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.

Pancreatic cancer cells intimately interact with a complex microenvironment that influences pancreatic cancer progression. The pancreas is innervated by fibers of the sympathetic nervous system (SNS) and pancreatic cancer cells have receptors for SNS neurotransmitters which suggests that pancreatic cancer may be sensitive to neural signaling. In vitro and non-orthotopic in vivo studies showed that neural signaling modulates tumour cell behavior. However the effect of SNS signaling on tumor progression within the pancreatic microenvironment has not previously been investigated. To address this, we used in vivo optical imaging to non-invasively track growth and dissemination of primary pancreatic cancer using an orthotopic mouse model that replicates the complex interaction between pancreatic tumor cells and their microenvironment. Stress-induced neural activation increased primary tumor growth and tumor cell dissemination to normal adjacent pancreas. These effects were associated with increased expression of invasion genes by tumor cells and pancreatic stromal cells. Pharmacological activation of β-adrenergic signaling induced similar effects to chronic stress, and pharmacological β-blockade reversed the effects of chronic stress on pancreatic cancer progression. These findings indicate that neural β-adrenergic signaling regulates pancreatic cancer progression and suggest β-blockade as a novel strategy to complement existing therapies for pancreatic cancer.

Chronic stress accelerates metastasis - the main cause of death in cancer patients - through the activation of β-adrenoceptors (βARs). We have previously shown that β2AR signaling in MDA-MB-231HM breast cancer cells, facilitates invadopodia formation and invasion in vitro. However, in the tumor microenvironment where many stromal cells also express βAR, the role of β2AR signaling in tumor cells in metastasis is unclear. Therefore, to investigate the contribution of β2AR signaling in tumor cells to metastasis in vivo, we used RNA interference to generate MDA-MB-231HM breast cancer cells that are deficient in β2AR. β2AR knockdown in tumor cells reduced the proportion of cells with a mesenchymal-like morphology and, as expected, reduced tumor cell invasion in vitro. Conversely, overexpression of β2AR in low metastatic MCF-7 breast cancer cells induced an invasive phenotype. Importantly, we found that knockdown of β2AR in tumor cells significantly reduced the impact of stress on metastasis in vivo. These findings highlight a crucial role for β2AR tumor cell signaling in the adverse effects of stress on metastasis, and indicate that it may be necessary to block β2AR on tumor cells to fully control metastatic progression.

The efficacy of protein-based therapeutics with indications in the treatment of lymphatic diseases is expected to be improved by enhancing lymphatic disposition. This study was therefore aimed at examining whether PEGylation can usefully be applied to improve the lymphatic uptake of interferon α2 and whether this ultimately translates into improved therapeutic efficacy against lymph-resident cancer. The lymphatic pharmacokinetics of interferon α2b (IFN, 19 kDa) and PEGylated interferon α2b (IFN-PEG12, 31 kDa) or α2a (IFN-PEG40, 60 kDa) was examined in thoracic lymph duct cannulated rats. IFN was poorly absorbed from the SC injection site (Fabs 36%) and showed little uptake into lymph after SC or IV administration (≤1%). In contrast, IFN-PEG12 was efficiently absorbed from the SC injection site (Fabs 82%) and approximately 20% and 8% of the injected dose was recovered in thoracic lymph over 30 hours after SC or IV administration respectively. IFN-PEG40, however, was incompletely absorbed from the SC injection site (Fabs 23%) and showed similar lymphatic access after SC administration to IFN-PEG12 (21%). The recovery of IFN-PEG40 in thoracic lymph after IV administration, however, was significantly greater (29%) when compared to IV IFN-PEG12. The anti-tumour efficacy of interferon against axillary metastases of a highly lymph-metastatic variant of human breast MDA-MB-231 carcinoma was significantly increased by SC administration of lymph-targeted IFN-PEG12 when compared to the administration of IFN on the ipsilateral side to the axillary metastasis. Optimal PEGylation may therefore represent a viable approach to improving the lymphatic disposition and efficacy of therapeutic proteins against lymphatic diseases.

IntroductionFor efficient metastatic dissemination, tumor cells form invadopodia to degrade and move through three-dimensional extracellular matrix. However, little is known about the conditions that favor invadopodia formation. Here, we investigated the effect of β-adrenoceptor signaling - which allows cells to respond to stress neurotransmitters - on the formation of invadopodia and examined the effect on tumor cell invasion.MethodsTo characterize the molecular and cellular mechanisms of β-adrenergic signaling on the invasive properties of breast cancer cells, we used functional cellular assays to quantify invadopodia formation and to evaluate cell invasion in two-dimensional and three-dimensional environments. The functional significance of β-adrenergic regulation of invadopodia was investigated in an orthotopic mouse model of spontaneous breast cancer metastasis.Resultsβ-adrenoceptor activation increased the frequency of invadopodia-positive tumor cells and the number of invadopodia per cell. The effects were selectively mediated by the β2-adrenoceptor subtype, which signaled through the canonical Src pathway to regulate invadopodia formation. Increased invadopodia occurred at the expense of focal adhesion formation, resulting in a switch to increased tumor cell invasion through three-dimensional extracellular matrix. β2-adrenoceptor signaling increased invasion of tumor cells from explanted primary tumors through surrounding extracellular matrix, suggesting a possible mechanism for the observed increased spontaneous tumor cell dissemination in vivo. Selective antagonism of β2-adrenoceptors blocked invadopodia formation, suggesting a pharmacological strategy to prevent tumor cell dissemination.ConclusionThese findings provide insight into conditions that control tumor cell invasion by identifying signaling through β2-adrenoceptors as a regulator of invadopodia formation. These findings suggest novel pharmacological strategies for intervention, by using β-blockers to target β2-adrenoceptors to limit tumor cell dissemination and metastasis.