Thermal necrosis in orthopedic bone tumors: experimental research

Abstract

Introduction:

The extent of surgical resection in orthopedic oncology differs according to tumor biology. While malignant bone tumors are operatively managed with wide resection, benign bone tumors and metastatic carcinomas are often treated through intralesional excision and adjuvant modalities, including the elimination of residual neoplastic cells through thermal necrosis. This study investigates in vitro temperature thresholds for thermal necrosis in common orthopedic bone tumors.

Methodology:

Eleven cell lines, including metastatic carcinomas to bone (A549, A498, FU-UR-1, PC3, MDA-MB-231, TT, MCF7, and K1), giant cell tumor of bone, osteosarcoma (HG-63), and control non-neoplastic cells (HEK293) were cultured. Cells were exposed to thermal stress at varying times and temperatures and evaluated for survival and viability with crystal violet and MTT assays.

Results:

Both the MTT and crystal violet assay demonstrated statistically superior rates of viability and survival for A549 (lung carcinoma), FU-UR-1 (renal carcinoma), K1 (thyroid carcinoma), and MG-63 (osteosarcoma) cell lines compared to control (HEK293 cells) at 60°C. Additionally, the MTT assay demonstrated superior viability for PC3 (prostate carcinoma), MCF7 (breast carcinoma), and A498 (renal carcinoma) compared to control. All cell lines demonstrated significantly decreased survival and viability in temperatures more than 90°C.

Conclusion:

This study demonstrated in vitro thresholds for thermal necrosis for cell lines of common orthopedic tumors of bone. The A549 (lung carcinoma), K1 (thyroid carcinoma), and FU-UR-1 (renal carcinoma) cell lines demonstrated greater resistance to heat stress compared to non-neoplastic control cells. Temperatures in excess of 90°C are necessary to reliably reduce cell survival and viability to less than 10%.

Introduction

Highlights

• Surgical management of orthopedic tumors of bone differs according to tumor biology, and adjuvant modalities, such as thermal stress, can be helpful in obtaining oncologic control and reducing local recurrence.

• MTT and crystal violet assays were performed to investigate in vitro temperature thresholds for thermal necrosis in several orthopedic tumor cell lines.

• A549 (lung), FU-UR-1 (renal), PC3 (prostate), MCF7 (breast), A498 (renal), and K1 (thyroid) cancers, and MG-63 (osteosarcoma) had statistically superior rates of survival compared to control (HEK293 cells) at 60°C.

• All cell lines demonstrated significantly decreased survival and viability in temperatures more than 90°C. Temperatures in excess of 90°C are necessary to reliably reduce cell survival and viability to less than 10%.

When surgically addressing aggressive or malignant bone tumors, orthopedic oncologists must understand the extent of oncologic resection that each specific diagnosis requires. For primary sarcomas of bone, the oncologic goal is a wide surgical resection, removing the tumor en-bloc, with a margin of normal tissue surrounding the sarcoma¹. There are, however, many situations and diagnoses that are better served with a less aggressive approach. For certain conditions treated by the orthopedic oncologist, an intralesional curettage and intraoperative adjuvant treatment may sufficiently address the tumoral tissue prior to any required orthopedic augmentation or stabilization^2–5. For example, benign, aggressive bone tumors and metastatic carcinomas to bone often require surgical intervention, but a wide surgical excision would frequently be unnecessarily morbid. During an intralesional procedure, the tumor can be debulked through curettage; however, this does not remove all of the neoplastic cells. In order to further decrease the tumor burden in these cases, and thereby minimize the risk of recurrence of a benign-aggressive tumor or a metastatic cancer, the surgeon may elect to use an intraoperative adjuvant in an attempt to kill residual pathological cells that remain in the tumor bed. These adjuvants may include various methods of heating the tissue to induce thermal necrosis^6–9, treating the tumor bed with denaturing chemicals such as phenol^10,11, or cooling the tissue with liquid nitrogen^12,13. To date, the medical literature is equivocal on the efficacy of these various adjuvants in killing the neoplastic cells and decreasing tumor recurrence.

Using heat as a surgical adjuvant in the treatment of benign active and aggressive bone lesions has been described in the literature and is a part of the surgical armamentarium of the orthopedic oncologist. Radiofrequency ablation (RFA) has become standard care in the treatment of osteoid osteomas, a benign but painful bone tumor^14–16. RFA generates heat through radiowaves, achieving local temperatures up to 100° C^17–19. Likewise, argon beam coagulation has been proposed for the adjuvant treatment of benign, aggressive neoplasms, such as aneurysmal bone cysts and giant cell tumors (GCT) of bone¹⁰. Additionally, some orthopedic oncologists advocate for the use of polymethyl methacrylate (PMMA) in the treatment of metastatic carcinoma to bone as well as benign-aggressive tumors. For PMMA, it has been proposed, in part, that the temperature of the curing polymer conducts heat to the surrounding tissues during the exothermic setting reaction, which may kill any residual tumor cells; once set, the structure of the cement would then provide long-term stability to the affected bone^9,20,21. The temperature of setting PMMA is variable, but some formulations have been shown to have surface temperatures that reach 60° C for up to 5 minutes²². All of these modalities have different temperature profiles and may be able to achieve the stated goal of tumor necrosis differently. The extent to which various neoplastic tissues are sensitive to heat, however, has not been fully investigated, and any variation between different cell lines in regard to temperature-induced necrosis remains unknown.

The purpose of this study was to investigate the temperatures required to induce thermal necrosis in cell lines that are involved in common orthopedic bone tumors. The primary goal was to determine the ability of heat to produce thermal-induced-necrosis across a range of temperatures that are clinically applicable; we aimed to produce data representing the percentage of each cell line that was able to survive through a range of temperatures, up to 100°C. The secondary goal was to determine if there are differences between the neoplastic cell lines with regard to heat-induced necrosis, indicating whether certain tumor processes may be more or less sensitive to heat as an adjuvant treatment.

Methods

Eleven cell lines were selected and obtained from The American Type Culture Collection. Cell lines were chosen based on their known involvement in producing bone tumors, including a GCT of bone cell line, an osteosarcoma cell line, and eight additional cell lines representing the carcinomas that most commonly metastasize to bone (lung, renal, breast, prostate, and thyroid). A final cell line was tested as a control and as a non-neoplastic comparison; this line of cells (human embryonic kidney cells, HEK293) is well established in research and the medical literature for its reliable growth and stability during laboratory testing. In reference to the standardization of these cell lines, cells were chosen on account of similar growth timelines, culture criteria, and known clinical association with orthopedic tumors of bone. The specific cell lines are listed in Tables 1 and 2.

Table 1.

Cell lines utilized for heat sensitivity testing along with percentage of cell survival following heating to given temperatures for 5 min as determined by crystal violet assay.

Cell lines from ATCC	Disease process	60°C	90°C
HEK293	None	53%	9%
GCT	Giant cell tumor of bone	43%	3%
MG-63	Osteosarcoma	49%	4%
A549	Lung carcinoma	75%*	5%
A498	Kidney carcinoma	28%*	6%
FU-UR-1	Kidney clear cell carcinoma	60%	4%
PC3	Prostate adenocarcinoma	57%	8%
MDA-MB-231	Breast adenocarcinoma	55%	6%
MCF7	Breast adenocarcinoma	45%	6%
K1	Thyroid papillary carcinoma	57%	7%

^*Statistically different from HEK293 for each given temperature.

Note: TT data for the Crystal Violet Assay is not presented, as this cell line was not adherent to the plate under control tests and was thus deemed to be incompatible with the process of the assay.

Table 2.

Cell Lines utilized for heat sensitivity testing along with percentage of cells remaining active and viable following heating to given temperatures for 5 min as determined by MTT assay.

Cell lines from ATCC	Disease process	60°C	90°C
HEK293	none	10%	3%
GCT	Giant cell tumor of bone	10%	0%
MG-63	Osteosarcoma	25%*	0%
A549	Lung carcinoma	18%*	6%
A498	Kidney carcinoma	32%*	12%*
FU-UR-1	Kidney clear cell carcinoma	17%*	0%
PC3	Prostate adenocarcinoma	24%*	9%*
MDA-MB-231	Breast adenocarcinoma	9%	7%
MCF7	Breast adenocarcinoma	17%*	2%
TT	Thyroid medullary carcinoma	12%	0%
K1	Thyroid papillary carcinoma	22%*	0%

^*Statistically different from HEK293 for each given temperature.

Note: The values listed as ‘0%’ in this table had slightly negative values on the raw MTT test results; the raw values are presented in Figure 2. These negative numbers are corrected to 0% in this table, as the negative percentages reflect a byproduct of the testing procedure due to the correction of the results through subtraction of the background controls, under the assumption that cellular activity cannot be a negative number.

The cells were cultured in their appropriate media at 37°C, 95% humidity, and 5% CO₂. To test the ability of each cell line to withstand a heat stress, two testing methods were employed: a crystal violet assay and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The two tests were selected to increase the robustness of the results, as they measure cell death through two different processes, and both processes have been validated in the existing literature for these cell lines. The crystal violet assay measures the detachment of adherent cells from the plate after cell death, with the dye binding to the nucleic acids of the remaining cells to allow for quantification. In this fashion, dead cells are presumed to slough from the plate, while the surviving cells remain adherent to the testing plate and are thereby quantifiable. The MTT assay, on the other hand, quantifies the metabolic activity of the cells through their ability to reduce the tetrazole in the assay to a fomazan. The amount of formazan produced reflects the extent to which the cell lines are still viable.

Each cell line and plate were subjected to heating at 37, 42, 47, 50, 60, 90, or 100°C for 5 or 10 minutes using a dry heating block. A fresh plate and cell sample was used for each temperature tested, each time duration, and each assay performed. Three trials were run for each cell line, temperature, and heat time.

Methodology: Crystal violet assay

Crystal violet assays were performed in triplicates according to the previously described protocol (https://www.biovision.com/documentation/datasheets/K329.pdf). In short, 10 cm plates of adherent cells at 60–70% confluency were trypsinized and spun down. The cell density was verified by using a hemocytometer. On average, 60% of the resultant cell pellet was resuspended in 12 ml growth media and plated in a 96-well clear flat-bottom plate at 150 µl/well, and 4 wells per plate. Sixteen hours after plating, each cell line reached 60% confluency, and each plate was subjected to heating at 37, 42, 47, 50, 60, 90, or 100°C for 5 or 10 min using a dry heating block. The media was also exchanged for an equal volume of warmed media at each corresponding temperature. The media was then removed and each well was washed with 200 µl washing solution, then incubated for 20 min at room temperature with 50 µl crystal violet staining solution. The stain was then removed, and each well was washed, dried, and solubilized, and the absorbance of each was measured at 570 nm. The readings were then corrected by subtracting the absorbance of the background controls.

Methodology: MTT assay

MTT assays were performed in triplicates according to a previously published protocol with minor modifications (https://biotium.com/wp-content/uploads/2013/07/PI-30006.pdf). In short, 96-well plates of each cell line were prepared and treated using the same process as for the crystal violet assay. After heat treatment, plates were treated immediately or with a delay of 24 h with 10 µl of MTT solution depending on the experimental design. The plates were incubated at 37°C for 4 h before 100 µl of 10% SDS in 0.01 M HCl was added to each well to solubilize the formazan salt. The solution was then incubated overnight in a humidified 5% CO₂ atmosphere, and the absorbance was measured at 570 nm with a spectrophotometer, with correction by subtraction of background controls.

Methodology: Data analysis

Once collected, the data were collated and organized into line graphs. Differences between temperature-induced-necrosis rates between cell lines were assessed by comparing cell survival (crystal violet assay) and cell viability (MTT assay) between each cell line at 60 and 90°C with Analysis of Variance (ANOVA) tests and Tukey post-hoc analysis, with a P-value set at 0.05 for statistical significance. Additional temperature values were not analyzed in the data review in an effort to minimize random ‘significant’ results through excessive data manipulation (‘p-hacking’)²³.

Results

The results of heating the respective cell lines to temperatures ranging from 37 to 100°C on cell line survival (crystal violet assay) and viability (MTT assay) are presented in Figures 1 and 2, respectively. Likewise, Table 1 lists the percentage of each cell line surviving a heat stress of 60 and 90°C for 5 min, as determined by the crystal violet assay, while Table 2 lists the percentage of each cell line remaining viable for those same temperatures, as measured by the MTT assay.

Figure 1.

Cell survival after 5 (A) or 10 (B) min of heat stress. Crystal violet assay was used to assess cell survival for a range of 9 cell lines representing the most common cancers found in the bone, with HEK283T as a comparator.

Figure 2.

Cell viability at 24 h post 5 (A) or 10 (B) min of heat stress. MTT assay was used to assess cell viability for a range of 10 cell lines representing the most common cancers found in the bone, with HEK283T as a comparator.

Results: Crystal violet assay

While decreased survival was noted across all cell lines starting at 60°C, there was some variation between the survival of the tested cell lines (Tables 1 and 2, Figs 1A, B). The percentage of cells surviving the 60°C stress at 5 min ranged from a high of 75% to a low of 28% (A549 and A498, respectively), and ranged from 67 to 12% for the 10 min 60°C stress. When comparing the neoplastic cell lines to the non-neoplastic control (HEK293), the A549 line (lung carcinoma) had a more robust survival than the HEK293 control, with 75% of the A549 line surviving the 5 min heat stress at 60°C compared to 53% survival of the HEK293 cells (P<0.01). When challenged with a 10 min stress, the A549 (Lung), FU-UR-1 (Renal), MG-63 (osteosarcoma), and K1 (Thyroid) lines were more likely to survive at the 60°C stress than the HEK293 control, with survival rates of 67, 58, 53, and 50%, compared to 38%, respectively (P<0.01). Only the A498 (Renal) cell line demonstrated a statistically increased susceptibility to heat than the non-neoplastic control, with a 28% cell line survival in the A498 group following a 5 min heat stress at 60°C compared to the 53% survival of the HEK293 line (P<0.01). These differences disappeared by the 90°C trial, with all cell lines demonstrating survival below 10% of their starting values, with no significant differences noted between the cell lines for both the 5 and 10 min trials.

As depicted in Figs 1A, B, there are two aberrations within the data of the Crystal Violet Assay. First, in comparison to their control groups where temperatures were maintained at 37°C, the data demonstrate increased survival of all cell lines except for A549 (lung carcinoma) following the 47°C trial. This may represent an actual increase in cell number following the trial, as the assay was performed 24 h after the stress, or this may represent an aberration in the test mechanics, with some residual cellular debris altering the absorbance of the plates by not fully disengaging from the plates after cell necrosis. Second, the measured percent survival of all cell lines increases from the 90°C test to the 100°C trial; this is thought to reflect a mechanical aberration secondary to the evaporation of media during the 100°C trial, altering the background for the absorbance measurements.

Results: MTT assay

As measured by the MTT Assay, all cell lines demonstrated decreased cellular metabolic activity starting at 60°C, when heated to this temperature for 5 or 10 min (Figs 2A, B). The variation between the cell lines ranged from a high of 32% (A498) to a low of 9% (MDA-MB-231). The HEK293 line maintained only 10% of its metabolic activity after the 5 min 60°C stress. The A549 (Lung), A498 (Renal), PC3 (Prostate), MCF7 (Breast), FU-UR-1 (Renal), MG-63 (Osteosarcoma), and K1 (Thyroid) were more likely to be metabolically active than the HEK cell line (P<0.01), whereas the MDA-MB-231 (Breast), TT (Thyroid), and GCT were not significantly different compared to the non-neoplastic control. No cell line demonstrated significantly less metabolic activity than the control HEK293 line (Table 2, Fig. 2A) after a 60°C stress for 5 min. After the 90°C stress, only 3% of the HEK293 activity remained, comparable to all of the other cell lines, except A498 (Renal) and PC3 (Prostate), which demonstrated activity of 12 and 9%, respectively (P<0.05).

Unlike the data for the crystal violet assay, the data for the MTT assay did not demonstrate any increased metabolic activity in the cell lines following the 47°C or the 100°C trials, indicating that the rise in the absorbance in the Crystal Violet Assay reflected a mechanical aberration of that test and not actual increased cell line survival.

An aberration in the raw data for the MTT test was noted for some of the cell lines starting a 75°C. The absorbance for a few of these tests resulted as slightly negative numbers. By 90°C, this was seen in the GCT, MG-63 (osteosarcoma), FU-UR-1 (renal), and K1 (Thyroid) cell lines. These negative values are thought to result from the slight reduction in absorbance in the medium of the incubated cells due to pH changes resulting in color changes within the medium overnight when compared to the un-influenced control wells, with medium that was not affected by these pH changes. Assuming the cellular activity has a ‘left limit’ of zero, the negative values were corrected to 0% for data analysis and for display in Table 2, but the raw data was left for full clarification in Figure 2.

Discussion

The cell lines tested in this investigation were chosen due to the fact that they represent common neoplasms known to afflict bone, and these neoplasms are, at times, treated to some extent with heat as an adjuvant modality during interventions or surgery. All of the cell lines tested demonstrated decreased cell survival and activity with increased temperature stress. Of note, in the range of 47–75°C, there were differences observed between the tested cell lines in their susceptibility to heat. By 90°C, all cell lines demonstrated substantial decreases in survival and metabolic activity, when compared to their baseline controls, which were maintained at 37°C.

Taken as a whole, the data suggest that heat as an adjuvant in the surgical treatment of common bone tumors and malignancies requires tissue temperatures to reach 90°C in order to achieve a survival of less than 10% and cell viability of less than 5%, as measured by the crystal violet assay and MTT assay respectively. Within this overall effect, some variation is worth noting. Specifically, the A549 (Lung), K1 (Thyroid), and FU-UR-1 (Renal) cell lines demonstrated a resistance to damage by heat stress on both assays, maintaining 75, 57, and 60% survival at 60°C, respectively, and 18.0, 21.8, and 17.5% of baseline metabolic activity at 60°C, respectively. On the other hand, there were no cell lines that had significantly increased susceptibility to thermal stress relative to control, non-neoplastic tissue (HEK293), across both assays.

This would suggest that RFA, where tissue temperatures reach 100°C, could effectively treat any of these tumors, within the size range of its treatment effect. Using PMMA as a heat source during its setting phase; however, would not reasonably treat any of these neoplasms in isolation, as the surface setting temperatures of low and medium viscosity cement only reach up to 60°C for 5 min ²². In order to be a reliable solo-adjuvant, cell survival would need to theoretically approach 0%, as any surviving malignant cell would likely be able to re-populate. That said, relative cyto-reduction through a 60°C stress may have some benefit when combined with non-surgical treatment modalities, such as radiation, chemotherapy, and bisphosphonate treatments. These potential effects would require an in vivo study on the local recurrence rates of such neoplasms following adjuvant treatments following a standardized treatment algorithm. Clinical literature comparing recurrence rates following thermal ablation in comparison to other adjuvant treatments are limited in orthopedic literature, but the clinical utility of cementoplasty and thermal ablation is well-supported in the literature²⁴.

This study has several limitations. First, the cell lines used in this study may not accurately reflect the biologic behavior of similarly named neoplasms in vivo. That is, the lung cancer line used in this in vitro study may have biologic properties that substantively differ from any of the various iterations of metastatic lung carcinoma encountered in the clinical situation. Different lines of a given tumor can, in theory, vary in their sensitivity to heat stress, potentially depending on the distribution or size of cellular components, such as heat shock proteins and intermediate filaments. Second, this investigation is an in vitro study, which may underestimate the complexity of the true in vivo environment. For instance, our test may have underestimated the impact of the heat stress, as the potential secondary effects of hyperthermic injury could not be considered in this laboratory setting. These secondary effects may include alterations in perfusion due to microvascular damage and ischemia, as well as the in vivo initiation of a robust immune response through Kupffer cell activation and cytokine expression^25–32. Third, the methodology of the crystal violet assay may not accurately reflect the true necrosis rate, as the test is based on the detachment of adherent cells from the plate after cell death. The assay washes the adherent cells with a dye, which binds to the nucleic acids of the remaining cells, thereby allowing quantification. In running the trials, the cells may not have reliably detached from the plate upon cell death or the cells may have detached from the plate even though they were not dead. Additionally, artifact from the testing procedure may influence the percentages found. This was seen in the small rise in absorbance values with heating at 100°C; we surmised that this was likely due to the evaporation of water in the media, which concentrated the dye being measured (Figs 1A, B). In support of this supposition, the evaporative artifact was more evident with prolonged heating of 10 min (Fig. 1B) compared to 5 min (Fig. 1A).

The strengths of this study are the systematic, controlled approach to the heat stresses tested and the dual testing through both the crystal violet assay and the MTT assay, which provide complementary results and limit experimental bias. These tests serve as direct comparators to each other in assessing the validity of the results. Additionally, testing several lines of neoplastic tissues further specifies the types of tumors that may be most (or least) effectively treated with an adjuvant form of heat. Previous tests have found peak temperature and time exposed to be critical factors in inducing thermal necrosis in tumor tissue^28,30,32. This current study demonstrates that at least 5 min of heating at 60°C only consistently reduces neoplastic tumor cell viability by 40–50%. In order to achieve a reduction in tumor cell viability to under 10%, a heat stress of 90°C for at least 5 min is required.

Conclusion

While temperatures over 60°C were able to reliably reduce cell line viability and activity across several cell lines of various neoplastic processes, temperatures of 90°C for at least 5 min were required to reduce neoplastic cell survival to under 10% and neoplastic cell activity to nearly 0%. Temperatures short of this left substantial amounts of tumor viable and active, suggesting that heat as an adjuvant modality in the operating room likely has a limited role as a solo-treatment, unless sustained temperatures of 90°C or more can be achieved and maintained for more than 5 min to the entirety of the tumor.

Ethical approval

None.

Consent

None.

Sources of funding

None.

Author’s contribution

X.Q.W., J.B., and S.L.: contributed to experimental design and performed cell culture, heat stress experiments, and data analysis; K.J. and J.G.: contributed to writing of the manuscript and provided senior editorial oversight. All authors have read an approve the final version of the manuscript.

Conflicts of interest disclosure

The authors declare that there are no conflicts of interest.

Provenance and peer review

Not commissioned, externally peer reviewed.