A Hard Boiled Look at Metastatic Remodeling Molecules
By Jason Socrates
Bardi
The very worst kind of tumors are those drifters that pick
up and move to another part of the body, an event called metastasis.
When a tumor metastasizes, it does so by expressing proteins
that allow its cells to break free of the colony, enter the
bloodstream, survive in the circulation, and arrest in the
vessels of another organ. Then these founder cells express
more proteins that keep them alive, allow them to divide and
divide, bring them blood and nutrients, and help them grow
into new metastatic tumor colonies.
Professor James Quigley of the Department of Vascular Biology
calls the expression changes within a tumor that begins metastasis
remodeling.
"If a cell is going to spread into a neighboring tissue,"
he says, "there is going to be a lot of molecular remodeling...
We're after the early events. What are the molecules that
determine why a human tumor cell migrates to and survives
in a different organ—a different environment with different
growth factors, adhesion molecules, hormones, and glandular
products nearby?"
If one could identify these molecules, they would yield
information about both the basic biology of metastatic cancer
and possible therapeutic targets, and that is exactly what
Quigley is trying to do.
Under Glass, In Profile, Under Reason
This is easier said then done, though, because trying to
observe metastasis in the laboratory is not trivial. First,
scientists must study cells in vivo, because metastasis
is a complex, three-dimensional cascade event with too many
processes involved to be imitated in vitro. "Metastasis
cannot be mimicked at all in culture," says Quigley.
Second, some changes may not be morphological or otherwise
observable via some easily detected change or signal. In order
to detect the tumors with a microscope, a standard method,
one would have to wait for the tumors to grow large enough
to be seen through exhaustive searches of tissue sections,
a time-consuming processes. Using pigmented melanomas to increase
the visibility of the tumors helps, but these melanomas do
not necessarily have the desired metastatic properties.
Finally, there is the problem of false positives, which
arises from the fact that some of the most important molecules
that contribute to the phenotype of metastasis may not be
the ones that are most widely expressed. The observation that
a protein is up or down-regulated in cell metastasis does
not mean that protein is necessary for metastasis.
Plus, the metastatic phenotype may be brought about by extensive
combinations of subtle up and down regulations of proteins
that activate or suppress other proteins that are normally
there, in which case the more interesting observation would
be how the expression of the activator and suppressor proteins
change.
"We want to identify the molecules that are not just associated
or correlated with a given process, but functionally involved,"
Quigley says.
One way to get at these functionally involved molecules
is to reason out which proteins might be necessary for metastasis,
given everything we know about the basic biology, and study
those. Serine and metalloproteases, for instance, are necessary
to free a potentially metastatic cell from the collagens and
proteoglycans that make up the stromal tissues to which they
are bound. Quigley has worked on such proteases for years,
and he continues to devote a significant portion of his time
to them.
Quigley and his laboratory have also developed another method,
which Quigley calls an unbiased approach to identifying the
proteins involved in metastasis. This method has the advantage
of working without any preconceived notion as to what these
proteins are.
The Unbiased Way
This approach involves first generating many monoclonal
antibodies raised against "crude" tumor cell antigen populations—whole
cells and cell membranes—and then screening for those
that block the metastatic ability of the cell. Quigley reasons
that any antibody that arrests the metastasis must have as
its target some antigen involved in the process.
"[We do this] without having any idea as to what the nature
of the target of that antibody is," says Quigley. "Once we
screen for a blocker, then we try to identify its target."
The difficulty in using monoclonal antibodies against tumor
cells is that most of the antibodies raised will be against
immunodominant antigens—all the high-visibility proteins
that the immune system recognizes. But these antigens may
not be critical for a biological process like metastasis.
One would really like to make antibody against only those
minor or low-abundance antigens that are critical.
To accomplish this, Quigley uses a trick called "subtractive
immunization" that increases the proportion of non-immunodominant
antibody raised.
The trick with subtractive immunization is to first tolerize
an organism -with a non-metastatic tumor cell, using an immune
suppressant chemical like cyclophosphamide to kill off all
the immune cells that recognize the immunodominant antigens.
Once this tolerance is achieved, the next step is to challenge
the same organism with a second tumor cell. The second cell
should be similar in every way to the first except that it
is aggressively metastatic. "It will still have those same
common immunodominant antigens on the surface, but the tolerized
immune system won't mount a defense against them," says Quigley.
Instead, the immune system will produce antibodies against
those proteins that are unique to or enriched in the metastatic
cell. Some of the proteins may directly contribute to metastatic
spread. Then the B cells that make these antibodies can be
used to make antibody-producing cell lines, called hybridomas,
and the antibodies produced by these hybridomas can be screened
in an assay Quigley has developed to look for the phenotype
of blocking metastasis. Then the antigens of those antibodies
that do block metastasis can be isolated and sequenced.
Quigley calls this isolation "a hard chore," but once done,
these antigens should be cell molecules that are essential
components for metastasis and, perhaps, eventual targets for
therapeutics.
1 | 2 |
|
