Evolution's Spurs

As I've commented previously, it is pleasing when a new biological finding can be related to something both familiar and pleasing. In this case, Nature has, with perfect timing, carries a paper about one of my favorite garden plants.

I like to garden but my attention to it is somewhat erratic. As a result, for the ornamentals I have a strong bias towards plants which are perennial or nearly so; in theory you plant them once and enjoy for many years afterwards. There are a few catches, however. First, the hardiness guides in plant catalogs are only rough guides, and the local microenvironment determine whether a plant will actually thrive. As a result, I sometimes end up with very expensive annuals (perennials tend to sell for 2-10X the price of an annual). At a previous residence I couldn't get one wet soil-loving plant (Lobelia cardinalis) to overwinter until I put one directly under the downspout, though about 40 miles to the south I've seen it run rampant on the sides of cranberry bog irrigation ditches. Another species (Gaura lindheimeri) refuses to overwinter for me, but a gardener (and former MLNM employee) a few towns over has a magnificent specimen.

A good perennial garden also requires a certain attention to detail. In particular, many perennials have very restricted bloom times, since they must invest energy in surviving the winter and reappearing in the spring. Many annuals bloom all summer; annuals are grasshoppers, perennials ants. So to get color throughout the garden season, a mixture of plants is needed. Certain times of the mid-summer and early fall are awash in choices, but right after the bulbs fade in early summer can be challenging.

Columbines, species of the genus Aquilegia, are wonderful perennials. While the individual plants are short-lived, plants in a favorable environment will reseed vigorously. They are in bloom now, which is why the new paper's timing is so good. Many of the flowers are bicolor. The foliage is generally a neat mound, often with a bluish tint, making them attractive even when not in bloom. The seed heads are distinctive & visually interesting. Examples of two of mine, one established & one newly planted, show some of these features.

The signature feature of Aquilegia are the spurs. Depending on the species and variety, these can be nearly non-existent to quite large . I'll confess I had never pondered the biology behind the variation, but that is now remedied.

Figure 2 of the paper shows a remarkable correlation between who pollinates a columbine and the length of its spurs. Three major pollinators were explored: bumble bees, hummingbirds & hawk moths.

The key focus of the paper is distinguishing between two evolutionary models. Both Darwin & Wallace had proposed that such long spurs could evolve through a co-evolutionary race between polinator and flower. Longer spurs mean the pollinator must approach the flower more closely to reach the nectar, increasing pollen transfer. Longer tongues on the pollinators will be favored, as they can reach down longer tubes. This model would tend to suggest gradual, consistent changes in spur length.

A competing hypothesis is that spur length changes abruptly when the pollinator shifts. After long periods of stasis, the introduction of a new pollinator drives a short-term co-evolutionary race.

There is a lot of nice data, which I'm still chewing on, in the paper favoring the latter model. The authors used a large set of polymorphisms in the genome to generate a phylogeny. Regression analysis using this phylogeny showed that inferred pollinator shifts are correlated with large changes in spur length. Interestingly, their phylogeny suggests that there have been only two major shifts in pollinator strategy, each time going from a short-tongue to long-tongue pollinator (bumble bee -> hummingbird and hummingbird -> hawk moth). One interesting supporting piece of evidence: in Eurasia there are no hummingbirds, and there are also no Aquilegia known to be pollinated by hawk moths.

By a nice coincidence I was looking something else up & discovered that the Joint Genome Institute has commenced the sequencing of an Aquilegia species. Aquilegia's family is the first branching of the dicots, and therefore represents an important window into family-level evolution of plants.


One additional consideration in garden planning is what creatures your choices will attract: some are desirable, others not. I won't plant major hosts for Japanese beetles unless I really like them (raspberries escape the edict). On the other hand, bumble bees and hummingbirds are on my desired list (though I've never been able to attract a hummingbird) whereas I'm neutral on hawk moths. So, perhaps I should go to the garden center with a caliper in hand, and only get a few of the super-long spurs just to wonder at them.

Killer Co-evolution

To close up, for now, the story I've been spinning about cancer stroma from Day 1 & Day 2 of the Week of Science, I'll address a question which may have been prompted by yesterday's item about the symbiotic relationship between tumor and cancer stroma: How does this arise? What drives the stroma into being Benedict Arnold, and what chance is there to bring it back?

At the end of last year a paper came out in PNAS that looks at this question in a clever way. A challenge for studying cancer's interaction with its surrounding cellular environment is that it is very difficult to separate the two. How can you ever be sure you are looking at pure tumor or pure stroma?

The paper solves this problem by having the tumor come from one species and the stroma from another. Mouse xenograft models were built by injecting human tumor cell lines into immunodeficient mice. After tumors formed, the tumors were excised and then disaggregated into individual cells, and these cells sorted by flow cytometry. The tumor cells have higher DNA content than the mouse cells, so a DNA stain can sort one from the other. DNA from the mouse cells was then subjected to copy number analysis.

Copy number analysis is quite the rage these days, both for oncology and for looking at normal variation in the human genome. Most papers use array comparative genomic hybridization, or array CGH, to analyze copy number variation. This paper uses the closely related method ROMA, which differs in some key details but at a very high level is very similar. In short, the fragments from the genome are probed against a microarray which has markers spaced across each chromosome; by measuring the signals (and applying a lot of corrections, still being worked out), one can infer copy number changes ranging from complete losses of chromosome pieces to extreme amplifications.

ROMA provides another layer of filtration of the human tumor cells from mouse stromal cells, as the array probes shouldn't hybridize well cross-species. Normal tissue samples from the mice were used to normalize any murine copy number polymorphisms.

From seven tumors a number of genomic alterations were observed. This reinforces previous suggestions that the tumor stroma is co-evolving with the tumor, and that these changes are permanent since the genome itself is being altered. Two genes were observed to change copy number in models built from different tumor lines, while some other genes repeated in tumors built from the same line. However, no gene was universally observed to change copy number with the same cell line, suggesting that there are multiple co-evolutionary paths for successful tumor stroma.

This paper is just an crack into the field. In particular, they did not try to correlate their results with human clinical samples. The sample size here is very small, with only a few types of tumor lines tried. The functional roles of the altered genes was not explored. It is virtually a certainty (though I have no inside info) that such studies are ongoing -- especially since the lab involved has done all three of these in other papers. Of particular interest will be to better understand the mechanism of cancer stromal cell derangement. Is it purely an evolutionary selection for living near a tumor, or is the tumor somehow actively participating in the derangement by triggering mutagenic mechanisms or providing key survival signals?

A normal role for fibroblasts is to repair wounds, and hence the formation of tumor stroma may represent a repair attempt by the body which is co-opted by the tumor. Previous gene expression studies have identified a 'wound response signature' which is correlated with clinical outcome. Interestingly, the two genes reported to be the drivers of this signature did not show up in the ROMA analysis. This also suggests another line of experiment: do these mouse stromal cells exhibit the clinical signature?

Evolution, ecology & medicine all woven together -- it would be purely fascinating, if it weren't so deadly serious.

Cancer Cellular Ecology

This post kicks off my contribution to Just Science 2007. While it won't be a theme held to strictly, many of my planned entries will be about, or touch on, cancer.

Carl Zimmer had an excellent recent post on the evolutionary aspects of cancer; here I will take a stab at the cellular ecology of cancer. It is both a fascinating topic on its own, and something which later posts this week will refer back to. For this last reason, this post will also be sprinkled with teaser references to posts which will show up later in the week.

I don't remember when I first heard about cancer, but it was at a tender age. I don't remember the context either. My paternal grandmother succumbed to leukemia long before my parents met, at a time when leukemia was considered incurable -- though within a few years the first effect chemotherapeutic agents would appear. Or perhaps it was hearing about the boy a street over who died of childhood leukemia. Most certainly I knew by 2nd grade, as that is when the kindly custodian at my elementary school died of cancer. So sometime I heard the word, and I was not one to withhold questions.

The answer I got that first time, and for many times later, is that cancer is part of the body gone haywire, an uncontrolled & chaotic growth that eventually crowds out normal tissue. One analogy is that of a weed which takes over the garden. It's a very simple answer -- suitable for an inquisitive elementary school student -- and it's also the model that long held sway. But the modern view is much more complex. That complexity extends the mystery of cancer, but also offers new opportunities for treating it.

First off, the modern view is of a tumor with some internal complexity; we now believe that many, and perhaps all, malignant tumors have at least two classes of cells: cancer stem cells (more later this week) and the bulk of the tumor. But furthermore, the tumor recruits other cells to assist it. Depending on the tumor type, these could include endothelial cells to build new blood vessels (a process called angiogenesis), fibroblasts which become the tumor stroma, and immune cells which may be co-opted to provide useful signals to the tumor. There's a fascinating story of the intersection of tumor, endothelial cells & stroma that will follow later. Other interactions may depend on the tumor type.

For example, take the interaction of multiple myeloma & the bone marrow. Normal bone marrow contains a host of different cell types & interacts with the nearby bone. Normal bone is maintained by a healthy balance between two opposed cellular factions: osteoclasts which break bone and osteoblasts which build bone. Both are derived from Greek, with osteoclast being my favorite because of it's onomatopoetic root 'clastos' (to break).

Multiple myeloma results from the derangement of a plasma cell, the final stage in B-cell development (free review). B-cells are the antibody producing cells, and go through a complex series of transformations. A unifying theory of B-cell malignancies (B-cell leukemias, B-cell lymphomas & multiple myleoma) is that each represents a cell leaving the tracks at a certain stage of B-cell development. Myeloma represents the derangement of the final stage, a plasma cell, whose normal job is to secrete large quantities of a single antibody. One of the clinical hallmarks of multiple myeloma is the overabundance of a single antibody species in the blood. An even more devastating effect is bone destruction; on some of the X-rays the patient's skullcase literally looked like swiss cheese.

This clinical sign has a relatively straightforward cellular explanation: Myeloma cells stimulate the numbers of osteoclasts. This benefits the myeloma cells via osteoclasts secreting various growth factors favorable to myeloma cells. Myeloma cells reciprocate both by stimulating mature osteoclasts and by encouraging the common progenitor of osteoclasts and osteoblasts to more often mature into osteoclasts. Myelomas may also send hostile signals to osteoblasts, encouraging them to commit suicide. A new garden metaphor appears: myelomas cultivate their surroundings & fertilize their soil.

Many of the active drugs for myeloma, including my former employer's drug Velcade, may work by both targeting the myeloma cells but also targeting these interactions with their cellular microenvironment. If a drug can block the stimulation of osteoclasts, or attenuate the inhibition of osteoblasts, then useful clinical benefits might ensue -- such as reduced bone damage but also perhaps dampening the stimulatory signals from the osteoclasts to the myeloma cells.

Many existing drugs may target other cellular ecological interactions in tumors. In particular, many drugs may antagonize angiogenesis, the formation of new blood vessels to feed the tumor. The first drug targeting angiogenesis specifically, Avastin, appeared about two years ago, and more will undoubtedly appear.

An important implication of this sort of thinking is questioning the very way we study cancer. Much early stage research is based on tumor cell lines in culture -- cell lines which do not have any cellular partners present. Mouse models using human cell lines may not recapitulate these cellular interactions, as the mouse and human cells may be communicating inefficiently due to different molecular accents. Even looking at humans, pharmacogenomics studies which extract signal only from tumor may be missing much of the picture. In addition, most cancer models and many early stage cancer clinical trials are based on reductions in the size of the tumor as the figure-of-merit. Therapies targeting the cellular ecology might not produce rapid, drastic changes in tumor volume. New models must be (and have been) developed & validated, and they are often more complex and take longer or more resources to execute. Complexity breeds complexity -- and we have no idea how many more complexities we will encounter.

But in the end, we have no choice. We can try to make simple models of what cancer is, but if you are trying to treat a complex disease you need an appropriately complex model. But if you're trying to explain cancer to a second grader, perhaps you need to fall back on the 'cancer is like a weed' explanation.

[Note: Blogger has an odd way of timestamping posts you save as drafts, so I'm adding this note to try to get this popped into the Week of Science]
[updating again to try to push this through]