Synergistic activation by p38MAPK and glucocorticoid signaling mediates induction of M2-like tumor-associated macrophages expressing the novel CD20 homolog MS4A8A.

Tumor-associated macrophages (TAMs) represent alternatively activated (M2) macrophages that support tumor growth. Previously, we have described a special LYVE-1(+) M2 TAM subset in vitro and in vivo; gene profiling of this TAM subset identified MS4A8A as a novel TAM molecule expressed in vivo by TAM in mammary carcinoma and malignant melanoma. In vitro, Ms4a8a mRNA and MS4A8A protein expression was strongly induced in bone marrow-derived macrophages (BMDMs) by combining M2 mediators (IL-4, glucocorticoids) and tumor-conditioned media (TCM). Admixture of MS4A8A(+) TCM/IL-4/GC-treated BMDM significantly enhanced the tumor growth rate of subcutaneously transplanted TS/A mammary carcinomas. Upon forced overexpression of MS4A8A, Raw 264.7 macrophage-like cells displayed a special gene signature. Admixture of these MS4A8A(+) Raw 264.7 cells also significantly enhanced the tumor growth rate of subcutaneously transplanted mammary carcinomas. To identify the signaling pathways involved in synergistic induction of MS4A8A, the major signaling cascades with known functions in TAM were analyzed. Although inhibitors of NF-κB activation and of the MAPK JNK and ERK did not show relevant effects, the p38α/β MAPK inhibitor SB203580 strongly and highly significantly (p > 0.001) inhibited MS4A8A expression on mRNA and protein level. In addition, MS4A8A expression was restricted in M2 BMDM from mice with defective GC receptor (GR) dimerization indicating that classical GR gene regulation is mandatory for MS4A8A induction. In conclusion, expression of MS4A8A within the complex signal integration during macrophage immune responses may act to fine tune gene regulation. Furthermore, MS4A8A(+) TAM may serve as a novel cellular target for selective cancer therapy. A recombinant Ms4a8a cDNA was amplified by PCR (primer: Ms4a8a-SpeI-fw 5’ATCGAATTCACTAGTAGCAAAGAGTTGGGAACCGGAGCAAGA3’ and Ms4a8a-NotI-rv: 5’ATATGCGGCCGCTAGAGCATCTTTAT3’) from Ms4a8a cDNA RZPDp981B0530D (IMAGE ID 905005), purified on agarose gel and subcloned after digestion with SpeI and NotI restriction enzymes into the expression vector pEF6/V5-His Topo (Invitrogen) according to standard molecular biology protocols. After confirming sequence identity, we transfected RAW264.7 cells with Ms4a8a vector DNA using Lipofectamine 2000 (Invitrogen) transfection reagent. Transfectants were selected by resistance to blasticidin (Invitrogen). Raw264.7Ms4a8a clone K8 with recombinant Ms4a4a expression were propagated. As a negative control, a vector-transfected RAW264.7mock (clone K3) was selected under parallel culture conditions.

肿瘤相关巨噬细胞（Tumor-associated macrophages, TAMs）指的是能够促进肿瘤生长的选择性活化（M2型）巨噬细胞。此前本团队已在体内外分别鉴定出一类特殊的LYVE-1阳性M2型TAM亚群；通过对该TAM亚群进行基因表达谱分析，发现MS4A8A是一种新型的TAM分子，可在乳腺癌与恶性黑色素瘤体内的TAM中表达。在体外实验中，联合使用M2型极化介质（IL-4、糖皮质激素）与肿瘤条件培养基（tumor-conditioned media, TCM），可强力诱导骨髓来源巨噬细胞（bone marrow-derived macrophages, BMDMs）表达Ms4a8a mRNA与MS4A8A蛋白。将经MS4A8A阳性的TCM/IL-4/GC处理的BMDMs与肿瘤细胞混合接种，可显著加快皮下移植的TS/A乳腺癌的生长速率。当强制过表达MS4A8A时，Raw 264.7巨噬细胞样细胞会呈现出独特的特征性基因表达谱。将这类MS4A8A阳性的Raw 264.7细胞与肿瘤细胞混合接种，同样可显著提升皮下移植乳腺癌的生长速度。为明确参与MS4A8A协同诱导过程的信号通路，本研究对已知在TAM中发挥功能的主要信号级联反应进行了分析。尽管NF-κB活化抑制剂、MAPK家族的JNK与ERK抑制剂未展现出显著调控效果，但p38α/β MAPK抑制剂SB203580可强力且极显著（p>0.001）地抑制MS4A8A在mRNA与蛋白水平的表达。此外，糖皮质激素受体（glucocorticoid receptor, GR）二聚化缺陷小鼠来源的M2型BMDMs中MS4A8A的表达受到显著限制，这表明经典的GR基因调控通路是MS4A8A诱导所必需的。综上，在巨噬细胞免疫应答过程中，MS4A8A的表达参与复杂的信号整合调控，可起到微调基因表达的作用。此外，MS4A8A阳性的TAM有望成为选择性癌症治疗的新型细胞靶点。本研究通过PCR扩增获得重组Ms4a8a cDNA，所用引物序列为：Ms4a8a-SpeI-fw：5’ATCGAATTCACTAGTAGCAAAGAGTTGGGAACCGGAGCAAGA3’与Ms4a8a-NotI-rv：5’ATATGCGGCCGCTAGAGCATCTTTAT3’，扩增模板为来自RZPDp981B0530D（IMAGE ID 905005）的Ms4a8a cDNA；扩增产物经琼脂糖凝胶纯化后，按照标准分子生物学实验流程，使用SpeI与NotI限制性内切酶酶切，随后亚克隆至表达载体pEF6/V5-His Topo（Invitrogen公司）中。经测序确认序列正确性后，本研究使用Lipofectamine 2000（Invitrogen公司）转染试剂将Ms4a8a载体DNA转入RAW264.7细胞。通过杀稻瘟菌素（blasticidin，Invitrogen公司）抗性筛选获得稳定转染的细胞株。将表达重组Ms4a4a的Raw264.7Ms4a8a克隆K8进行传代培养；作为阴性对照，同时在平行培养条件下筛选得到空载体转染的RAW264.7mock细胞株（克隆K3）。