Effects of Cell Adhesion Peptides, pH, and Matrix Shape on Maintenance of Breast Cancer Stem Cells in an Engineered Hydrogel Matrix

Metastasis, resistance to chemo- and radiotherapy, and eventual relapse has been attributed to a tumor subpopulation known as cancer stem cells (CSCs). CSCs are regulated in their tumor microenvironment by various factors. Synthetic hydrogels can be used to investigate the effects of individual environmental factors on CSCs by providing inert 3D matrices. In this thesis, poly ethylene glycol diacrylate (PEGDA) hydrogel with 5kpa modulus has been used as a culture system to study the effect of; I) integrin and heparin binding peptides, 2) pH, and 3) the shape of the microenvironment on breast CSCs maintenance and tumorsphere formation in PEGDA. Human breast cancer cells were encapsulated in PEGDA hydrogels and the effect of the peptides, pH, and the shape of the environment on tumorsphere formation was investigated by fluorescent microscopy, qRT-PCR and DNA content assay. All peptides including RGD, RYD, IKLLI, LIGRKK, VAPG, WQPPRARI, and SPPRRARV affected breast cancer cells by reducing their capability of sphere formation. Among peptides, RGD, RYD, and WQPPRARI were the most effective peptides in reducing sphere formation of breast CSCs. Moreover, different shapes of micropatterned PEGDA including circle, square, and rectangle did not influence CSCs maintenance and behavior in forming tumorsphere. Breast CSCs formed spherical tumors regardless of the shape of the micropatterned PEGDA and had the minimum surface area for a given volume. Furthermore, breast CSCs showed more resistance to acidic pH compared to non-stem breast cancer cells and normal breast epithelial cells

Structural and functional characterization of an intradiol ring-cleavage dioxygenase from the polyphagous spider mite herbivore Tetranychus urticae Koch

Genome analyses of the polyphagous spider mite herbivore Tetranychus urticae (two-spotted spider mite) revealed the presence of a set of 17 genes that code for secreted proteins belonging to the "intradiol dioxygenase-like" subgroup. Phylogenetic analyses indicate that this novel enzyme family has been acquired by horizontal gene transfer. In order to better understand the role of these proteins in T. urticae, we have structurally and functionally characterized one paralog (tetur07g02040). It was demonstrated that this protein is indeed an intradiol ring-cleavage dioxygenase, as the enzyme is able to cleave catechol between two hydroxyl-groups using atmospheric dioxygen. The enzyme was characterized functionally and structurally. The active site of the T. urticae enzyme contains an Fe3+ cofactor that is coordinated by two histidine and two tyrosine residues, an arrangement that is similar to those observed in bacterial homologs. However, the active site is significantly more solvent exposed than in bacterial proteins. Moreover, the mite enzyme is monomeric, while almost all structurally characterized bacterial homologs form oligomeric assemblies. Tetur07g02040 is not only the first spider mite dioxygenase that has been characterized at the molecular level, but is also the first structurally characterized intradiol ring-cleavage dioxygenase originating from a eukaryote

Molecular Basis of Xenobiotic Metabolism and Resistance in \u3ci\u3eTetranychus Urticae\u3c/i\u3e

Invasive pest species are a major cause of crop loss around the world and adversely affect the agricultural industry. We are focusing on Tetranychus urticae (two-spotted spider mite; TSSM), which is a polyphagous pest that targets more than 1100 plant species. The TSSM alone causes a loss of roughly $1.6 billion per year globally due to acaricide resistance. It is developing resistance to most acaricides due to rapid growth and reproduction. Therefore, new acaricides are needed to combat TSSM resistance and infestation [1]. To address this problem, four enzymes of TSSM, intradiol ring cleavage dioxygenases, a glutathione S-transferase, a β cyanoalanine synthase, and uridine diphosphate glycosyltransferases, are characterized. These enzymes contribute to the detoxification system of TSSM and are potential protein targets to develop new acaricides. Intradiol ring-cleavage dioxygenases are involved in the breakdown and metabolism of toxic aromatic compounds [2]. Glutathione S-transferases conjugate reduced glutathione to xenobiotics for detoxification and have been associated with insecticide resistance [3]. The β cyanoalanine synthase is known for detoxification of cyanide and silencing this gene in TSSM reduces the survival of the mites on cyanogenic plants [4]. Uridine diphosphate glycosyltransferases (UGTs) catalyze the covalent addition of sugar moieties from UDP sugar donors to xenobiotics to facilitate their elimination from cells [5]. UGTs are known for the detoxification of acaricides such as abamectin. Here we have structurally and functionally characterized these proteins, particularly focusing on revealing the crystal structure of enzymes with the intention of exposing unique properties that will allow for the design of new acaricides and control of TSSM

Dependence of tumorsphere growth on culture medium in 2D versus 3D for MDA231 cells.

<p>Number density (A) and mRNA expression of CD44 (B), ABCG2 (C), and EGFR (D) markers for MDA231 cells as a function of incubation time. Groups included cells on 2D adherent plates and cultured in RPMI-1640 medium (2D-RPMI), cells on 2D plates and cultured in CSC medium (2D-CSC), and cells encapsulated in the 5 kPa PEGDA gel and cultured in CSC medium (3D-CSC). An Asterisk in (A) indicates a statistically lower (p<0.05) cell number in the test group compared to 2D groups at the same time point. An Asterisk in (B-D) indicates a statistically higher (p<0.05) mRNA expression in the test group compared to 2D groups at the same time point. The p-values for the asterisks in (A-D) are listed in Tables E-H in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132377#pone.0132377.s001" target="_blank">S1 File</a>. Error bars correspond to means±1 SD for n = 3.</p

CSC fraction of MDA231 cells encapsulated in the patterned 5 kPa gel.

<p>MDA231 cells cultured on adherent tissue culture plate (TCP, A), non-adherent tissue culture plate (suspension, B), by encapsulation in Matrigel (C), encapsulation in un-patterned 5 kPa PEGDA gel (D), and encapsulation in 50 μm patterned 5 kPa PEGDA gel; (F) CSC fraction of the MDA231 cells in (A-E) as the sub-population of cells in the fourth quadrant (CD44⁺/CD24^-).</p

Optimum 3D Matrix Stiffness for Maintenance of Cancer Stem Cells Is Dependent on Tissue Origin of Cancer Cells

<div><p>Introduction</p><p>The growth and expression of cancer stem cells (CSCs) depend on many factors in the tumor microenvironment. The objective of this work was to investigate the effect of cancer cells’ tissue origin on the optimum matrix stiffness for CSC growth and marker expression in a model polyethylene glycol diacrylate (PEGDA) hydrogel without the interference of other factors in the microenvironment.</p><p>Methods</p><p>Human MCF7 and MDA-MB-231 breast carcinoma, HCT116 colorectal and AGS gastric carcinoma, and U2OS osteosarcoma cells were used. The cells were encapsulated in PEGDA gels with compressive moduli in the 2-70 kPa range and optimized cell seeding density of 0.6x10⁶ cells/mL. Micropatterning was used to optimize the growth of encapsulated cells with respect to average tumorsphere size. The CSC sub-population of the encapsulated cells was characterized by cell number, tumorsphere size and number density, and mRNA expression of CSC markers.</p><p>Results</p><p>The optimum matrix stiffness for growth and marker expression of CSC sub-population of cancer cells was 5 kPa for breast MCF7 and MDA231, 25 kPa for colorectal HCT116 and gastric AGS, and 50 kPa for bone U2OS cells. Conjugation of a CD44 binding peptide to the gel stopped tumorsphere formation by cancer cells from different tissue origin. The expression of YAP/TAZ transcription factors by the encapsulated cancer cells was highest at the optimum stiffness indicating a link between the Hippo transducers and CSC growth. The optimum average tumorsphere size for CSC growth and marker expression was 50 μm.</p><p>Conclusion</p><p>The marker expression results suggest that the CSC sub-population of cancer cells resides within a niche with optimum stiffness which depends on the cancer cells’ tissue origin.</p></div

Dependence of tumorsphere growth on conjugation of CD44 binding peptide to the gel.

<p>(A) DAPI (blue) and phalloidin (red) stained images of MDA231 (A+B), HCT116 (C+D), and U2OS (E+F) cells encapsulated in the un-patterned gel with optimum modulus (5 kPa for MDA231, 25 kPa for HCT116, and 50 kPa for U2OS) without CD44BP (A+C+E) and with CD44BP conjugation (B+D+F) after 9 days incubation (scale bar in A-F is 200 μm). The initial seeding density of all cell types in the gel was 0.6x10⁶ cells/mL. Cell number (G), tumorsphere size (H), and tumorsphere number (I) for MDA231, HCT116, and U2OS cells encapsulated in the gel without (blue) and with (green) conjugated CD44BP and with conjuagted mutant-CD44BP (mCD44BP, orange) after 9 days incubation. mRNA expression of CSC markers for MDA231 (J, CD44 and EGFR), HCT116 (K, CD44 and TGF-β), and U2OS (L, CD44 and CD133) encapsulated in the gel without conjugation, with mCD44BP, and with CD44BP conjugation. An Asterisk in (G-L) indicates a statistically lower cell number, sphere number and size, and marker expression for the test group compared to those groups without CD44BP conjugation and with mutant CD44BP conjugation of the gel for a given cell type. The p-values for the asterisks in (G-L) are listed in Tables A-F in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132377#pone.0132377.s002" target="_blank">S2 File</a>. Error bars correspond to mean±1 SD for n = 3.</p

Dependence of the optimum gel modulus for CSC growth on tissue origin of cancer cells.

<p>(A) DAPI (blue) and phalloidin (red) stained images of MCF10A, MCF7, MDA231, HCT116, U2OS, and AGS cancer cells encapsulated in the un-patterned gels after 2, 6, and 9 days of incubation (scale bars in A are 50 μm). Cell number (B), tumorsphere number (C), tumorsphere diameter (D), and tumorsphere size distribution (E) as a function of cancer cell type after 9 days of encapsulation for gel moduli of 2 (pink), 5 (blue), 25 (brown), 50 (purple), and 70 (green) kPa. The gel modulus in (A and E) was the optimum PEGDA modulus of 5 kPa for MCF7 and MDA231 cells; 25 kPa for HCT116 and AGS; 50 kPa for U2OS. (F) mRNA expression of CSC markers for MCF7 (CD44 and ABCG2), MDA231 (CD44 and EGFR), HCT116 (CD44 and TGF-β), U2OS (CD44 and CD133), and AGS (CD44 and OCT4) after 6 and 9 days of encapsulation for gel moduli of 2 (pink), 5 (blue), 25 (brown), 50 (purple), and 70 (green) kPa. An asterisk in (B-D) indicates a statistically higher (p<0.05) cell number, sphere number and size for the test modulus compared to all other moduli for a given cell type. An asterisk in (F) indicates a statistically higher (p<0.05) marker expression level for the test modulus compared to all other moduli for a given cell type and at a given time. The p-values for the asterisks in (B-D,F) are listed in Tables I-U in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132377#pone.0132377.s001" target="_blank">S1 File</a>. Error bars correspond to mean±1 SD for n = 3.</p

Dependence of tumorsphere growth on niche size for MDA231 cells.

<p>(A) Procedure for cell encapsulation in the micropatterned gel. CellTracker stained images of MDA231 cells encapsulated in the 5 kPa gel with circular patterns with diameter of 50 (B), 75 (C), 100 (D), 150 (E), and 250 (F) μm after 2 days of incubation (scale bars in B-F are 200 μm). DAPI (blue) and phalloidin (red) stained images of 7 representative tumorspheres formed by the encapsulated cells in (B-F) from different sections of the patterned gels after 14 days of incubation with 50 (G), 75 (H), 100 (I), 150 (J), and 250 (K) μm patterns (scale bars in G-K are 50 μm). Note that the 7 representative tumorspheres in (G-K) are from multiple patterns in the gel sample, not a single pattern, to show size range and shape of tumorspheres for a given pattern size. Cell number (L), average tumorsphere size (M), CD44 expression (N), and ABCG2 expression (O) of the cells in (B-F) with incubation time for 50 (pink), 75 (blue), 100 (red), 150 (green), 250 (brown) μm patterns, and un-patterned gel (purple). Error bars in (L-O) correspond to mean±1 SD for n = 3.</p