans as metareactants—or positionally defined three-dimensional graphs of constituent chemicals made available for ongoing functionalization, transformation, and readout—is stimulating innovation across biology and medicine

The genetics of anthracycline-induced cardiotoxicity in cancer patients

INTRODUCTION: Breast cancer makes up 25% of all cancers diagnosed worldwide. Despite an increasing yearly incidence, there has been a significant decrease in mortality owing to early diagnosis and advances in treatment. Anthracycline-based chemotherapy is a relatively low cost yet highly effective anti-cancer treatment, increasing survival from 30% to >80%, presently. However, treatment efficacy is marred by the increased risk of anthracycline-induced cardiotoxicity (ACT) - estimated at 10-26%. Internationally, there has been evidence of ACT having a genetic basis. Currently in South Africa, there is little information on ACT in cancer patients and survivors, and no information on the genetic basis of this phenomenon. Our recruitment sites in Cape Town - Groote Schuur Hospital (GSH) and Tygerberg Hospital (TBH), routinely treat hundreds of patients, notably with breast cancer, with anthracycline-based therapy every year, and provided the environment to assess ACT, as well as genetic factors which may influence this adverse drug reaction. Left ventricular ejection fraction (LVEF) acts as a surrogate measure of cardiac function in the public health-care setting. OBJECTIVES: To provide insight into the clinical management of breast cancer patients on anthracycline-based treatment with a focus on the prevalence of ACT. To provide an index of genetic susceptibility to ACT and potentially contribute to a personalized medicine approach for a genetically diverse population. METHODOLOGY: In the retrospective part of the study, the clinical records of cancer patients treated with anthracyclines from 2011- 2016 at the Oncology Clinic at GSH were analysed. Clinical co-morbidities such as hypertension, diabetes, pre-existing cardiac disease and smoking as well as type and dose of anthracyclines, cardiac function and patient status were assessed. In the prospective study, breast cancer patients treated with anthracyclines, with a pre and post-treatment LVEF measure were recruited at GSH and TBH from 2013 to 2016. Patients were consented for access to both clinical information and biological material. Demographics, clinical risk factors and chemotherapeutic regimen data were analysed. LVEF, biomarkers and clinical status were also assessed in terms of reflecting ACT. In some instances certain clinical information was not available (i.e. LVEF) and out of necessity, a statistical correlation model or classifier was created in order to use available clinical data to derive missing clinical measures. Patients' DNA were analysed for seven genetic variants in the following six genes ABCC1 (rs246221); ABCC2 (rs17222723; rs8187710); HNMT (rs17583889); NCF4 (rs1883112): RAC2 (rs13058338) and RARG (rs2229774), and tested for correlation with clinical status and cardiac injury. Finally, a corollary study was conducted on a subset of patients in an attempt to determine whether cardiac biomarkers may be more sensitive measures of cardiotoxicity. RESULTS & DISCUSSION: In the retrospective cohort (n=402) 19.7% of patients showed diminished cardiac function. Logistic regression showed that the following predictors: type of first line chemotherapy, and total dose significantly contributed to the ACT phenotype as measured by change in LVEF. In the prospective patients (n=272), 14% were affected with ACT, with an increased likelihood of cardiotoxicity in the Indigenous African population. Logistic regression showed that both total anthracycline dose and change in LVEF were predictive of ACT. In the association study of prospective patients, only the RARG rs2229774 variant was significantly associated with patient ACT status (p=0.049, Chi-Square Test). Forty-two patients were assessed for the β-Natriuretic Peptide (BNP) biomarker and showed limited utility in correlating clinical status and/or LVEF decrease in all patients except Indigenous Africans indicating potential increased susceptibility of population group to ACT. LVEF was found to be unreliable as significant LVEF decreases did not always correlate with cardiac impairment and vice-versa. Changes in routine clinical patient management and overburdening of the nuclear medicine department also translated to only one LVEF measure being obtained in some instances. The statistically derived classifier for missing indicators of heart function was useful, but will require refinement. CONCLUSIONS AND RECOMMENDATIONS: Despite the inability of genotype as a predictor of ACT in this study, the increased susceptibility in the Indigenous African population to ACT as well as increased BNP levels after chemotherapy requires a closer look. The interrogation of lndigenous African patient genomes for novel variants of susceptibility to ACT are recommended; this requires building up of a substantial cohort from this population group, which would likely require collaboration with health care institutions in one of the other provinces of South Africa e.g. Eastern Cape, KwaZulu-Natal and/or Gauteng. Both this study and literature recommend the need for clinical trials for new and existing drugs on local African populations for both safety and efficacy. Furthermore, the BNP biomarker may be better suited to the prediction of irreversible cardiac damage rather than early cardiotoxicity. Troponin, released in response to cardiomyocyte death, may be a more sensitive biomarker in predicting ACT. Similarly, the inherent variability and lack of sensitivity of LVEF as a measure of cardiac function warrants the consideration of alternatives such as echocardiography or tissue-doppler imaging. Findings derived from this study indicate the need for refined patient management of ACT in a South African population to potentially allow for treatment with minimised risk and event-free breast cancer survival

CD20+ T cells: phenotype, origin and presence in the tumour microenvironment

CD20 is well-known as a lineage marker for B cells in human, although the presence of CD20+ T cells has been reported previously (Schuh et al., 2016). In my experiments we sorted memory CD8+CD20+ and CD20- T cells and subsequently analysed TCRβ CDR3 sequences to determine clonal overlap between populations. I was able to demonstrate TCRβ CDR3 amino acid sequences within the CD20+ T cells that were not found in the CD20- T cells population, suggesting the presence of unique T cell clones. This data suggests possible thymic origin of at least some CD20+ T cells, or an early expression of CD20+ following activation of naïve T cells. This does not however refute the trogocytosis theory where some T cells acquire CD20 from B cell. I analysed a number of published mass cytometry datasets and determined that memory CD20+ T cells express higher levels of CD127, CD27, CD28, PD-1 and CD57 especially by the effector memory (Tem) and effector memory CD45RA revertant (Temra) subsets, suggesting a possible late differentiation state of the CD20+ T cells. Further phenotyping revealed a novel population of CD20+Vα7.2+CD161+ cells in the peripheral blood of healthy donors, which are potentially CD20+ mucosal-invariant T (MAIT) cells. In colon cancer, CD20+ T cells were found within tumour tissue, adjacent normal tissue and showed fewer CD20+ T cells expressing the marker CD39. This may indicate lower numbers of CD20+ T cells within tumour-reactive T cell pool and requires further investigation. Using mass cytometry datasets from colorectal cancer patients, and melanoma patients receiving anti- programmed cells death (PD-1) or anti-cytotoxic T lymphocyte-associated protein-4 (CTLA-4), CD20+ T cells showed similar phenotypes to those found in healthy peripheral blood. Importantly, they produced higher levels of cytokines compared to the CD20- T cells including; TNF-α, IFN-γ, IL-2, IL-17, and MIP-1β. My data, including both primary data and analysis of published datasets, indicates that CD20+ T cells can originate as a separate lineage, in addition to acquiring CD20 by trogocytosis. CD20+ T cells were found to be more highly differentiated, with increased cytokine production and contained a high frequency of CD20+ Vα7.2+CD161+ cells. Although there was no detectable difference in cancer, future work will be required to determine if they play any role within the tumour microenvironment or other pathological scenarios

Developing Zebrafish as an In Vivo Model to Screen Compounds for Anti-Cancer Activity in Human Breast Cancer

Breast Cancer (BC) is the most frequently diagnosed cancer; 1:8 women are at risk of developing BC in her lifetime. Cancer metastasis causes the majority of deaths in BC patients. Moreover, side effects of traditional chemotherapeutic drugs (TCD) impair the quality of life of these patients. Discovery and development of safe and effective new therapies is imperative for the treatment of BC and targeting metastasis. The goal herein is to further expand the applicability of in vivo xenotransplantation of human BC cells in transgenic zebrafish to screen potential chemotherapeutics for toxicity and efficacy. For xenotransplantation, MCF-7, BT-474, and MDA-MB-231 BC cells were used to canvas the benign and malignant types of BC, respectively. Using fluorescently-labeled cells, the cytotoxic effect of doxorubicin, 4-hydroxytamoxifen, and paclitaxel were determined using a cell viability assay. Test compounds (fractions of Tinospora crispa and potent microtubule inhibitors) were also tested for in vitro cytotoxicity. Maximally tolerated concentrations (MTCs; \u3e80% survival) and no observed adverse effect level (NOAELs) for developmental morphology were determined in zebrafish following a 96 hr waterborne exposure to 1 - 50 µM of doxorubicin, 4-hydroxytamoxifen, paclitaxel, and curcumin or 10 - 800 nM of mertansine, ansamitocin P-3, and monomethyl auristatin E (MMAE). NOAELs for paclitaxel, mertansine, ansamitocin P-3, and MMAE were 25, 400, 50, and 400 nM, respectively. Zebrafish were xenotransplanted with MCF-7, BT-474, and MDA-MB-231 cell lines to observe the effects of exposure to microtubule inhibitors on the proliferation of cancer cells. After xenotransplanting 50-100 BC cells/larva at 2 days post-fertilization, cell growth and migration were imaged at 1 and 5 days post-injection using fluorescent microscopy. Paclitaxel (25 nM) and mertansine (10 and 200 nM) significantly reduced the proliferation of MCF-7 cell xenografts compared to controls, confirming the use of this model for MCF-7 cell xenografts. In contrast, MDA-MB-231 xenografts did not respond to the same concentrations of PTX or mertansine. Here, we developed an optimized assay and quantitative approach to screen novel anti-cancer agents against breast cancer xenotranplants

SRC HOMOLOGY 2 DOMAIN-CONTAINING 5’-INOSITOL PHOSPHATASE-2 (SHIP2) IS AN EFFECTOR OF LYMPHATIC DYSFUNCTION

The lymphatic system is essential for the transport of excess fluid, protein, and foreign materials from interstitial tissues to lymph nodes; for immune surveillance, and to maintain fluid homeostasis. Dysregulated lymphatics can be attributed to pathological conditions including tumor metastasis, inflammation, chronic wounds, obesity, blood vascular disorders, and lymphedema. Of these, lymphedema is the most extreme of lymphatic disorders and is represented by a spectrum of symptoms ranging from mild, subtle presentation to severe, disfiguring, overt presentation. Lymphedema is more manageable in the early stages of disease but severely reduces quality of life with progression. Due to lack of molecular knowledge and inadequate imaging techniques to safely, rapidly and non-invasively visualize the lymphatics, lymphedema remains under diagnosed and progresses to the irreversible stage if not diagnosed early. Candidate gene studies have identified a myriad of genes responsible for lymphedema, however, majority of patients do not harbor mutations in these putative genes, indicating many more unknown genes contribute to the pathology of this disease. In an effort to identify new polymorphisms that possibly effect lymphatic dysfunction, we combined investigational, non-invasive near-infrared fluorescence lymphatic imaging (NIRFLI) and next generation sequencing (NGS), to phenotype and genotype human subjects with familial lymphedema. We discovered that mutations in src homology 2-domain containing 5’-inositol phosphatase-2 (SHIP2), encoded by INPPL1, are associated with lymphatic abnormalities. SHIP2 is a phosphatidyIinositol (3,4,5) triphosphate (PIP3) 5’-phosphatase that negatively controls PIP3 levels thereby inhibiting the PI3K/AKT signaling, a pathway implicated in various lymphatic disorders. Our studies confirm this inhibitory role of SHIP2 against PI3K/AKT in lymphatic endothelial cells, and identify SHIP2 as a potential regulator of MAPK/ERK signaling, another pathway also recently identified as important in lymphatic malformations. Pharmacological inhibition of SHIP2 impedes lymphatic contractility and impairs the normal wound healing processes of lymphangiogenesis and angiogenesis in mice. These studies suggest that SHIP2 could have a previously unidentified effector role in lymphatic dysfunction. In elucidating the roles of SHIP2 in lymphatic dysfunction, the work presented herein expands our understanding of molecular basis of lymphatic failure which could have clinical implications affecting populations with lymphatic disorders, including the ever increasing population of cancer survivors who experience the chronic, disfiguring and incurable lymphedema

Model Informed Drug Development and Precision Dosing for Drug-Drug-Gene-Interactions: Application of Physiologically-Based Pharmacokinetic Modeling

The global demand for pharmaceuticals is continuously growing. As a result, one can observe an increase in adverse drug reactions, which pose a critical risk to patients. The primary triggers for adverse drug reactions are drug-drug- and drug-gene-interactions. Model-informed drug discovery and development as well as model-informed precision dosing can help to mitigate the risks of drug-drug and drug-gene interactions. Thus, this work aimed to improve and to apply physiologically-based pharmacokinetic modeling strategies in the context of model-informed drug discovery and development as well as model-informed precision dosing. For this purpose, best practices for data digitization as an essential step in the development process of most physiologically-based pharmacokinetic models have been established. Moreover, models for zoptarelin doxorubicin and simvastatin were developed and evaluated. The zoptarelin doxorubicin model was used to guide the development process of this drug. In contrast, the simvastatin model was utilized in a drug-drug-gene interaction network to generate 10368 dose recommendations for different interaction scenarios, which were made available in a digital decision support system. In conclusion, the work can be seen as a beacon project to illustrate how physiologically-based pharmacokinetic modeling of drug-drug and drug-gene interactions can be applied in model-informed drug discovery and development as well as in model-informed precision dosing.Der globale Arzneimittelbedarf steigt kontinuierlich an. Infolgedessen kommt es vermehrt zu unerwünschten Arzneimittelwirkungen, die eine Gefahr für Patienten darstellen. Eine wichtige Rolle beim Auftreten unerwünschter Arzneimittelwirkungen spielen Arzneimittel-Arzneimittel- und Arzneimittel-Gen-Wechselwirkungen. Um das Risiko solcher Wechselwirkungen zu minimieren, kann die modellgestützte Arzneimittelentwicklung und Präzisionsdosierung angewendet werden. Das Ziel dieser Arbeit war es, physiologie-basierte pharmakokinetische Modelle zum Zweck der modellgestützten Arzneimittelentwicklung und Präzisionsdosierung einzusetzen. Dafür wurde die Datendigitalisierung als wesentlicher Bestandteil der Entwicklung neuer physiologie-basierter pharmakokinetischer Modelle untersucht. Außerdem wurden Modelle für Zoptarelin Doxorubicin und Simvastatin entwickelt. Das Zoptarelin Doxorubicin Modell wurde verwendet, um die Entwicklung dieses Medikaments zu unterstützen. Mittels des Simvastatin Modells wurden in einem Interaktionsnetzwerk 10368 Dosisempfehlungen für verschiedene Szenarien generiert und in einem digitalen Entscheidungsunterstützungssystem verfügbar gemacht. Zusammenfassend kann die Arbeit als Leuchtturmprojekt gesehen werden, das zeigt, wie die physiologie-basierte pharmakokinetische Modellierung von Arzneimittel-Arzneimittel- und Arzneimittel-Gen-Wechselwirkungen in der modellgestützte Arzneimittelentwicklung und Präzisionsdosierung angewendet werden kann