Ryan P. Dawes

The antidepressant desipramine and α2-adrenergic receptor activation promote breast tumor progression in association with altered collagen structure.

Emotional stress activates the sympathetic nervous system (SNS) and release of the neurotransmitter norepinephrine to promote breast tumor pathogenesis. We demonstrate here that the metastatic mammary adenocarcinoma cell line 4T1 does not express functional adrenergic receptors (AR), the receptors activated by norepinephrine, yet stimulation of adrenergic receptor in vivo altered 4T1 tumor progression in vivo. Chronic treatment with the antidepressant desipramine (DMI) to inhibit norepinephrine reuptake increased 4T1 tumor growth but not metastasis. Treatment with a highly selective α2-adrenergic receptor agonist, dexmedetomidine (DEX), increased tumor growth and metastasis. Neither isoproterenol (ISO), a β-AR agonist, nor phenylephrine, an α1-AR agonist, altered tumor growth or metastasis. Neither DMI- nor DEX-induced tumor growth was associated with increased angiogenesis. In DMI-treated mice, tumor VEGF, IL-6, and the prometastatic chemokines RANTES, M-CSF, and MIP-2 were reduced. Tumor collagen microstructure was examined using second harmonic generation (SHG), a nonabsorptive optical scattering process to highlight fibrillar collagen. In DMI- and DEX-treated mice, but not ISO-treated mice, tumor SHG was significantly altered without changing fibrillar collagen content, as detected by immunofluorescence. These results demonstrate that α2-AR activation can promote tumor progression in the absence of direct sympathetic input to breast tumor cells. The results also suggest that SNS activation may regulate tumor progression through alterations in the extracellular matrix, with outcome dependent on the combination of adrenergic receptor activated. These results underscore the complexities underlying SNS regulation of breast tumor pathogenesis, and suggest that the therapeutic use of adrenergic receptor blockers, tricyclic antidepressants, and adrenergic receptor agonists must be approached cautiously in patients with breast cancer. Cancer Prev Res; 6(12); 1262–72. ©2013 AACR.

Sympathetic innervation, norepinephrine content, and norepinephrine turnover in orthotopic and spontaneous models of breast cancer.

Activation of the sympathetic nervous system (SNS) drives breast cancer progression in preclinical breast cancer models, but it has yet to be established if neoplastic and stromal cells residing in the tumor are directly targeted by locally released norepinephrine (NE). In murine orthotopic and spontaneous mammary tumors, tyrosine hydroxylase (TH)+ sympathetic nerves were limited to the periphery of the tumor. No TH+ staining was detected deeper within these tumors, even in regions with a high density of blood vessels. NE concentration was much lower in tumors compared to the more densely innervated spleen, reflecting the relative paucity of tumor TH+ innervation. Tumor and spleen NE concentration decreased with increased tissue mass. In mice treated with the neurotoxin 6-hydroxydopamine (6-OHDA) to selectively destroy sympathetic nerves, tumor NE concentration was reduced approximately 50%, suggesting that the majority of tumor NE is derived from local sympathetic nerves. To evaluate NE utilization, NE turnover in orthotopic 4T1 mammary tumors was compared to spleen under baseline and stress conditions. In non-stressed mice, NE turnover was equivalent between tumor and spleen. In mice exposed to a stressor, tumor NE turnover was increased compared to spleen NE turnover, and compared to non-stressed tumor NE turnover. Together, these results demonstrate that NE in mammary tumors is derived from local sympathetic nerves that synthesize and metabolize NE. However, differences between spleen and tumor NE turnover with stressor exposure suggest that sympathetic NE release is regulated differently within the tumor microenvironment compared to the spleen. Local mammary tumor sympathetic innervation, despite its limited distribution, is responsive to stressor exposure and therefore can contribute to stress-induced tumor progression.

Second-harmonic generation scattering directionality predicts tumor cell motility in collagen gels

Abstract. Second-harmonic generation (SHG) allows for the analysis of tumor collagen structural changes throughout metastatic progression. SHG directionality, measured through the ratio of the forward-propagating to backward-propagating signal (F/B ratio), is affected by collagen fibril diameter, spacing, and disorder of fibril packing within a fiber. As tumors progress, these parameters evolve, producing concurrent changes in F/B. It has been recently shown that the F/B of highly metastatic invasive ductal carcinoma (IDC) breast tumors is significantly different from less metastatic tumors. This suggests a possible relationship between the microstructure of collagen, as measured by the F/B, and the ability of tumor cells to locomote through that collagen. Utilizing in vitro collagen gels of different F/B ratios, we explored the relationship between collagen microstructure and motility of tumor cells in a “clean” environment, free of the myriad cells, and signals found in in vivo. We found a significant relationship between F/B and the total distance traveled by the tumor cell, as well as both the average and maximum velocities of the cells. Consequently, one possible mechanism underlying the observed relationship between tumor F/B and metastatic output in IDC patient samples is a direct influence of collagen structure on tumor cell motility.

Semi-solid tumor model in Xenopus laevis/gilli cloned tadpoles for intravital study of neovascularization, immune cells and melanophore infiltration.

Tumors have the ability to grow as a self-sustaining entity within the body. This autonomy is in part accomplished by the tumor cells ability to induce the formation of new blood vessels (angiogenesis) and by controlling cell trafficking inside the tumor mass. These abilities greatly reduce the efficacy of many cancer therapies and pose challenges for the development of more effective cancer treatments. Hence, there is a need for animal models suitable for direct microscopy observation of blood vessel formation and cell trafficking, especially during early stages of tumor establishment. Here, we have developed a reliable and cost effective tumor model system in tadpoles of the amphibian Xenopus laevis. Tadpoles are ideally suited for direct microscopy observation because of their small size and transparency. Using the thymic lymphoid tumor line 15/0 derived from, and transplantable into, the X. laevis/gilli isogenic clone LG-15, we have adapted a system that consists in transplanting 15/0 tumor cells embedded into rat collagen under the dorsal skin of LG-15 tadpole recipients. This system recapitulates many facets of mammalian tumorigenesis and permits real time visualization of the active formation of the tumor microenvironment induced by 15/0 tumor cells including neovascularization, collagen rearrangements as well as infiltration of immune cells and melanophores.

Chronic cigarette smoke exposure drives spiral ganglion neuron loss in mice

Tobacco use is associated with an increased risk of hearing loss in older individuals, suggesting cigarette smoke (CS) exposure may target the peripheral auditory organs. However, the effects of CS exposure on general cochlear anatomy have not previously been explored. Here we compare control and chronic CS exposed cochleae from adult mice to assess changes in structure and cell survival. Two-photon imaging techniques, including the imaging of second harmonic generation (SHG) and two-photon excitation fluorescence (TPEF) from native molecules, were used to probe the whole cochlear organ for changes. We found evidence for fibrillar collagen accumulation in the spiral ganglion and organ of Corti, consistent with fibrosis. Quantitative TPEF indicated that basal CS-exposed spiral ganglion neurons experienced greater oxidative stress than control neurons, which was confirmed by histological staining for lipid peroxidation products. Cell counts confirmed that the CS-exposed spiral ganglion also contained fewer basal neurons. Taken together, these data support the premise that CS exposure induces oxidative stress in cochlear cells. They also indicate that two-photon techniques may screen cochlear tissues for oxidative stress.

Abstract # 1825 Beta-adrenergic receptor signaling constrains mammary tumor progression and modulates tumor-associated exosomes

Pre-clinical evidence has linked stress exposure to tumor progression via sympathetic nervous system (SNS) activation and beta-adrenergic receptor (beta-AR) signaling. However, retrospective epidemiological studies suggest the association between stress and breast cancer is more ambiguous. To interrogate the impact of stress on the most common type of clinical breast cancer, hormone receptor-positive ductal carcinoma, we used MMTV-PyMT mice, a spontaneous model of metastatic breast cancer. MMTV-PyMT mice were exposed to a stressor featuring chronic social isolation and acute restraint. The combined stressor elicited a prolonged activation of the SNS compared to non-tumor bearing FVB/NJ mice, the MMTV-PyMT background strain. Intriguingly, stress exposure diminished MMTV-PyMT tumor growth, in conjunction with decreased immunosuppressive myeloid-derived suppressor cell frequency and reduced TGF-beta content in circulating tumor-associated exosomes. Blockade of peripheral beta-ARs with the beta-blocker nadolol abrogated stress-induced tumor suppression and restored MDSC frequency and TGF-beta exosomal content. Furthermore, in non-stressed mice, nadolol treatment increased tumor growth and metastasis. These findings suggest (1) mammary tumor progression dysregulates SNS outflow, (2) beta-AR signaling can impede tumor progression, and (3) circulating exosomes may represent biomarkers for the tumor response to beta-AR activation. This work implies that therapeutic outcomes can be optimized by exploiting nervous system regulation of cancer in a patient-specific manner. Funded by NCI Contract No. HHSN261200800001E, DOD W81XWH-13-1-0439.

Psychosocial stress suppresses tumor progression in a murine model of spontaneous, metastatic breast cancer

Late-stage breast cancer remains an intractable problem, with ∼ 40,000 deaths annually in the United States. Pre-clinical evidence suggests psychosocial stress accelerates cancer progression. Notably, these studies feature injectable tumor models, often in immunocompromised animals exposed to prolonged stress. We have employed a model of spontaneous, metastatic breast cancer, MMTV-PyMT mice, to more closely mimic clinical disease progression. Mice were exposed to a combined stressor featuring chronic social isolation and acute restraint, which activated the hypothalamic-pituitary-adrenal (HPA)-axis and sympathetic nervous system (SNS). Stressed mice were socially isolated during premalignancy. Two weeks later, isolated mice were exposed to 3xa0days of 2-hour restraint. We hypothesized this stressor would exacerbate tumor progression. Instead, 2 weeks post-restraint, stressed mice had smaller tumors, reduced splenic/lung CD11b+Gr-1+ myeloid-derived suppressor cells, and reduced pro-tumor MMP-2 and TGF-beta1/2 in plasma exosomes. By 3 weeks post-restraint, HPA-axis and SNS activation were still apparent in stressed mice, but no alterations in tumor size, immune populations, exosomal proteins, or lung metastases were detected. Stress-induced reduction in tumor growth suggests an inhibitory neurohormonal pathway linking stress and cancer progression. Psychosocial stress exposure had no long-term impact on progression, despite evidence for an altered neurohormonal environment, suggesting receptor desensitization/resistance may compensate for elevated neurohormones. A deeper understanding of biobehavioral pathways in cancer progression is needed before targeting stress neuroendocrine signaling therapeutically. Funded in part by NCI Contract No. HHSN261200800001E .

Second-harmonic generation reveals a relationship between metastatic potential and collagen fiber structure

Second Harmonic Generation (SHG) of collagen signals allows for the analysis of collagen structural changes throughout metastatic progression. The directionality of coherent SHG signals, measured through the ratio of the forward-propagating to backward propagating signal (F/B ratio), is affected by fibril diameter, spacing, and order versus disorder of fibril packing within a fiber. As tumors interact with their microenvironment and metastasize, it causes changes in these parameters, and concurrent changes in the F/B ratio. Specifically, the F/B ratio of breast tumors that are highly metastatic to the lymph nodes is significantly higher than those in tumors with restricted lymph node involvement. We utilized in vitro analysis of tumor cell motility through collagen gels of different microstructures, and hence different F/B ratios, to explore the relationship between collagen microstructures and metastatic capabilities of the tumor. By manipulating environmental factors of fibrillogenesis and biochemical factors of fiber composition we created methods of varying the average F/B ratio of the gel, with significant changes in fiber structure occurring as a result of alterations in incubation temperature and increasing type III collagen presence. A migration assay was performed using simultaneous SHG and fluorescent imaging to measure average penetration depth of human tumor cells into the gels of significantly different F/B ratios, with preliminary data demonstrating that cells penetrate deeper into gels of higher F/B ratio caused by lower type III collagen concentration. Determining the role of collagen structure in tumor cell motility will aid in the future prediction metastatic capabilities of a primary tumor.

20. ß2-Adrenergic agonist treatment inhibits 4T1 breast tumor metastasis to the lung

Solid tumors are composed of neoplastic cells and non-cancerous stromal cells consisting of immune cells, fibroblasts, and endothelial cells. To determine how s2-adrenergic receptor (s2-AR) activation of stromal cells influences cancer progression in the absence of tumor cell s2-AR activation, we utilized the mammary adenocarcinoma 4T1, a murine model of metastatic breast cancer. 4T1 cells do not express functional s-AR and are unable to respond to s-AR agonists or the endogenous s-AR ligand, norepinephrine. BALB/c female mice were injected IP with 5xa0mg/kg salmeterol, a long-acting s2-AR-selective agonist, or saline beginning 2xa0days before tumor cell injection and continued daily until 24xa0h before sacrifice. In BALB/c mice, salmeterol treatment significantly reduced metastasis to the lungs in association with decreased tumor myeloid derived suppressor cells (CD11b+Gr-1+) and a non-significant reduction in tumor growth. To test if the inhibitory effects of salmeterol were dependent on T cell function, BALB/c SCID mice were treated with salmeterol. In the absence of mature T cells, salmeterol treatment did not reduce lung metastases or tumor growth. These results provide evidence that s2-AR activation can inhibit tumor growth and metastasis through host s2-AR-expressing stromal cells including T cells and myeloid derived suppressor cells. Targeting stromal cell s2-AR may inhibit metastasis in the context of aggressive breast cancer.

71. A novel combined psychosocial stress paradigm alters tumor VEGF and circulating exosomes in a spontaneous, metastatic model of breast cancer

Metastatic breast cancer carries significant risk of morbidity and mortality with limited treatment options. Psychosocial stress exposure has been linked to tumor growth and metastasis; therefore understanding the underlying neurohormonal mechanisms may yield new therapeutic options and lead to the development of more effective disease interventions. Here we investigated the impact of social isolation combined with acute restraint stress on spontaneous tumor development in MMTV-PyMT mice. Mammary tumor progression in this mouse model of metastatic breast cancer mimics that of invasive ductal carcinoma in humans. Female MMTV-PyMT mice were singly housed at 8xa0weeks of age during the hyperplastic stage of tumor development. Restraint stress was initiated after 2xa0weeks of isolation. Control mice remained in their home cages throughout the experiment. Twenty-four hours after the final restraint exposure, stressed mice exhibited elevated circulating corticosterone and splenic normetanephrine (a norepinephrine metabolite), which correlated with increased tumor VEGF and decreased splenic neutrophils. There was no difference in total tumor burden at this early carcinoma stage. Circulating CD63+ exosomes, extracellular vesicles that drive cancer pathogenesis, were decreased. These results demonstrate the dual stressor elicited potent hypothalamic pituitary axis (HPA) and sympathetic nervous system (SNS) activation in conjunction with alterations in the premetastatic tumor that may facilitate progression.