This tissue section from a normal mammary gland has been stained to show myoepithelial cells (red) and luminal epithelial cells (green). Credit: Mark LaBarge |
In biology, the key to a healthy life is organization. Cells that properly
organize themselves into communities live long and prosper, whereas
disorganized cells can become cancerous. A study by researchers with the
Lawrence Berkeley National Laboratory (Berkeley Lab) of the different types of
cells that make up the human breast shows that not only do cells possess an
innate ability to self-organize into communities, but these communities of
different types of cells can also organize themselves with respect to one
another to form and maintain healthy tissue. Understanding this ability of
different types of cell communities to self-organize into tissue may help
explain how the processes of stem cell differentiation and tissue architecture
maintenance are coordinated. It might also lead to a better understanding of
what goes wrong in cancer.
Mark LaBarge, a cell and molecular biologist in Berkeley Lab’s Life Sciences
Division, and Mina Bissell, a Berkeley Lab Distinguished Scientist also with
the Life Sciences Division, carried out a unique study of normal human mammary
epithelial cells that had been enriched into pools of the two principal lineages
that make up breast tissue—the milk-producing luminals and the myoepithelials
that blanket them. In healthy breast tissue, these two lineages organize
themselves into an ordered bi-layer. To observe and quantify changes in the
distribution of these cell lines with respect to one another over time,
LaBarge, Bissell and a team of collaborators used a unique “micropatterning”
technique, in which the cells were confined to a three-dimensional cylindrical
geometry.
“We demonstrated that while bi-layered organization in mammary epithelium is
driven mainly by the lineage-specific differential expression of the E-cadherin
adhesion protein, the expression of the P-cadherin adhesion protein makes
additional contributions that are specific to the organization of the
myoepithelial layer,” LaBarge says. “Disruption of these adherens junction
proteins or the actomyosin network that supports them either prevented the
formation of the bi-layer, or caused a loss of pre-formed bi-layers. This is
the first reported evidence that the two principle lineages of adult human
mammary gland possess intrinsic and reversible characteristics that guide their
organization into a bi-layer.”
Throughout a person’s life, the various tissues in his or her body will be
replenished and repaired by drawing upon a reservoir of adult stem cells. As
new cells replace old ones or are used to construct new tissue, the
architecture of that specific tissue must be maintained. Otherwise, cancer or
other diseases can arise. This process requires that lineage-specific
progenitor cells or their differentiated progeny be able to reach their
ultimate destination within the tissue. This task is particularly daunting for
breast cell lineages because the mammary gland undergoes cyclical changes in
its architectural structure, showing as much as a 10-fold expansion in
preparation for lactation followed by return to normal size. During these
cycles, the precise bi-layered branching organization throughout the gland, in
which a layer of secretory luminal epithelial cells (LEPs) is surrounded by a
layer of contractile myoepithelial cells (MEPs), must be maintained.
These image show the distribution of cell lineages in human mammary epitheial cells over time in the presence of (top) an anti–E-cadherin agent and (bottom) an anti–P-cadherin agent. LEPs are stained green, and MEPs are stained red. Credit: Mark LaBarge |
“We hypothesized that mammary epithelial cells possess lineage-specific
intrinsic abilities to self-organize into domains of lineage specificity, which
would help explain how, for instance, the mammary stem cell-enriched zone in
the ducts is maintained separately from the rank-and-file LEPs and MEPs, and
how LEPs and MEPs form and maintain bi-layers,” LaBarge says. “The phenomenon
of self-organization has not been well studied in humans, perhaps because of
the challenges of working with primary materials and a paucity of tractable
culture systems for maintaining cell types from normal adult tissues.”
Initially, LaBarge, Bissell and their collaborators used a classical
self-organization assay, in which heterogeneous aggregates of dissociated cells
from embryonic tissues were cultured on non-adherent agarose-coated surfaces,
to observe organization amongst cells divided into low and high cadherin
expression groups. While somewhat effective, there was a “tremendous variation”
in the size and shape of the aggregations of cells that, among other factors,
made watching the same cells over time “out of the question,” according to
LaBarge. To meet this challenge, he and his colleagues engineered a microwell
culture platform that could confine mixtures of human mammary epithelial cells
to a 3D cylindrical geometry.
“Suddenly, we could work with small numbers of rare cells and we could watch
them in action over time and perturb the system in meaningful ways,” LaBarge
says, “which could all be quantified and displayed in an unbiased manner.”
In addition to the micropatterned assays, LaBarge and Bissell also made use
of a cell culture system invented by Martha Stampfer and Jim Garbe, both with
Berkeley Lab’s Life Sciences Division. This unique cell culture system made it
possible for LaBarge and Bissell to carry out their study using normal human
adult epithelia.
“Without the Stampfer and Garbe system, our experiments would likely have
been one-offs that were subject to the genetic makeup of the host,” LaBarge says.
“Instead, we were able to perform the experiments many times on the same lot of
isogenic LEPs and MEPs to arrive at statistically significant conclusions.”
LaBarge says the discovery of the important roles played by E-cadherin and
P-cadherin proteins in the organization of human LEPs and MEPs into a bi-layer
was a major surprise.
“For the formation of the breast tissue bi-layer, the LEP and MEP progenitor
cells need a way to get instructions, or else the differentiated LEP and MEP
cells need to find their correct home,” he says. “Modulation of LEP and MEP activity
seems to get the cells to where they ultimately need to be, but, as other
studies have suggested, there is clearly much more to maintaining a breast
tissue bi-layer than just adherens like LEP and MEP.”