Inatani Hiroyuki, Yamamoto Norio, Hayashi Katsuhiro, Kimura Hiroaki, Takeuchi Akihiko, Miwa Shinji, Higuchi Takashi, Abe Kensaku, Taniguchi Yuta, Yamada Satoshi, Asai Kiyofumi, Otsuka Takanobu, Tsuchiya Hiroyuki
Department of Orthopaedic Surgery, Kanazawa University Graduate School of Medical Science, 13-1, Takara-machi, Kanazawa-shi, Ishikawa-ken, 920-8640, Japan.
Department of Orthopaedic Surgery, Nagoya City University Graduate School of Medical Science, Nagoya-shi, Japan.
Clin Orthop Relat Res. 2017 Jun;475(6):1693-1701. doi: 10.1007/s11999-017-5259-z. Epub 2017 Feb 2.
The p53 protein in mesenchymal stem cells (MSCs) regulates differentiation to osteogenic or adipogenic lineage. Because p53 function is depressed in most malignancies, if MSCs in malignancy also have p53 hypofunction, differentiation therapy to osteogenic or adipogenic lineage may be an effective treatment. We therefore wished to begin to explore this idea by evaluating atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) cells, because murine double minute 2 (MDM2) gene amplification, which leads to p53 hypofunction, is found in almost all ALT/WDLs.
QUESTIONS/PURPOSES: We compared osteogenic and adipogenic differentiation potency between MSCs isolated and cultured from normal adipose tissues and ALT/WDLs from the same patients.
During tumor resections in six patients with ALT/WDL, we analyzed 3 mL of tumor, and for comparison, we harvested a similar amount of normal-appearing subcutaneous adipose tissue from an area remote from the tumor for comparison. Adipogenic differentiation potency was quantitatively assessed using spectrometry after oil red O staining. Osteogenic differentiation potency was semiquantitatively assessed by measuring a specific colored area after alkaline phosphatase (ALP) and alizarin red S staining. ALP is related to preosseous cellular metabolism, and alizarin red is related to calcium deposits in cell culture. There were three observers for each assessment, and each assessment (including induced-differentiation and histologic analysis) was performed in duplicate. We then analyzed the mechanism of the difference of osteogenic differentiation potency using the MDM2-specific inhibitor Nutlin-3 at various concentrations.
In terms of adipogenic differentiation potency, contrary to our expectations, more fatty acid droplets were observed in MSCs derived from normal fat than in MSCs derived from ALT/WDL, although we found no significant difference between MSCs derived from ALT/WDL and MSCs derived from normal fat; the mean differentiation potency values (normal adipose tissue versus ALT/WDL) (± SD) were 0.34 (SD, ± 0.13; 95% CI, 0.24-0.44) versus 0.25 (SD, ± 0.10; 95% CI, 0.18-0.33; p = 0.22). By contrast, we found greater osteogenic differentiation potency in MSCs derived from ALT/WDL than in MSCs derived from normal fat. The mean differentiation potency values (normal adipose tissue versus ALT/WDL) (±SD) based on ALP staining was 1.0 versus 17 (SD, ± 36; 95% CI, -2.8 to 38; p = 0.04). However, we found no differences based on alizarin red S staining; mean differentiation potency value (normal adipose tissue versus ALT/WDL) (± SD) was 1.0 versus 4.2 (SD, ± 4.8; 95% CI, 1.3-7.2; p = 0.58). The gap of osteogenic differentiation potency between MSCs from normal adipose tissue and ALT/WDL was decreased as MDM2-inhibitor Nutlin-3 concentration increased.
MSCs derived from ALT/WDL had higher osteogenic differentiation potency based on ALP staining, which disappeared as Nutlin-3 concentration increased, suggesting that could be caused by amplified MDM2 in ALT/WDL. Future laboratory studies might mechanistically confirm the gene and protein expression, and based on the mechanism of the gap of differentiation potency, if p53 contrast between MSCs in tumor and normal tissue could be stimulated, less-toxic and more-effective differentiation therapy to MSCs in malignancies might be developed.
间充质干细胞（MSCs）中的p53蛋白调节向成骨或成脂谱系的分化。由于p53功能在大多数恶性肿瘤中受到抑制，如果恶性肿瘤中的MSCs也存在p53功能低下，那么向成骨或成脂谱系的分化疗法可能是一种有效的治疗方法。因此，我们希望通过评估非典型脂肪瘤/高分化脂肪肉瘤（ALT/WDL）细胞来开始探索这一想法，因为几乎所有ALT/WDL中都发现了导致p53功能低下的小鼠双微体2（MDM2）基因扩增。
问题/目的：我们比较了从正常脂肪组织和同一患者的ALT/WDL中分离培养的MSCs之间的成骨和成脂分化能力。
在6例ALT/WDL患者的肿瘤切除过程中，我们分析了3 mL肿瘤组织，作为对照，我们从远离肿瘤的区域采集了相似量的外观正常的皮下脂肪组织。油红O染色后通过光谱法对成脂分化能力进行定量评估。通过测量碱性磷酸酶（ALP）和茜素红S染色后的特定染色面积对成骨分化能力进行半定量评估。ALP与骨前体细胞代谢有关，茜素红与细胞培养中的钙沉积有关。每次评估有三名观察者，每次评估（包括诱导分化和组织学分析）均重复进行两次。然后我们使用不同浓度的MDM2特异性抑制剂Nutlin-3分析成骨分化能力差异的机制。
在成脂分化能力方面，与我们的预期相反，在来自正常脂肪的MSCs中观察到的脂肪酸滴比来自ALT/WDL的MSCs中更多，尽管我们发现来自ALT/WDL的MSCs和来自正常脂肪的MSCs之间没有显著差异；平均分化能力值（正常脂肪组织对ALT/WDL）（±标准差）分别为0.34（标准差，±0.13；95%可信区间，0.24 - 0.44）和0.25（标准差，±0.10；95%可信区间，0.18 - 0.33；p = 0.22）。相比之下，我们发现来自ALT/WDL的MSCs的成骨分化能力高于来自正常脂肪的MSCs。基于ALP染色的平均分化能力值（正常脂肪组织对ALT/WDL）（±标准差）为1.0对17（标准差，±36；95%可信区间，-2.8至38；p = 0.04）。然而，基于茜素红S染色我们未发现差异；平均分化能力值（正常脂肪组织对ALT/WDL）（±标准差）为1.0对4.2（标准差，±4.8；95%可信区间，1.3 - 7.2；p = 0.58）。随着MDM2抑制剂Nutlin-3浓度增加，来自正常脂肪组织和ALT/WDL的MSCs之间的成骨分化能力差距减小。
基于ALP染色，来自ALT/WDL的MSCs具有更高的成骨分化能力，随着Nutlin-3浓度增加这种能力消失，这表明可能是由ALT/WDL中扩增的MDM2所致。未来的实验室研究可能会从机制上证实基因和蛋白表达，并且基于分化能力差距的机制，如果能够刺激肿瘤和正常组织中MSCs之间的p53差异，可能会开发出对恶性肿瘤中MSCs毒性更小、更有效的分化疗法。
