Norepinephrine induces anoikis resistance in high-grade serous ovarian cancer precursor cells

Abstract

High-grade serous carcinoma (HGSC) is the most lethal gynecological malignancy in the United States. Late diagnosis and the emergence of chemoresistance have prompted studies into how the tumor microenvironment, and more recently tumor innervation, may be leveraged for HGSC prevention and interception. In addition to stess-induced sources, concentrations of the sympathetic neurotransmitter norepinephrine (NE) in the ovary increase during ovulation and after menopause. Importantly, NE exacerbates advanced HGSC progression. However, little is known about the role of NE in early disease pathogenesis. Here, we investigated the role of NE in instigating anchorage independence and micrometastasis of preneoplastic lesions from the fallopian tube epithelium (FTE) to the ovary, an essential step in HGSC onset. We found that in the presence of NE, FTE cell lines were able to survive in ultra-low-attachment (ULA) culture in a β-adrenergic receptor-dependent (β-AR-dependent) manner. Importantly, spheroid formation and cell viability conferred by treatment with physiological sources of NE were abrogated using the β-AR blocker propranolol. We have also identified that NE-mediated anoikis resistance may be attributable to downregulation of colony-stimulating factor 2. These findings provide mechanistic insight and identify targets that may be regulated by ovary-derived NE in early HGSC.

Keywords: Cancer; Cell biology; Cell migration/adhesion; Obstetrics/gynecology; Oncology.

Figure 1. NE induces spheroid formation and anoikis resistance in FTE cells.

(A) Bright-field images of the HGSC cell line SKOV3 as well as 3 immortalized human FTE cell lines (FT237, FT240, and FT246) cultured under ultra-low-attachment (ULA) conditions for 24 hours with vehicle or 10 μM NE. (B) Fold-change in average area for NE-treated spheres relative to vehicle-treated spheres as visualized in A. (C) ReadyProbe viability staining of cells cultured as in A. These images were taken at 10× original magnification and scale bars represent 100 µm. (D) Absolute quantification of dead cells (shown in green in C) per spheroid (shown in blue in C). (E) Representative flow cytometry plots for propidium iodide + annexin V staining, with quantification of cell populations relative to vehicle-treated cells in F, as cultured in A. Each experiment was conducted in technical triplicate for each biological replicate (n = 3). All statistical analyses for this figure were conducted with Student’s t tests. Data are represented as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 2. Viable FTE precursor spheroids adhere to and displace OSE.

(A) Fluorescence images after 24 hours of coculture with 2D immortalized human OSE cells (HIO80-L2G, green) and CellTracker Red–stained FTE cells that were pretreated in ULA culture for 24 hours ± 10 μM NE. These images were taken at 4× original magnification, and insets are uniformly magnified regions of interest. (B) Quantification of the number of multicellular FTE cell adhesions (TxRed) on the GFP⁺ OSE monolayer. (C) Quantification of scar area in the OSE monolayer relative to the area of the adhered FTE cells. Each experiment was conducted in technical triplicate for each biological replicate (n = 3). All statistical analyses for this figure were conducted with Student’s t tests. Data are represented as mean ± standard deviation. *P < 0.05, **P < 0.01.

Figure 3. NE-induced anoikis resistance occurs in an ADRβ2-dependent manner.

(A and B) Bright-field images and relative area quantification of FTE spheres cultured in ULA culture for 24 hours ± 10 μM NE (V, NE, respectively) and/or nonselective beta blocker, propranolol (NE+P, P, respectively). (C and D) Fluorescence images of ReadyProbe viability staining for spheroids (blue) and quantification of dead cells (green) cultured as in A. (E) Bright-field images for FTE cells cultured in ULA plates ± 10 μM NE after transfection with siNTC, siADRβ1, or siADRβ2. (F) Quantification of NE-treated spheroid area relative to vehicle-treated cells. (G) Fluorescence images of ReadyProbe viability staining for spheroids (blue) and quantification of dead cells (green) (H) in each siRNA condition. These images were all taken at 10× original magnification and scale bars represent 100 µm. Each experiment was conducted in technical triplicate for each biological replicate (n = 3). Statistical analyses were conducted with (B and D) 1-way ANOVA with Tukey’s correction for multiple comparisons or (F and H) 2-way ANOVA with Holm-Šídák correction for multiple comparisons. Data are represented as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4. Propranolol abrogates morphological and survival changes imparted by NE-rich human FF.

(A and B) Bright-field images and area measurements for FT246 cells treated for 24 hours in ULA culture with 18 different FF samples collected from 17 different patients (10% volume). (C) Linear regression for correlation between FT246 aggregate area and relative NE concentration quantified via mass spectrometry analysis. (D) Fluorescence images of total cells (blue) and dead cells (green), quantified in E, for each FT246 cell aggregate, cultured as in A. (F) Linear regression for correlation between the number of dead cells in FT246 aggregates and relative NE concentration per sample. (G and H) Bright-field images and area measurements for FT246 cells treated with vehicle, NE, FF2, FF6, or FF16 in the presence or absence of 10 μM propranolol. (I and J) Fluorescence images and dead cell quantification for cells as cultured in G. These images were all taken at 10× original magnification and scale bars represent 100 µm. Each experiment was conducted in technical triplicate for each biological sample (n = 1).

Figure 5. NE activates ADRβ-dependent transcriptional changes.

(A) Experimental design for bulk RNA sequencing of FT237 and FT246 cells grown under ULA conditions for 4 and 24 hours, with or without 10 μM NE and/or 10 μM propranolol. (B) Volcano plots of differentially expressed transcripts upregulated (magenta) and downregulated (cyan) upon NE treatment relative to vehicle-treated cells at 24 hours. CSF2, colony-stimulating factor 2. (C) Volcano plots of differentially expressed transcripts upregulated (magenta) and downregulated (cyan) upon propranolol and NE treatment relative to NE treatment alone at 24 hours. (D) Heatmap of common transcripts between FT237 and FT246 cells whose NE-induced expression levels are abrogated upon cotreatment with propranolol at 24 hours; all transcript values are normalized to untreated 2D control cells. N, norepinephrine; NP, norepinephrine + propranolol; P, propranolol. (E) Ingenuity Pathway Analysis results for disease-related pathways enriched in NE-treated cells at the 24-hour time point.

Figure 6. CSF2 knockdown recapitulates NE-induced anoikis resistance phenotype.

(A) RT-qPCR validation of CSF2 expression in FT237 and FT246 upon 24-hour treatment with vehicle, 10 μM propranolol, and/or 10 μM NE under ULA conditions. (B) Representative bright-field images of respective siNTC/siCSF2-transfected FTE cell lines cultured for 24 hours under ULA conditions ± 10 μM NE, quantified in C. (D) Representative fluorescence images of total cells (blue) and dead cells (green) in siNTC/siCSF2-transfected cells cultured as in B, quantified in E. These images were all taken at 10× original magnification and scale bars represent 100 µm. Each experiment was conducted in technical triplicate for each biological replicate (n = 3). All statistical analyses for this figure were conducted with 1-way ANOVA with either (A) Tukey’s or (C and E) Dunnett’s corrections for multiple comparisons. Data are represented as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.