Host epithelial geometry regulates breast cancer cell invasiveness
This paper delves into how mechanical properties of cells affect tumor proliferation and invasiveness in epithelial breast tissue. They use finite element analysis to simulate stress levels and then validate their model with experimentation. They find that tumor cells invade more in regions of high mechanical stress (and contractility). They also looked into several other factors which can affect tumor invasiveness but those are the major ones and the major contribution of the paper as a whole.
My notes aren’t super helpful probably, but here they are. Essentially a regurgitation of the paper.
- “More than 90% of all human mammary carcinomas originate in the epithelial ducts rather than the surrounding connective tissue” and the majority of these arise from the terminal ductal lobular unit (this is basically the functional part of the breast at the end of the branches).
- The specific location of the tumor can affect the patient’s outcome significantly
- People with tumors in the upper lateral quadrant of the breast have better prognosis than those with tumors in the other three quadrants
- This indicates that these architecture-dependent variatons in tumor development are affected by the microenvironment of the tumor in determining the phenotypic outcome of genetic mutations.
- “the tumor microenvironment can suppress or induce a malignant phenotype in cells with a preexisting malignant genotype”
- While certain microenvironment can provide tumor-supressive signals, the loss of tissue homeostasis can induce the development of an aberrant tumor microenvironment that can act as a potent tumor promoter
A tumor can affect its environment to cause promotion
- A developing tumor itself can affect its micronenvironment though by changing mechanical forces.
- Normally, cells exert contractile forces against their surrounding microenvironment. Failure to properly balance these forces can actually result in tumor promotion.
- This could happen because cells can recognize an increase in ECM stiffness and respond by generating increasing traction forces on their surroundings
- this actually enhances tumor growth, survival, and invasion of tumor cells by promoting focal adhesion maturation and signaling through actomyosin contractility.
- Basically, invasion, mobility, etc. are assisted because the cell-junctional integrity is disrupted due to the traction forces changing and the cytoskeletal remodeling which is happening.
Rho GTPase effector
- The signaling molecule Rho GTPase is a key effector of these processes (identified since Rho activity is elevated in tumors and elevated signaling in Rho can regulate cell proliferation and invasion).
- Increased Rho signaling can also affect the allignment of collagen fibers and prime the microenvironment for subsequent invasion by tumor cells + Rho GTPase is found in a lot of other signaling pathways.
In vitro experimentation
- To determine how the phenotype of tumor cell varies with their location, the authors used 3D microlithography-based approaches in an engineered epithelial host tissue.
- “Our results show that mammary tumor cells proliferate or invade preferentially in regions of endogenously generated mechanical stress”
- Tumor cell phenotype can be controlled by altering the contractility of the host epithelial tissue through the expression of dominant-negative or constitutively active RhoA
- At regions of high mechanical stress where the tumor can be activated, the tumors actually require signaling through FAK (focal adhesion kinase) to invade the cell
- Where there’s low mech. stress (inhibiting tumor cell invasion), if you artificially induce assembly of focal adhesions by promoting integrin clustering you can actually drive the invasive phenotype anyways.
Note to self, maybe I should stop basically rewriting the paper…
Setup and engineered ducts
- Again, used a microlithography approach to engineer surrogate ducts composed of normal mammary epithelial cells that contained a single tumor cell.
- Varied the init. position of tumor cells in the ducts and monitored phenotype.
- Found that proliferation of tumor cell lines were affected by position
- Previously non-invasive cell lines
- SKBR3, MDA-MB-361, MDA-MB-453
- Proliferated at significantly higher rate when they were located at the ends of the tissue surrogates, compared with when the tumor cells were located elsewhere within the duct.
- Previously non-invasive cell lines
- Proliferation rates of invasive brease tumor lines were independent of their initial location within the tissue
- SCg6, 4T1, MDA-MB-231, and Hs578T
- These cells showed dramatic differences in invasiveness depending on location (see above figure).
- When grown as homogeneous cultures, the cells invaded randomly into the surrounding collagen gel (as would be expected of invasive cells)
- However, when put into a nonmalignant host tissue surrogate, they invaded much more readily at the end rather than in the duct (to me personally, this is pretty intuitive because they would be more exposed to the collagen rather than sheltered from it, but I guess this goes to prove their point about cell environments affecting tumor cell invasiveness.
- Tumor cell invasion required signaling through epidermal growth factor receptor and the catalytic activity of matrix metalloproteinases, because inhibiting these with AG1478 and GM6001, respectively, blocked invasion
- This would also make sense that the cells at the end invaded? Wouldn’t it? Since the epidermal growth factor receptors would be exposed on the ends?
Tissue geometry in affecting cellular phenotype
- By establishing regional differences in endogeneous mechanical stress or concentration gradients of diffusible molecules (TGF-$\beta$), different tissue geometries can affect cellular phenotypes
- Used finite element method (FEM) to simulate two tissue geometries that could distinguish between these signals and reveal which regulated tumor cell invasion
- Simulated contraction of an epithelial tissue within a collageneous matrix
- used FEM to compute maximum principal stress in the tissue
- represents a coordinate-invariant description of the stress at each point
- Calculated that the proximal ends had more mechanical stress than the distal ends
- Measured this experimentally by tracking movement of beads embedded in the tissue.
- Comparison validated the FEM model
- model predicted regions of high displacement at the tips and correctly predicted lower displacements at the proximal ends than at the distal ends (because the tissues are in opposition to each other and thus pulling on the matrix between them in opposite directions) but failed to capture some of the larger spatial patterns of displacement in the matrix
- Then engineered paires of ducts containing a single tumor cell.
- Tumor cells invaded at regions of high stress as predicted (proximal and distal ends)
- Again made a tissue with a central bump and predicted the high stress areas and predicted correctly that tumor cells would invade more there
- However the difference between the bump and the ends in invasiveness was not great even though the stress levels were different. This indicates a threshold level of stress which causes invasiveness.
Altering Host Tissue Contractility Directs Tumor Cell Invasiveness.
- Mechanical stress is generated by the actin cytoskeleton and transmitted between cells through cadherin-mediated intercellular adhesions.
- Reducing mechanical stress by inhibiting the Rho effector, blocked invasion of the tumor cells from the end tissues (Fig. 3 A, B, and L).
- Similarly, inhibiting actomyosin-mediated contractility using blebbistatin, a selective inhibitor of nonmuscle myosin II ATPase activity, blocked tumor cell invasion from the tissue ends (Fig. 3 C and L)
- However, increasing cytoskeletal contractility and mechanical stress using the phosphatase inhibitor calyculin A did not further increase invasion from the ends + Suggests that a max. level of invasion was already induced by the microenvironment of this region of surrogate tissues (enforces the idea that there is a stress threshold)
- Selectively modulated the contractility of the non-malignant host tissue without alterning the tumor cell embedded within.
- Low levels of Rho activity prevent myosin light chain phosphorylation and cytoskeletal contraction.
- Transducing host epithelial cells with an adenovirus encoding dominant-negative RhoA ($RhoA^N19$) reduced the levels of endogenous contractility generated within the host tissue without causing tissues to dissociate.
- BASICALLY, what this means is that they made the cells unable to contract as much, meaning they don’t move around as much meaning the tumors can’t weasle their way in?
- This actually also reduced the subsequent invasion of tumor cells from the ends of the tissue (Figure 3 D and L)
- Conversly, putting an active RhoA in there resulted in an increase in contractility generated by the tissues, which in turn resulted in tumor cells invading from all directions (Fig 3 E and L).
- The transmission of this mechanical stress can be disrupted between cells by expressing a dominant-negative mutant of E-cadherin that lacks the $\beta$-catenin-binding domain ($E\delta$) and thereby prevents coupling to the actin cytoskeleton (gives the cells some slack?)
- Preventing the transmission of mechanical stress reduces the invasion of tumor cells (Fig 3 F and L). And prevents preferential proliferation of noninvasive tumor cell lines.
- The effects did not result from a loss of apicobasal polarity within the host (proved by staining for ZO-1)
- apicobasal polarity is a type of cell polarity specific to epithelial cells, referring to a specialised apical membrane facing the outside of the body or lumen of internal cavities, and a specialised basolateral membrane localised at the opposite side, away from the lumen. The two domains are often physically separated by adherens junctions complexes.
- Additionally, the effects could not be rescued by simultaneously expressing an active RhoA complex (Fig. 3 G and L).
- This suggests that the tumorigenic phenotype is modulated by variations in mechanical stress that are generated by actomyosin-mediated contractility of the normal host epithelium and transmitted through intercellular adhesions within the host tissue.
Tumor Cell Invasion Is Regulated Through Focal Adhesions and Integrin Clustering
- The authors wanted to figure out whether FAK played a role in regulating the response of tumor cells to host tissue contractility
- So they expressed a dominant-negative mutant that lacks teh kinase domain (FAK-Dter).
- “Transducing tumor cells with an adenovirus encoding FAK-Dter significantly inhibited their ability to invade (Fig. 3 H and L). Conversely, forcing the assembly of focal adhesions specifically in tumor cells by selectively expressing an auto-clustering mutant of β1-integrin (β1V737N) induced invasion from all locations within the tissue surrogates (Fig. 3 I and L).”
- Integrins basically just trigger cell pathways in the cell from binding events on the exoskeleton, from what I understand. It seems kind of weird to me that they try the whole integrin thing when they’re trying to prove FAK’s role, but this may be due to my lack of knowledge of the whole cell signaling pathway.
- “However, inducing focal adhesion assembly in tumor cells did not rescue the inhibition of invasion resulting from expression of EΔ in the host cells (Fig. 3 J and L) or treatment with blebbistatin (Fig. 3 K and L).”
- Apparently this data shows that integrin clustering and FAK activation are required, but not sufficient, for invasion from the high-stress ends.
Predictions of Mechanical Stress Correspond with Tumor Development in Vivo.
- They’re trying to predict how the tumor cells would react in vivo to mechanical stresses
- First they try to scan a tumor in a mouse
- “Micro- computed tomography (μCT) was used to create a 3D rendering of the mouse mammary gland (Fig. 4A)”
- “The data generated by μCT were converted to a solid model that re- capitulated the native architecture of the gland (Fig. 4B), consisting of a hollow epithelial tree embedded in a 3D ECM.”
- At first, the ECM was modeled as a homogeneous linearly elastic solid with the material properties of collagen
- Using FEM, they calculated the max. principal stress throughout the scanned geometry. They predicted high stress at the ends of the epithelial tree compared with the ducts (Fig. 4 C and D).
- To test whether tumors grow more at these points of high mechanical stress, they “created mice transgenic for an inducible form of the kRas oncogene, under the control of the mouse mammary tumor virus (MMTV) promoter.”
- This allowed them to kickstart the cancer when the epithelial ducts had developed. Sure enough, “When kRas was expressed in adult mammary glands, tumors developed prefer- entially at the ends of mammary epithelial ducts (Fig. 4 G–I), whereas control mice failed to develop tumors (Fig. 4 E and F).”
Stuff I still don’t fully get and need to look into more:
- Focal adhesions
- Integrin clustering
- basically all cell pathways that they mention
- Tumor cell proliferation