Abstract
The progression of prostate cancer (PC) into neuroendocrine prostate cancer (NEPC) is a major challenge in treating PC. In NEPC, the PC cells undergo neuroendocrine differentiation (NED); however, the exact molecular mechanism that triggers NED is unknown. Peripheral nerves are recently shown to promote PC. However, their contribution to NEPC was not studied well. In this study, we explored whether sympathetic neurosignaling contributes to NED. We found that human prostate tumors from patients that later developed metastases and castration-resistant prostate cancer (CRPC), a stage preceding to NEPC, have high sympathetic innervations. Our work revealed that high concentrations of the sympathetic neurotransmitter norepinephrine (NE) induces NED-like changes in PC cells in vitro, evident by their characteristic cellular and molecular changes. The NE-mediated NED was effectively inhibited by the AdrĪ²2 blocker propranolol. Strikingly, propranolol along with castration also significantly inhibited the development and progression of NEPC in vivo in an orthotopic NEPC model. Altogether, our results indicate that the NE-AdrĪ²2 axis is a potential therapeutic intervention point for NEPC.
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Introduction
Prostate cancer (PC) is the second leading cancer diagnosed and the fifth leading cause of cancer-related deaths in men worldwide [1]. The mortality associated with PC often results from the development of treatment-resistant neuroendocrine PC (NEPC) [2]. In NEPC, prostate adenocarcinoma cells transdifferentiate into neuroendocrine cells [3, 4]. However, the exact mechanism of neuroendocrine differentiation (NED) is unknown and, hence, effective therapies for NEPC are currently lacking. The average survival period of NEPC patients is around 7ā15 months [2].
Recent studies demonstrated that peripheral nerves promote PC [5,6,7]. For example, denser autonomic innervation is associated with poor prognosis of PC [5, 6]. In addition, several studies showed that the sympathetic neurotransmitter norepinephrine (NE) promotes PC migration and metastasis by activating adrenergic Ī²-receptors (AdrĪ²s) [8, 9]. Interestingly, a recent study demonstrated that AdrĪ²2 indeed promotes NED [10]. However, whether NE directly contributes to NED is not known. Understanding whether NE has any direct contribution to NED would expand potential therapeutic intervention points for NEPC spanning around NE biosynthesis, metabolism and NE-AdrĪ² axis. In this study, we explored whether NE has any direct potential to induce NED. Strikingly, we found that NE at supraphysiological concentrations induces the essential morphological and molecular features required for NED of PC cells. We revealed that the NE-mediated NED involves AdrĪ²2 signaling. Overall, our study indicates that targeting the NE-AdrĪ²2 axis may prevent NEPC development and progression.
Results
Human prostate tumors are densely innervated by sympathetic nerves
We examined the sympathetic nerve distribution in eight human prostate adenocarcinoma and corresponding normal adjacent tissues to understand whether sympathetic axonogenesis occurs in prostate tumors. Tyrosine hydroxylase (TH) was used for staining the sympathetic nerves. Co-staining experiments with TH and the pan-neuronal marker Ī²III tubulin confirmed that TH specifically stains nerve fibers (Supplementary Fig. S1). We found that four out of the eight adenocarcinoma samples analyzed had higher sympathetic innervations (Fig. 1A and Table 1). The associated clinical information data from ACRB revealed that three out of those four patients that demonstrated higher sympathetic innervations in tumors had developed metastasis later. Most importantly, two of them developed castration-resistant prostate cancer (CRPC) (Table 1). Although this is a low sample size, it indicates the propensity of sympathetic signaling in facilitating PC progression. Increased tumor innervations also indicate that active axonogenesis occurs in PC. We found that the newly formed axons make specific contacts with cancer cells, indicating their direct interaction with each other (Fig. 1B). We then examined the expression of AdrĪ²1, AdrĪ²2, and AdrĪ²3 receptors in prostate tumors and PC cell lines and found that AdrĪ²2 is relatively highly expressed in them, suggesting that AdrĪ²2 may be the receptor mediating the sympathetic nerveātumor interaction (Fig. 1C, D). Quantification of AdrĪ²2 expression in tumor samples and corresponding normal adjacent tissues revealed that AdrĪ²2 is overexpressed in tumors (Fig. 1E, F). Overall, our results suggest that newly formed sympathetic fibers may play critical roles in PC progression, in particular its transition to advanced stages such as CRPC, by establishing a direct interaction with cancer cells.
NE induces NED-like morphological changes in PC cells
Based on our observation that prostate tumors acquire higher sympathetic innervations, we asked whether the sympathetic neurotransmitter NE has any direct role in promoting PC, especially its advancement into NEPC. NED is a pre-requisite for NEPC. Therefore, we focus our studies on NEās potential contribution to NED. We used both androgen receptor negative (ARā) DU145 and AR+ LNCaP cells, representing two spectra of PC cells, for our experiments. A previous pre-clinical study reported that early-stage prostate tumors achieve high concentrations (supraphysiological) of NE from local nerves [6]. Therefore, we considered physiologically relevant (10āĀµM) and supraphysiological (50āĀµM and above) concentrations of NE for our initial experiments. Strikingly, we observed that NE at all supraphysiological concentrations (50ā300āĀµM) induced NED-like morphological changes in PC cells, evident from the development of neurite-like extensions and compact cell bodies (Fig. 2A, B). Although neurite-like extensions were evident in DU145 cells at 50āĀµM NE treatment, compact cell bodies were much apparent only from 100āĀµM NE onwards. At the same time, both increased neurite-like extensions and compact cell bodies appeared in LNCaP cells in response to 50āĀµM NE onwards. We did not find such morphological changes in PC cells at a lower concentration of NE, such as 10āĀµM, even after 96āh treatment (Supplementary Fig. S2). We also noted that, at a dose of 300āĀµM NE and above, the cells lose viability and detach from the surface (see below for additional details). Overall, our result indicates that NE induces NED-like morphological changes in PC cells, regardless of their AR status.
NE-induced NED-like changes does not alter the viability of PC cells
We next examined the viability of NE-treated cells to determine at what supraphysiological concentrations it induces NED-like morphological changes, while retaining the cell viability intact. For this, we performed an 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT)-based cell viability assay in DU145 cells after treating them with NE in a dose- and time-dependent manner. Our assay revealed a CC50 value of 494, 272, 207, and 214āĀµM at 24, 48, 72, and 96āh NE treatment, respectively (Supplementary Fig. S3A). Combined with our previous result, it indicates that a dose range of 50ā200āĀµM NE induces NED-like morphological changes, and at the same time, retains more than 50% cell viability for a period of 24ā96āh. Our examination of propidium iodide (PI) live/dead cell staining, where the dead cells only would take up the dye, also did not show difference in PI staining between the control and up to 200āĀµM NE treatment over a period of 48āh, further confirming that NE at supraphysiological concentrations up to 200āĀµM is not cytotoxic to PC cells (Supplementary Fig. S3B). Although this result demonstrated that a single pulse of up to 200āĀµM NE induces NED in PC cells without affecting their viability, prostate tumors are likely to receive continuous pulses of NE from denser sympathetic innervations. Therefore, we tested the effect of frequent pulses of NE on NED by replenishing the culture media with fresh NE (50ā200āĀµM) every 24āh for 7 days. We found NED-like morphology, such as neurite-like extensions and compact cell bodies, in these cultures at all the time points (96 and 168āh) and concentrations (50, 100, 200āĀµM) tested (Supplementary Fig. S4). Importantly, our PI live/dead cell staining did not show cytotoxicity in these cultures, indicating that even continuous pulses of supraphysiological NE is not cytotoxic, but can trigger, and perhaps maintain, transdifferentiation of PC cells.
NE induces characteristic molecular changes involved with NED in PC cells
We next asked whether the NED-like morphology induced by NE is true NED by examining the NED-characteristic molecular changes in PC cells. For this, we examined the mRNA expression of the well-known NED markers chromogranin A (CHGA), chromogranin B (CHGB), and synaptophysin (SYP) in DU145 and LNCaP cells after NE treatment. We found a dose-dependent upregulation of CHGA, CHGB, and SYP mRNAs in DU145 cells at 24āh, whereas there was a slight fluctuation in their levels at 48āh (Fig. 3A-B). Strikingly, NE at the lower spectrum of the supraphysiological concentrations tested, such as 50āĀµM, consistently induced the upregulation of CHGB and SYP at both treatment time points. Similarly, both 50āĀµM and 100āĀµM NE induced the upregulation of all three markers in LNCaP cells at 24āh and 48āh, confirming that NE induces NED-characteristic molecular changes in PC cells (Fig. 3C-D).
We then examined the protein expression of CHGB and SYP by immunostaining in PC cells after NE treatment. We observed the characteristic granular staining of both CHGB and SYP in DU145 cells after 50āĀµM NE. However, they were absent in control cells, indicating that NE specifically upregulates the NED markers expression (Fig. 4A). The LNCaP cells showed mild basal expression of these markers, evident from their uniform cytoplasmic staining in control cells. However, 50āĀµM NE accentuated their staining intensity in the cells confirming that NE induces the expression of these NED markers (Fig. 4B). Overall, our result indicates that NE, even at a lower spectrum of the supraphysiological concentration, initiates true NED in PC cells.
AdrĪ²s are involved with NE-mediated NED
We next examined whether AdrĪ²2 play a role in NE-mediated NED. While 50āĀµM NE induced the upregulation of CHGB and SYP mRNAs in both DU145 and LNCaP cells indicating initiation of transdifferentiation, pre-treatment with propranolol (an AdrĪ²1 and AdrĪ²2 antagonist) inhibited their upregulation (Fig. S5A, B). Similarly, immunostaining showed that propranolol prevents NE-mediated induction of CHGB and SYP remarkably in DU145, whereas its effect was moderate in LNCaP cells (Fig. S5C, D). As our earlier results showed that AdrĪ²2 has a comparatively higher expression in these cells, the propranolol-mediated inhibition indicates that NE induces NED through activation of AdrĪ²2 receptors.
Propranolol inhibits the development and progression of NEPC
We next examined the effect of AdrĪ²2 inhibition in NEPC development. We tested this by inhibiting AdrĪ²2 using propranolol in an orthotopic NEPC model. We used the well-established NEPC cell line, LASCPC-01, to generate the NEPC orthotopic model. Control animals were treated with saline, whereas two other groups were treated with either castration or castrationā+āpropranolol. We performed castration to mimic androgen deprivation therapy (ADT), which is a standard treatment approach in the PC clinics prior to advanced stages, such as NEPC. Propranolol treatment lasted for 21 days. Interestingly, we found that all animals in the saline control and castration-alone group developed tumors; however, only one out of the four animals in the castrationā+āpropranolol group developed tumor (Fig. 5A, B). Our results thus showed that, while castration alone had a significant effect in lowering the tumor size, its combination with propranolol provides enhanced protection in terms of NEPC development and progression, indicating that AdrĪ²2 inhibition is an ideal therapeutic strategy for NEPC.
Discussion
Nerve dependence of PC has gained much attention recently [7, 11, 12]. For example, studies showed that depletion of autonomic nerves suppress PC. Follow-up studies revealed a critical involvement of stromal cells in nerve-dependent PC growth [5]. For instance, selective knockdown of AdrĪ²2 or AdrĪ²3 in stromal cells was shown to suppress PC in animal models [5, 6]. Sympathetic nerve-mediated AdrĪ²2 activation in endothelial cells and resulting enhanced angiogenesis also indirectly promotes nerve-dependent PC growth [6]. Nerves directly alter PC cell dynamics by inducing perineural invasion where PC cells use nerves as physical cues to invade and metastasize [13]. Nerves are also equipped with tumor regulatory machineries, such as active tumor suppressor and DNA repair networks, in modulating tumor dynamics [14,15,16]. However, whether nerves play a direct role in promoting NEPC has not been studied. In this study, we report the potential role of NE-AdrĪ²2 axis in inducing NED and NEPC.
A previous study demonstrated that a potent AdrĪ²2 agonist isoproterenol induces proliferation of PC cells [17]. Therefore, we initially examined whether NE induces PC cell proliferation, but our experiments failed to demonstrate significant cell proliferation in response to 10ā30āĀµM NE, a standard dose used in the literature (data not shown). This led to our attention to a pre-clinical study, which showed that high-grade prostatic intraepithelial neoplasia (HGPIN), an early-stage PC, acquires higher levels of NE compared to healthy prostate [6]. For example, it was shown that mouse HGPIN expresses ~120āng/mg protein of NE [6]. Inclined to this finding, we and others observed that human prostate tumors possess denser sympathetic innervations [5]. We then estimated that a 40āmg human prostate tumor expresses a total of 4āmg protein. Considering that a complete human prostate tumor weighs a minimum of 1ā2āg, the total protein concentration that could be achieved in the tumor is around 100ā200āmg, which can consist of 12ā24āĪ¼g of NE. Then, 100āĪ¼M of NE in 1āml culture offers 16āĪ¼g NE, which closely matches the tumor-level, supraphysiological, NE. Interestingly, our experiments using the supraphysiological levels of NEinduced NED in both AR+ and ARā PC cells. As NED often leads to NEPC, our finding indicates that aberrant sympathetic activity, and the resulting increased NE signaling, might contribute to NEPC.
Androgen (testosterone) is a major growth factor for PC and ADT is a standard and effective treatment for PC [18]. However, PC eventually develops resistance to ADT, undergoes NED, and emerges as treatment-resistant NEPC. Our findings that supraphysiological NE could induce NED of PC cells suggest that NE might serve as an alternate growth factor for PC, when androgen is deprived, resulting in the transition of PC into NEPC. DU145 cells we used in our experiments lack AR and LNCaP cells, although they express AR, were cultured in androgen-lacking media. Thus, our culture conditions simulated an ADT environment and the effect of NE at these culture conditions in inducing NED supports our argument. We did not, however, examine the effect of NE in inducing NED in androgen enriched conditions.
We found that NE-mediated NED is dependent on AdrĪ²2. A recent study using a subcutaneous PC xenograft model also demonstrated that AdrĪ²2 is involved with NED [10]. In general, cutaneous structures have less autonomic innervations compared to internal organs and, hence, the triggering force of AdrĪ²2 activation in subcutaneous PC models is not clear and less likely to be NE. Therefore, NE being the major catecholamine available in the prostate, our finding of its potential to induce NED warrants special attention. Our experiments showed that propranolol inhibits NE-driven NED. Although propranolol targets both AdrĪ²1 and AdrĪ²2, our mRNA analysis showed that AdrĪ²2 is highly expressed, compared to AdrĪ²1, in PC cells and prostate tumors, and hence we believe that AdrĪ²2 might be critical for NE-driven NED.
Overall, our study, for the first time, showed that supraphysiological concentrations of NE facilitates NED of PC cells through activation of AdrĪ²2. The prostate is supplied by hypogastric and pelvic nerves, providing adrenergic and cholinergic innervations, respectively. The actions of the cholinergic neurotransmitter acetylcholine (Ach) and nerve-derived neurotrophic factors, such as nerve growth factor and brain-derived neurotrophic factor in the prostate milieu might influence the action of NE on PC cells in vivo. Further investigations on the fate of cancer cells in response to the co-ordinated actions of NE, Ach, and neurotrophins may reveal an in-depth understanding of the nature of NE-mediated NED in vivo. Having said that, our in vivo studies provide strong evidence that AdrĪ²2 inhibition along with castration has superior benefit in managing NEPC. Overall, our findings indicate that NE- AdrĪ²2 axis is an ideal therapeutic intervention point for NEPC.
Materials and methods
Cells, chemicals, and reagents
The PC cell lines DU145 (ATCC HTB81) and LNCaP (ATCC CRL1740) were purchased from ATCC. NE bitartrate (A9512), propranolol hydrochloride (537075) and PI (P4170) were procured from Millipore Sigma. Trizol (15596018), cDNA synthesis kit, Dulbeccoās modified Eagle medium (DMEM)/F12 media (11330032), and fetal bovine serum (12483020) were purchased from Life Technologies. Antibiotic/antimycotic solution (SV3007901) was purchased from HyClone. All other chemicals used were of analytical grade.
Nerve innervation studies in human prostate tumors
Human tissue studies were performed after receiving approval from the Biomedical Research Ethics Board at the University of Saskatchewan. Fresh, frozen human prostate adenocarcinoma tissues were procured from the Alberta Cancer Research Biobank (ACRB). They were fixed in Zamboniās fixative overnight at 4āĀ°C and further incubated overnight in 20% sucrose solution. The tissues were then embedded in optimal cutting temperature (OCT) compound and allowed to freeze, followed by 12āĀµm thick sections taken on slides. The generated sections were blocked for 30āmin using 5% donkey serum containing 0.3% Triton X-100. The sections were then co-labeled with primary antibodies against TH (rabbit pAb; AB152, Sigma) and Ī²III tubulin (chicken pAb; AB9354, Sigma) for 1āh, followed by incubation with a cocktail of anti-rabbit Alexa FluorĀ® 488 Conjugate (A11034, ThermoFisher Scientific) and anti-chicken Alexa FluorĀ® 647 (A21449, ThermoFisher Scientific) secondary antibodies for 1āh at room temperature. Sections were then mounted using slow-fade DAPI (S36973, Life Technologies) and images captured using Axio Observer 7 (inverted bright-field/fluorescence microscope, Carl Zeiss, Germany). Quantification of nerve fibers was done manually in a blinded manner by tracing the fibers using Fiji software.
To investigate nerve-cancer cell interface, some sections were also co-labeled with primary antibodies against TH and cytokeratin (mouse mAb; MA1-82041, ThermoFisher Scientific, CA) for 1āh, followed by incubation with a cocktail of anti-rabbit Alexa FluorĀ® 488 Conjugate and anti-mouse Alexa FluorĀ® 546 (A21045, ThermoFisher Scientific, CA) secondary antibodies for 1āh at room temperature.
Morphological assessment of NED features
The PC cells were cultured in DMEM/F12 media containing 10% fetal bovine serum and penicillin and streptomycin cocktail (50āU/ml) at 37āĀ°C and 5% CO2 conditions. For the morphological assessment of NED occurrence, 5āĆā104 cells/well were seeded in six-well plates and defined treatments were given after overnight incubation. The characteristic NED features, such as compact cell bodies and neurite-like extensions, were then evaluated at 24 and 48āh using Axio Observer 7.
Cell viability
Cell viability was assessed using both MTT assay (quantitative method) and PI staining (qualitative method). MTT assay was performed as done previously [19]. Briefly, 5āĆā103 cells/well were seeded in a 96-well plate and incubated overnight. The cells were then given specific treatments and incubated for 24ā96āh. The media was then removed and 1āmg/ml of MTT solution (100āĀµl) was added to each well followed by 4āh incubation at 37āĀ°C and 5% CO2 conditions. After the incubation, the MTT solution was removed and the formazan crystals formed were dissolved in 100āĀµl of dimethyl sulfoxide. The absorbance of the solution was then read at 570 and 630ānm (for background correction) using SpectraMax M2 (Molecular Devices, San Jose, CA). The percentage change in cell viability was calculated compared to corresponding controls.
For PI staining, 5āĆā104 cells/well were seeded in a six-well plate and incubated overnight at 37āĀ°C and 5% CO2 conditions. The cells were then treated with varying concentrations of NE for 8, 24, and 48āh, followed by the media removed and 750āĀµl of freshly prepared PI solution (10āĀµg/ml) added to the plate. The plates were then incubated at 37āĀ°C for 10āmin and the images captured using Axio Observer 7.
Real-time quantitative reverse-transcription PCR
Total RNA was isolated using TRIzol reagent (15596018, Life Technologies) and quantified using ND-1000 spectrophotometer (NanoDrop Technologies, USA). Then, 500āng of total RNA was converted into cDNA using a cDNA synthesis kit (4368813, Applied Biosystems) as per the manufacturerās instructions. The cDNAs were amplified using the specific primers mentioned below, and by using PowerUpā¢ SYBRā¢ Green Master Mix (A25741, Applied Biosystems). The cDNA amplifications were done in QuantStudioā¢ 3 Real-Time PCR System (Applied Biosystems). All reactions were performed in triplicate, and the gene expressions were normalized to the house keeping gene, RPLP.
Primer | Sequence (5ā²ā3ā²) |
---|---|
Chromogranin A Forward | GGGATACCGAGGTGATGAAATG |
Chromogranin A Reverse | TCTCCTCGGAGTGTCTCAAA |
Chromogranin B Forward | GGATGAGGAGGACAAGAGAAAC |
Chromogranin B Reverse | CCCTCTCTTCCTCACTTTCTTC |
Synaptophysin Forward | CGTGTTTGCCTTCCTCTACT |
Synaptophysin Reverse | GCATGGGCCCTTTGTTATTC |
AdrĪ²1 Forward | CAA TGT GCT GGT GAT CG |
AdrĪ²1 Reverse | CCA GGG ACA TGA TGA AGA |
AdrĪ²2 Forward | AGA CCT GCT GTG ACT TCT |
AdrĪ²2 Reverse | CTG AAA GAC CCT GGA GTA GA |
AdrĪ²3 Forward | GCT GGT TGC CCT TCT TT |
AdrĪ²3 Reverse | GCA TAA CCT AGC CAG TTC AG |
RPLP Forward | AGCCCAGAACACTGG TCT |
RPLP Reverse | ACTCAG GATTTCAATGGTGCC |
Immunofluorescence
NE-treated cells were fixed for 15āmin using 4% paraformaldehyde and then blocked for 30āmin using 5% donkey serum containing 0.3% Triton X-100. The cells were then incubated with the primary antibodies against CHGB (1ā:ā100; rabbit; PA5-52605, ThermoFisher Scientific) or SYP (1ā:ā100; rabbit; MA5-14532, ThermoFisher Scientific) for 3 and 1āh, respectively, followed by incubation with anti-rabbit Alexa FluorĀ® 488 Conjugate (1ā:ā100, A11034, ThermoFisher Scientific) secondary antibody for 1āh at room temperature. Cells were then mounted using slow-fade DAPI (S36973, Life Technologies) and examined using Axio Observer 7.
For AdrĪ²2 and cytokeratin expression in tumor and adjacent normal tissues, the tissues were incubated with primary antibodies against AdrĪ²2 (1ā:ā100; rabbit; PA5-14117, ThermoFisher Scientific) and cytokeratin (1ā:ā100; mouse; MA1-82041; ThermoFisher Scientific) for 1āh at room temperature, followed by incubation with a cocktail of anti-rabbit Alexa FluorĀ® 488 Conjugate (1ā:ā100, A11034, ThermoFisher Scientific) and anti-mouse Alexa FluorĀ® 546 (1ā:ā100, A21045, ThermoFisher Scientific) for 1āh at room temperature. Cells were then mounted using slow-fade DAPI (S36973, Life Technologies) and examined using Axio Observer 7.
Western blotting
Total protein from tumor and normal adjacent tissues were isolated using RIPA buffer (Thermo Scientific) containing protease and phosphatase inhibitor cocktail (Thermo Scientific). Thirty micrograms of proteins were then allowed to resolve in an SDS-polyacrylamide gel electrophoresis gel and the resolved proteins were transferred onto a polyvinylidene difluoride membrane. The membrane was then incubated with the primary antibody against AdrĪ²2 (1ā:ā1000; rabbit; PA5-14117, ThermoFisher Scientific) for 1āh followed by goat anti-rabbit horseradish peroxidase (HRP) conjugate (1ā:ā3000; Biorad) and then developed using ECL reagent (Biorad). The membrane was also re-probed with Ī²-actin antibody (1ā:ā2000; mouse; sc-47778, Santa Cruz Biotechnology) for 1āh followed by goat anti-mouse HRP conjugate (1ā:ā3000; 170-6516, Biorad) and then developed using ECL reagent. The blots were visualized and images captured, using a Geldoc (Biorad).
AdrĪ²2 inhibition studies in vitro
For AdrĪ²2 inhibition studies, DU145 and LNCaP cells were pre-treated for 1āh with 50āĀµM propranolol (AdrĪ²1/AdrĪ²2 antagonist). The cells were then treated with 50āĀµM NE either for 6āh (for DU145 mRNA expression studies) or 24āh.
Orthotopic PC model
All animal experiments were conducted after obtaining approval from the animal ethics committee at the University of Saskatchewan. Adult male athymic nude mice (Crl:NU(NCr)-Foxn1, Charles River, Canada) weighing 20ā25āg were used for the study and equal number of animals were randomly allocated into different groups. Tumor induction, treatment, daily monitoring of animals, and final tumor volume reading were done by the same person, with the tumor volume reading done in the presence of an additional person who had no prior knowledge of the experimental layout. The NEPC cells, LASCPC-01, were obtained from ATCC. For castration and orthotopic injection of the cells into the prostate, the mice were first anesthetized with isoflurane and injected with buprenorphine (0.05āmg/kg). A small incision was made in the abdomen, urinary bladder was exposed, and the testicles were located. Castration was performed as described previously [20], and by ligating the testicular arteries followed by removing the testicles by cutting the other end of the arteries along with the attached fat pad. Orthotopic injection was performed by injecting 2āĆā105 LASCPC-01 cells in 20āĀµl (1ā:ā1 RPMI media and Cultrex basement membrane extract (R&D Systems)) into the exposed prostate. An additional 30ās was provided before removing the syringe from the prostate to minimize cell leaking. The animalās skin was sutured back and allowed to recover from anesthesia. The animals were weighed once a week. Treatment with either vehicle (saline) or propranolol (20āmg/kg) intraperitoneally was started 7 days after the tumor inoculation procedure. The treatment lasted for 21 days, and then the animals were sacrificed, and tumor sizes recorded.
Statistics
A minimum of three replicates were performed for each experiment and the exact number of replicates is given in the corresponding figure legend, excluding the samples exempted from the analysis due to technical inaccuracy. Data are presented as meanāĀ±āSEM unless otherwise described in the figure legend. The statistical significance was calculated using standard Studentās t-test or one-way analysis of variance followed by Dunnettās post hoc test, wherever appropriate, using GraphPad PrismĀ® 8.0 software. Each group in an experiment is statistically analyzed in similar manner to keep uniform variations between the experimental groups. P-value of <0.05 was considered statistically significant.
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Acknowledgements
This work was supported by the Start-up research fund and the College of Medicine Research Award (CoMRAD) fund, from the College of Medicine, U of S, to AK. SS is supported by the Biomedical summer research fellowship from the College of Medicine, U of S. TC is supported by the Mitacs fellowship. FV and HE are supported by funds from the College of Medicine, U of S.
Author contributions
SD performed experiments, analyzed, and interpreted the results, and composed the first draft of the manuscript. MB, SS, HE, and TC performed experiments, analyzed and interpreted the data, and made intellectual contributions. FV helped with orthotopic NEPC studies, provided intellectual contributions to the work, and helped with revising the manuscript draft. AK formulated the research question, provided sufficient resources, guided and supervised the progress of the work, and wrote the final version of the manuscript.
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Dwivedi, S., Bautista, M., Shrestha, S. et al. Sympathetic signaling facilitates progression of neuroendocrine prostate cancer. Cell Death Discov. 7, 364 (2021). https://doi.org/10.1038/s41420-021-00752-1
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DOI: https://doi.org/10.1038/s41420-021-00752-1
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