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Directed invasion and migratory modes of cancer cells in structured 3D collagen matrices
Directed invasion and migratory modes of cancer cells in structured 3D collagen matrices
Cancer is a disease responsible for a large number of deaths in society. Specifically, cancer cell migration is associated with malignancy and difficult to treat. Migration is influenced by mechanical and chemical properties of the environment as well as the cellular phenotype and behavior. Cancer cells can adapt to the properties of their environment by adjusting their phenotype and they can alter the tissue around them. In the following, the influence of fiber stiffness, confinement, and adhesion properties on cancer cell migration in porous collagen gels is investigated. Soft collagen gels with short fibers hinder migration and promote a round, non-invasive phenotype. Longer and stiffer collagen fibers lead to an adhesive phenotype and confined migration due to their more adhesive properties. With TGF-β, adhesion is lowered and the cancer cells switch from the adhesive phenotype to highly motile amoeboid phenotypes. In stiff collagen gels with pores of about cell size the highest migration speeds and longest displacements are achieved by cells with an amoeboid phenotype. The mechanical properties of collagen gels directly influence the phenotype and subsequently migration, which is most efficient with an amoeboid phenotype and in gels with stiff fibers, cell sized pores, and low adhesion. This was also investigated for cancer cell aggregates in highly oriented collagen gels. By using a microfluidic channel setup the collagen fibers were aligned tangentially and radially with respect to the spheroid surface. The alignment can be described by finite element simulations. This specific orientation of the collagen matrix influenced invasion from the cancer cell spheroid, creating a strong bias of invasion towards radial as compared to tangential fiber orientation. Brownian diffusion model simulations suggest a completely blocked migration perpendicular to fibers, allowing migration only along fibers. The actual invasion is slowed down in areas with tangentially oriented fibers, but it is still possible. Furthermore a new method to detect proliferating tumor cells in situ by using multiple consecutive click reactions with dendrimeric molecules and clickable dyes is presented. Cells are grown in the presence of ethinyl‐dU (EdU), where the EdU becomes part of the genome during proliferation. After the cells were fixed and permeabilised, the incorporated alkynes of the EdU are functionalized with azide‐containing fluorophores through the CuI‐catalysed alkyne–azide click reaction. These azide‐ and alkyne‐modified dendrimers allow the establishment of sandwich‐type detection assays, which have improved sensitivities, signal intensities and signal‐to‐noise ratios in reference to comparable techniques. The RNA‐FISH‐based detection of RNA was also improved in the following by increasing the number of fluorophores per oligonucleotide probe. Currently, the RNA-FISH detection method uses sets of single‐fluorophore‐containing oligonucleotide probes that hybridize to the mRNA of interest. This reliable detection of transcription events and the localization and quantification of particular mRNA allows disease states to be characterized more directly. For the early detection of virus infections, when spreading of the virus in- and outside of the organism can be controlled much better, this is particularly important (e. g. Sars-CoV-2). To receive a reliable signal, a large number of probe strands (>30) is required, but the more oligonucleotide probes are used, the higher the off‐target binding effects, which create background noise. Through the use of click chemistry and alkyne‐modified DNA oligonucleotides multiple‐fluorophore‐containing probes were prepared. These triply labeled probes allow reliable detection and direct visualization of mRNA with only a very small number (5–10) of probe strands. In an in situ experiment this lead to lower background noise and a better signal to noise ratio, whereby viral transcripts can be detected 4 hours after infection. RNA viruses induce formation of subcellular organelles that provide microenvironments beneficial to their replication. These replication factories of rotaviruses are protein-RNA condensates, formed via liquid-liquid phase separation. Through phase separation rotavirus proteins NSP5 and NSP2 form RNA-rich condensates, which can be reversibly dissolved in vitro by aliphatic diols. These RNA-protein condensates became less dynamic and impervious to aliphatic diols during infection, indicating a transition from a liquid to solid state. The selective enrichment of viral transcripts seems to be a unique feature of these condensates, but other aspects of assembly are similar to the formation of cytoplasmic ribonucleoprotein granules. By targeting these complex RNA-protein condensates, that underlie replication of RNA viruses, promising novel therapeutic approaches could be developed.
Invasion, Migration, Collagen, Cancer, Cells
Geiger, Florian
2021
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Geiger, Florian (2021): Directed invasion and migratory modes of cancer cells in structured 3D collagen matrices. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Cancer is a disease responsible for a large number of deaths in society. Specifically, cancer cell migration is associated with malignancy and difficult to treat. Migration is influenced by mechanical and chemical properties of the environment as well as the cellular phenotype and behavior. Cancer cells can adapt to the properties of their environment by adjusting their phenotype and they can alter the tissue around them. In the following, the influence of fiber stiffness, confinement, and adhesion properties on cancer cell migration in porous collagen gels is investigated. Soft collagen gels with short fibers hinder migration and promote a round, non-invasive phenotype. Longer and stiffer collagen fibers lead to an adhesive phenotype and confined migration due to their more adhesive properties. With TGF-β, adhesion is lowered and the cancer cells switch from the adhesive phenotype to highly motile amoeboid phenotypes. In stiff collagen gels with pores of about cell size the highest migration speeds and longest displacements are achieved by cells with an amoeboid phenotype. The mechanical properties of collagen gels directly influence the phenotype and subsequently migration, which is most efficient with an amoeboid phenotype and in gels with stiff fibers, cell sized pores, and low adhesion. This was also investigated for cancer cell aggregates in highly oriented collagen gels. By using a microfluidic channel setup the collagen fibers were aligned tangentially and radially with respect to the spheroid surface. The alignment can be described by finite element simulations. This specific orientation of the collagen matrix influenced invasion from the cancer cell spheroid, creating a strong bias of invasion towards radial as compared to tangential fiber orientation. Brownian diffusion model simulations suggest a completely blocked migration perpendicular to fibers, allowing migration only along fibers. The actual invasion is slowed down in areas with tangentially oriented fibers, but it is still possible. Furthermore a new method to detect proliferating tumor cells in situ by using multiple consecutive click reactions with dendrimeric molecules and clickable dyes is presented. Cells are grown in the presence of ethinyl‐dU (EdU), where the EdU becomes part of the genome during proliferation. After the cells were fixed and permeabilised, the incorporated alkynes of the EdU are functionalized with azide‐containing fluorophores through the CuI‐catalysed alkyne–azide click reaction. These azide‐ and alkyne‐modified dendrimers allow the establishment of sandwich‐type detection assays, which have improved sensitivities, signal intensities and signal‐to‐noise ratios in reference to comparable techniques. The RNA‐FISH‐based detection of RNA was also improved in the following by increasing the number of fluorophores per oligonucleotide probe. Currently, the RNA-FISH detection method uses sets of single‐fluorophore‐containing oligonucleotide probes that hybridize to the mRNA of interest. This reliable detection of transcription events and the localization and quantification of particular mRNA allows disease states to be characterized more directly. For the early detection of virus infections, when spreading of the virus in- and outside of the organism can be controlled much better, this is particularly important (e. g. Sars-CoV-2). To receive a reliable signal, a large number of probe strands (>30) is required, but the more oligonucleotide probes are used, the higher the off‐target binding effects, which create background noise. Through the use of click chemistry and alkyne‐modified DNA oligonucleotides multiple‐fluorophore‐containing probes were prepared. These triply labeled probes allow reliable detection and direct visualization of mRNA with only a very small number (5–10) of probe strands. In an in situ experiment this lead to lower background noise and a better signal to noise ratio, whereby viral transcripts can be detected 4 hours after infection. RNA viruses induce formation of subcellular organelles that provide microenvironments beneficial to their replication. These replication factories of rotaviruses are protein-RNA condensates, formed via liquid-liquid phase separation. Through phase separation rotavirus proteins NSP5 and NSP2 form RNA-rich condensates, which can be reversibly dissolved in vitro by aliphatic diols. These RNA-protein condensates became less dynamic and impervious to aliphatic diols during infection, indicating a transition from a liquid to solid state. The selective enrichment of viral transcripts seems to be a unique feature of these condensates, but other aspects of assembly are similar to the formation of cytoplasmic ribonucleoprotein granules. By targeting these complex RNA-protein condensates, that underlie replication of RNA viruses, promising novel therapeutic approaches could be developed.