软骨肉瘤以对常规化疗和放疗治疗方案具有耐药性而著名，这对于无法手术切除的肿瘤患者尤为不利。最近的研究表明，通过托拉唑帕利抑制聚(ADP-核糖)聚合酶 (PARP) 的作用可以增加软骨肉瘤细胞系对化疗药物（替莫唑胺）或放疗的敏感性，无论异柠檬酸脱氢酶 (IDH) 基因突变情况如何。由于二维培养的细胞系具有局限性，可能无法准确地代表药物治疗的临床反应，我们旨在使用更具代表性的三维海藻酸钠小球软骨肉瘤模型。在将治疗剂在动物或人类身上测试之前，在体外测试治疗剂的有效性至关重要；因此，我们旨在确定 PARP 抑制剂在降低软骨肉瘤小球生存能力方面的有效性。利用更严格、更复杂的体外模型可以改进未来的治疗选择，以进一步在动物模型中进行调查，从而提高效率、减少不必要的动物使用，并节省时间和成本。 (1) 托拉唑帕利治疗是否能减缓或阻止软骨肉瘤小球的生长，治疗持续时间是否会改变药物的效果？ (2) 托拉唑帕利是否与替莫唑胺联合治疗以降低软骨肉瘤小球的生存能力？ (3) 托拉唑帕利是否与放疗治疗联合降低软骨肉瘤小球的生存能力？我们使用三个代表性的传统软骨肉瘤细胞系 (CH2879 [IDH 野生型]、JJ012 [IDH1 突变] 和 SW1353 [IDH2 突变]) 分别作为海藻酸钠小球培养，并使用托拉唑帕利 (0.001 至 10 µM)、替莫唑胺 (0.01 至 100 µM) 或这些药物的组合，对不同阶段的小球生长进行了 3、7 和 14 天的处理，以代表不同阶段的小球生长。这些细胞系被选择来代表各种 IDH 基因突变状态，并且已经在小球培养方面进行了验证。替莫唑胺的选择是因为它与 PARP 抑制剂联合使用时的成功，不同于其他常用的化疗药物。使用三种细胞存活力分析方法评估对小球生存能力的影响。此外，评估了小球数量、形态、增殖和凋亡。采用 137C辐射源进行的γ-放射线联合托拉唑帕利（5至10 nM）后的效果评估为存活分数，通过计算小球数量（三个）来评估低浓度托拉唑帕利（5至10 nM）与替莫唑胺或放疗的治疗协同作用。托拉唑帕利处理可降低三个细胞系小球在 14 天后的生存能力 (IC50 ± SD 为 CH2879:0.1 ± 0.03 µM，倍数变化:220；JJ012：12 ± 1.4 µM，倍数变化:4.8；SW1353：1.0 ± 0.2 µM，倍数变化:154)，与成熟小球三天的处理相比。经过 14 天的处理后，替莫唑胺和托拉唑帕利的 Excess over Bliss 分数表明两者联合治疗的协同作用有效（Excess over Bliss 分数: CH2879 59% [lower 95% CI 52%]，JJ012 18% [lower 95% CI 8%]，和 SW1353 55% [lower 95% CI 25%])。托拉唑帕利和放疗的治疗协同作用只在 JJ012 小球中存在，剂量为 4 Gy 的 4Gƴ辐射剂量（Excess over Bliss 分数: 22% [lower 95% CI 6%]）。在我们的研究中，长期 PARP 抑制比短期治疗更为有效，只有三个软骨肉瘤小球系列中的一个对联合 PARP 抑制和放疗敏感。这些发现表明后续的动物研究应该专注于长期 PARP 抑制，而 temozolomide 与托拉唑帕利的联合使用比联合放疗成功的机会更高。联合托拉唑帕利和替莫唑胺的治疗组合可以有效地降低软骨肉瘤小球和小球生长的生存能力，无论 IDH 基因突变状态如何，这为在动物软骨肉瘤模型中复制这种治疗组合提供了理论依据。版权所有© 2022 作者。由 Wolters Kluwer Health，Inc. 代表骨科协会出版。

Chondrosarcomas are well known for their resistance to conventional chemotherapy and radiotherapy treatment regimens, which is particularly detrimental in patients who have unresectable tumors. Recently, inhibition of poly(ADP-ribose) polymerase (PARP) by talazoparib was shown to sensitize chondrosarcoma cell lines to chemotherapy (temozolomide) or radiotherapy, irrespective of isocitrate dehydrogenase (IDH) mutation status. Because two-dimensionally grown cell lines have limitations and may not accurately represent the clinical response to drug treatment, we aimed to use a more representative three-dimensional alginate spheroid chondrosarcoma model. It is important to test therapeutic agents in vitro before testing them in animals or humans; therefore, we aimed to determine the effectiveness of a PARP inhibitor in reducing the viability of chondrosarcoma spheroids. Using a more stringent, complex in vitro model refines future therapeutic options for further investigation in animal models, increasing efficiency, reducing unnecessary animal use, and saving time and cost.(1) Does talazoparib treatment slow or inhibit the growth of chondrosarcoma spheroids, and does an increased treatment duration change the drug's effect? (2) Does talazoparib work in synergy with temozolomide treatment to reduce the viability of chondrosarcoma spheroids? (3) Does talazoparib work in synergy with radiotherapy treatment to reduce the viability of chondrosarcoma spheroids?Three representative conventional chondrosarcoma cell lines (CH2879 [IDH wildtype], JJ012 [IDH1 mutant], and SW1353 [IDH2 mutant]) were cultured as alginate spheroids and treated with talazoparib (0.001 to 10 µM), temozolomide (0.01 to 100 µM), or combinations of these drugs for 3, 7, and 14 days, representing different stages of spheroid growth. The cell lines were selected to represent a variety of IDH mutation statuses and were previously validated in spheroid culturing. Temozolomide was chosen because of its previous success when combined with PARP inhibitors, dissimilar to other commonly used chemotherapies. The effect on spheroid viability was assessed using three cell viability assays. Additionally, spheroid count, morphology, proliferation, and apoptosis were assessed. The effect of talazoparib (5 to 10 nM) combined with ƴ-radiation applied using a 137 C source (0 to 6 Gy) was assessed as surviving fractions by counting the number of spheroids (three). The therapeutic synergy of low-concentration talazoparib (5 to 10 nM) with temozolomide or radiotherapy was determined by calculating Excess over Bliss scores.Talazoparib treatment reduced the spheroid viability of all three cell lines after 14 days (IC 50 ± SD of CH2879: 0.1 ± 0.03 µM, fold change: 220; JJ012: 12 ± 1.4 µM, fold change: 4.8; and SW1353: 1.0 ± 0.2 µM, fold change: 154), compared with 3-day treatments of mature spheroids. After 14 days of treatment, the Excess over Bliss scores for 100 µM temozolomide and talazoparib indicated synergistic efficacy (Excess over Bliss scores: CH2879 59% [lower 95% CI 52%], JJ012 18% [lower 95% CI 8%], and SW1353 55% [lower 95% CI 25%]) of this combination treatment. A stable synergistic effect of talazoparib and radiotherapy was present only in JJ012 spheroids at a 4Gƴ radiation dose (Excess over Bliss score: 22% [lower 95% CI 6%]).In our study, long-term PARP inhibition was more effective than short-term treatment, and only one of the three chondrosarcoma spheroid lines was sensitive to combined PARP inhibition and radiotherapy. These findings suggest subsequent animal studies should focus on long-term PARP inhibition, and temozolomide combined with talazoparib has a higher chance of success than combination with radiotherapy.Combination treatment of talazoparib and temozolomide was effective in reducing the viability of chondrosarcoma spheroids and spheroid growth, regardless of IDH mutation status, providing rationale to replicate this treatment combination in an animal chondrosarcoma model.Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Association of Bone and Joint Surgeons.