Akatsu T, Ono K, Katayama Y, Tamura T, Nishikawa M, Kugai N, Yamamoto M, Nagata N
The Third Department of Internal Medicine, National Defense Medical College, Tokorozawa, Saitama, Japan.
J Bone Miner Res. 1998 Mar;13(3):400-8. doi: 10.1359/jbmr.1998.13.3.400.
Osteoclastic bone resorption increases at the site of bone metastasis, but little is known about how tumor cells induce osteoclast (OC) recruitment in the bone marrow microenvironment. To clarify this point, we examined the effects of various mouse tumor cells on OC recruitment using cocultures of tumor cells and mouse marrow cells. The mouse mammary tumor cell lines, MMT060562 (MMT), BALB/c-MC, Jyg-MC(A), or other nonmammary tumor cell lines, LLC and B16, were cocultured with mouse marrow cells, and OC recruitment from marrow cells was determined by counting the number of tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP(+) MNCs) formed. Of the tumor cells examined, MMT and BALB/c-MC stimulated OC formation, but other tumor cells did not. OC formation with MMT was dependent on the number of MMTs inoculated, and only ten cells per well were sufficient to induce OC development. OCs appeared on day 4, and the number reached a maximum on days 5-8 and decreased thereafter. TRAP(+) MNCs induced by MMT satisfied the major criteria of OCs, such as the presence of calcitonin receptors and the ability to resorb calcified tissues. The majority of OCs were formed adjacent to the stromal cells, which were positive for alkaline phosphatase. When spleen cells were cocultured with MMT, no OCs were formed. In contrast, when osteoblastic cells were added to cocultures of spleen cells and MMT, many OCs were formed. The cultured media (CM) of MMT induced OC formation in mouse marrow cultures. Neither parathyroid hormone-like nor interleukin 1-like activity was present in the CM. MMT constitutively produced prostaglandin E2 (PGE2) and OC formation in cocultures was completely inhibited by indomethacin. Fractionation of the CM of MMT by ultrafiltration indicated that the OC-inducing activities were present not only in the fraction with molecular weight below 3 kDa but also in the fraction with molecular weight above 3 kDa. OC-inducing activity with high molecular weight was eluted around 50 kDa by Bio-Gel P-60 column chromatography. The active fractions also possessed leukemia inhibitory factor (LIF) activity, and OC-inducing activity of the peak fraction was inhibited in the presence of anti-LIF neutralizing antibody. The results of this study indicated that MMTs release PGE2 and LIF, which in turn stimulate OC formation via a stromal cell-dependent pathway. These culture systems will help to clarify the mechanisms by which tumor cells induce OC formation in a bone marrow microenvironment.
在骨转移部位破骨细胞介导的骨吸收增加，但关于肿瘤细胞如何在骨髓微环境中诱导破骨细胞（OC）募集却知之甚少。为阐明这一点，我们使用肿瘤细胞与小鼠骨髓细胞共培养的方法，研究了各种小鼠肿瘤细胞对OC募集的影响。将小鼠乳腺肿瘤细胞系MMT060562（MMT）、BALB/c-MC、Jyg-MC(A)，或其他非乳腺肿瘤细胞系LLC和B16与小鼠骨髓细胞共培养，通过计数形成的抗酒石酸酸性磷酸酶阳性多核细胞（TRAP(+) MNCs）数量来确定骨髓细胞中OC的募集情况。在所检测的肿瘤细胞中，MMT和BALB/c-MC刺激了OC形成，而其他肿瘤细胞则没有。MMT诱导的OC形成取决于接种的MMT数量，每孔仅10个细胞就足以诱导OC发育。OC在第4天出现，数量在第5 - 8天达到最大值，此后减少。MMT诱导的TRAP(+) MNCs满足OC的主要标准，如存在降钙素受体和吸收钙化组织的能力。大多数OC在碱性磷酸酶阳性的基质细胞附近形成。当脾细胞与MMT共培养时，未形成OC。相反，当将成骨细胞添加到脾细胞与MMT的共培养体系中时，形成了许多OC。MMT的培养基（CM）在小鼠骨髓培养中诱导OC形成。CM中不存在甲状旁腺激素样或白细胞介素1样活性。MMT持续产生前列腺素E2（PGE2），共培养中OC的形成被吲哚美辛完全抑制。通过超滤对MMT的CM进行分级分离表明，OC诱导活性不仅存在于分子量低于3 kDa的组分中，也存在于分子量高于3 kDa的组分中。通过Bio-Gel P - 60柱色谱法，高分子量的OC诱导活性在约50 kDa处被洗脱。活性组分还具有白血病抑制因子（LIF）活性，并且在存在抗LIF中和抗体的情况下，峰值组分的OC诱导活性受到抑制。本研究结果表明，MMT释放PGE2和LIF，进而通过基质细胞依赖途径刺激OC形成。这些培养系统将有助于阐明肿瘤细胞在骨髓微环境中诱导OC形成的机制。
