Hemangiosarcoma is a rapidly growing, highly invasive cancer arising from the lining of blood vessels of any tissue.1 More than half of all dogs with hemangiosarcoma primary tumors arise within the spleen.2–4 Because of its aggressive biological behavior, dogs with hemangiosarcoma are associated with a poor prognosis.1 Despite surgical removal of the primary tumor, median survival time in dogs with splenic hemangiosarcoma is 2 months or less.2,3 Adding chemotherapy with vincristine, doxorubicin, epirubicin, cyclophosphamide, anthracycline, or metronomic-based protocols may prolong survival time medians to 6 to 12 months.2,5,6
Hemangiosarcoma occurs in 0.3% (39/12,635)7 to 2% (23/1,168)8 of all dogs. Affected dogs are typically older than 5 years, but the disease can occur at younger ages.3,9 German Shepherds, Golden Retrievers, and Labrador Retrievers are overrepresented compared to other breeds with splenic hemangiosarcoma.2,3,9 In addition to age and breed, gonad removal also increases the likelihood of developing splenic hemangiosarcoma. In comparison to intact female dogs, spayed female dogs have 2 to 10 times the risk of developing hemangiosarcoma.10–12
Luteinizing hormone (LH) is secreted by the anterior pituitary gland in brief low-amplitude pulses in sexually intact female and male dogs to stimulate the synthesis of estrogen and testosterone, respectively.13 These gonadal steroid hormones then negatively feedback to decrease the secretion of LH. However, following the removal of the gonads with spaying or castrating, there is a loss of negative feedback and LH is continuously secreted at supraphysiologic concentrations. For example, in intact female dogs, plasma concentrations of LH range from 0.81 to 5.4 ng/mL, whereas in spayed female dogs, plasma concentrations of LH range from 6.4 to 64.3 ng/mL.14 In intact male dogs, serum concentrations of LH range from 0.8 to 11.2 ng/mL, whereas in castrated male dogs, serum concentrations of LH range from 7.2 to 88.2 ng/mL.15 The effect of continuous and supraphysiologic LH concentrations on gonadectomized dogs was not seriously considered until LH receptors (LHR) were found in cells of the canine bladder,16 skin,17,18 adrenal gland,19 thyroid gland,20 lymph node,21 and musculoskeletal tissues,22 as well as lymphoma21 and splenic hemangiosarcoma.23 In isolated canine T-lymphoma cells, LHR activation resulted in dose-dependent cell proliferation24 and increased LHR gene expression.25 To the authors’ knowledge, the effect of LHR activation in isolated canine splenic hemangiosarcoma cells has not yet been studied.
The objective of the current research was to examine LHR expression and response to LHR activation in isolated canine splenic hemangiosarcoma cell lines in vitro. It was hypothesized that the isolated canine splenic hemangiosarcoma cells would express LHR and the activation of these receptors with LH circulating concentrations reported for spayed and castrated dogs would result in cell proliferation.
Materials and Methods
Source of animal tissues
Three immortalized cell lines (No. EMN011-FP, No. EMN016-FP, and No. EMN018-FP; Kerafast Inc), each isolated from different dogs with a primary splenic hemangiosarcoma, were used in this research. The signalment of the dogs was as follows: 9-year-old spayed female Golden Retriever (EFS), 8-year-old intact female Portuguese Water Dog (GRACE-HSA), and 7-year-old, intact male Dalmatian (DAL-4).
Immunocytochemistry
Splenic hemangiosarcoma cells (500,000/dish) were plated on sterile 22-mm2 coverslips in 35-mm diameter dishes. Cells were cultured at 37°C in a humidified 5% CO2 incubator with media (No. Cn211-500; Cell Application Inc) containing 10% protein (No. 10803-034; Avantor Seradigm), 10,000 µg/mL penicillin, and 10,000 µg/mL streptomycin. Media were replaced every 1 to 2 days until reaching 70% to 80% confluence, at which point the cells were fixed onto the coverslips using 70% ethanol with 50 mM glycine (pH 2.0) for 30 minutes at −20°C. The coverslips were stored at 4°C in PBS with 1% sodium azide until assayed.
Routine immunocytochemical localization was performed between August 20, 2021, and September 2, 2021, as previously described.26 Briefly, rabbit polyclonal anti-human LHR antibody (No. NLS1436; Novus Biologicals) was applied to the coverslips at a 1:200 dilution for incubation overnight at 4°C. Negative controls were treated in the same manner except that a universal negative antibody (No. NC495H; Biocare Medical) was applied to coverslips. Coverslips were then washed in PBS, and a fluorescein isothiocyanate-conjugated horse anti-rabbit antibody (No. NB 7159; Novus Biologicals) was applied at a 1:200 dilution in a dark room for a 2-hour incubation at 20°C. Coverslips were then washed in PBS, inverted, and mounted onto slides with mounting medium containing 4′,6-diamidino-2-phenylindole (No. H-1500; Vector Laboratories). Three randomly selected fields from each cell line were captured using fluorescence microscopy (No. DMI6000B; Leica Microsystems) at X400 magnification. This experiment was performed in triplicate for each cell line.
Cell proliferation assay
The cell proliferation experiments were performed between September 13, 2021, and January 29, 2022. After cells were passaged, cell concentrations for each cell line were determined with a hemocytometer and cells were diluted in media (No. Cn211-500; Cell Application Inc) containing 10% protein (No. 10803-034; Avantor Seradigm), 10,000 µg/mL penicillin, and 10,000 µg/mL streptomycin as well as containing 3 concentrations (0, 7.5, and 75 ng/mL) of recombinant canine LH (cLH; No. QP6304; EnQuire BioReagents). One hundred thousand cells per well were plated in triplicate in 96-well plates (No. CLS3841; Sigma Aldrich). Plates were incubated at 37°C in 5% CO2 for 48 hours. A commercial cell growth determining kit (No. 10009365; Cayman Chemical) was used for this experiment. Briefly, 10 µL of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide was added to each well and the plates were incubated at 37°C in 5% CO2 for 3.5 hours. After incubation, 100 µL of the detergent reagent was added to all wells. The plates were then swirled gently for 60 seconds and returned to the incubator for 24 hours at which point the plates were read on a plate reader (SpectraMax 190; Molecular Devices, LLC) at 570 nm. Wells containing only media (no cells) served as a blank for the spectrophotometer. This experiment was performed in triplicate.
Data analysis
All analyses were performed with standard software (GraphPad Prism version 8.4.3; GraphPad Software Inc). For immunocytochemistry, the mean ± SD percentage of LHR-positive cells for each cell line was compared by 1-way ANOVA. For the cell proliferation assay, the absorbance of each triplicate was averaged and the absorbance of the average of the wells containing only media (no cells, blank) was subtracted from the average absorbance. The percentage relative to the control (0 ng/mL cLH) was calculated, and the results are reported as mean ± SD percentage of the control. Cell counts at each cLH concentration were compared within cell lines using a 1-way ANOVA. In addition, a 2-way ANOVA was performed followed by a Dunnett post hoc test to compare the effect of cLH concentration and cell line. Significance was defined as P < .05.
Results
For isolated canine splenic hemangiosarcoma cells, the percentage of cells positive for LHR was 17.2 ± 4.5%, 11.8 ± 3.1%, and 6.9 ± 2.5% in EFS, GRACE-HSA, and DAL-4, respectively. The EFS cell line expressed more LH receptors compared with the DAL-4 cell line (P = .014; Figure 1).
Immunocytochemical localization performed between August 20 and September 2, 2021, showing the expression of luteinizing hormone receptors (arrows) in canine splenic hemangiosarcoma cell lines from a spayed female Golden Retriever (EFS; A), an intact female Portuguese Water Dog (GRACE-HSA; B), and an intact male Dalmatian (DAL-4; C). Fluorescein isothiocyanate stain; bar = 10 µm. Insets (negative controls)—bar = 50 µm.
Citation: American Journal of Veterinary Research 83, 12; 10.2460/ajvr.22.07.0120
Immunocytochemical localization performed between August 20 and September 2, 2021, showing the expression of luteinizing hormone receptors (arrows) in canine splenic hemangiosarcoma cell lines from a spayed female Golden Retriever (EFS; A), an intact female Portuguese Water Dog (GRACE-HSA; B), and an intact male Dalmatian (DAL-4; C). Fluorescein isothiocyanate stain; bar = 10 µm. Insets (negative controls)—bar = 50 µm.
Citation: American Journal of Veterinary Research 83, 12; 10.2460/ajvr.22.07.0120
Immunocytochemical localization performed between August 20 and September 2, 2021, showing the expression of luteinizing hormone receptors (arrows) in canine splenic hemangiosarcoma cell lines from a spayed female Golden Retriever (EFS; A), an intact female Portuguese Water Dog (GRACE-HSA; B), and an intact male Dalmatian (DAL-4; C). Fluorescein isothiocyanate stain; bar = 10 µm. Insets (negative controls)—bar = 50 µm.
Citation: American Journal of Veterinary Research 83, 12; 10.2460/ajvr.22.07.0120
Activation of LHR with cLH significantly increased cell counts in EFS and DAL-4 cell lines (P = .024 and P = .016, respectively; Figure 2). However, there was not a significant increase in cell count in the GRACE-HSA cell line at either cLH concentration. There was a significant effect of cLH concentration and cell line (P = .001 and P = .001, respectively).
Cell proliferation responses of each of the 3 cell lines described in Figure 1 after 48 hours of incubation without recombinant canine luteinizing hormone (cLH; 0 ng/mL [control; solid bars]) or with 7.5 ng/mL [white bars] or 75 ng/mL [checkered bars]) of cLH, reported as percentages of cells relative to the control for each cell line. Each bar represents the mean, and each whisker represents the upper SD. Solid brackets indicate results differed significantly between cLH concentrations within cell lines. Dotted brackets indicate differences between cell lines at the same cLH concentration. P values for these comparisons are provided above each bracket.
Citation: American Journal of Veterinary Research 83, 12; 10.2460/ajvr.22.07.0120
Cell proliferation responses of each of the 3 cell lines described in Figure 1 after 48 hours of incubation without recombinant canine luteinizing hormone (cLH; 0 ng/mL [control; solid bars]) or with 7.5 ng/mL [white bars] or 75 ng/mL [checkered bars]) of cLH, reported as percentages of cells relative to the control for each cell line. Each bar represents the mean, and each whisker represents the upper SD. Solid brackets indicate results differed significantly between cLH concentrations within cell lines. Dotted brackets indicate differences between cell lines at the same cLH concentration. P values for these comparisons are provided above each bracket.
Citation: American Journal of Veterinary Research 83, 12; 10.2460/ajvr.22.07.0120
Cell proliferation responses of each of the 3 cell lines described in Figure 1 after 48 hours of incubation without recombinant canine luteinizing hormone (cLH; 0 ng/mL [control; solid bars]) or with 7.5 ng/mL [white bars] or 75 ng/mL [checkered bars]) of cLH, reported as percentages of cells relative to the control for each cell line. Each bar represents the mean, and each whisker represents the upper SD. Solid brackets indicate results differed significantly between cLH concentrations within cell lines. Dotted brackets indicate differences between cell lines at the same cLH concentration. P values for these comparisons are provided above each bracket.
Citation: American Journal of Veterinary Research 83, 12; 10.2460/ajvr.22.07.0120
Discussion
Previous research has documented the presence of LH receptors (LHR) in cells of canine bladder,16 skin,17,18 adrenal gland,19 thyroid gland,20 lymph node,21 and musculoskeletal tissues,22 as well as lymphoma21 and splenic hemangiosarcoma.23 In the current study, it was hypothesized that the isolated canine splenic hemangiosarcoma cells would also express LHR. To the authors’ knowledge, this is first study to show LHR expression in isolated canine splenic hemangiosarcoma cells.
Previous research21 from our laboratory has shown that the percentage of circulating T-lymphocytes that expressed LHR was significantly higher in gonadectomized dogs (16.6%) than in sexually intact groups (10.5%). In addition, isolated canine neoplastic T-lymphocytes had increased LHR expression following activation in vitro.25 In the current study, the isolated canine hemangiosarcoma cells expressing the highest percentage of LHR were from a spayed female dog, which would have had a high circulating LH concentration at tumor removal. Together, these findings illustrate that high circulating LH concentration following gonad removal increase LHR expression in normal and neoplastic canine cells.
An additional hypothesis of the current study was that activation of LHR in isolated canine splenic hemangiosarcoma cells with cLH would result in the proliferation of hemangiosarcoma cells. In human leiomyoma cells, activation of LHR with human chronic gonadotropin increased cell proliferation in vitro.27 Previous research24 in our laboratory demonstrated LHR activation resulted in dose-dependent cell proliferation of isolated canine T-lymphoma cells. In the current study, activation of LHR with an LH concentration similar to what has been reported in spayed and castrated dogs (75 ng/mL) induced cell proliferation in canine splenic hemangiosarcoma cells in vitro. It is important to note that the cell line from the spayed female Golden Retriever (EFS) had the greatest proliferative response and the highest number of cells expressing LHR. However, the lack of a proliferative response to LHR activation in the cell line from the intact female Portuguese Water Dog (GRACE-HSA) warrants further investigation. It would also be interesting to investigate if circulating LH concentrations and LHR expression in splenic hemangiosarcoma of dogs differ between highly affected breeds (eg, Golden Retrievers) and less frequently affected breeds (eg, Portuguese Water Dogs).
The current research supports the need for in vivo clinical studies aimed at reducing circulating plasma LH concentrations in spayed or castrated dogs. Previous clinical studies have reduced circulating LH concentrations in gonadectomized dogs using gonadotropin-releasing hormone agonist (GnRH agonists)28 or immunization29,30 to restore continence to incontinent spayed dogs as well as to restore a normal hair coat in dogs with puppy coat syndrome.31 We hypothesize that reducing LH concentrations in dogs with splenic hemangiosarcoma or other neoplastic diseases with cellular LHR expression would prolong survival time following diagnosis and conventional oncology treatment.
Acknowledgments
This research was funded by the American Kennel Club Canine Health Foundation. Graduate student support was funded by the Libyan Ministry of Higher Education and Scientific Research.
The authors declare that there were no conflicts of interest.
This research was presented in part as an abstract at the 2022 Society for Theriogenology Annual Conference, Bellevue, Washington, July 2022; and the 2022 Northwest Reproductive Sciences Symposium, Stevenson, Washington, May 2022.
The authors thank Alexa Dietz for assistance with the cell proliferation assay.
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