Department of Pathology, Amsterdam University Medical Centers, Loc. AMC, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands.
Lymphoma and Myeloma Center Amsterdam - LYMMCARE, and Cancer Center Amsterdam (CCA), Amsterdam, The Netherlands.
J Hematol Oncol. 2021 Jan 12;14(1):11. doi: 10.1186/s13045-021-01031-3.
The survival and proliferation of multiple myeloma (MM) cells in the bone marrow (BM) critically depend on interaction with stromal cells expressing the chemokine CXCL12. CXCL12 regulates the homing to the BM niche by mediating the transendothelial migration and adhesion/retention of the MM cells. The gamma isoform of CXCL12 (CXCL12γ) has been reported to be highly expressed in mouse BM and to show enhanced biological activity compared to the 'common' CXCL12α isoform, mediated by its unique extended C-terminal domain, which binds heparan sulfate proteoglycans (HSPGs) with an extraordinary high affinity. Here, we investigated the expression of CXCL12γ in human BM and studied its functional role in the interaction of MM cells with BM stromal cells (BMSCs).
We assessed CXCL12γ mRNA and protein expression by human BMSCs using qPCR, flow cytometry, and immunohistochemistry. CRISPR-Cas9 was employed to delete CXCL12γ and the heparan sulfate (HS) co-polymerase EXT1 in BMSCs. To study the functional roles of BMSC-derived CXCL12γ and HSPGs in the interaction of MM cells with BMSCs cells, MM cell lines and primary MM cells were co-cultured with BMSCs.
We observed that CXCL12γ is expressed in situ by reticular stromal cells in both normal and MM BM, as well as by primary BMSC isolates and BMSC lines. Importantly, upon secretion, CXCL12γ, unlike the CXCL12α isoform, was retained on the surface of BMSCs. This membrane retention of CXCL12γ is HSPG mediated, since it was completely annulated by CRISPR-Cas9-mediated deletion of the HS co-polymerase EXT1. CXCL12γ expressed by BMSCs and membrane-retained by HSPGs supported robust adhesion of MM cells to the BMSCs. Specific genetic deletion of either CXCL12γ or EXT1 significantly attenuated the ability of BMSCs to support MM cell adhesion and, in addition, impaired their capacity to protect MM cells from bortezomib-induced cell death.
We show that CXCL12γ is expressed by human BMSCs and upon secretion is retained on their cell surface by HSPGs. The membrane-bound CXCL12γ controls adhesion of MM cells to the stromal niche and mediates drug resistance. These findings designate CXCL12γ and associated HSPGs as partners in mediating MM-niche interaction and as potential therapeutic targets in MM.
多发性骨髓瘤（MM）细胞在骨髓（BM）中的存活和增殖，严重依赖于表达趋化因子 CXCL12 的基质细胞的相互作用。CXCL12 通过介导 MM 细胞的跨内皮迁移和黏附/保留，调节其归巢到 BM 龛位。与“常见”的 CXCL12α 同工型相比，CXCL12 的γ同工型（CXCL12γ）已被报道在小鼠 BM 中高度表达，并通过其独特的延伸 C 末端结构域介导，该结构域与硫酸乙酰肝素蛋白聚糖（HSPGs）具有极高的亲和力。在这里，我们研究了 CXCL12γ 在人类 BM 中的表达，并研究了其在 MM 细胞与 BM 基质细胞（BMSCs）相互作用中的功能作用。
我们使用 qPCR、流式细胞术和免疫组织化学评估了 CXCL12γ 在人类 BMSCs 中的 mRNA 和蛋白表达。CRISPR-Cas9 被用于敲除 BMSCs 中的 CXCL12γ 和硫酸乙酰肝素（HS）共聚合酶 EXT1。为了研究 BMSC 衍生的 CXCL12γ 和 HSPGs 在 MM 细胞与 BMSCs 细胞相互作用中的功能作用，我们将 MM 细胞系和原代 MM 细胞与 BMSCs 共培养。
我们观察到 CXCL12γ 原位表达于正常和 MM BM 的网状基质细胞，以及原代 BMSC 分离物和 BMSC 系中。重要的是，与 CXCL12α 同工型不同，CXCL12γ 在分泌后保留在 BMSCs 的表面。这种 CXCL12γ 的膜保留是 HSPG 介导的，因为它完全被 CRISPR-Cas9 介导的 HS 共聚合酶 EXT1 的缺失所消除。BMSCs 表达的 CXCL12γ 和 HSPG 膜保留支持 MM 细胞与 BMSCs 的牢固黏附。BMSCs 中 CXCL12γ 或 EXT1 的特异性基因缺失显著减弱了 BMSCs 支持 MM 细胞黏附的能力，并且还损害了它们保护 MM 细胞免受硼替佐米诱导的细胞死亡的能力。
我们表明，CXCL12γ 由人类 BMSCs 表达，并且在分泌后通过 HSPGs 保留在其细胞膜上。膜结合的 CXCL12γ 控制 MM 细胞与基质巢的黏附，并介导药物耐药性。这些发现将 CXCL12γ 及其相关的 HSPGs 指定为介导 MM 巢相互作用的伙伴，并作为 MM 的潜在治疗靶点。
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