Lymphatic Cancer Metastasis Model
Larry H. Bernstein, MD, FCAP, Curator
LPBI
How Stress Affects Cancer’s Spread
A mouse study reveals how chronic stress remodels lymphatic vasculature to facilitate the spread of tumor cells.
| March 1, 2016
http://www.the-scientist.com/?articles.view/articleNo/45476/title/How-Stress-Affects-Cancer-s-Spread
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Green fluorescently-tagged nanospheres flow through a lymph vessel from an unstressed mouse (top) and a mouse that has been administered the stress hormone norepinephrine (bottom). Scale bar: 20 μmNATURE COMMUNICATIONS, LE ET AL.
Stress is implicated in increased tumor progression risk and poor survival in cancer patients. A number of recent studies have linked these effects to the promotion of tumor cell dissemination through the bloodstream via stress-induced pathways. Now, a mouse study led by researchers in Australia has revealed the mechanisms by which stress modulates cancer’s spread through another transport network open to tumor cells—the lymphatic system. The findings were published today (March 1) in Nature Communications.
Chronic stress in mice remodels lymph vasculature to promote tumour cell dissemination
Caroline P. Le, Cameron J. Nowell, Corina Kim-Fuchs, Edoardo Botteri, …., Andreas Möller, Steven A. Stacker & Erica K. Sloan
Nature Communications 7, Article number:10634 doi:10.1038/ncomms10634
Chronic stress induces signalling from the sympathetic nervous system (SNS) and drives cancer progression, although the pathways of tumour cell dissemination are unclear. Here we show that chronic stress restructures lymphatic networks within and around tumours to provide pathways for tumour cell escape. We show that VEGFC derived from tumour cells is required for stress to induce lymphatic remodelling and that this depends on COX2 inflammatory signalling from macrophages. Pharmacological inhibition of SNS signalling blocks the effect of chronic stress on lymphatic remodelling in vivo and reduces lymphatic metastasis in preclinical cancer models and in patients with breast cancer. These findings reveal unanticipated communication between stress-induced neural signalling and inflammation, which regulates tumour lymphatic architecture and lymphogenous tumour cell dissemination. These findings suggest that limiting the effects of SNS signalling to prevent tumour cell dissemination through lymphatic routes may provide a strategy to improve cancer outcomes.
In everyday life, we encounter stressful experiences that pose a threat to physiological homeostasis. These threats trigger stress responses, including activation of the sympathetic nervous system (SNS), which leads to elevated local and systemic levels of catecholaminergic neurotransmitters that signal to cells1. Stress-induced SNS signalling is important to enhance alertness and physiological functions for rapid reaction to threat2. However, chronic periods of stress can be detrimental to health by increasing inflammation and promoting the progression of diseases including cancer3, 4, 5, 6. Clinical studies have linked experience of stressful events to poor cancer survival7, 8. This is supported by preclinical studies that show chronic stress promotes cancer progression3, 4, 6. These studies found that stress recruits inflammatory cells to tumours and increases the formation of blood vessels3, 6, which may provide routes for tumour cell dissemination. In addition to dissemination through blood vessels, cancer cells also escape from tumours through lymphatic vasculature9, 10, 11.
The lymphatic system plays an important role in immune function and therefore can influence the trajectory of disease progression. Under normal physiological conditions, the lymphatic system maintains homeostasis by directing cells and solutes from the interstitial fluid of peripheral tissues through lymphatic vessels and into lymph nodes, where they undergo immune examination12, 13. In addition, the lymphatic system aids in the resolution of inflammation by transporting immune cells away from sites of infection14. In cancer, the lymphatic system contributes to disease progression by providing a pathway for tumour cell escape while also being a rich source of chemokines that can promote the invasive properties of tumour cells15. Furthermore, tumour-draining lymph nodes and associated lymphatic endothelium have been shown to develop an immunosuppressive environment, which promotes immune tolerance to the cancer and facilitates tumour growth and spread16, 17, 18. The importance of the lymphatic system in cancer progression is supported by vast clinical data that show tumour-associated lymphatic vessel density (LVD), tumour cell invasion into lymphatic vasculature and the presence of tumour cells in lymph nodes are each associated with increased clinical tumour stage and reduced disease-free survival19, 20,21.
The lymphatic system is innervated by fibres of the SNS22, and acute SNS activity has been shown to increase lymphatic vessel contraction23, 24 and lymphocyte output into lymphatic circulation25. However, little is known about whether stress-induced SNS signalling affects tumour lymphatic vasculature and the consequences this may have on cancer progression.
In this study, we show that chronic stress increases intratumoural LVD while also inducing dilation and increasing flow in lymphatic vessels that drain metastatic tumour cells into lymphatic circulation. Inhibition of COX2 activity blocked the effect of stress on lymphatic vascular remodelling, and showed a key role for macrophage-mediated inflammation in the effects of stress. In addition, we show a critical role for tumour cell-derived VEGFC in the effects of stress on lymphatic vasculature. In both clinical and preclinical studies we demonstrate that disrupting SNS regulation of lymphatics, by blocking β-adrenoceptor signalling, protects against lymphatic dissemination and cancer progression. These findings identify stress signalling as a regulator of lymphatic remodelling and provide evidence for the feasibility of clinically targeting SNS regulation of lymphatics to prevent tumour cell dissemination through lymphatic routes.
Figure 1: Chronic stress remodels tumour-associated lymphatic architecture to promote lymph node metastasis.
http://www.nature.com/ncomms/2016/160301/ncomms10634/images_article/ncomms10634-f1.jpg
(a) Schematic representation of the chronic stress paradigm. (b) Quantification and representative images of tumour LVD (LYVE-1+, green; nuclear, blue) immunostaining of MDA-MB-231 orthotopic tumours. Scale bar, 200 μm (n=5). (c) Quantification of MDA-MB-231 primary tumour size in control or stressed BALB/c nu/nu mice over time (n=5 at each time point). (d) Quantification and representative images of tumour-draining lymphatic vessel diameter (LV, blue) in mice with MDA-MB-231 tumours. Scale bar, 1 mm (n
7). (e) Left: skin flap preparation after injection of Patent Blue V dye into the primary tumour (PT) showing the dye taken up into the tumour-draining LV and into the tumour-draining axillary lymph node (AxLN). The LV is adjacent to a blood vessel (BV). Right top panel: epifluorescence image of mCherry-tagged MDA-MB-231 tumour cells (TCs, red) that had spontaneously disseminated from orthotopic PT and were present in the tumour-draining LV that contained microspheres (green) and was adjacent to an autofluorescent BV. Right lower panel: corresponding maximum projection of multiphoton image. Scale bar, 100 μm (Supplementary Movie 1). (f) Representative in vivo bioluminescence image of orthotopic MDA-MB-231 breast cancer model showing PT, and spontaneous metastasis to draining lymph node (LN) and lung 21 days after tumour cell injection. (g) Representative images of LN and lung metastasis and quantification of metastasis by ex vivo bioluminescence (BLI) imaging in control versus stressed mice with MDA-MB-231 tumours (n=5). (h) Metastasis in vivo over time (n=5 at each time point). (i) LN metastasis in mice that were negative or positive for tumour cells in collecting lymphatic vessels (n13). (j) Ex vivo quantification of bioluminescence from LN at day 28 of 66cl4 tumour progression from control or stressed mice (n=5). (k) Area of lymph node metastasis when primary tumour diameter reached 12 mm in control or stressed MMTV-PyMT mice (n8). Experiments were completed 2–4 times. All data represent mean±s.e. **P<0.01 and ***P<0.001 by Student’s t-test or Mann–Whitney U-test (post hocBonferroni correction).
Figure 7: Stress-induced lymphatic remodelling
http://www.nature.com/ncomms/2016/160301/ncomms10634/images_article/ncomms10634-f7.jpg
Stress remodels lymphatic vasculature through a tumour neural-inflammatory axis to promote lymphogenous tumour cell dissemination and metastasis. Tumour cell-derived VEGFC is necessary for stress-enhanced lymphatic remodelling but is not directly activated by β-adrenoceptor signalling. Tumour-associated macrophages respond to β-adrenoceptor signalling to produce inflammatory molecules such as PGE2, which may then signal to tumour cells to produce VEGFC required for lymphatic remodelling. These effects may be clinically blocked using BBs, anti-VEGFC therapeutics (αVEGFC) or COX2 inhibitors (COX2i). E, epinephrine; NE, norepinephrine; β-AR, β-adrenoceptor.
These findings suggest that it may be important to identify stressed individuals who may be particularly susceptible to lymphogenous dissemination. One approach may be through transcriptional profiling using a stress signature55. Alternatively, as cancer is often a highly stressful experience, it is plausible that SNS intervention may be generally useful to improve cancer outcome. In support of that contention, we found here that clinical BB use was linked to a significant reduction in lymph node metastasis (and reduced distant metastasis) in a cancer cohort without prior evaluation of stress levels.
Stress regulation of lymphatic vasculature may have evolved to promote survival during times of threat. Co-ordinated regulation of the fight-or-flight stress response with increased lymphatic function may have provided an evolutionary advantage by enhancing immune surveillance and activating a rapid immune response to physical threat. However, the findings presented here demonstrate that SNS-regulated lymphatic function can have adverse effects in the context of chronic diseases such as cancer. Importantly, these findings identify multiple points of clinical intervention to limit these adverse effects of stress.
“Stress not only affects your well-being, but it also affects your biology,” said study coauthor Erica Sloan, a cancer researcher at Monash University in Melbourne. “Our study particularly highlights the early steps of tumor cell dissemination into the lymphatic system.”
“This is an excellent contribution,” said Kari Alitalo, a professor of translational cancer biology at the University of Helsinki, Finland, who was not involved in the study. “It’s certainly a very refreshing, novel aspect of biology that they explore in this paper.”
Chronic stress, mediated partly through the sympathetic nervous system, has been associated in cancer patients with a number of physiological changes that promote metastasis (the spread of cancer), including the promotion of blood vessel formation and the recruitment of inflammatory cells like macrophages.
To investigate whether stress could also induce changes in lymph vasculature, the researchers subjected various types of mammary tumor–bearing mice—including strains genetically engineered to develop tumors spontaneously, as well as animals given tumor transplants—to a paradigm designed to induce chronic stress: confinement in a tight space. Comparing stressed mice to controls that bore the same cancerous tumors but had been kept in normal cage conditions, the researchers found no difference in primary tumor growth, but significant differences in lymph vasculature architecture and the frequency of metastases.
“We found that stress helps to build new lymphatic freeways out of the tumor [and] modulates how quickly lymph flows through lymph vessels,” said Sloan, adding that “stress increases the speed limit on these little lymphatic highways and helps cells transit more quickly out of the tumor.”
Since tumor cell dissemination is a key step in cancer metastasis, the team wanted to test whether dissemination through the lymphatic system could be reduced by blocking stress signaling pathways. The researchers turned to beta-blockers—cheap, widely available drugs commonly used to treat hypertension—which inhibit signaling of norepinephrine (or noadrenaline), a stress hormone already implicated in cancer progression risk.
Administering beta-blockers to tumor-bearing mice, the researchers were able to minimize changes in the density of lymph vessels at the primary tumor site, and subsequently reduce metastasis to the lymph nodes. By contrast, artificially stimulating norepinephrine receptors increased both lymph vessel density and metastasis. Through a series of further experiments, the team demonstrated important roles for macrophages involved in inflammatory signaling and a set of tumor-secreted vascular endothelial growth factors (VEGFs) in the regulation of lymph vasculature remodeling and tumor cell dissemination.
“It’s an important step in understanding how stress pathways can influence metastasis,” said Anil Sood, a professor of translational research at MD Anderson Cancer Center in Houston, Texas, who was not involved in the research. “It really helps us to understand the possible mechanisms by which sympathetic nervous system pathways can affect how lymphatics may be remodeled.”
The study also included an analysis of observational data from a cohort of nearly 1,000 breast cancer patients in Milan, which corroborate the team’s findings in mice: patients taking beta-blockers showed a significantly lower incidence of lymph node and distant metastases, even once potentially confounding factors such as age and treatment type had been taken into account.
But Alitalo cautioned against drawing strong conclusions from these data. “Stress biology is complex,” he said. “In laboratory conditions with mice, it’s easier to define and measure stress. In real life, these things fluctuate a lot, especially in cancer patients.” He added that beta-blockers show “no specificity to the lymphatic system, so [their] effects as such could be transduced via a variety of pathways.”
Sloan and colleagues are now working to further resolve the molecular mechanisms involved in stress-induced remodeling of the tumor microenvironment in mice, and are investigating potential interactions between beta-blockers and standard cancer treatments, with a view to using the drugs to tackle stress-related metastasis risk in the clinic.
“This is something that, when we treat cancer, we should be considering,” Sloan said. “By actually addressing stress in the patient, we’re giving our cancer therapies a better chance to work.”
C.P. Le et al., “Chronic stress in mice remodels lymph vasculature to promote tumour cell dissemination,” Nature Communications, http://dx.doi.org:/10.1038/ncomms10634,2016.
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