Doctor of Philosophy / Food Science Institute / Channa B. Rajashekar / Weiqun Wang / As health-promoting agents, phytochemicals are biosynthesized in the plants that typically respond to environmental stresses. This study focused on the analysis of phytochemical contents in vegetables in response to environmental changes of high tunnel and light spectra. A potential anticancer activity was further studied by developing a novel 3D cell culture model. Three specific studies were conducted as follows. Study 1: High tunnel production has been applied in mid-west for many years due to the advantages of extending growing season and increasing crop yield. Previous studies, however, showed high tunnel resulted in reduction of phenolic contents in vegetables. Therefore, the first study was to confirm the effect of high tunnel on phenolic contents in two varieties of lettuce (‘Two Star’ and ‘Red Fire’) and carotenoid contents in two varieties of tomatoes (‘Mountain Fresh’ and ‘Celebrity’). Phenolics in lettuce and carotenoids in tomato were isolated and quantitated, respectively, by HPLC. High tunnel resulted in a significant reduction of phenolic contents in ‘Two Star’ but not in ‘Red Fire’ lettuce when compared with open field. A significant decrease of carotenoid contents in ‘Celebrity’ but not in ‘Mountain Fresh’ tomato was also observed. Therefore, this study confirmed that high tunnel application reduced phenolic or carotenoid contents in one of the two lettuce or tomato varieties, suggesting the effect of high tunnel production is variable and genotype specific. Study 2: Light is an important environmental factor influenced not only photosynthesis but also phenolic biosynthesis in vegetables. The objective of this study was to investigate the effect of supplemental light spectra including red, far-red, and blue light on phenolic contents in two varieties of lettuce (green-leaf variety ‘Two Star’ and red-leaf variety ‘Red Fire’) and two varieties of spinach (‘Avon’ and ‘Bloomsdale’). The phenolics were extracted and quantitated by HPLC. Far-red and blue light but not red light resulted in an increase of phenolic contents in ‘Two Star’ lettuce. In ‘Red Fire’ lettuce, a significant increase in phenolic contents were observed when exposed to red light, while far-red and blue light reduced phenolic contents. Supplemental lighting did not alter flavonoid contents in two varieties of spinach. Taking together, the results showed that supplemental lighting and its spectral quality had significant effect on the phytochemical contents of lettuce but not spinach, and the impact varied depending upon the variety or species. Study 3: Traditionally, cancer research is primarily relied on in vitro 2D monolayer cell culture and in vivo animal model studies. Given a flat 2D cell culture that usually lacks 3D microenvironmental cell-cell interaction and considering an animal model that is typically expensive and time-consumed, an alternative 3D cell culture has been promising. This pilot study was to develop a novel 3D hydrogel cell culture model of human hepatocarcinoma HepG2 cells or colorectal adenocarcinoma SW480 cells by treating with chlorogenic acid (CGA) at 0-40 μM. The results showed both HepG2 and SW480 cells grew much better in 3D hydrogel culture system than 2D by extended exponential phase and high proliferation. CGA treatment resulted in a dose- and time-response inhibition of HepG2 and SW480 growth in exponential phase, while HepG2 cells were more susceptible than SW480 cells. Establishment of this novel 3D hydrogel culture model for future phytochemical function may bridge the gap between 2D cell culture and in vivo animal model studies. Taken together, this dissertation of three studies focused on phytochemicals from quantitation analysis in vegetables in response to environmental factors of high tunnel and light spectra to a novel 3D hydrogel cell culture development for potential phytochemical anti-cancer function. The conclusions, i.e., (1). high tunnel application reduced phenolic or carotenoid contents in special genotype of lettuce or tomato varieties; (2). lighting and its spectral quality had significant effect on the phytochemical contents of lettuce but not spinach; (3). establishment of a novel 3D hydrogel culture model for phytochemical treatment may bridge the gap between 2D cell culture and in vivo animal model studies, could be of particular significance in health-promoting phytochemical research and functional food application. Study 1: High tunnel production has been applied in mid-west for many years due to the advantages of extending growing season and increasing crop yield. Previous studies, however, showed high tunnel resulted in reduction of phenolic contents in vegetables. Therefore, the first study was to confirm the effect of high tunnel on phenolic contents in two varieties of lettuce (‘Two Star’ and ‘Red Fire’) and carotenoid contents in two varieties of tomatoes (‘Mountain Fresh’ and ‘Celebrity’). Phenolics in lettuce and carotenoids in tomato were isolated and quantitated, respectively, by HPLC. High tunnel resulted in a significant reduction of phenolic contents in ‘Two Star’ but not in ‘Red Fire’ lettuce when compared with open field. A significant decrease of carotenoid contents in ‘Celebrity’ but not in ‘Mountain Fresh’ tomato was also observed. Therefore, this study confirmed that high tunnel application reduced phenolic or carotenoid contents in one of the two lettuce or tomato varieties, suggesting the effect of high tunnel production is variable and genotype specific. Study 2: Light is an important environmental factor influenced not only photosynthesis but also phenolic biosynthesis in vegetables. The objective of this study was to investigate the effect of supplemental light spectra including red, far-red, and blue light on phenolic contents in two varieties of lettuce (green-leaf variety ‘Two Star’ and red-leaf variety ‘Red Fire’) and two varieties of spinach (‘Avon’ and ‘Bloomsdale’). The phenolics were extracted and quantitated by HPLC. Far-red and blue light but not red light resulted in an increase of phenolic contents in ‘Two Star’ lettuce. In ‘Red Fire’ lettuce, a significant increase in phenolic contents were observed when exposed to red light, while far-red and blue light reduced phenolic contents. Supplemental lighting did not alter flavonoid contents in two varieties of spinach. Taking together, the results showed that supplemental lighting and its spectral quality had significant effect on the phytochemical contents of lettuce but not spinach, and the impact varied depending upon the variety or species. Study 3: Traditionally, cancer research is primarily relied on in vitro 2D monolayer cell culture and in vivo animal model studies. Given a flat 2D cell culture that usually lacks 3D microenvironmental cell-cell interaction and considering an animal model that is typically expensive and time-consumed, an alternative 3D cell culture has been promising. This pilot study was to develop a novel 3D hydrogel cell culture model of human hepatocarcinoma HepG2 cells or colorectal adenocarcinoma SW480 cells by treating with chlorogenic acid (CGA) at 0-40 M. The results showed both HepG2 and SW480 cells grew much better in 3D hydrogel culture system than 2D by extended exponential phase and high proliferation. CGA treatment resulted in a dose- and time-response inhibition of HepG2 and SW480 growth in exponential phase, while HepG2 cells were more susceptible than SW480 cells. Establishment of this novel 3D hydrogel culture model for future phytochemical function may bridge the gap between 2D cell culture and in vivo animal model studies. Taken together, this dissertation of three studies focused on phytochemicals from quantitation analysis in vegetables in response to environmental factors of high tunnel and light spectra to a novel 3D hydrogel cell culture development for potential phytochemical anti-cancer function. The conclusions, i.e., (1). high tunnel application reduced phenolic or carotenoid contents in special genotype of lettuce or tomato varieties; (2). lighting and its spectral quality had significant effect on the phytochemical contents of lettuce but not spinach; (3). establishment of a novel 3D hydrogel culture model for phytochemical treatment may bridge the gap between 2D cell culture and in vivo animal model studies, could be of particular significance in health-promoting phytochemical research and functional food application.

Acute myeloid leukemia (AML) presents the deadliest form of blood cancer which leads to abrupt, premature deaths. Current therapeutic treatment options in AML are unspecific, resulting in high relapse rates and poor clinical responses in patients. Therapy-resistant, stem cell-like AML cells are believed to be protected by proximal stromal cells in their microenvironment, the leukemic stem cell niche. In part A of this work, an innovative first-of-its-kind arrayed endoribonuclease-prepared siRNA (esiRNA) screen was established for the targeted identification of stromal-derived, AML-supportive genes. Immortalized bone-marrow derived mesenchymal stromal cells (SCP-1) were subjected to individual esiRNA-mediated target gene knockdowns (KD) and subsequently cocultured with AML cell lines MV4-11, OCI-AML3, MOLM-13 and HL-60. AML proliferation and therapy resistance to cytostatic agents Cytarabine or Daunorubicin and tyrosine kinase inhibitor Midostaurin were assessed in direct cocultures. In SCP-1, several secreted, membrane-associated and intracellular molecules were identified which, upon esiRNA-mediated KD, resulted in proliferation inhibition and enhanced treatment response of cocultured AML cells. Carbonic anhydrase 9 (CA9), a stabilizer of intracellular pH, was identified as a supportive factor in proliferation and resistance of leukemic cells to Daunorubicin treatment whilst CA9-KD exerted only a comparably low toxicity in SCP-1 cells. Excitingly, published data by Chen and colleagues (Blood, 2017, Vol. 130, Suppl. 1, 2521) indicated an upregulation of CA9 in hypoxic ex vivo cultures of leukemic cells, measured an anti-leukemic effect of pharmacological CA9 inhibition and identified a synergistic effect on leukemic cells via combinatorial treatment of CA9-inhibition and Cytarabine under hypoxic culture conditions. Taken together, an arrayed esiRNA screen identified CA9 and other stromal-derived factors which potentially open up new avenues for selective therapeutic treatments targeting the leukemic microenvironment in AML. Currently, preclinical leukemia research relies on artificial suspension cultures of AML cells and highly sophisticated, patient-derived xenograft (PDX) mouse models that are marked by suboptimal translation of findings of PDX experiments into the clinic. Recent developments in complex three-dimensional (3D) hydrogel star-shaped poly(ethylene glycol) (starPEG)-heparin cocultures of leukemic and stromal cells of human origin showed promising results in proliferation and drug response studies. Therefore, in part B of this work, a high throughput screening (HTS)-compatible 3D hydrogel culture setup of human stromal cells was established in 384-well plates. Implementation of design of experiments (DoE) enabled an efficient, cost-effective optimization of hydrogel monocultures of human umbilical vein endothelial cells (HUVECs). Optimized culture conditions favored angiogenic sprouting of hydrogel-embedded HUVECs which responded to angiogenic inhibitors Axitinib, AZD4547 and Bevacizumab in a dose-dependent manner. A coculture with bone marrow derived MSCs altered the angiogenic network formation of endothelial CD31+ vessel-like structures. The hydrogel coculture was further stabilized by extensive hydrogel degradation and ECM deposition of MSCs. Stromal MSC networks were illustrated as highly interconnected and elongated F-Actin filament structures (CD31- F-Actin+) that were closely associating with CD31+ F-Actin+ endothelial vessel-like structures. Excitingly, the established 3D hydrogel HTS platform of primary human stromal cells enables future addition of patient-derived leukemic cells for targeted leukemic vulnerability screens in an ex vivo cell culture model of the perivascular stem cell niche. / Akute myeloische Leukämie (AML) gilt als die tödlichste Form der Blutkrebserkrankungen, welche untherapiert zum abrupten, vorzeitigen Tod führt. Etablierte therapeutische Verfahren der AML sind unspezifisch, welche durch heterogene Behandlungseffekte gekennzeichnet sind und zu hohen Rückfallquoten führen. Man vermutet, dass therapie-resistente, stammzellähnliche leukämische Zellen von proximal residierenden Stromazellen in ihrem Mikromilieu, in der sogenannten leukämischen Stammzellnische, vor therapeutischen Behandlungen geschützt werden. In Teil A dieser Arbeit wurde ein innovativer, neuartiger Screen basierend auf Endoribonuklease-generierten kleinen, interferierenden Ribonukleinsäuren (esiRNAs) für eine gezielte Identifikation von AML-supportiven, stromalen Faktoren etabliert. Immortalisierte, mesenchymale Stromazellen aus dem Knochenmark (SCP-1) wurden in einem Array mit spezifischen esiRNAs transfiziert, um esiRNA-basierende inhibierende Effekte (Knockdown) auf die Genexpression von Zielgenen in SCP-1 zu studieren und indirekte Auswirkungen auf Proliferationsrate und Therapieresistenz von kokultivierten leukämischen Zelllinien, MV4-11, OCI-AML3, MOLM-13 und HL-60, bei Behandlung mit Cytarabin, Daunorubicin und Midostaurin, zu studieren. Mehrere sezernierte, membranständige und intrazelluläre Faktoren wurden in SCP-1 identifiziert, deren esiRNA-vermittelter Knockdown zu einer Proliferationsminderung sowie verstärkten Toxizitätseffekten von applizierten Therapeutika in Leukämiezellen führten. Beispielhaft wurde Carboanhydrase (CA9), ein Enzym welches den intrazellularen pH einer Zelle stabilisert, als Target identifiziert. Ein Knockdown von CA9 in SCP-1 resultierte in einer Proliferationsminderung von kokultivierten Leukämiezellen, welche des Weiteren in einer Behandlung mit Daunorubicin verstärkt abgetötet wurden. Publizierte Daten von Chen et al. (Blood, 2017, Vol. 130, Suppl. 1, 2521) zeigten, dass CA9 in hypoxischen ex vivo Kulturen in leukämischen Zellen hochreguliert war und, dass dessen pharmakologische Inhibition einen anti-leukämischen Effekt aufwies. Zudem wurde ein synergistischer Therapieffekt, bei einer Kombinationstherapie mit einem CA9-Inhibitor und Cytarabin, auf AML Zellen in hypoxischer Zellkultur festgestellt. Zusammenfassend wurden in einem esiRNA-Screen CA9 und weitere stromal-exprimierte Faktoren identifiziert, die das Potential besitzen neuartige Therapiestrategien zu ermöglichen, welche auf die leukämische Stammzellnische als Zielstruktur ausgerichtet sind. In der präklinischen Forschung von hämatologischen Erkrankungen werden vorrangig artifizielle zweidimensionale Suspensionskulturen von Leukämiezellen verwendet oder ausgefeilte, patienten-derivierende Xenograft (PDX) Mausmodelle eingesetzt. Bedauerlicherweise weisen Erkenntnisse aus Mausmodellen eine geringe Translationseffizienz in die klinische Forschung auf. Neuste Entwicklungen mit komplexen, dreidimensionalen Hydrogelkulturen, bestehend aus sternförmigem Polyethylenglykol (starPEG) und Heparin, von stromalen und leukämischen Zellen humanen Ursprungs zeigten vielversprechende Ergebnisse in präklinischen Proliferations- und Vulnerabilitätsstudien. Daher wurde in Teil B dieser Arbeit ein hochdurchsatzfähiges dreidimensionales Kultursystem von humanen Stromazellen in Hydrogelen entwickelt. Per statistischer Versuchsplanung wurde eine effiziente, kostengünstige Optimierung von etablierten Hydrogelkulturen für die Hochdurchsatz-kompatible Kultur von humanen venösen Endothelzellen aus Nabelschnuren (HUVECs) durchgeführt. Optimierte Kulturbedingungen führten zur Angiogenese von Hydrogel-eingebetteten HUVECs, welche des Weiteren auf die Angiogenese-Inhibitoren Axitinib, AZD4547 und Bevacizumab in einer konzentrationsabhängigen Weise mit verminderter Bildung von gefäßähnlichen Strukturen reagierten. Eine Kokultur von HUVECs mit primären, mesenchymalen Stromazellen aus dem Knochenmark (MSCs) beeinflusste die Bildung von CD31+ gefäßähnlichen Strukturen. Die Hydrogel-Kokultur wurde des Weiteren durch verstärkte Degradation des Hydrogels und Deposition von Komponenten der extrazellulären Matrix via MSCs verändert und dadurch zusätzlich stabilisiert. Geformte Netzwerkstrukturen von MSCs und HUVECs wurden mittels F-Actin Färbung identifiziert, wodurch ersichtlich wurde, dass Strukturen von MSCs (CD31- F-Actin+) in enger räumlicher Distanz zu HUVEC Strukturen (CD31+ F-Actin+) gebildet wurden. Spannenderweise ermöglicht die, in dieser Arbeit etablierte, Hochdurchsatz-kompatible Kokultur von humanen Stromazellen die Möglichkeit auch leukämische Zellen in die Hydrogelmatrix einzubetten. Eine humane AML-Stroma Kokultur in Hydrogelen wird gezielte Vulnerabilitätsscreens von AML Zellen in einem komplexen ex vivo Zellkulturmodel der perivaskulären Stammzellnische ermöglichen.