Synthetic biomaterials are constantly being developed and play central roles in contemporary strategies in regenerative medicine and tissue engineering as artificial extracellular microenvironments. Such scaffolds provide 2D- and 3D-support for interaction with cells and thus convey spatial and temporal control over their function and multicellular processes, such as differentiation and morphogenesis. A model fibrillar system with tunable viscoelastic properties, comprised of 2 native ECM components like collagen type I and the GAG heparin, is presented here. Although the individual components comply with the adhesive, mechanical and bioinductive requirements for artificial reconstituted ECMs, their interaction and structural characterization remains an intriguing conundrum. The aim of the work was to analyze and structurally characterize a xenogeneic in vitro cell culture scaffold reconstituted from two native ECM components, collagen type I and the highly negatively charged glycosaminoglycan heparin. Utilizing a broad spectrum of structural analysis it could be shown that pepsin-solubilized collagen type I fibrils, reconstituted in vitro in the presence of heparin, exhibit an unusually thick and straight shape, with a non-linear dependence in size distribution, width-to-length ratio, and morphology over a wide range of GAG concentrations. The experiments imply a pronounced impact of the nucleation phase on the cofibril morphology as a result of the strong electrostatic interaction of heparin with atelocollagen. Heparin is assumed to stabilize the collagen-GAG complexes and to enhance their parallel accretion during cofibrillogenesis, furthermore corroborated by the heparin quantitation data showing the GAG to be intercalated as a linker molecule with a specific binding site inside the cofibrils. In addition, the exerted morphogenic effect of the GAG, appears to be influenced by factors as degree of sulfation, charge, and concentration. Further detailed structural analysis of the PSC-heparin gels using TEM and SFM showed a hierarchy involving 3 different structural levels and banding patterns in the system: asymmetric segment longspacing (SLS) fibrils and symmetric segments with an average periodicity (AP) of 250 - 260 nm, symmetric fibrous longspacing (FLS IV) nanofibrils with AP of 165 nm, and cofibrils exhibiting an asymmetric D-periodicity of 67 nm with a striking resemblance to the native collagen type I banding pattern. The intercalation of the high negatively charged heparin in the cofibrils was suggested as the main trigger for the hierarchical formation of the polymorphic structures. We also proposed a model explaining the unexpected presence of a symmetric and asymmetric form in the system and the principles governing the symmetric or asymmetric fate of the molecules. The last section of the experiments showed that the presence of telopeptides and heparin both had significant effects on the structural and mechanical characteristics of in vitro reconstituted fibrillar collagen type I. The implemented structural analysis showed that the presence of telopeptides in acid soluble collagen (ASC) impeded the reconstitution of D-periodic collagen fibrils in the presence of heparin, leaving behind only a symmetric polymorphic form with a repeating unit of 165 nm (FLS IV). Further x-ray diffraction analysis of both telopeptide-free and telopeptide-intact collagen fibrils showed that the absence of the flanking non-helical termini in pepsin-solubilized collagen (PSC) resulted in a less compact packing of triple helices of atelocollagen with an increase of interhelical distance from 1.0 to 1.2 nm in dried samples. The looser packing of the triple helices was accompanied by a decrease in bending stiffness of the collagen fibrils, which demonstrated that the intercalated heparin cannot compensate for the depletion of telopeptides. Based on morphological, structural and mechanical differences between ASC and PSC-heparin fibrils reported here, we endorsed the idea that heparin acts as an intrafibrillar cross-linker which competed for binding sites at places along the atelocollagen helix that are occupied in vivo by telopeptides in the fibrillar collagen type I. The performed studies are of particular interest for understanding and gaining control over a rather versatile and already exploited xenogeneic cell culture system. The reconstituted cofibrils with their unusual morphology and GAG intercalation – a phenomenon not reported in vivo – are expected to exhibit interesting biochemical behavior as a biomaterial for ECM scaffolds. Varying the experimental conditions, extent of telopeptide removal, and heparin concentration provides powerful means to control the kinetics, structure, dimensions, as well as mechanical properties of the system which is particularly important for predicting a certain cell behavior towards the newly developed matrix. The GAG intercalation could be interesting for studies with required long-term 'release upon demand' of the GAG, as well as native binding and stabilization of growth factors, cytokines, chemokines, thus providing a secondary tool to control cell signaling and fate, and later on tissue morphogenesis. / Synthetische Biomaterialien werden stetig weiterentwickelt und spielen als künstliche Mikroumgebungen eine zentrale Rolle in den modernen Strategien der regenerativen Medizin und des Tissue Engineerings. Solche sogenannten Scaffolds liefern eine 2D- und 3D-Struktur zur Interaktion mit Zellen und üben somit eine räumliche und zeitliche Kontrolle auf ihre Funktion und multizelluläre Prozesse aus, wie die Differenzierung und Morphogenese. Obwohl häufig die adhäsiven, mechanischen und bioinduzierenden Eigenschaften von Einzelkomponenten aus natürlichen Bestandteilen der extrazellulären Matrix (ECM) rekonstituierten Trägerstrukturen bekannt sind, bleiben die funktionalen und strukturellen Auswirkungen in Mehrkomponentensystemen eine faszinierende Fragestellung. Das Ziel der Arbeit war die Analyse und die strukturelle Charakterisierung einer xenogenen in vitro Zellkultur-Trägerstruktur, die aus den zwei nativen ECM Komponenten Kollagen Typ I und das stark negativ geladene Glykosaminoglykan (GAG) Heparin rekonstituiert wurde. Unter Nutzung eines breiten Spektrums von Methoden zur strukturellen Analyse konnte gezeigt werden, dass im Beisein von Heparin rekonstituierte Pepsin-gelöste Kollagen Typ I Fibrillen eine ungewöhnlich dicke und gerade Form, mit nichtlinearen Abhängigkeiten der Größenverteilung, des Breite-zu-Länge Verhältnises und der Morphologie für eine Reihe von GAG Konzentrationen, aufweisen. Die Experimente deuten auf eine besondere Wirkung der Nukleierungsphase auf die Kofibrillmorphologie hin, als Folge der starken elektrostatischen Inteaktionen Heparins mit Atelokollagen. Es wird angenommen, dass Heparin die Komplexe aus Kollagen-GAG stabilisiert, die parallele Anlagerung während der Kofibrillogenese verbessert und dass überdies, belegt durch Heparin Quantitätsdaten, als Verbindungsmolekül mit einer spezifischen Anbindungsstelle innerhalb der Kofibrillen eingelagert wird. Darüber hinaus scheint der ausgeübte morphogene Effekt des GAGs Heparins von Faktoren wie Grad der Sulfatierung, Ladung und Konzentration abzuhängen. Weitere detailierte Strukturanalysen der PSC - Heparin Gele mit TEM und SFM zeigten eine Hierarchie mit drei unterschiedlichen strukturellen Ebenen und Bandmustern im System: asymmetrisch segmentierte, weitabständige Fibrillen (SLS) und symmetrische Segmente mit einem AP von 250-260 nm, symmetrische fibrose weitabständige (FLS IV) Nanofibrillen mit einem AP von von 165 nm und Kofibrillen asymmetrischer D-Periodizität von 67 nm, die eine erstaunliche Ähnlichkeit zum natürlichen Kollagen Typ I Bandmuster haben. Die Einlagerung des sehr negativ geladenen Heparins in die Kofibrillen wurde als Hauptauslöser der hierarchischen Formation der polymorphen Strukturen betrachtet. Wir schlugen ebenso ein Model vor, welches sowohl das unerwartete Vorhandensein symmetrischer und asymmetrischer Formen im System als auch die Regeln erklärt, die das symmetrische oder asymmetrische Schicksal der Moleküle steuern. Der letzte Abschnitt der Experimente zeigte, dass die Anwesenheit der Telopeptide und Heparins eine signifikante Wirkung auf die strukturellen und mechanischen Charakteristika der in vitro rekonstituierten Kollagen Typ I Fibrillen hatte. Die durchgeführten Strukturanalysen zeigten außerdem, dass die Anwesenheit der Telopeptide in säurelöslichem Kollagen (ASC) die Rekonstitution D-periodischer Kollagenfibrillen mit Heparin verhinderte, sodass nur symmetrisch polymorphe Formen mit einer Wiederholeinheit von 165 nm möglich waren (FLS IV). Weitere Messungen der Telopeptid-freien und Telopeptid-intakten Kollagenfibrillen mit Röntgendiffraktometrie ergaben, dass die Abwesenheit der nicht-helix-strukturierten Enden in Pepsin-gelöstem Kollagen (PSC) zu einer weniger kompakten Anordnung der Tripelhelices von Atelokollagen führte. Der interhelix Abstand erhöhte sich von 1,0 zu 1,2 nm für getrocknete Proben. Das zeigt, dass die losere Anordnung der Tripelhelices einhergeht mit der Verringerung der Biege-Elastizitäts-module der Kollagenfibrillen,. Basierend auf den hier vorgestellten morphologischen, strukturellen und mechanischen Unterschieden zwischen ASC und PSC-Heparin Fibrillen wird die Idee unterstützt, dass Heparin als intrafibrillärer Vernetzer fungiert und an Bindungsstellen der Helix bindet, welche in vivo bei Kollagen Typ I Fibrillen durch Telopeptide besetzt sind. Die durchgeführten Studien sind von besonderem Interesse für das Verständnis und die Steuerung eines sehr vielseitigen und bereits verwendeten xenogenes Zellkultursystem für das Tissue Engineering. Von den rekonstituierten Kofibrillen mit ihrer ungewöhnlichen Morphologie und GAG Einlagerung - ein in vivo nicht bekanntes Phänomen - erwartet man, dass sie ein intressantes biochemisches Verhalten als Biomaterial für ECM Scaffolds zeigen. Variationen der experimentellen Bedingungen, des Ausmaßes der Telopeptidentfernung und der Heparinkonzentration liefern vielfältige Möglichkeiten um die Kinetik, Struktur, Dimension sowie die mechanischen Eigenschaften des Systems zu kontrollieren. Damit sollte es möglich sein, ein bestimmtes Zellverhalten gegenüber der neu entwickelten Matrix vorherzusagen. Die GAG-Einlagerung bietet interessante Optionen für eine langfristige Freisetzung des GAGs 'on demand', sowie die native Bindung und Stabilisierung von Wachstumsfaktoren, Cytokinen, Chemokinen, womit zusätzlich Zellsignalisierung und -schicksal und später Gewebemorphogenese kontrolliert werden kann.

L’athérosclérose est caractérisée par l’accumulation de lipoprotéines de basse densité (LDL) liées aux protéoglycanes de la paroi artérielle. Des anticorps chimériques (ch) qui se lient aux glycosaminoglycanes (GAG) ont été générés. L'hypothèse est que la vaccination avec le chP3R99, un anticorps chimérique mutant de l'hybridome P3, pouvait interférer avec la rétention des LDL par l’induction d’une cascade d’anticorps anti-idiotypiques dirigés contre les GAG. Des souris mâles déficientes en apolipoprotéine E ont été soumises à une diète hypercholestérolémique et ont reçu 5 injections sous-cutanées de 50 μg de vaccin chP3R99 ou de vaccin chP3S98 (un mutant de faible réactivité). Les injections ont été effectuées à chaque semaine ou aux 2 semaines. Au moment du sacrifice, l'aorte perfusée avec du PBS a été excisée et analysée après coloration au Oil Red-O. Les résultats ont été exprimés en pourcentage de lésions sur la superficie totale de l'aorte. La réactivité contre le chP3R99, chP3S98, l’héparine, le sulfate de dermatane et de chondroïtine des sérums de souris immunisées a été mesurée par ELISA. De plus, la liaison de l'anticorps chP3R99 aux GAG dans la lésion d'athérosclérose a été observée par un appareil de visualisation in vivo.Nos résultats montrent que l’immunogénicité des anticorps chP3R99 est supérieure à celle des anticorps chP3S98 et que le sérum des souris immunisées avec le chP3R99 présente des anticorps anti-idiotypiques dirigés contre les GAG. Cet effet est associé à une réduction de 42 % (p < 0.01) du pourcentage de lésions athérosclérotiques chez les souris vaccinées. L'utilisation d’une immunisation active avec l’anticorps chP3R99 pourrait constituer une approche thérapeutique pour le traitement de l'athérosclérose. / Atherosclerosis is characterized by the accumulation of low density lipoprotein (LDL) associated with the proteoglycans of the arterial wall. Chimeric (ch) antibodies that react against glycosaminoglycans (GAG) were generated. We tested the hypothesis that vaccination with chP3R99, a mutant chimeric antibody of the P3 hybridoma, could interfere with the retention of LDL by inducing a cascade of anti-idiotypic antibodies directed against the GAG.Male mice deficient in apolipoprotein E fed with a hypercholesterolemic diet were given five subcutaneous injections of 50 μg of chP3R99 or chP3S98 (a mutant with low reactivity) vaccine. The injections were performed every week or every two weeks. After sacrifice, the aorta was perfused with PBS, excised and analyzed after staining with Oil Red-O. The results were expressed as a percentage of lesions areas on the total area of the aorta. The reactivity of the sera obtained was tested against the chP3R99, chP3S98, heparin, dermatan and chondroïtin sulfate from obtained immunized mice by ELISA. The anti-idiotypic response was measured by blocking the anti-isotypic reactivity by a nonspecific IgG, hR3. In addition, the antibody chP3R99 binding to GAG in the atherosclerotic lesion was shown by an in vivo molecular imaging device. Our results show that the immunogenicity of the antibodies chP3R99 is higher than chP3S98 and that sera from mice immunized with chP3R99 present anti-idiotypic antibodies directed against the GAG. This effect is associated with a 42 % reduction (p <0.01) of atherosclerotic lesions in vaccinated mice. The use of active immunization with antibodies chP3R99 may constitute a new therapeutic approach for the treatment of atherosclerosis.

Glycosaminoglycans (GAGs) play an important role in many biological processes in the extracellular matrix. In a theoretical approach, structures of monosaccharide building blocks of natural GAGs and their sulfated derivatives were optimized by a B3LYP6311ppdd//B3LYP/ 6-31+G(d) method. The dependence of the observed conformational properties on the applied methodology is described. NMR chemical shifts and proton-proton spin-spin coupling constants were calculated using the GIAO approach and analyzed in terms of the method's accuracy and sensitivity towards the influence of sulfation, O1-methylation, conformations of sugar ring, and ω dihedral angle. The net sulfation of the monosaccharides was found to be correlated with the 1H chemical shifts in the methyl group of the N-acetylated saccharides both theoretically and experimentally. The ω dihedral angle conformation populations of free monosaccharides and monosaccharide blocks within polymeric GAG molecules were calculated by a molecular dynamics approach using the GLYCAM06 force field and compared with the available NMR and quantum mechanical data. Qualitative trends for the impact of sulfation and ring conformation on the chemical shifts and proton-proton spin-spin coupling constants were obtained and discussed in terms of the potential and limitations of the computational methodology used to be complementary to NMR experiments and to assist in experimental data assignment.

Thrombin is the key protease that regulates hemostasis; the delicate balance between procoagulation and anticoagulation of blood. In clotting disorders, like deep vein thrombosis or pulmonary embolism, procoagulation is up-regulated, but propagation of clotting can be inhibited with drugs targeting the proteases involved, like thrombin. Such drugs however, have serious side effects (e.g., excessive bleeding) and some require monitoring during the course of treatment. The reason for these side effects is the mechanism by which the drugs’ act. The two major mechanisms are direct orthosteric and indirect allosteric inhibition, which will completely abolish the protease’s activity. Herein we sought an alternative mechanism called allosteric, partial inhibition, that has shown promise to truly regulate coagulation. Partial inhibition through allosteric mechanisms are well described for membrane-bound and oligomeric proteins. However proteases, specifically monomeric proteases (i.e., thrombin), have not shown this phenomenon until now. A small library of coumarin-based sulfated allosteric modulators (CSAMs) was synthesized to target a surface region called exosite 2; mainly composed of highly positively charged residues surrounded by hydrophobic patches. Studies revealed a non-competitive mechanism of binding with a range of IC50s between 0.2-58 µM combined with inhibitory efficacies (ΔY) between 22-73%; indicative of allosteric, partial inhibition. The KD was determined for the most potent compound (3g; IC50 = 0.2 µM, ΔY = 47%) at 0.15 µM. 3g was observed to bind at exosite 2 through unfractionated heparin competition and thrombin mutant studies. Additional computational studies were in agreement with the mutant and competition studies, showing the sulfate of 3g binding within a pocket containing R126 and R233. Fluorescence quenching and antithrombin inactivation rate studies described a conformational change to thrombin’s active site in the presence of 3g, supporting reduction of thrombin’s catalytic efficiency, without complete inhibition of thrombin’s proteolytic activities. Coupled enzyme assays and gel electrophoresis showed that in the presence of 3g, hydrolysis of fibrinogen (IC50 = 0.51 µM, ΔY = 94%) and protein C activation (IC50 = 1.7 µM, ΔY = 91%) is fully inhibited. Alternatively, FXIII activation was shown to be only partially inhibited by the presence of 3g, and FXI activation did not show any significant activation or inhibition. 3g was also shown to be active in human plasma and whole blood, but requiring much higher concentrations to induce an anticoagulant effect. Mice studies looking at the effects of 3g in vivo showed that even at high concentrations, showed no abnormal bleeding or any other irregularities. This work highlights a novel occurrence regarding thrombin’s allosteric functionality against multiple endogenous substrates. This library of compounds may be useful in the future development of allosteric inhibitors and probes that pose little to no risk of bleeding events by inducing partial inhibition.

Connor O’Hara July 29, 2019 Inhibition of Cancer Stem Cells by Glycosaminoglycan Mimetics In the United States cancer is the second leading cause of death, with colorectal cancer (CRC) being the third deadliest cancer and expected to cause over 51,000 fatalities in 2019 alone.1 The current standard of care for CRC depends largely on the staging, location, and presence of metastasis.2 As the tumor grows and invades nearby lymph tissue and blood vessels, CRC has the opportunity to invade not only nearby tissue but also metastasize into the liver and lung (most commonly).3 The 5-year survival rate for metastasized CRC is <15%, and standard of care chemotherapy regimens utilizing combination treatments only marginally improve survival.3-5 Additionally, patients who have gone into remission from late-stage CRC have a high risk of recurrence despite advances in treatment.6-7 The Cancer Stem-like Cell (CSC) paradigm has grown over the last 20 years to become a unifying hypothesis to support the growth and relapse of tumors previously regressed from chemotherapy (Figure 1).8 The paradigm emphasizes the heterogeneity of a tumor and its microenvironment, proposing that a small subset of cells in the tumor are the source of tumorigenesis with features akin to normal stem cells.9 The CSCs normally in a quiescent state survive this chemotherapy and “seed” tumor redevelopment.10 First observed in acute myeloid lymphoma models, CSCs have since been identified in various other cancers (to include CRC) by their cell surface antigens and unique properties characterizing them from normal cancer cells.11-12 These include tumor initiation, limitless self-renewal capacity to generate clonal daughter cells, as well as phenotypically diverse, mature, and highly differentiated progeny.13-14 Previously our lab has identified a novel molecule called G2.2 (Figure 2) from a unique library of sulfated compounds showing selective and potent inhibition of colorectal CSCs in-vitro.15 G2.2 is a mimetic of glycosaminoglycans (GAGs) and belongs to a class of molecules called non-saccharide GAG mimetics (NSGMs). Using a novel dual-screening platform, comparisons were made on the potency of G2.2 in bulk monolayer cells, primary 3D tumor spheroids of the same cell line, and subsequent generations of tumor spheroids. This work has shown in-vitro the fold-enhancement of CSCs when culturing as 3D tumor spheroids. Spheroid culture serves as a more accurate model for the physiological conditions of a tumor, as well as the functional importance of upregulating CSCs. Evaluation of G2.2 and other NSGMs was performed in only a few cell lines, developing a need to better understand the ability of G2.2 to inhibit spheroids from a more diverse panel of cancer cells to better understand G2.2’s mechanism. The last few decades have seen the advancement in fundamental biological and biochemical knowledge of tumor cell biology and genetics.16 CRC, in particular, has served as a useful preclinical model in recapitulating patient tumor heterogeneity in-vitro.17 Recent work has characterized the molecular phenotypes of CRC cell lines in a multi-omics analysis, stratifying them into 4 clinically robust and relevant consensus molecular subtypes (CMS).18-19 Our work was directed to screen a panel of cells from each of the molecular subtypes and characterize the action of G2.2 and 2nd generation lipid-modified analogs, synthesized to improve the pharmacokinetic properties of the parent compound. Four NSGMs, namely G2.2, G2C, G5C, and G8C (Figure 2) were studied for their ability to inhibit the growth of primary spheroids across a phenotypically diverse panel. Compound HT-29 IC50 (μM) Panel Average IC50 (μM) G2.2 28 ± 1 185 ± 55 G2C 5 ± 2 16 ± 15 G5C 8 ± 2 63 ± 19 G8C 0.7 ± 0.2 6 ± 3 Primary spheroid inhibition assays were performed comparing the potency of new NSGMs to G2.2. Fifteen cell lines were evaluated in a panel of colorectal adenocarcinoma cell lines with several cell lines representing each CMS. Primary spheroid inhibition assays revealed 3 distinct response with regard to G2.2’s ability to inhibit spheroid growth. Cells from CMS 3 and 4, which display poor clinical prognosis, metabolic dysregulation, and enhanced activation of CSC pathways, showed the most sensitivity to G2.2 (mean IC50 = 89 ± 55 μM). Mesenchymal CMS 4 cell lines were over 3-fold more sensitive to treatment with G2.2 when compared to CMS 1 cell lines. Resistant cell lines were composed entirely of CMS 1 and 2 (mean IC50 = 267 ± 105 μM). In contrast, all lipid-modified analogs showed greater potency than the parent NSGM in almost every CRC cell line. Of the three analogs, G8C showed the greatest potency with a mean IC50 of less than 15 μM. Of the CRC spheroids studied, HT-29 (CMS 3) was most sensitive to G8C (IC50 = 0.73 μM). To evaluate the selectivity of NSGMs for CSC spheroid inhibition, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) cytotoxicity assays were performed on monolayer cell culture, and the fold-selectivity of NSGM for spheroids was analyzed. Data shows that NSGMs preferentially target CSC-rich spheroids compared with monolayer cellular growth, with G2.2 having over 7-fold selectivity for spheroid conditions. This fold selectivity was enhanced in CMS 3/4, supporting the idea that G2.2 targets a mesenchymal and stem-like phenotype. To further validate this selectivity, limiting dilution assays were performed across the panel to determine the tumor-initiating capacity of each cell line. Cell lines which showed a sensitive response to G2.2 were over 2-fold more likely to develop into spheroids, validating the previous hypothesis. Further characterization was performed analyzing the changes G2.2 induced on CSC markers, as well as the basal expression of a unique pair of cancer cells. Western blots showed a reduction in self-renewal marker across all CMS after treatment with G2.2, and that cell lines sensitive to G2.2-treatment overexpress mesenchymal and stem-like markers. G2.2-resistant cell lines show an epithelial phenotype, lacking this expression. The positive results observed in these studies enhance the understanding of G2.2 and analogs, and further evaluation with additional cell lines of various tissues would improve the knowledge thus far gained. However, all experiments described take valuable time to perform and analyze. Thus, there became a need to develop a high-throughput screening (HTS) platform for our assays that standardized analysis and enhanced productivity. Initial development of the method for this assay are underway, and recent evidence from these evaluations of breast cancer spheroids suggests that G2.2 and analogs may be tissue-specific compounds for the treatment of cancer. Future work entails refining the application of this method for evaluation of the NCI-60 (National Cancer Institute) tumor cell panel. Overall, these results make several suggestions concerning the NSGMs evaluated against the panel. First, G2.2 selectively targets CSCs with limited toxicity to monolayer cells of the same cell line. Further, G2.2 has the greatest potency with CMS 3/4, whose mesenchymal phenotypes are associated with poor clinical prognosis and enrichment of CSCs. Supporting evidence include that sensitive cell lines are highly tumorigenic and show enhanced expression of mesenchymal/CSC markers compared to resistant cell lines. Lipid-modification of G2.2 enhances in-vitro potency against spheroid growth, with nM potency reached in the most sensitive cell lines. Evidence in the development of a HTS platform also suggests these NSGMs show tissue specificity to cancers of the intestine. Further work characterizing the mechanism of NSGMs in a broader multi-tissue panel will enhance our understanding of the compounds as a potential therapy to dramatically improve patient survival through specific targeting of tumorigenesis. References 1. Colorectal Cancer Facts & Figures 2017-2019. American Cancer Society 2017. 2. Compton, C. C.; Byrd, D. R.; Garcia-Aguilar, J.; Kurtzman, S. H.; Olawaiye, A.; Washington, M. K. Colon and rectum. In AJCC Cancer Staging Atlas, 2nd ed.; Ed. Springer Science: New York, 2012; pp 185–201. 3. Van Cutsem, E.; Cervantes, A.; Adam, R.; Sobrero, A.; Van Krieken, J. H.; Aderka, D.; Aranda Aguilar, E.; Bardelli, A.; Benson, A.; Bodoky, G.; et al. ESMO consensus guidelines for the management of patients with metastatic colorectal cancer. Ann. Oncol. 2016, 27, 1386–422. 4. Siegel, R. L.; Miller, K. D.; Fedewa, S. A.; Ahnen, D. J.; Meester, R. G. S.; Barzi, A.; Jemal, A. Colorectal cancer statistics, 2017. CA Cancer J. Clin. 2017, 67, 177–193. 5. Moriarity, A.; O'Sullivan, J.; Kennedy, J.; Mehigan, B.; McCormick, P. Current targeted therapies in the treatment of advanced colorectal cancer: a review. Ther. Adv. Med. Oncol. 2016, 8, 276–293. 6. Seidel, J.; Farber, E.; Baumbach, R.; Cordruwisch, W.; Bohmler, U.; Feyerabend, B.; Faiss, S. Complication and local recurrence rate after endoscopic resection of large high-risk colorectal adenomas of >/=3 cm in size. Int. J. Colorectal Dis. 2016, 31, 603–611. 7. Pugh, S. A.; Shinkins, B.; Fuller, A.; Mellor, J.; Mant, D.; Primrose, J. N. Site and stage of colorectal cancer influence the likelihood and distribution of disease recurrence and postrecurrence survival: data from the FACS randomized controlled trial. Ann. Surg. 2016, 263, 1143–1147. 8. Batlle, E.; Clevers, H. Cancer stem cells revisited. Nat. Med. 2017, 23, 1124–1134. 9. Hanahan, D.; Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 2011, 144, 646–674. 10. Tirino, V.; Desiderio, V.; Paino, F.; De Rosa, A.; Papaccio, F.; La Noce, M.; Laino, L.; De Francesco, F.; Papaccio, G. Cancer stem cells in solid tumors: an overview and new approaches for their isolation and characterization. FASEB J. 2013, 27, 13–24. 11. Bonnet, D.; Dick, J. E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat. Med. 1997, 3, 730–737. 12. Desai, A.; Yan, Y.; Gerson, S. L. Concise reviews: cancer stem cell targeted therapies: toward clinical success. Stem Cells Transl. Med. 2019, 8, 75–81. 13. Munro, M. J.; Wickremesekera, S. K.; Peng, L.; Tan, S. T.; Itinteang, T. Cancer stem cells in colorectal cancer: a review. J. Clin. Pathol. 2018, 71, 110–116. 14. Zhou, Y.; Xia, L.; Wang, H.; Oyang, L.; Su, M.; Liu, Q.; Lin, J.; Tan, S.; Tian, Y.; Liao, Q.; Cao, D. Cancer stem cells in progression of colorectal cancer. Oncotarget 2018, 9, 33403–33415. 15. Patel, N. J.; Karuturi, R.; Al-Horani, R. A.; Baranwal, S.; Patel, J.; Desai, U. R.; Patel, B. B. Synthetic, non-saccharide, glycosaminoglycan mimetics selectively target colon cancer stem cells. ACS Chem. Biol. 2014, 9, 1826–1833. 16. Punt, C. J.; Koopman, M.; Vermeulen, L. From tumour heterogeneity to advances in precision treatment of colorectal cancer. Nat. Rev. Clin. Oncol. 2017, 14, 235–246. 17. Mouradov, D.; Sloggett, C.; Jorissen, R. N.; Love, C. G.; Li, S.; Burgess, A. W.; Arango, D.; Strausberg, R. L.; Buchanan, D.; Wormald, S.; et al. Colorectal cancer cell lines are representative models of the main molecular subtypes of primary cancer. Cancer Res. 2014, 74, 3238–3247. 18. Guinney, J.; Dienstmann, R.; Wang, X.; de Reynies, A.; Schlicker, A.; Soneson, C.; Marisa, L.; Roepman, P.; Nyamundanda, G.; Angelino, P.; et al. The consensus molecular subtypes of colorectal cancer. Nat. Med. 2015, 21, 1350–1356. 19. Berg, K. C. G.; Eide, P. W.; Eilertsen, I. A.; Johannessen, B.; Bruun, J.; Danielsen, S. A.; Bjornslett, M.; Meza-Zepeda, L. A.; Eknaes, M.; Lind, G. E.; et al. Multi-omics of 34 colorectal cancer cell lines - a resource for biomedical studies. Mol. Cancer 2017, 16, 116–132.

SerpinA12 (vaspin) is thought to be mainly expressed in adipose tissue and has multiple beneficial effects on metabolic, inflammatory and atherogenic processes related to obesity. KLK7 (kallikrein 7) is the only known protease target of vaspin to date and is inhibited with a moderate inhibition rate. In the crystal structure, the cleavage site (P1-P1′) of the vaspin reactive centre loop is fairly rigid compared with the flexible residues before P2, possibly supported by an ionic interaction of P1′ glutamate (Glu379) with an arginine residue (Arg302) of the β-sheet C. A P1′ glutamate seems highly unusual and unfavourable for the protease KLK7. We characterized vaspin mutants to investigate the roles of these two residues in protease inhibition and recognition by vaspin. Reactive centre loop mutations changing the P1′ residue or altering the reactive centre loop conformation significantly increased inhibition parameters, whereas removal of the positive charge within β-sheet C impeded the serpin–protease interaction. Arg302 is a crucial contact to enable vaspin recognition by KLK7 and it supports moderate inhibition of the serpin despite the presence of the detrimental P1′ Glu379, which clearly represents a major limiting factor for vaspin-inhibitory activity. We also show that the vaspin-inhibition rate for KLK7 can be modestly increased by heparin and demonstrate that vaspin is a heparin-binding serpin. Noteworthily, we observed vaspin as a remarkably thermostable serpin and found that Glu379 and Arg302 influence heat-induced polymerization. These structural and functional results reveal the mechanistic basis of how reactive centre loop sequence and exosite interaction in vaspin enable KLK7 recognition and regulate protease inhibition as well as stability of this adipose tissue-derived serpin.

Synthetic biomaterials are constantly being developed and play central roles in contemporary strategies in regenerative medicine and tissue engineering as artificial extracellular microenvironments. Such scaffolds provide 2D- and 3D-support for interaction with cells and thus convey spatial and temporal control over their function and multicellular processes, such as differentiation and morphogenesis. A model fibrillar system with tunable viscoelastic properties, comprised of 2 native ECM components like collagen type I and the GAG heparin, is presented here. Although the individual components comply with the adhesive, mechanical and bioinductive requirements for artificial reconstituted ECMs, their interaction and structural characterization remains an intriguing conundrum. The aim of the work was to analyze and structurally characterize a xenogeneic in vitro cell culture scaffold reconstituted from two native ECM components, collagen type I and the highly negatively charged glycosaminoglycan heparin. Utilizing a broad spectrum of structural analysis it could be shown that pepsin-solubilized collagen type I fibrils, reconstituted in vitro in the presence of heparin, exhibit an unusually thick and straight shape, with a non-linear dependence in size distribution, width-to-length ratio, and morphology over a wide range of GAG concentrations. The experiments imply a pronounced impact of the nucleation phase on the cofibril morphology as a result of the strong electrostatic interaction of heparin with atelocollagen. Heparin is assumed to stabilize the collagen-GAG complexes and to enhance their parallel accretion during cofibrillogenesis, furthermore corroborated by the heparin quantitation data showing the GAG to be intercalated as a linker molecule with a specific binding site inside the cofibrils. In addition, the exerted morphogenic effect of the GAG, appears to be influenced by factors as degree of sulfation, charge, and concentration. Further detailed structural analysis of the PSC-heparin gels using TEM and SFM showed a hierarchy involving 3 different structural levels and banding patterns in the system: asymmetric segment longspacing (SLS) fibrils and symmetric segments with an average periodicity (AP) of 250 - 260 nm, symmetric fibrous longspacing (FLS IV) nanofibrils with AP of 165 nm, and cofibrils exhibiting an asymmetric D-periodicity of 67 nm with a striking resemblance to the native collagen type I banding pattern. The intercalation of the high negatively charged heparin in the cofibrils was suggested as the main trigger for the hierarchical formation of the polymorphic structures. We also proposed a model explaining the unexpected presence of a symmetric and asymmetric form in the system and the principles governing the symmetric or asymmetric fate of the molecules. The last section of the experiments showed that the presence of telopeptides and heparin both had significant effects on the structural and mechanical characteristics of in vitro reconstituted fibrillar collagen type I. The implemented structural analysis showed that the presence of telopeptides in acid soluble collagen (ASC) impeded the reconstitution of D-periodic collagen fibrils in the presence of heparin, leaving behind only a symmetric polymorphic form with a repeating unit of 165 nm (FLS IV). Further x-ray diffraction analysis of both telopeptide-free and telopeptide-intact collagen fibrils showed that the absence of the flanking non-helical termini in pepsin-solubilized collagen (PSC) resulted in a less compact packing of triple helices of atelocollagen with an increase of interhelical distance from 1.0 to 1.2 nm in dried samples. The looser packing of the triple helices was accompanied by a decrease in bending stiffness of the collagen fibrils, which demonstrated that the intercalated heparin cannot compensate for the depletion of telopeptides. Based on morphological, structural and mechanical differences between ASC and PSC-heparin fibrils reported here, we endorsed the idea that heparin acts as an intrafibrillar cross-linker which competed for binding sites at places along the atelocollagen helix that are occupied in vivo by telopeptides in the fibrillar collagen type I. The performed studies are of particular interest for understanding and gaining control over a rather versatile and already exploited xenogeneic cell culture system. The reconstituted cofibrils with their unusual morphology and GAG intercalation – a phenomenon not reported in vivo – are expected to exhibit interesting biochemical behavior as a biomaterial for ECM scaffolds. Varying the experimental conditions, extent of telopeptide removal, and heparin concentration provides powerful means to control the kinetics, structure, dimensions, as well as mechanical properties of the system which is particularly important for predicting a certain cell behavior towards the newly developed matrix. The GAG intercalation could be interesting for studies with required long-term 'release upon demand' of the GAG, as well as native binding and stabilization of growth factors, cytokines, chemokines, thus providing a secondary tool to control cell signaling and fate, and later on tissue morphogenesis. / Synthetische Biomaterialien werden stetig weiterentwickelt und spielen als künstliche Mikroumgebungen eine zentrale Rolle in den modernen Strategien der regenerativen Medizin und des Tissue Engineerings. Solche sogenannten Scaffolds liefern eine 2D- und 3D-Struktur zur Interaktion mit Zellen und üben somit eine räumliche und zeitliche Kontrolle auf ihre Funktion und multizelluläre Prozesse aus, wie die Differenzierung und Morphogenese. Obwohl häufig die adhäsiven, mechanischen und bioinduzierenden Eigenschaften von Einzelkomponenten aus natürlichen Bestandteilen der extrazellulären Matrix (ECM) rekonstituierten Trägerstrukturen bekannt sind, bleiben die funktionalen und strukturellen Auswirkungen in Mehrkomponentensystemen eine faszinierende Fragestellung. Das Ziel der Arbeit war die Analyse und die strukturelle Charakterisierung einer xenogenen in vitro Zellkultur-Trägerstruktur, die aus den zwei nativen ECM Komponenten Kollagen Typ I und das stark negativ geladene Glykosaminoglykan (GAG) Heparin rekonstituiert wurde. Unter Nutzung eines breiten Spektrums von Methoden zur strukturellen Analyse konnte gezeigt werden, dass im Beisein von Heparin rekonstituierte Pepsin-gelöste Kollagen Typ I Fibrillen eine ungewöhnlich dicke und gerade Form, mit nichtlinearen Abhängigkeiten der Größenverteilung, des Breite-zu-Länge Verhältnises und der Morphologie für eine Reihe von GAG Konzentrationen, aufweisen. Die Experimente deuten auf eine besondere Wirkung der Nukleierungsphase auf die Kofibrillmorphologie hin, als Folge der starken elektrostatischen Inteaktionen Heparins mit Atelokollagen. Es wird angenommen, dass Heparin die Komplexe aus Kollagen-GAG stabilisiert, die parallele Anlagerung während der Kofibrillogenese verbessert und dass überdies, belegt durch Heparin Quantitätsdaten, als Verbindungsmolekül mit einer spezifischen Anbindungsstelle innerhalb der Kofibrillen eingelagert wird. Darüber hinaus scheint der ausgeübte morphogene Effekt des GAGs Heparins von Faktoren wie Grad der Sulfatierung, Ladung und Konzentration abzuhängen. Weitere detailierte Strukturanalysen der PSC - Heparin Gele mit TEM und SFM zeigten eine Hierarchie mit drei unterschiedlichen strukturellen Ebenen und Bandmustern im System: asymmetrisch segmentierte, weitabständige Fibrillen (SLS) und symmetrische Segmente mit einem AP von 250-260 nm, symmetrische fibrose weitabständige (FLS IV) Nanofibrillen mit einem AP von von 165 nm und Kofibrillen asymmetrischer D-Periodizität von 67 nm, die eine erstaunliche Ähnlichkeit zum natürlichen Kollagen Typ I Bandmuster haben. Die Einlagerung des sehr negativ geladenen Heparins in die Kofibrillen wurde als Hauptauslöser der hierarchischen Formation der polymorphen Strukturen betrachtet. Wir schlugen ebenso ein Model vor, welches sowohl das unerwartete Vorhandensein symmetrischer und asymmetrischer Formen im System als auch die Regeln erklärt, die das symmetrische oder asymmetrische Schicksal der Moleküle steuern. Der letzte Abschnitt der Experimente zeigte, dass die Anwesenheit der Telopeptide und Heparins eine signifikante Wirkung auf die strukturellen und mechanischen Charakteristika der in vitro rekonstituierten Kollagen Typ I Fibrillen hatte. Die durchgeführten Strukturanalysen zeigten außerdem, dass die Anwesenheit der Telopeptide in säurelöslichem Kollagen (ASC) die Rekonstitution D-periodischer Kollagenfibrillen mit Heparin verhinderte, sodass nur symmetrisch polymorphe Formen mit einer Wiederholeinheit von 165 nm möglich waren (FLS IV). Weitere Messungen der Telopeptid-freien und Telopeptid-intakten Kollagenfibrillen mit Röntgendiffraktometrie ergaben, dass die Abwesenheit der nicht-helix-strukturierten Enden in Pepsin-gelöstem Kollagen (PSC) zu einer weniger kompakten Anordnung der Tripelhelices von Atelokollagen führte. Der interhelix Abstand erhöhte sich von 1,0 zu 1,2 nm für getrocknete Proben. Das zeigt, dass die losere Anordnung der Tripelhelices einhergeht mit der Verringerung der Biege-Elastizitäts-module der Kollagenfibrillen,. Basierend auf den hier vorgestellten morphologischen, strukturellen und mechanischen Unterschieden zwischen ASC und PSC-Heparin Fibrillen wird die Idee unterstützt, dass Heparin als intrafibrillärer Vernetzer fungiert und an Bindungsstellen der Helix bindet, welche in vivo bei Kollagen Typ I Fibrillen durch Telopeptide besetzt sind. Die durchgeführten Studien sind von besonderem Interesse für das Verständnis und die Steuerung eines sehr vielseitigen und bereits verwendeten xenogenes Zellkultursystem für das Tissue Engineering. Von den rekonstituierten Kofibrillen mit ihrer ungewöhnlichen Morphologie und GAG Einlagerung - ein in vivo nicht bekanntes Phänomen - erwartet man, dass sie ein intressantes biochemisches Verhalten als Biomaterial für ECM Scaffolds zeigen. Variationen der experimentellen Bedingungen, des Ausmaßes der Telopeptidentfernung und der Heparinkonzentration liefern vielfältige Möglichkeiten um die Kinetik, Struktur, Dimension sowie die mechanischen Eigenschaften des Systems zu kontrollieren. Damit sollte es möglich sein, ein bestimmtes Zellverhalten gegenüber der neu entwickelten Matrix vorherzusagen. Die GAG-Einlagerung bietet interessante Optionen für eine langfristige Freisetzung des GAGs 'on demand', sowie die native Bindung und Stabilisierung von Wachstumsfaktoren, Cytokinen, Chemokinen, womit zusätzlich Zellsignalisierung und -schicksal und später Gewebemorphogenese kontrolliert werden kann.