Abstract
Background/Aim: Eribulin is a microtubule inhibitor used in the treatment of various malignancies, including soft-tissue sarcoma. However, the development of eribulin resistance is a recalcitrant clinical problem. The present study demonstrates that super eribulin-resistant HT1080 human fibrosarcoma cells become highly malignant but can be eradicated synergistically by the combination of eribulin and methionine restriction in nude mice.
Materials and Methods: The cell viability of parental HT1080 cells and super eribulin-resistant HT1080 was determined following eribulin treatment using the WST-8 reagent. In vitro invasion assays, comprising wound-healing of cell monolayers, were performed to determine the degree of malignancy. Tumor growth to determine malignancy and tumor-growth sensitivity to eribulin, or a methionine-restricted diet, or their combination were analyzed in athymic nude mice.
Results: The IC50 of eribulin for parental HT1080 cells was 0.15 nM, whereas for super eribulin-resistant HT1080 cells, the IC50 of eribulin was 18 nM, a 120-fold increase. Super eribulin-resistant HT1080 cells had a more rapid wound-healing closure rate in vitro than their parental HT1080 cells, indicating increased malignancy. Similarly, in vivo, untreated super eribulin-resistant HT1080 tumors grew faster than parental HT1080 tumors, confirming their high malignancy. The combination of a methionine-restricted diet and eribulin synergistically arrested super eribulin-resistant HT1080 tumors. Methionine restriction sensitized these highly resistant and malignant cells to eribulin.
Conclusion: Methionine restriction combined with eribulin represents a promising strategy to effectively treat eribulin-resistant high-malignancy fibrosarcoma, that can be immediately applied to the clinic.
- HT1080
- fibrosarcoma
- super eribulin-resistance
- high malignancy
- methionine addiction
- Hoffman effect
- methionine restriction
- eribulin
- synergy
- nude mice
Introduction
Soft tissue sarcomas (STS) are heterogeneous, but rare (1). STS which acquire drug resistance in the clinic become a recalcitrant cancer with no improvement over decades (2). Eribulin is a synthetic analog of halichondrin B, which inhibits microtubule dynamics (3) and is approved for second-line treatment for advanced STS (4).
The development of eribulin resistance significantly limits its clinical efficacy. Eribulin resistance in STS is incompletely understood and results in very poor patient prognosis with no improvements made for better therapeutic outcomes (4).
All cancer cell types, including STS, are methionine addicted, known as the Hoffman effect, relying on exogenous methionine for survival and proliferation (5-13) even though cancer cells synthesize large amounts of methionine via vitamin B-12-dependent methionine synthesase, determined in vivo and in vitro (7, 12, 13). Methionine addiction of cancer is targeted by methionine restriction (MR) by both recombinant methioninase (rMETase) and a methionine-restricted diet, or medium, in preclinical models and in the clinic (14-18).
In the present study, we developed in vitro a super eribulin-resistant HT1080 (SER-HT1080) human fibrosarcoma cell line model to investigate the effects of eribulin resistance on tumor malignancy. MR was used to overcome eribulin resistance and enhance antitumor efficacy in vivo.
Materials and Methods
Cell culture. The HT1080 human fibrosarcoma cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Grand Island, NY, USA) and 1 IU/ml penicillin/streptomycin (Thermo Fisher Scientific).
Reagents and diet. Eribulin was acquired from Eisai Inc. (Nutley, NJ, USA). The methionine-deficient diet (TD. 210562, Inotiv, Inc., West Lafayette, IN, USA) contained 1.2 g/kg L-methionine compared to 8.2g/kg L-methionine in a normal diet.
Establishment of super eribulin-resistant HT1080 cells. Super eribulin-resistant HT1080 cells (SER-HT1080) were developed by culturing parental HT1080 cells in progressively step-wise increasing concentrations (0.15-6 nM) of eribulin over six months.
IC50 determination of eribulin. Cell viability was assessed using the WST-8 reagent (Dojindo Laboratory, Kumamoto, Japan). Cells were cultured in 96-well plates (3,000 cells/well) in DMEM (100 μl/well) and incubated at 37°C overnight. Cells (HT1080 and SER-HT1080) were treated with increasing concentrations of eribulin, between 0.5 nM and 16 nM for 72 h. At the end of the culture period, 10 μl of the WST-8 solution was added to each well and the plates were additionally incubated for 1 h at 37°C. Absorbance was measured with a microplate reader (SUNRISE: TECAN, Mannedorf, Switzerland) at 450 nm. Drug sensitivity curves were obtained with Microsoft Excel for Mac 2016 ver. 15.52 (Microsoft, Redmond, USA) and half-maximal inhibitory concentration (IC50) values were calculated using ImageJ ver. 1.53k (National Institutes of Health, Bethesda, MD, USA). Experiments were performed twice, each in triplicate.
Wound healing invasion assay. HT1080 and SER-HT1080 cells were seeded in 6-well plates containing DMEM and 10% FBS and incubated overnight at 37°C. Wounds were created by scratching the cell monolayers with a 200 μl pipette tip. Subsequent to scratching, the plates were rinsed twice with phosphate-buffered saline (PBS; #MB1039-1X, BioPioneer Inc., San Diego, CA, USA) to remove detached cells, and then incubated at 37°C in DMEM with 10% FBS. The wound width was measured using light microscopy (Olympus IX71, Olympus Corporation, Tokyo, Japan) at four and eight hours after wounding. Each experiment was conducted in triplicate and repeated twice.
Mice. Athymic nu/nu nude mice (4-6 weeks) were obtained from AntiCancer Inc. (San Diego, CA, USA). All animal experiments were performed in accordance with a protocol sanctioned by the Institutional Animal Care and Use Committee (IACUC) of AntiCancer Inc. The procedures complied with the National Institutes of Health (NIH) guidelines for the Care and Use of Laboratory Animals and conformed to the ARRIVE guidelines 2.0.
Establishment of subcutaneous HT1080 tumors and SER-HT1080 tumors. Nude mice were injected in the right flank with HT1080 or SER-HT1080 cells (1×106) to induce subcutaneous tumor formation. One month after cell injection, the resultant subcutaneous tumors were excised, sectioned into 3-4 mm3 fragments, and re-implanted into the right flank of additional nude mice. Two weeks post-implantation, the tumor-bearing mice were treated as described below.
Treatment protocol. The SER-HT1080 nude mouse models were randomized into four groups once the tumor volume exceeded 100 mm3. The parental HT1080 nude mouse models were divided into two groups. Group 1: untreated control (SER-HT1080), Group 2: eribulin treated [intravenous (iv), 1.5 mg/kg (once a week, two weeks)] (SER-HT1080); Group 3: methionine-restricted diet (SER-HT1080); Group 4: eribulin treatment [iv, 1.5 mg/kg (once a week, two weeks)] plus a methionine-restricted diet (SER-HT1080); Group 5: untreated control (HT1080); Group 6 eribulin treated [iv, 1.5 mg/kg (once a week, two weeks)] (HT1080). Each group comprised five mice. Tumor volume and body weight were measured twice a week. The calculation of tumor volume was performed using the formula: tumor volume (mm)=length (mm)×width (mm)×width (mm)×1/2.
Statistical analysis. Statistical analyses were conducted using EZR software (Jichi Medical University, Saitama, Japan) (16). The relationships between variables were analyzed using the Tukey-Kramer method, with a p-value of ≤0.05 considered statistically significant.
Results
Eribulin IC50 on SER HT1080 increased 120-fold compared to parental HT1080 cells. The eribulin IC50 on HT1080 cells was 0.15 nM [data from (17)]. The eribulin IC50 of SER-HT1080 cells was 18 nM (120 fold increase) (Figure 1).
Eribulin IC50 on HT1080 and super eribulin-resistant HT1080 (SER-HT1080) cells (mean±SD, n=3). A) Sensitivity of HT1080 cells to eribulin [data from (17)]. B) Sensitivity of SER-HT1080 cells to eribulin, which have a 120-fold increased eribulin IC50. Please see Materials and Methods for details.
SER-HT1080 cells acquired greater malignancy determined by 2.78 fold more rapid in vitro wound-healing compared to parental HT1080. A wound-healing assay in vitro was used to determine the invasion capability of HT1080 and SER-HT1080 cells. SER-HT1080 cells had a 2.78 fold more rapid wound repair than their parental HT1080 cells (p<0.05) (Figure 2). These results suggest that SER-HT1080 cells have a greater level of malignancy in vitro.
In vitro wound-healing assay to determine relative malignancy of super-eribulin -resistant HT1080 (SER-HT1080) compared to parental cells. SER-HT1080 cells have more rapid wound healing than parental HT1080. A) Black lines indicate the boundaries of the wound of the cells grown in Dulbecco’s Modified Eagle’s Medium (DMEM). Wounds were made by scratching the cell monolayers with a 200 μl pipette tip. An Olympus IX71 microscope was used to observe wound closure. B) Bar graphs compare the wound closure ratio at 8 hours for HT1080 and SER-HT1080. Magnification: 40×. Scale bar: 100 μm. Please see Materials and Methods for details.
Tumor growth rate of SER-HT1080 increased 2.36-fold compared to parental HT1080 in nude mice, indicating increased malignancy. The volume of SER-HT1080 tumors without treatment increased 2.36 fold compared to parental HT1080 tumors during 14 days growth in nude mice, indicating that SER-HT1080 tumors had more malignancy (Figure 3).
Comparison of super eribulin-resistant HT1080 and parental HT1080 tumor growth in nude mice. A) The volume of untreated super eribulin-resistant HT1080 (SER-HT1080) tumors increased more than HT1080 tumors (2.36-fold) over 14 days growth in nude mice. B) Bar graphs represent tumor volume of SER-HT1080 and HT1080 after 14 days growth in nude mice. These results indicate that SER-HT1080 tumors had more malignancy. Tumors were grown subcutaneously in nu/nu nude mice. Please see Materials and Methods for details.
SER-HT1080 is eribulin resistant in vivo. The volume of HT1080 tumors treated with eribulin was 4.82 fold slower compared to SER-HT1080 tumors treated with eribulin, indicating that SER-HT1080 tumors were more resistant to eribulin in vivo than parental HT1080 tumors (Figure 4).
Comparison of super eribulin-resistant and parental HT1080 tumor growth during eribulin treatment. A) Super eribulin-resistant HT1080 (SER-HT1080) tumors treated with eribulin [intravenous, 1.5 mg/kg (once a week, two weeks)] grew more than eribulin-treated HT1080 tumors in nude mice (4.82 fold) over a 14-day treatment period. B) Bar graphs represent tumor volume of eribulin-treated SER-HT1080 and HT1080 after 14 days growth in nude mice. These results indicate that SER-HT1080 tumors were more resistant to eribulin. Tumors were grown subcutaneously in nude mice. Please see Materials and Methods for details.
The combination of eribulin and methionine restriction synergistically arrested tumor growth of SER-HT1080. The combination of the methionine-restricted diet plus eribulin synergistically arrested the SER-HT1080 tumors (p<0.05 compared to all other groups) without causing more than 10% weight loss (Figure 5 and Figure 6).
Efficacy of eribulin and a methionine restricted diet (MR) and their combination on super eribulin-resistant HT1080 tumors in nude mice. A) Eribulin+methionine restriction (MR) had high synergy on super eribulin-resistant HT1080 (SER-HT1080) tumor growth in nude mice. B) Bar graphs represent tumor growth of SER-HT1080 and HT1080 at day 14, treated with eribulin or MR or their combination, showing high synergy of the combination. Tumors were grown subcutaneously in nude mice. Please see Materials and Methods for treatment protocol and other details.
Effect of eribulin or a methionine-restricted diet or their combination on body weight of nude mice with super eribulin-resistant HT1080 (SER-HT1080) and parental HT1080 tumors. Tumors were grown subcutaneously in nude mice. Please see Materials and Methods for treatment protocol and other details. Data are shown as the mean±standard deviation.
Discussion
The present study demonstrated that SER-HT1080 human fibrosarcoma cells acquire increased malignancy during in vitro selection for eribulin resistance, as shown by enhanced invasion ability in vitro and accelerated tumor growth in nude mice. These findings are consistent with previous reports that drug-resistant cancer cells often acquire more aggressive phenotypes, posing significant problems in cancer treatment (19).
We have previously shown MR and all types of chemotherapy are highly synergistic on all major types of cancer (16). Regarding HT1080, we have shown the following chemotherapy drugs are synergistic with MR: doxorubicin (20), ifosfamide (21), eribulin (17, 22), trabectedin (23, 24), gemcitabine (25) and docetaxel (26).
In the present study, the combination of MR and eribulin arrested SER-HT1080 tumor growth, indicating high synergy of MR and eribulin, suggesting a new paradigm of treating drug-resistant sarcoma.
Conclusion
The present study highlights the increased malignancy of SER-HT1080 cells and the potential of methionine restriction to overcome eribulin resistance and high malignancy. Methionine restriction, in combination with eribulin, has immediate clinical potential for eribulin-resistant soft tissue sarcoma.
Acknowledgements
This article is dedicated to the memory of A.R. Moossa, MD, Sun Lee, MD, Richard W. Erbe, MD, Professor Gordon H. Sato, Professor Li Jiaxi, Masaki Kitajima, MD, Joseph R. Bertino, MD, Shigeo Yagi, PhD, J.A.R Mead, Ph.D., Eugene P. Frenkel, MD, Professor Lev Bergelson, Professor Sheldon Penman, Professor John R. Raper, Joseph Leighton, MD and John Mendelsohn, MD. The Robert M. Hoffman Foundation for Cancer Research provided funds for the present study.
Footnotes
Authors’ Contributions
SM and RMH designed the study. HQ, KM, BMK, MB, NY, KH, HK, SM, KI, TH, HT and SD critically reviewed the manuscript. SM performed experiments. SM was a major contributor to writing the manuscript and RMH revised the article. All Authors read and approved the final manuscript.
Conflicts of Interest
The Authors declare no competing interests in relation to this study.
- Received January 16, 2025.
- Revision received February 15, 2025.
- Accepted February 17, 2025.
- Copyright © 2025, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
