Cell and Tissue Engineering: 3D Effects John Eichorst BIOE 598 April 7, 2008

Outline of Talk: Review Article 1)Discuss the motivation for examining cells in 3D systems 2)Look at some of the common types of 3D models currently being researched 3)Examine the effects that 3D ECMs have on morphology and behavior of cells -polarity, migration, stress, local oxygen concentration, branching morphogenesis 4)Finally, discuss the clinical applications of research using 3D ECM models -Stem Cell -Cancer Research Modeling Tissue Morphogenesis and Cancer in 3D: Cell 130, , (2007) Yamada, Kenneth M. and Edna Cukierman

Why are we thinking about 3D environments? Short Answer: Cells cultured in 2D settings can differ greatly from those grown in 3D environments: -morphology -cell-cell interactions -cell-matrix interactions -differentiation

Tissues can be harvested in vivo and later cultured in vitro. Often, they retain their original 3D structure. In many organs, epithelial clefts and resulting bud formations can occur leading to interesting 3D structures during branch morphogenesis in many organs. Initiation of Branching morphogenesis Shallow clefts form in a globular bud As the clefts deepen new buds are formed Cell 130, , (2007) modified from Nature 423, (2003) Branching Morphogenesis in salivary glands

In Vitro Cell. Dev. Biol. Anim. 42, (2006) Cells from a tissue culture can be placed into a 3D structure as single cells or as a larger aggregate from a tissue. As shown in the procedure above, researchers in 2006 tried to simulate the environment by including other cell types (keratinocytes and fibroblasts) in the 3D model. In these spheroids, each cell inside the spheroid is subject to a different microenvironment within the sphere itself. Interestingly, the authors noted that EMT6 tumors showed different drug(etoposide) resistances as function of the dimension of the culture they were studied in. In Vitro Cell. Dev. Biol. Anim. 42, (2006) WM793 Melanoma Spheroids

General conclusions describing the differences between the cell’s behavior in 2D and in 3D matrices are summarized below. In addition, the choice of the design of the 3D model and the resulting unique microenvironment that the cell experiences can also introduce substantial variation in the cell’s behavior. Human Fibroblasts in 3D vs. 2D Environment Cell 130, , (2007)

Cell signaling and behavior in response to the extra-cellular environment can be greatly affected by the 3D model being used. There is also evidence that hypoxia can to some extent control the proliferation and differentiation of certain stem cells including embryonic stem cells, neuronal stem cells, hematopoietic stem cells … HIF-α proteins are typically translated and rapidly degraded by the hydroxylation of two proline residues by proline hydrolyase enzymes. Lack of oxygen stabilizes HIF-α resulting in the expression of the subunit HIF-β in the nucleus and ultimately the expression of various genes. HIF proteins, by being able to promote the expression of the various proteins listed in the figure in hypoxic locations in tumors has been considered as one of the potential causes of tumor progression. Cell 129, , (2007)

Various 2D and 3D models can be designed to contain a variety of stiffness in the ECM that the cell is exposed to. Although not necessarily 3D, the design of the 2D construct was intended to model the diverse environment encountered by cells from both a mechanical and signaling perspective. Single Cell Migration of Adult Human Dermal Fibroblasts Actin Cytoskeleton Remodeling of Adult Human Dermal Fibroblasts Biomaterials 28, , (2007)

Cell and tissue polarity can also vary in 3D matrices depending on the cell type and the microenvironment that they experience. Epithelial cells are often polarized and aggregate in spherical 3D forms as shown, to function properly and secrete products based on this organization. This spherical form is not possible on 2D tissue cultures that tend to flatten the cells. However, as shown on the right, Mesenchymal cells lose their “dorsal-ventral” polarity when placed in the 3D matrix and appear more as an elongated spindle shape, similar to a fibroblast. Cell 130, , (2007)

Branching morphogenesis has also been studied in 3D model systems in order to try to understand the formation of glands and organs. Nature 423, (2003) Initiation of Branching morphogenesis Shallow clefts form in a globular bud As the clefts deepen new buds are formed The hypothesis of the paper was that the local expression of fibronectin mRNA s in areas called “presumptive cleft regions” ultimately left to the formation of a cleft in the tissue resulting in the branching morphogenesis.

-Controlling the differentiation of stem cells into different lineages by examining their interaction with various 3D ECMs. -Embryonic stem cells have been lead to differentiate into epithelial cells as a function of the 3D environment (collagen matrix) -Culturing embryonic stems cells with fibroblasts was noted as generating a neural lineage -Culturing with keratinocytes lead to epithelial differentiation. -Growing stem cells in 2D culture has been shown to promote differentiation into blood vessels -Matrix stiffness as well as been shown to be able to select for different lineages in in stem cell differentiation. Guiding the differentiation of stem cells into specific lineages by manipulating their extra-cellular environment could have definite clinical applications as shown by the summary of some of the current research below. Cell 130, , (2007)

3D ECM models can also be applied to study the growth of cancer and proliferation of tumor cells. Cell 130, , (2007) MT1-MMP, which typically degrades the ECM proteins to encourage the invasion and proliferation of tumor cells. -However, in a 3D collagen gel model, researchers noted that the elimination of all extracellular proteases did not affect cell invasion. Gene expression to some extent as hinted before, can be a function of a tumor cell’s microenvironment as implied by the previous description of hypoxic cells. The microenvironment of a tumor with respect to the stiffness of the ECM it encounters as well as the features of the stroma have been implicated as possible factors determining the tumor’s progression.

Conclusions: 3D cell cultures can begin to more accurately model the environment experienced by cells in vivo. The striking differences between the behavior of cells in 2D and in 3D environments imply appealing questions about how these differences can be quantified and related to the knowledge of in vivo systems for scientific and clinical applications. Areas of Research with Clinical Applications: -Controlling or directly modulating the differentiation of stem cells -Engineering cells to be able to be introduced into tissues, having them respond to their local environment in a way beneficial to the tissue itself -Replacing damaged tissue -Develop high throughput methods for drug screening for in vitro settings -development of “therapeutic targets” and methods for cancer treatment

Research Article: Science 294, , (2001)

Outline of Paper The purpose of the paper is to study the structure and function of 3D matrix adhesions formed from the interaction of fibroblastic cells with a 3D extra- cellular matrix. The experiments described in the paper examine the following: 1)Physiological responses of the cell to the 3D environment 2)Determine the composition of the 3D matrix adhesions 3) Study the cell’s response to rigidity and lack of de novo protein synthesis in a 3D model

Focal Adhesions Focal Adhesion Kinase Paxillin Vinculin α v β 3 integrin Direct immunofluorescence staining from 2D Culture of mouse fibroblast Fibrillar Adhesions α 5 integrin - important fibronectin receptor Tensin The definition of a 3D matrix adhesion is related to the co-localization of the α 5 integrin, Paxillin and Fibronectin. There are more components of these adhesions. However, those listed were the subject of the initial experiments.

The next set of experiments explored in more depth how the 3D matrix adhesions were associated with the physiological responses of the cell in 2D and 3D environments using human fibroblasts. The 3D set of experiments: -“tissue derived 3D matrices” derived from detergent extracted mouse embryo sections -“cell derived 3D matrices” from naturally deposited 3D ECMs of NIH-3T3 fibroblasts -3D collagen gel lattices The 2D set of experiments: -fibronectin -laminin -collagen 1 -2D matrix -> 3D matrix that was mechanically compressed. -2D mix -> 2D coatings of solubilized, cell derived 3D matrix

Attachment (a.u.) = the relative number of attached cells compared to fibronectin sample after plating = condition where α 5 integrin was inhibited Below are the results presented for the 10 min cell attachment assay for the experimental setup described previously Cell attachment was determined by staining the cells with bisenzimide H3342 fluorochrome, for nuclear staining?

In addition to cell attachment, the cell morphology also changed depending on the conditions of the various substrates used The fibroblasts, themselves, were stained with Dil to generate the images. The images were then thresholded for the representation in the figure. Interestingly, the fibroblasts in the 3D matrix (3D mat, control) achieved the elongated morphology after 5 hours. The fibroblasts cultured in the 2D matrices were noted as achieving the same morphology after 18 hours.

Sixteen paths were positioned at the same starting point to generate the figures. The motility of the cells was tracked for 5 hours. This experiment also described the cell motility as a function of the dimension of the substrate as well as α 5 integrin. Summary of the data in previous 3 plots

The next question asked was about how the previously described fibrillar and focal adhesions respond to a similar set of ECM substrates. 3 substrates were designed to study the 3D matrix adhesions Human fibroblasts were cultured overnight on a tissue-derived 3D matrix. Mouse tissue was used for this matrix A cell-derived 3D matrix was also used to culture the fibroblast cells and later flattened to form a 2D matrix A 3D matrix was also made containing solely fibronectin fibrils to study the fibroblasts As a result of the integrin dependent behavior noticed in the previous experiments, a substrate of entirely fibronectin was used to determine to some extent, the interplay between the matrix composition and the dimension of the environment.

As mentioned before, the definition of a 3D matrix adhesion is related to the co-localization of the α 5 integrin, Paxillin and Fibronectin. Other observations mentioned in paper included that rigidifying the cell-derived (although tissue-derived is shown above for 3D) caused a lack of 3D matrix adhesions. Only the tissue derived 3D matrix showed indication of the localization consistent with 3D matrix adhesion. Insets of Images: Red/Purple indicate focal adhesions. Turqoise indicates fibrillar adhesions. White describes the combination of the adhesion structures indicating the overall 3D matrix adhesion

The effects of both de novo protein synthesis and rigidity were studied to determine their effects on the formation of 3D matrix adhesions. Inhibitor of protein synthesis To obtain the rigidified cell condition, the cell was treated by the covalent fixation with glutaraldehyde prior to plating. As mentioned before, cell rigidity in the 3D matrix substantially reduced the formation of the 3D matrix adhesion. However, the inhibition of the de novo protein synthesis has very little effect in this regard. Human fibroblasts were plated in cell-derived 3D matrices.

The spatial localization of other proteins and signaling molecules related to cell adhesion were also studied to determine composition of the 3D matrix adhesions. Human fibroblasts were again cultured on a 2D substrate (fibronectin), a mechanically compressed cell-derived matrix and a 3D matrix. The data in the table describe the results of the immunofluorescence staining examined at the adhesion sites in the 3D matrix. Green = α 5 integrin Red = Paxillin

Paxililin [pY 31 ] = phospho-paxillin 31 FAK [pY 397 ] = tyrosine phosphorylated FAK As before, FAK and paxillin co-localized with the α 5 integrin in the 3D matrix adhesions. Only phospho-paxillin 31 co-localized with the α 5 integrin. FAK[pY 397 ] was absent from the 3D matrix adhesions as shown by the lack of co-localization with the α 5 integrin. Planar fibronectin surface Fluorescently labeled antibodies also pointed to other components involved in the formation of 3D matrix adhesions.

In a similar experiment, confocal images were also acquired from the immunofluorescence staining of a section of a E13.5 mouse embryo As also shown in the previous images, phospho-FAK 397 does not co-localize well with the α 5 integrin while the blue and green image in B infer that FAK and α 5 integrin do co-localize.

Phosphorylation Levels of FAK, Paxillin, and ERK in 3D Matrix and in 2D fibronectin The last question discussed concerning the final experiment asked if it was possible that the lack of FAK[pY 397 ] could be the result of the inactivity of FAK at stable adhesion sites. FAK phosphorylation on tyrosine 397 generated by the signaling interaction with integrins is required in many signaling pathways associated with focal contacts. Therefore, is it possible that there is independent regulation of FAK and paxillin phosphorylation throughout the 3D matrix adhesion? The western blot analysis was performed to show that the lack of FAK phosphorylation can occur in 3D matrices with relatively little effect on paxilin or ERK1/2.

Questions: 1)The definition of the 3D matrix adhesion given in the initial section of the paper does not seem consistent with the other molecules found in the 3D matrix adhesions. Why did they concentrate so much on α 5 integrin and paxillin? 2)How are the 3D matrix adhesions organized to allow for motility and is there coordination specific for random or directed movement? 3) Generally, are there multi-faceted functions of the 3D matrix adhesions that may distinguish from those in 2D cultures.