Saturday, 22 August 2009

Evidence for Systemic Spread prior to the Establishment of Primary Tumors

ResearchBlogging.orgIt is widely believed that metastasis is a late event in cancer progression. This view is based on several clinical and experimental observations. First, most cancer patients die from metastasis and not the primary disease. Second, early surgery is often the only cure. Third, somatic genetic changes accumulate during local progression (Fearon and Vogelstein, 1990) which was extrapolated to systemic progression. Fourth, repeated rounds of in vivo selection led to cell lines with increased metastasis formation (Kang et. al 2003; Minn et. al 2005). However, in a recent study titled Systemic Spread is an Early Step in Breast Cancer, published in Cell in 2008, Hüsemann, Geigl, Schubert et. al report that tumor cells can disseminate systemically from the earliest epithelial alterations in HER-2 and PyMT transgenic mice and from ductal carcinoma in situ in women. 

The mouse model they work with is the BALB/c mice transgenic for the activated rat HER-2/neu gene (BALB-Neu T mice). This model mimics progression and gene expression profiles of human breast cancer. Females hemizygous for the rat HER-2 gene under control of the MMTV (mouse mammary tumor virus) promoter develop  invasive mammary cancer while their HER-2 negative siblings (wild-type BALB/c mice) remain tumor free. Typically, in the BALB-Neu T model, the mammary epithelia starts to express the oncogene at about weeks 3 to 4 (which coincides with the onset of puberty). Hyperplasia can be detected microscopically at weeks 7-9. Progress to in situ  carcinomas occurs between weeks 14-18 and at weeks 23-30, invasive cancers become apparent (see Fig. 1a)

Fig. 1a; Left Panel: Whole mount of the mammary gland at week 9 showing absence of tumor in branching ductal tree; Middle Panel: Histological Section at week 9 showing side buds displaying the morphology of atypical ductal hyperplasia; Right Panel: Histology of invasive cancer at week 30 (Adapted from Hüsemann, Geigl, Schubert et. al (2008))

The principle goal was to determine when cells expressing the HER-2 transgene disseminate. The authors chose the lung and bone marrow as the sites to look out for metastasis since the HER-2 receptor is not expressed in either of these two organs. In addition to HER, they also used anti-cytokeratin (CK) antibodies to check whether disseminated cells were epithelial in origin. Surprisingly in BALB-Neu T mice, CK+ and HER-2+ cells became detectable 4-9 weeks when meticulous analysis could only detect atypical ductal hyperplasia (ADH) (see Fig. 1b) 

Fig. 1b; Left Panel: Increase in tumor area at primary site. Triangles indicate mean value, whiskers indicate 95% confidence interval and solid line indicates best fitted curve; Right Panel: Number of CK+ cells (red dots) and HER+ cells (blue dots) per 5000 bone marrow cells. Triangles, whiskers and solid line indicate same. Note the presence of disseminated cells in the bone marrow even when the primary tumor area is zero.(Adapted from Hüsemann, Geigl, Schubert et. al (2008)) 

Further, lung micrometastasis from mammary tissue was confirmed by demonstration of mammary-specific alpha casein and lactalbumin transcripts (see Fig. 1c)

Fig. 1c; Left Panel: Lung micrometastasis at week 27, detected using anti-HER-2 antibody;Right Panel: Eight HER-2 + and two samples of normal lung tissues, analyzed for mammary gland specific transcripts. "+" indicates normal mammary gland and "-" indicates mock control (Adapted from Hüsemann, Geigl, Schubert et. al (2008)) 

Despite exponential growth at the primary tumor site, the number of CK+ cells and HER-2+cells in the bone marrow rose marginally over the course of time. Cells singly positive for HER-2 and CK were not congruent. Not all tumor cells expressed both markers (see Fig. 1d)

Fig. 1d; Left Panel: CK+, HER-2- cell; Middle Panel: CK-, HER-2+ cell; Right Panel: CK+, HER-2+ cell (Adapted from Hüsemann, Geigl, Schubert et. al (2008))

This suggests, either the existence of heterogeneous tumor cell populations that disseminate to distant sites or different cellular states of the disseminated tumor cells (DTCs)

More Evidence for Early Dissemination

Could the HER-2+, CK+ cells have disseminated from extra-mammary tissues expressing the transgene? The authors took mammary gland fragments from 3-12 week old transgenic mice (displaying only atypical ductal hyperplasia) and transplanted the them into 3 week old wild-type siblings.  Wild type bone marrow was screened at different time points (see Fig. 2)

Fig. 2; Left Panel: Week 14 post-transplantation, CK+ cells in bone marrow of wild-type siblings; Right Panel: HER-2+ cells in the bone marrow (Adapted from Hüsemann, Geigl, Schubert et. al (2008))

Although CK+ and HER-2+ cells were observed in the bone marrow, the levels were lower than transgenic BALB-Neu T mice, but were definitely above rare false positives. Additionally, just as in the transgenic mice, there was no significant increase in disseminated cells from ADH stages to invasive cancer, in the wild-type siblings. 

The malignant nature of these DTCs was established by Comparative Genomics Hybridization (see Fig. 3)

Fig. 3; Top Panel: Karyogram (left) and CGH profile (right) of sorted mouse metaphase of a single HER-2+ cell disseminated from a transplanted mammary gland and subsequently isolated from bone marrow of a recipient wild-type mouse; Bottom Panel: Sorting of mouse metaphase of a single leukocyte in a balanced CGH profile; Green and red bars indicate genomic gains and losses respectively. (Adapted from Hüsemann, Geigl, Schubert et. al (2008))

Evidence that early disseminated cells can grow into metastases

The onset of metastasis relative to primary tumor growth was assessed . Histological sections of the lungs were analyzed and micrometastases could be detected from weeks 20 to 21 onward, a time point at which mostly in-situ carcinomas are present at the primary sites (see Fig. 4)

Fig. 4; Progression of lung metastasis in BALB-Neu T mice, with and without the removal of the primary tumor. Left Panel: Increase in tumor area over time in BALB-Neu T mice (same as Fig. 1b); Right Panel: Size of the largest lung metastasis detected in individual mice. Blue squares indicate average size of metastases from non-operated mice at various time points and red triangles indicate average size from operated animals at 10-15 weeks after surgery. Whiskers indicate 95% confidence intervals (Adapted from Hüsemann, Geigl, Schubert et. al (2008))

Since metastases need time to grow, their increase in size paralleling that of the primary lesion supports the conclusion that, at least in some cases, founder cells of metastasis had disseminated earlier and had started to proliferate.

Some other experiments conducted by the group, demonstrate that bone marrow disseminated cells that do not grow into metastases can be released from growth arrest. Also, there does not appear to be an association between the number of disseminated cells and the stage of the tumor. Large tumors do not necessarily seed more.

Overall the paper presents some compelling evidence for the occurrence of dissemination, even before the primary tumor is established. This would indeed force the field to rethink it's current strategies in combating cancer.


Hüsemann, Y., Geigl, J., Schubert, F., Musiani, P., Meyer, M., Burghart, E., Forni, G., Eils, R., Fehm, T., & Riethmüller, G. (2008). Systemic Spread Is an Early Step in Breast Cancer Cancer Cell, 13 (1), 58-68 DOI: 10.1016/j.ccr.2007.12.003



Geneva Kimball said...

I've had many pet rabbits (over 55 years) who have develped tumours. If a large tumour is surgically removed from, say, a muscle area like the shoulder, it always seemed that a bunch of tumours would emerge soon after, and effect essential organs that would kill the poor bunny within weeks. However, if the large tumour is carefully aspirated regularly instead, so as to minimize discomfort, the rabbit could live for a year or more. Just thought I'd tell people about this. My Vet and I think primary tumour management rather than eradication, is the best bet for best outcomes in rabbits with cancerous growths. Has anyone studied this approach formally?

Geneva Kimball said...

Just to be a bit clearer about this, I've read that Dr. Jonah Folkman (name?) predicted (or observed) similar evidence in discussing hypotheses related to his angiogenisis theory.
It just seems logical that if there are tiny seeds of cancer spread systemically long before a tumour mass is detected (ie., that metastisis happens quite earlier than wishful thinking has it) then it is interesting to think that the primary tumour is fundamentally keeping these seeds of invisible tumours in check. As if there are "pecking orders of tumours"
Obviously, I'm no scientist, I just like to imagine potential "societies" of inter-dependent cell types with unique intercellular communication modes.
It's like imagining another dimension in a way...

Varun said...

I think you mean Judah Folkman. I'm not sure he observed or reported this. I might be wrong on that.

Your second point is not very clear to me. If a cancer cell has already disseminated to another organ, then how is it under the control of the primary tumor anymore?

The intercellular communication idea raises a good point. In the bone for example, they did find disseminated cells but none of these cells metastasized like the disseminated cells in the lung. This would indicate that there is some sort of signaling between the disseminated cell and the bone environment which is keeping the cancer cell in check

Geneva Kimball said...

Thankyou Varun. I really appreciated reading your comments about the bone study.
Ah yes Judah Folkman. I am a bit imprecise--and with names especially.
I actually meant that Dr. Folkman predicted remote communication of cancer cells, not these scientists' observations of early systemic spread, although that may have been something he considered in passing in an interview as I recall. So anyway, regarding how these remote cells could be conrolled from a different organ site?
Now that is precisely what I think would need a good scientific mind to consider. Obviously if the tumour is large enough to begin to grow its own structures that provide blood supply, then perhaps the signalling is via a level of growth hormone that the tumour is sensitive to--with cancers in some cell structure or organs being more sensitive to the (organic) chemical signal. However because of other favorable conditions--say a high level of local oxygenation (or some specific preference of the cancer type) or by not being accessible by soldier/T cells (as in brain tumours), the cancer cells defy the signals that normal inhibit growth and proliferate aggressively.So your confusion is precisely what I am saying might not yet be observed yet. I'm also thinking that such a signal is sent out intermittently by the primary, or at least is not constant in its strength. Which makes it harder to discover of course. Has anyone observed whether tumours grow in spurts not attributable to the math of cell division??
A mysterious ebb and flow of checking signal chemistry might even explain temporary cancer remissions in lieu of aggressive medical treatment.
Just a few extra thoughts...All this interests me because my mother died of lung cancer 2 years ago, and while she was ill I tried to read up on cancer theories and digest the cell science pages of Grey's Anatomy. (My newly gained knowledge was superficial of course, so maybe I am sounding very idiotic with these ideas.)
But at least my personal REAL LIFE experience with rabbits and their cancers is quite true and I hope is of interest to someone reading this..

Varun said...

Okay I think I understand the point you're trying to make. It's about how cancer cells could communicate with other cell types to establish favourable conditions. I was under the impression that you were considering cancer cells in secondary sites communicating with cancer cells in the primary tumor.

Very good point indeed and I'll answer them one at a time. In fact a lot of work has been done in this area. I'll Take two examples

1) Angiogenesis: In breast carcinomas, it has been established that cancer cells of the primary tumor secrete a factor called SDF-1 (Stromal cell derived factor 1). This SDF-1 is a factor which actually attracts a certain cell type called Endothelial precursor cells (from the bone). These endothelial precursor cells, once they reach the tumor assemble to form minute blood vessels that start providing oxygen to the tumor cells

2) Defense: This second example is a really cool one. As you mentioned, we have T-cells in our body. But there are different kinds of them. There is the "T-helper" cell which as the name suggests helps in the activation of an immune response and there are "T-cytotoxic" cells (killer cells) that actually do all the killing. There is a third kind "T-regulatory" cells. Now these are needed to actually ensure that there is no immune response against "self" molecules (molecules inherently present in the body). It turns out that in some ovarian cancers, the cancer actually secretes certain factors which recruits these T-regulatory cells to the cancer site which in turn inhibit T-cytotoxic cells from doing their normal job of killing

Hope that answers some of your questions. There are tons of examples. I'll write some reviews on a few more interesting cancer papers over the next couple of days.

I'd be interested in knowing what you do with rabbits. Do you work with them or do you just have them as pets?

PS: Geneva is a very interesting first name (am I mistaken?) :)

I live very close to Geneva (40 minutes)

Geneva Kimball said...

Thanks Varun.
I was waiting to see if anyone else might post. I found your blog through a mention in the New York Times btw.
I'm way too tired to write anything that could make sense. But about Geneva-my parents (My mom is a Florence) named all of us after cities (They loved to travel). My oldest brothr is Oslo...but I digress...
Some News: U.S. Senator Ted Kennedy died of brain cancer a few hours ago... He had been diagnosed in May 2008. He had undergone at least one surgery to help with seizures.. But his options were few.
bfn. Geneva

Varun said...

Yes I read about Senator Ted Kennedy passing away. My condolences are with the American people.

This blog has been mentioned in the New York Times? Wow!

Alejandro Montenegro-Montero said...

Hi Varun,

Nice post.
I selected it as one of my “picks of the week” of posts aggregated at RB in molecular biology, over at my blog

Varun said...

Thank you :)