19406207capr130079-sup-110002_2_supp_2181541_mbmjfy.pptx (2.95 MB)

Data Supplement from The Antidepressant Desipramine and α₂-Adrenergic Receptor Activation Promote Breast Tumor Progression in Association with Altered Collagen Structure

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posted on 2023-04-03, 19:23 authored by Mercedes J. Szpunar, Kathleen A. Burke, Ryan P. Dawes, Edward B. Brown, Kelley S. Madden

Supplemental Fig. S1. 4T1 Cells Do Not Express Functional a2-AR. High dose DEX-induced effects on 4T1 (A) proliferation and (B) VEGF production in vitro are not blocked by the a2-AR antagonist yohimbine (YOH). (C) The alpha2-AR agonists clonidine (CLON) and dexmedetomidine (DEX) do not inhibit forskolin (FORSK)-induced cAMP in 4T1 cells. (D) CLON and DEX reduced FORSK-induced cAMP in human foreskin fibroblasts (HFF). Groups labeled with same letter are not significantly different from each other, but are significantly different from groups labeled with different letters, p{less than or equal to}0.01, Tukey's post-hoc test. Supplemental Fig. S2. DMI Elevates Spleen Norepinephrine (NE) and the NE Metabolite, Normetanephrine (NMN). Spleen (A) NE and (B) NMN were increased 3 days post-DMI pellet implantation. (C) Spleen weight was decreased day 3 post-DMI implantation. *** indicates significant differences versus corresponding placebo group, Holm-Sidak multiple comparison test, p<0.001. Supplemental Fig. S3. DMI-induced Tumor Growth is Not Associated with Altered Tumor Angiogenesis. Immunocytochemical staining of CD31+ blood vessels in tumor slices were quantified by two different methods: (A) percentage of pixels above threshold and (B) calculating the area of traced vessels per cell density based on nuclear DAPI staining (not shown). (C) A representative image of CD31+ blood vessels. Scale bar = 50 µm. Supplemental Fig. S4. TNF-a Does Not Induce 4T1 Proliferation in Vitro. Results are expressed as mean {plus minus} SD of triplicate wells. Supplemental Fig. S5. Tumor F4/80+ Macrophage Density Is Not Significantly Changed with A) DMI or B) DEX treatment. (C) Representative image of F4/80+ macrophages in a 4T1 tumor section by immunofluorescence; scale bar = 100 µm. Statistical analysis by Mann-Whitney (M-W) nonparametric test.

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ARTICLE ABSTRACT

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.