Data_Sheet_1_Tumor-Associated Macrophages in Canine Oral and Cutaneous Melanomas and Melanocytomas: Phenotypic and Prognostic Assessment.docx

The tumor microenvironment is a complex system, where neoplastic cells interact with immune and stromal cells. Tumor-associated macrophages (TAMs) are considered among the most numerically and biologically noteworthy cellular components in tumors and the attention on this cellular population has been growing during the last decade, both for its prognostic role and as a potential future therapeutic target. Melanoma, particularly the oral form, despite being one of the most immunogenic tumors, bears a poor prognosis in dogs and humans, due to its highly aggressive biological behavior and limited therapeutic options. The aims of this study are to characterize and quantify TAMs (using CD163, CD204, Iba1, and MAC387) in canine melanocytic tumors and to evaluate the association of these markers with diagnosis, histologic prognostic features, presence of metastases, and outcome, and to provide preliminary data for possible future therapies targeting TAMs. Seventy-two melanocytic tumors (27 oral melanomas, 25 cutaneous melanomas, 14 cutaneous melanocytomas, and 6 oral melanocytomas) were retrospectively selected and submitted to immunohistochemistry and double immunofluorescence. Double immunolabeling revealed that most CD163+ and CD204+cells co-expressed Iba1, which labeled also dendritic cells. Iba1 was instead rarely co-expressed with MAC387. Nevertheless, the expression of macrophagic markers showed a mild to moderate association among the four markers, except for CD204 and MAC387. The number of CD163+, CD204+, and MAC387+ cells was significantly higher in oral melanomas compared to oral melanocytomas (p < 0.001; p < 0.05 and p < 0.01, respectively), whereas Iba1 was differentially expressed in cutaneous melanomas and melanocytomas (p < 0.05). Moreover, CD163, IBA1 and MAC387 expression was associated with nuclear atypia and mitotic count. The number of CD163+cells was associated with the presence of metastases and tumor-related death in oral melanocytic tumors (p < 0.05 and p = 0.001, respectively).

肿瘤微环境（tumor microenvironment）是一个复杂的系统，肿瘤细胞在此与免疫细胞及基质细胞发生相互作用。肿瘤相关巨噬细胞（tumor-associated macrophages, TAMs）被认为是肿瘤中数量与生物学特性均最为显著的细胞组分之一；近十年来，学界对这类细胞群的关注度持续攀升，这既因其具备预后价值，也因其有望成为未来潜在的治疗靶点。
黑色素瘤，尤其是口腔型黑色素瘤，尽管属于免疫原性最强的肿瘤之一，但由于其生物学行为极具侵袭性且治疗手段有限，犬类与人类患者的预后均较差。
本研究旨在对犬黑色素细胞性肿瘤中的TAMs（采用CD163、CD204、Iba1及MAC387进行标记）开展表征与定量分析，评估这些标记物与诊断、组织学预后特征、转移状态及患者转归的关联，并为未来靶向TAMs的潜在治疗方案提供初步数据。
本研究回顾性筛选了72例黑色素细胞性肿瘤（其中口腔黑色素瘤27例、皮肤黑色素瘤25例、皮肤黑色素细胞瘤14例、口腔黑色素细胞瘤6例），所有样本均接受了免疫组织化学染色与双重免疫荧光染色。
双重免疫标记结果显示，绝大多数CD163阳性与CD204阳性细胞共表达Iba1，而Iba1亦可标记树突状细胞；与之相反，Iba1与MAC387的共表达情况较为罕见。尽管如此，四种标记物的巨噬细胞表达水平均呈轻度至中度相关，仅CD204与MAC387除外。
与口腔黑色素细胞瘤相比，口腔黑色素瘤中的CD163阳性、CD204阳性及MAC387阳性细胞数量均显著升高（分别为p < 0.001、p < 0.05及p < 0.01）；而Iba1的表达在皮肤黑色素瘤与黑色素细胞瘤中存在差异（p < 0.05）。
此外，CD163、Iba1及MAC387的表达与核异型性及核分裂象计数相关。
在口腔黑色素细胞性肿瘤中，CD163阳性细胞的数量与转移状态及肿瘤相关死亡均存在显著关联（分别为p < 0.05及p = 0.001）。