Effect of timing of bisphosphonate administration on canine osteosarcoma cells undergoing radiation therapy

Abstract

The effects of radiation therapy may be potentiated by combining radiation therapy with secondary therapies. Clinically, radiation therapy has been combined with bisphosphonates for treatment of canine appendicular osteosarcoma for years. The objective of this study was to determine if the timing of administration of bisphosphonates in relation to radiation therapy alters clonogenic survival or cell viability of canine osteosarcoma cells in vitro. Canine osteosarcoma cells were treated before administration of radiation, concurrent with radiation, or after radiation. Reduction in clonogenic survival was identified when bisphosphonates were administered post-radiation compared with pre-radiation. No significant differences were identified for cell viability at any time points. Further investigation of the cellular effects of bisphosphonates on canine osteosarcoma cells is warranted. Consideration may be given to administering bisphosphonates 24 h after radiation to reduce replication of canine osteosarcoma cells and possibly prolong the analgesic effects of both treatments.

Introduction

For decades, the human medical field has exploited its knowledge of how radiation therapy (RT) affects individual cells by investigating other therapies that may create a cell that is more sensitive to the effects of RT or has decreased potential to recover from the effects of RT. By combining RT with other therapies that will result in a weakened tumor state, potential for greater local response to therapy may be achieved (1).

Timing of the administration of these secondary therapies is ultimately determined based on how they function at a cellular level. Therapies that cause alterations in cell cycle (2–4), induce damage to deoxyribonucleic acid (DNA) (3,5), reduce inherent radioresistance (4,6), or target hypoxic regions of tumors (7–10) will provide their best effects if present immediately before radiation therapy takes place. Other therapies that may alter a cell’s ability to recover from the effects of RT by reducing the tumor’s ability to repopulate (2,11) or by inhibiting repair from radiation damage (1,3,11) must be present after RT has been administered.

While there has only been minimal investigation to date of combining radiation therapy and bisphosphonates (BPs) for treating canine osteosarcoma (OSA), 1 in-vitro study has identified zoledronate (ZOL) as a possible radiosensitizing agent for treating OSA in humans (12). In this study, ZOL was administered 24 h before RT and was found to promote apoptosis, cause direct DNA damage, impair DNA repair, and alter cell-signaling pathways and the proportion of cells in each phase of the cell cycle (12). Administering ZOL before radiation, therefore, enhanced the effects of radiation therapy.

With various other treatment modalities for targeting the effects of RT to improve local outcomes and evidence of ZOL as a possible radiosensitizing agent (12), investigation into the timing of administration of BP and RT for treating canine OSA should be considered. The potential radiosensitizing effects of pamidronate (PAM) have not yet been studied, even though PAM is a common clinical drug combination with palliative RT for canine patients (13). Additionally, to the authors’ knowledge, there is no current research into the effects of BP when administered after radiation therapy.

While knowledge of the pharmacokinetics of bisphosphonates (BPs) in dogs is limited, it is known that plasma concentrations rise rapidly and early in BPs, followed by an early, rapid decline and that BPs are preferentially distributed to bone and excreted via the kidneys (14–16). This early but rapid decline in plasma concentrations suggests rapid uptake by bone, as initial urinary excretion is minimal (14). Prolonged bone retention is suspected, as urinary excretion remains incomplete even 12 mo after administration of a single dose of ZOL (14). With this rapid redistribution to bone, the radiosensitizing effects of BP could potentially be achieved by administering BP before RT, although the ideal interval between BP administration and RT remains unknown. As such, the clinical veterinary practice is to administer BPs on the same day as radiation therapy, probably due to its convenience (13).

The objective of this study was to determine if pre- or post-RT treatment of canine OSA cells with BP affects cell replication by evaluating clonogenic survival or a cellular metabolic reaction by evaluating cell viability compared to treatment with BP and RT administered concurrently. Our hypothesis was that timing of administration of BP (PAM or ZOL) in relation to timing of administration of RT would not affect inhibition of canine OSA cell growth in vitro.

Materials and methods

Cell culture

Two canine OSA cell lines were used for all experiments: D17 and Dharma. These cell lines differ from one another as D17 cells (17) originated from a metastatic pulmonary lesion and Dharma cells (18) were isolated from a primary appendicular lesion. D17 cells are commercially available and were obtained from Sigma-Aldrich/European Collection of Authenticated Cell Cultures (ECACC), while Dharma cells were obtained from a clinical patient and adapted to culture by Dr. Anthony Mutsaers. Cells were grown in standard cell culture dishes with Dulbecco’s modified Eagles medium (Hyclone DMEM; Fisher Scientific, Ottawa, Ontario), with 10% fetal bovine serum (FBS; Life Technologies, Burlington, Ontario) and 1% penicillin-streptomycin (PS; BioWhittaker, Mississauga, Ontario). Cells were incubated at 37°C in a 5% carbon dioxide (CO₂) humidified incubator.

Clonogenic survival assay

For each cell line and treatment condition, 6-well cell culture plates were plated with 500 D17 cells/well in 3 mL of media and 2000 Dharma cells/well in 3 mL of media. All plates were incubated until they were treated with either PAM or ZOL at 1 of 3 time points: pre-treatment (48 h before radiation), concurrent treatment (same day, 2 to 4 h before radiation), and post-treatment (24 h after radiation). Two doses of PAM (10 to 30 μM), 2 doses of ZOL (0.4 to 2 μM), and 1 control were used, with 2 wells/plate for each dose. All BP doses were chosen following optimization studies and fall within dose ranges used in previously reported in-vitro studies (12,19–21). At the predetermined timing interval, cell media were replaced by 3 mL of BP-containing media or standard culture media for control wells. One plate from each experiment received 4 Gy of radiation. Control plates (0 Gy) were transported to the radiation vault but remained outside during cell treatment. All time points were irradiated at the same time.

Plates remained incubated until termination of the experiment at 7 d, just before the control colonies became confluent. Cells were then fixed and stained with 0.5% crystal violet in 20% methanol before being counted using light microscopy. A colony was defined as an aggregate of ≥ 50 cells. Each experiment was done in triplicate.

Cell viability assay

For each cell line and treatment condition, a 96-well cell culture plate was plated with 500 D17 cells/well in 150 μL of media and 2000 Dharma cells/well in 150 μL of media. All plates were incubated until they were treated with either PAM or ZOL at 1 of 3 time points: pre-treatment (24 h before radiation), concurrent treatment (same day, 2 to 4 h before radiation), and post-treatment (24 h after radiation). All BP doses were chosen following optimization studies and fall within dose ranges used in previously reported in-vitro studies (12,19–21). Five doses of PAM (2 to 100 μM) or ZOL (0.4 to 10 μM) and 1 control were used, with each dose replicated in quadruplicate. At the predetermined timing interval, BP-containing media or standard culture media was added to each well to achieve the required BP dose. One plate from each experiment received a single dose (2 to 10 Gy) of radiation. Control plates (0 Gy) were transported to the radiation vault but remained outside during cell treatment. All time points were irradiated at the same time.

Plates were subsequently incubated for 7 d. On day 7 post-RT, 20 μL of resazurin solution was added to each well and 6 h later absorbance readings were obtained using a Synergy 2 spectrophotometer (BioTek, Winooski, Vermont, USA), at an excitation wavelength of 570 nm and emission wavelength of 600 nm. Each experiment was done in triplicate.

Radiation treatment setup

Photon radiation was delivered using a 6-MV linear accelerator (Clinac IX; Varian Medical Systems, Palo Alto, California, USA). Bolus was provided using solid water-equivalent plates above (4.5 cm thickness) and below (5 cm thickness) the cell culture plates. No lateral bolus was used. A single beam delivered radiation from above the plates and computerized planning was used to confirm dose distribution for the plate setup before proceeding with the experiments.

Statistical analysis

A general linear mixed model [3-way analysis of variance (ANOVA)] was used to test the fixed effects of BP (PAM or ZOL), RT, timing, and their interactions. Random effect of plate was accounted for in the model. Data were assessed for normality with a Shapiro-Wilk test and examination of the residuals. A log transform was applied, if the data were not normally distributed. A value of P < 0.05 was considered significant.

Results

Significantly fewer colonies grew for both D17 and Dharma cell lines treated with either PAM or ZOL when the cells were treated post-RT compared with pre-RT, independent of BP or RT dose (Figure 1). Additionally, for D17 cells treated with ZOL, all cells treated post-RT grew significantly fewer colonies than those cells treated on the same day as RT, independent of BP or RT dose (Figure 1).

Figure 1.

Clonogenic survival results for canine osteosarcoma (OSA) cells treated with pamidronate (PAM) or zoledronate (ZOL) + radiation therapy (RT), comparing timing of administration of bisphosphonates (BPs) in relation to RT, independent of BP or RT dosing. A — D17 OSA cells treated with PAM + RT. B — Dharma OSA cells treated with PAM + RT. C — D17 OSA cells treated with ZOL + RT. D — Dharma OSA cells treated with ZOL + RT. (N = 36 +/− SEM).

^* = P < 0.05 (pre- and post-comparisons).

^** = P < 0.05 (post- and concurrent-comparisons).

No significant differences in cell viability were identified among the different timing groups using D17 cells treated with either PAM or ZOL and Dharma cells treated with PAM, independent of BP or RT dose (Figure 2). A significant decrease in cell viability was identified, however, when Dharma cells were treated with ZOL on the same day as RT compared with those cells being treated post-RT, independent of BP or RT dose (Figure 2).

Figure 2.

Viability results for canine osteosarcoma (OSA) cells treated with pamidronate (PAM) or zoledronate (ZOL) + radiation therapy (RT), comparing timing of administration of bisphosphonates (BPs) in relation to RT, independent of BP or RT dosing. A — D17 OSA cells treated with PAM + RT. B — Dharma OSA cells treated with PAM + RT. C — D17 OSA cells treated with ZOL + RT. D — Dharma OSA cells treated with ZOL + RT. (N = 432 +/− SEM).

^** = P < 0.05 (post- and concurrent-comparisons).

Discussion

Timing of administration of bisphosphonate (BP) in relation to radiation treatment (RT) may be an important factor when evaluating the combined effects of treatment on canine OSA cells in vitro. Based on our previous and current work evaluating the combination of BP and RT, results vary among OSA cell lines, BPs, dosing combinations of BP and RT, laboratory assays, and timing of administration of each modality, therefore suggesting a multifactorial response to these treatments (22).

Decreased clonogenic survival was identified for both canine OSA cell lines when treated with either BP 24 h after RT compared to those treated with either BP 48 h before RT. These effects may be due to the weakened state of the OSA cells after RT as DNA damage secondary to RT has been shown to last at least 24 h (1,12). However, clonogenic survival after RT was not significantly different than the concurrent treatment group that received BP on the same day as RT, but before RT for D17 + PAM and Dharma + PAM or ZOL. These results together bring into question the previously suggested radiosensitizing effect of ZOL (12).

The significant decrease in clonogenic survival identified for the D17 (ZOL + RT) post-treatment group compared to the concurrent treatment group and the significant decrease in cell viability identified for the Dharma (ZOL + RT) concurrent treatment group compared to post-treatment group, may be outliers or may represent the increased potency of ZOL compared to PAM (15,23), as the same findings were not identified for the D17 (PAM + RT) or Dharma (PAM + RT) groups in their respective laboratory assay. A difference inherent in the cell lines should also be considered, as the same findings were not identified in the other cell line. Direct comparisons between PAM and ZOL can be considered for the colonization assay only due to similar dosing across cell lines.

While timing of administration of BP in relation to RT was significant for the clonogenic survival experiments, timing was not significant for the cell viability assays, aside from the Dharma (ZOL + RT) group. This may be due to the ability of BP to have a greater impact on RT-treated OSA cells biologically, such that a greater inhibition of cell replication and thus clonogenic survival can be achieved. Under the same circumstances, however, the cell’s metabolic activity is not altered significantly, as measured by the viability assay.

While post-RT treatment with BP resulted in increased inhibition of clonogenic survival on canine OSA cells, it failed to result in decreased cell viability, measured by metabolic activity. Further investigation into the cellular effects of ZOL and PAM on canine OSA cells is therefore warranted, which may help to determine an optimal timing for their administration in relation to RT (12).

Despite both cell lines behaving similarly in this study, other biologic effects, such as the tumor microenvironment, may play a larger role in how canine OSA cells respond to BP and RT treatments (24–28). Therefore, translation of this work into an orthotopic model of canine OSA may provide additional information about combining BP and RT and their optimal timing of administration in relation to one another.

Based on the results of this study, consideration may be given to administering bisphosphonates 24 h after radiation therapy, as this may have a more beneficial effect on reducing the replication of OSA cells, thus potentially prolonging the analgesic effects of both treatments.