Lymphoma is one of the most common cancers in dogs, occurring more frequently than human cases of lymphoma and leukemia combined.1 The clinical signs and treatment options for dogs with lymphoma differ depending on whether the lymphoma is derived from B or T lymphocytes. Even with therapy, dogs with T-cell lymphoma are at significantly higher risk of relapse and early death compared to dogs with B-cell lymphoma.2 In addition, the mean survival time for dogs with systemic multidrug chemotherapy for T-cell lymphoma is shorter than for dogs with B-cell lymphoma (6 to 8 months vs 12 months, respectively).3
Cell proliferation, adhesion, and transmigration play a crucial role in cancer metastasis. The effect of luteinizing hormone (LH) receptor activation on these processes has been investigated in various tissues.4–9 In women, ovarian cancer that developed during or after ovulation induction therapy with human chorionic gonadotropin (hCG; an LH receptor agonist) had a higher percentage of cells with LH receptor expression.4 Additionally, LH receptor stimulation of human ovarian cancer cells in vitro inhibited apoptosis5 and increased cell invasion.6 Activation of LH receptors with hCG in vitro significantly increased the viability and proliferation of human adrenocortical carcinoma cells (H295R)7 and significantly increased cell adhesion and invasion in human breast cancer cells.8 With respect to hematopoietic cells, Mierzejewska et al.9 reported that hematopoietic stem progenitor cells proliferated in vivo and in vitro in response to LH receptor activation.
LH receptors are also present in neoplastic canine lymphocytes.10 However, the response to LH receptor activation in neoplastic lymphocytes has not been studied. The purpose of the current study was to determine the effect of LH receptor activation in immortalized neoplastic canine T lymphocytes. It was hypothesized that increasing concentrations of an LH receptor agonist in vitro would result in increases in LH receptor gene expression as well as cell proliferation, adhesion, and transmigration in neoplastic canine T lymphocytes.
Methods
Sources of canine specimens
Cryopreserved immortalized canine neoplastic T-lymphocyte cell lines (CLC, CLK, and EMA) derived from adult female dogs with T-cell lymphoma (reproductive status unknown; 1 French Bulldog, 1 English Springer Spaniel, and 1 Great Pyrenees; age range, 6 to 8 years).11 Carp leukocyte culture and EMA cell lines were derived from pleural fluid containing malignant lymphoblastoid cells, and the CLK cell line was derived from ascitic fluid containing malignant lymphoblastoid cells. The immortalized cell lines had been previously phenotyped by Umeki et al.11 They found that all cell lines expressed CD45 and CD45RA and were negative for CD4, CD11b, CD14, CD21, CD34, T-cell receptor alpha and beta (TCRαβ), IgM, and IgG.11 Ettinger et al.10 evaluated the cell lines for LH receptor expression using flow cytometry and found that all 3 cell lines expressed LH receptors in varying percentages (45%, 35%, and 10% for CLC, CLK, and EMA cell lines, respectively).
An ovary and skin sample obtained at the time of ovariohysterectomy and mammary mass removal from a female 6-year-old German Shepherd was used as a positive control for LH receptor gene expression. Canine aortic endothelial cells (#Cn304–05; Cell Applications Inc) were used for the adhesion and transmigration assays.
Luteinizing hormone receptor gene expression assay
Each cell line was cultured at an initial concentration of 500,000 cells/mL in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) substitute (Fetal Bovine Essence 3100; VWR) and penicillin (100 U/mL)/streptomycin (100 μg/mL)/glutamine solution. Each cell line (run in triplicate) was incubated with hCG (treated; 4 or 400 U/mL) or with 0 U/mL hCG (control) for 72 hours at 37 °C in 5% CO2. Cells were removed from each subculture for total RNA (tRNA) isolation using a routine phenol-chloroform extraction following the manufacturer's protocol. Total RNA was also extracted in the same manner from the control tissue samples. RNA concentration and integrity was measured using spectrophotometry.
Complementary DNA (cDNA) was synthesized for each sample from tRNA (1 μg) following the manufacturer's instructions for the cDNA synthesis kit. In brief, tRNA (5 μL, 200 ng/μL) was added to a reaction mixture containing 10 mM deoxynucleotide triphosphate mix (1 μL), 0.5 μg/μL oligo(dT)12–18 (1 μL), and RNase-free water (5 μL), which was incubated at 65 °C for 5 minutes and then placed on ice for 2 minutes. A second reaction mixture containing 10X reverse transcription buffer (2 μL), 25 mM MgCl2 (4 μL), 100 mM dithiothreitol (2 μL), and RNase inhibitor (1 μL, 40 U/μL) was added to the reaction mixture and incubated at 42 °C for 2 minutes. Moloney murine leukemia virus reverse transcriptase (1 μL) was added to each mixture and incubated at 42 °C for 50 minutes. Additional cDNA synthesis was terminated by incubating the mixture at 70 °C for 15 minutes. RNase H (1 μL) was added to each mixture and incubated for 20 minutes at 37 °C. Synthesized cDNA was stored at −20 °C prior to quantitative PCR (qPCR) analysis.
Forward and reverse primers for the canine LH receptor (Figure 1) were designed using Primer-BLAST (NIH) and synthesized (Integrated DNA Technologies). In a preliminary experiment, primers for β-actin, glyceraldehyde 3-phosphate dehydrogenase, 18S rRNA, and β2 microglobulin12 were prepared by integrated DNA Technologies and tested for stable expression at increasing hCG concentrations in canine neoplastic T lymphocytes. The stability of these housekeeping genes (HKGs) was evaluated using software from geNorm, NormFinder, and BestKeeper. Only β2 microglobulin displayed stable expression in all 3 cell lines (data not shown) and was used as the HKG for canine LH receptor expression.
A—Forward and reverse primer sequences used for the gene expression study. B—Gene expression was confirmed by examination of PCR products using gel zymography. Lanes 1–6 luteinizing hormone (LH) receptor products for ovary, skin, T-cell lymphoma CLC cell line, T-cell lymphoma CLK cell line cells, T-cell lymphoma EMA cell line, and nuclease-free water; Lane M: DNA molecular marker; Lanes 7–12: β2 microglobulin products for ovary, skin, T-cell lymphoma CLC cell line, T-cell lymphoma CLK cell line cells, T-cell lymphoma EMA cell line, and nuclease-free water. C—Luteinizing hormone receptor relative to the housekeeping gene (β2 microglobulin) gene expression (mean ± SD) as a percentage of controls (0 U/mL human chorionic gonadotropin [hCG]) of immortalized neoplastic canine T lymphocytes (CLC, CLK, EMA) following 72 hours of LH receptor activation with increasing concentrations of hCG. Using 1-way analysis of variance, there was a significant increase in LH receptor gene expression in the CLC cell line (P < .05; bar), and there was a trend for an increase in LH receptor gene expression in the EMA cell line (P = .06). There was no increase in LH receptor gene expression in the CLK cell line (P = .37).
Citation: American Journal of Veterinary Research 86, 1; 10.2460/ajvr.24.07.0208
Qualitative PCR was performed to verify the size of the PCR products using gel electrophoresis (Figure 1). In brief, 2X Taq PCR Master Mix (10 μL), RNAse-free water (4 μL), forward primer (2 μL), reverse primer (2 μL), and cDNA (2μL) were combined. The PCR settings included an initial cycle at 95 °C for 5 minutes; then 40 cycles at 95 °C for 15 seconds, 57 °C for 15 seconds, and 72 °C for 15 seconds; and a final cycle at 72 °C for 5 minutes and 10 °C for 10 minutes. Following PCR, the PCR products (10 μL) with 6X loading buffer (2 μL) were added to an agarose gel made with tris-acetate EDTA buffer and gel stain (30 mL:6 μL). A DNA marker (25 to 500 bp) was used to verify band size.
Quantitative PCR was performed in duplicate on each sample following the SYBR Green qPCR kit manufacturer's instructions. In brief, forward primer (1 μL, 10 μM), reverse primer (1 μL, 10 μM), 2X qPCR master mix (10 μL), nuclease-free water (6 μL), and cDNA (2 μL) were used for each PCR reaction. The qPCR settings included an initial cycle at 95 °C for 20 seconds; 40 cycles at 95 °C for 3 seconds, then 57 °C for 30 seconds; and a final cycle at 95 °C for 15 seconds, 60 °C for 60 seconds, 95 °C for 15 seconds, and 60 °C for 15 seconds.
Cell proliferation assay
Each cell line was cultured in 96-well plates in RPMI 1640 media supplemented with 10% FBS substitute and penicillin (100 U/mL)/streptomycin (100 μg/mL)/glutamine solution. For each cell line, standard curves from 10 to 500,000 cells/well were plated in triplicate. Increasing concentrations of hCG (0, 4, 400, and 4,000 U/mL) were added to wells containing 100,000 cells plated in triplicate. Plates were incubated for 72 hours for hCG before cells were counted using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay kit following the manufacturer's instructions. Briefly, MTT (50 μg) was added to each well and incubated at 37 °C for 4 hours. Then, the MTT and media were replaced with 1-propanol (100 μL). The plates were gently rocked for 30 seconds and then incubated overnight at 37 °C. The absorbance was measured on a microplate reader at 570 nm the following day.
Cell adhesion assay
A commercial adhesion assay (CytoSelect Leukocyte-Endothelium Adhesion Assay; Cell Biolabs Inc) was used for this experiment. Each cell line was cultured in RPMI 1640 media supplemented with 10% FBS substitute and penicillin (100 U/mL)/streptomycin (100 μg/mL)/glutamine solution for 72 hours with increasing concentrations of hCG (0, 4, 400, and 4,000 U/mL). Four replicates of each neoplastic canine T-lymphocyte cell line were used in each assay, and assays were repeated 3 times. Canine aortic endothelial cells were cultured in a growth media specific for this cell type (#Cn211–500; Cell Applications Inc) to form a monolayer on 96-well plates. The endothelial cells were then activated with tumor necrosis factor α (25 ng/mL/well) for 12 hours. Neoplastic canine T lymphocytes were fluorescently labeled (CytoSelect LeukoTracker; Cell Biolabs Inc) and added to the endothelial monolayer. Following a 2-hour incubation, the nonadherent cells were removed by washing. Images of the adherent cells were captured at 400X magnification using fluorescent microscopy. The level of fluorescent attachment of neoplastic canine T lymphocytes to the endothelial monolayer was quantified with a fluorescence plate reader at 485 nm/528 nm.
Cell transmigration assay
A commercial transmigration assay (CytoSelect Leukocyte Transmigration Assay; Cell Biolabs Inc) was utilized to conduct the experiment. Each cell line was cultured in RPMI 1640 media supplemented with 10% FBS substitute and penicillin (100 U/mL)/streptomycin (100 μg/mL)/glutamine solution for about 72 hours with increasing concentrations of hCG (0, 4, 400, and 4,000 U/mL). Duplicate assays were run twice for each neoplastic canine T-lymphocyte cell line. Endothelial cells were cultured to form a monolayer on 24-well plate inserts (3-µm pore size) in their respective growth media, then activated with tumor necrosis factor α (25 ng/mL/well) for 12 hours. In serum-free RPMI media, neoplastic canine T lymphocytes were fluorescently labeled. Inserts with endothelial cell monolayers were transferred to new wells containing RPMI media with 10% FBS and the chemoattractant C-X-C motif chemokine ligand 12 (25 ng/mL). Wells with serum-free RPMI media without the chemoattractant accounted for background fluorescence. The hCG-treated neoplastic canine T-lymphocyte cells were added to each insert for an 8-hour incubation. Following this incubation, nonmigratory cells were removed, and migratory cells were lysed and quantified on a fluorescence plate reader at 485 nm/528 nm.
Statistical analysis
Gene expression of the LH receptor was determined using the 2-ΔΔCt method (where ΔΔCt = [Ct, LH receptor – Ct, HKG]treated – [Ct, LH receptor – Ct, HKG]control).13 Data were expressed as LH receptor gene expression relative to the HKG (β2 microglobulin), with the 0 U/mL of hCG (control) set to 100%.
For the cell proliferation assay, the absorbance recorded in the wells treated with 0 U/mL of hCG (control) was set to 100%. Data from the adhesion assay were corrected for the background fluorescence (wells without neoplastic T lymphocytes), and then the fluorescence of the 0 U/mL of hCG (control) was set to 100%. Outliers based on experimental error were excluded from each set of replicates. Data from the transmigration assay were corrected for the background fluorescence (serum-free wells), and then the fluorescence of the 0 U/mL of hCG (control) was set to 100%.
All data were expressed as a percentage of control (hCG, 0 U/mL). The effect of increasing hCG concentrations was compared using 1-way ANOVA followed by Dunnett post hoc analysis with GraphPad Prism (version 8; GraphPad Software Inc) statistical software. Significance was defined as a value of P < .05.
Results
In the CLC cell line, activation of the LH receptor with 4 U/mL and 400 U/mL of hCG increased LH receptor gene expression 1.7 and 36.4 times higher than the control group (0 U/mL hCG), which was significant when analyzed by 1-way ANOVA (P < .01) (Figure 1; Table 1). There was a trend toward a dose-dependent increase in LH receptor gene expression in the EMA cell line (P = .06). The effect of LH receptor activation did not significantly increase LH receptor gene expression in the CLK cell line (P = .37).
Summary of responses to luteinizing hormone receptor (LHR) activation in 3 immortalized canine neoplastic T-lymphocyte cell lines.
| Immortalized canine neoplastic T-lymphocyte cell lines | ||||
|---|---|---|---|---|
| hCG treatment (U/mL) | CLC | CLK | EMA | |
| LHR gene expression | 4 | Increased | No effect | Increased trend |
| 400 | Increased | No effect | Increased trend | |
| Cell proliferation | 4 | No effect | No effect | No effect |
| 400 | Increased | Increased | Increased | |
| 4,000 | Increased | Increased | Increased | |
| Cell adhesion | 4 | No effect | No effect | No effect |
| 400 | No effect | Increased | No effect | |
| 4,000 | Increased | No effect | No effect | |
| Cell transmigration | 4 | No effect | No effect | Increased |
| 400 | No effect | No effect | Increased | |
| 4,000 | No effect | Increased | No effect | |
CLC, CLK, and EMA are immortalized canine neoplastic T-lymphocyte cell lines. hCG = Human chorionic gonadotropin.
Activation of LH receptors with hCG induced a dose-dependent increase in cell proliferation in the CLC, CLK, and EMA cell lines (P < .05; Figure 2; Table 1). The effect of LH receptor activation with 4 U/mL resulted in no significant change in all 3 cell lines. However, at 400 U/mL there was a 22%, 10%, and 18% increase in cell proliferation compared to controls (P < .05). Additionally, there was a 39%, 17%, and 21% increase in cell proliferation at 4,000 U/mL compared to controls (P < .05).
Proliferation response (mean ± SD) of immortalized neoplastic canine T lymphocytes (CLC, CLK, EMA) following 72 hours of LH receptor activation with increasing concentrations of hCG. Treatment with hCG induced cell proliferation in all 3 cell lines in a dose-dependent manner. *P < .05 compared to hCG 0 U/mL (control) for each cell line.
Citation: American Journal of Veterinary Research 86, 1; 10.2460/ajvr.24.07.0208
Activation of LH receptors did not affect the adhesion response of EMA. However, for CLC and CLK, there was a significant difference between the mean adhesion responses due to a pair of hCG treatment conditions. Specifically, the adhesion response of CLC at 4,000 U/mL hCG (P = .01) and CLK at 400 U/mL hCG (P = .02) significantly differed from the baseline of 0 U/mL hCG (Figure 3; Table 1).
Adhesion response (mean ± SEM) of immortalized neoplastic canine T lymphocytes (CLC, CLK, EMA) following 72 hours of LH receptor activation with increasing concentrations of hCG. *P < .05 compared to hCG 0 U/mL (control) for each cell line.
Citation: American Journal of Veterinary Research 86, 1; 10.2460/ajvr.24.07.0208
Activation of LH receptors did not affect the transmigration response of CLC. However, for CLK and EMA there was a significant difference between the mean transmigration responses due to a pair of hCG treatment conditions. The transmigration response of CLK at 4,000 U/mL hCG (P = .02) significantly differed from the baseline of 0 U/mL hCG, whereas the response of EMA at 4 (P < .01) and 400 (P < .01) U/mL hCG significantly differed from the baseline (Figure 4).
Transmigration response (mean ± SEM) of immortalized neoplastic canine T lymphocytes (CLC, CLK, EMA) following 72 hours of LH receptor activation with increasing concentrations of hCG. *P < .05 compared to hCG 0 U/mL (control) for each cell line.
Citation: American Journal of Veterinary Research 86, 1; 10.2460/ajvr.24.07.0208
Discussion
The potential causes of canine lymphoma are complex, involving both genetic and environmental factors, such as breed and exposure to common household chemicals.14 Another factor that could contribute to the development of lymphoma in dogs is gonadectomy. Studies15,16 of early-neutered (< 12 months) Golden Retrievers and late-neutered (> 12 months) Viszlas have revealed that gonadectomy is associated with an increased risk of lymphoma in these breeds. Bennett et al.17 confirmed the association between gonadectomy and increased lymphoma risk in more than 30 breeds, including Bulldogs, Beagles, Labrador Retrievers, German Shepherds, and Rottweilers. Belanger et al.18 also reported an increase in lymphoma risk for gonadectomized compared to intact dogs.
Gonadectomy in dogs results in chronically elevated LH concentrations due to loss of negative feedback to the anterior pituitary.19 Previous research in our laboratory has demonstrated that circulating lymphocytes from healthy dogs of different breeds express LH receptors, and gonadectomy increases the percentage of circulating T lymphocytes expressing LH receptors (16.6% compared to 10.5% in sexually intact dogs).10 In addition, our laboratory has demonstrated that immortalized neoplastic T lymphocytes express LH receptors, although the percentage of cells expressing LH receptors differed between cell lines (45%, 35%, and 10% of the cells expressed LH receptors for the CLC, CLK, and EMA cell lines, respectively).10 In the current study, LH receptor gene expression increased in the CLC cell line. It is not likely that the increase in gene expression was related to the higher percentage of CLC cells expressing LH receptors since a similar effect was not observed in the CLK cell line. In other hematopoietic stem cell lines, an increase in LH receptor gene expression following hCG treatment has been shown.9
In the current study, activation of LH receptors in immortalized canine neoplastic T lymphocytes induced significant cell proliferation in all 3 cell lines at the 400 and 4,000 U/mL hCG treatments. This is supported by work in mouse and human adrenocortical carcinoma cell lines treated with increasing concentrations of hCG that reported similar cell proliferation responses.7,20 In addition, in human ovarian cancer cells, LH receptor activation upregulated insulin-like growth factor-1, which inhibited cisplatin-induced apoptosis and resulted in increased cell proliferation.21
Previous research in human breast cancer cells has shown that LH treatment induced cell adhesion via increasing the phosphorylation of an actin-binding protein and focal adhesion modulator.8 In the current study, activation of LH receptors by hCG increased cell adhesion to an endothelial monolayer in the CLC and CLK cell lines. It is not known if the increased cell adhesion observed in these 2 immortalized canine neoplastic T-cell lines is mediated by the same mechanism as the previous study. However, it is important to note that both the CLC and CLK cell lines expressed CD18, whereas the EMA cell line did not.11 CD18 is commonly referred to as a leukocyte adhesion molecule.22 It would be interesting to investigate what, if any, role LH receptor activation plays with CD18 in leukocytes.
Also in the current study, activation of LH receptors by hCG in CLK and EMA increased cell transmigration. It is interesting to note that EMA and CLK express T-cell receptor γ (TCRγ), whereas CLC does not.11 TCRγ is a membrane-bound receptor, similar to the LH receptor, and has been shown to respond to immune challenges.23 Given that LH receptor activation is correlated with a phosphorylation cascade, these intermediates could also play a role in TCRγ activation. The variability in transmigration responses between cell lines could also be due to the size and shape of the cells. Modeling of circulating tumor cell transmigration across the endothelium showed that deformability of the tumor cell facilitates transmigration. Specifically, cell shape and surface area increase were more relevant factors in deformability than elasticity. For instance, ellipsoidal cells could more easily pass through slits between endothelial cells than spherical cells.24 This aligns with findings in the current study given that EMA and CLK were relatively smaller than CLC and exhibited poikilocytosis (data not shown).
A potential limitation of the current study was that only 3 immortalized neoplastic T-lymphocyte cell lines were used. These cell lines were provided by researchers at Yamaguchi University as there were no commercially available canine neoplastic T-lymphocyte cell lines available when the current research was initiated. To the best of the investigators’ knowledge, this remains true. It is notable that the activation of LH receptors with increasing concentrations of hCG induced somewhat different responses in each cell line. This could be due to the variability in responses to treatment generally seen with T-cell lymphoma.
Considering T-cell lymphoma's resistance to traditional cancer treatments, a thorough understanding of cell line characteristics is vital to developing new treatments. Large clinical studies of dogs phenotyped for T-cell lymphoma could be planned in which LH receptor expression is included with the phenotyping. This would allow for in vivo comparison of various LH receptor expression profiles in neoplastic T lymphocytes and their corresponding response rates to various chemotherapeutic regimens.
The identification of potential neoplasia-promoting effects of LH receptor activation in lymphocytes lends support for additional studies of the usefulness of treatments that reduce LH concentrations in gonadectomized dogs with lymphoma. Treatment with deslorelin implants (Suprelorin) have been shown to downregulate LH secretion in gonadectomized dogs, restoring coat condition and continence.25,26 Downregulating LH secretion in dogs presenting with T-cell lymphoma could be a complementary treatment with traditional chemotherapy regimens.
In conclusion, the current study has demonstrated that LH receptors within canine neoplastic T lymphocytes are functional, and their activation can result in increased gene expression of LH receptors as well as increased cell proliferation, adhesion, and transmigration. This evidence is significant because following gonadectomy in dogs, LH is continuously overproduced at concentrations 10 to 100 times higher than those found in intact dogs.27 The combined effects of supraphysiologic concentrations of circulating LH and activation of LH receptors on lymphocytes may explain why lymphoma is more common following gonadectomy in some dogs.
Acknowledgments
The immortalized neoplastic T lymphocytes were provided by the Laboratory of Veterinary Internal Medicine, Department of Veterinary Medicine, Yamaguchi University (Yamaguchi, Japan). The authors thank Dr. Camryn Flint for work on the proliferation assay.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.
Funding
This project was supported by the American Kennel Club Canine Health Foundation (grant No. 02751-A), International Youth Exchange Foundation of Henan Academy of Agricultural Sciences Fund, and Oregon University College of Agricultural Sciences Beginning Scientist Program and Continuing Scientist Program.
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