簡介：以往的研究已經證實免疫腫瘤微環境在惡性胸膜間皮瘤(MPM)中的預後作用。腫瘤浸潤巨噬細胞是形成MPM腫瘤微環境中的白細胞的主要成分。巨噬細胞可分為兩種主要表型：M1型巨噬細胞，具有抗腫瘤發生功能，M2型巨噬細胞具有促腫瘤發展的促生功能。巨噬細胞表型影響MPM的生物學行為，並可能提供潛在的治療靶點。因此，我們目標在空間上描繪MPM腫瘤組織中巨噬細胞表達的五種標誌物的表達，並評估其與患者存活關係。

Introduction:Previous studies have demonstrated the prognostic role of the immunological tumor microenvironment in malignant pleural mesothelioma (MPM). Tumor-infiltrating macrophages form the major component of the leucocytes in MPM tumor microenvironment. Macrophages can be divided into two main phenotypes: type M1 macrophages have an anti-tumorigenic function and type M2 macrophages a pro-tumorigenic function promoting tumor development. The macrophage phenotypes affect the biological behavior of MPM and may provide potential therapeutic targets. Thus, we aimed to spatially profile the expression of five markers expressed by macrophages in MPM tumor tissue and assess their association with patient survival.

方法：該研究隊列由組織微陣列組成，包括來自76名芬蘭MPM患者(71名上皮樣和5名雙相)的樣本，其中包括18名存活至少60個月的患者。分析了巨噬細胞表達的標誌物的位置特異性組織表達(CD68[Dako;M0876]，CMAF[Abcam;ab199424]，pSTAT1[Cell Signaling Technology;8826]，HLA-DRA1[Abcam;ab20181]和CD163[Abcam;ab188571])使用多重熒光免疫組織化學和基於數字像素的圖像分析。測量單個標誌物表達和標誌物組合作為與總組織，基質或腫瘤麵積成比例的麵積。單變量Cox回歸用於評估巨噬細胞表達與死亡時間(全因死亡率)之間的關聯。

Methods:The study cohort consisted of tissue microarrays including samples from 76 Finnish MPM patients (71 epithelioid and 5 biphasic), including 18 patients who survived at least 60 months. We analyzed location-specific tissue expression of markers expressed by macrophages (CD68 [Dako; M0876], CMAF [Abcam; ab199424], pSTAT1 [Cell Signaling Technology; 8826], HLA-DRA1 [Abcam; ab20181] and CD163 [Abcam; ab188571]) using multiplexed fluorescence immunohistochemistry and digital pixel-based image analysis. Single marker expressions and marker combinations were measured as proportional areas to total tissue, stromal, or tumor area. Univariate Cox regression was used to assess the association between macrophage expression and time to death (all-cause mortality).

結果：在單變量Cox回歸分析中，M2型促腫瘤發生巨噬細胞(CD163+CMAF+HLA-DRA1-)與較短存活時間相關(HR=9.54，p=0.03)，而M1型抗腫瘤發生巨噬細胞(CD68+pSTAT1+HLA-DRA1+)與較長存活時間相關(HR=0.87，p=0.03)。此外，表達pSTAT1的免疫原性腫瘤細胞(CK5+pSTAT1+CD68-CD163-HLA-DRA1-)的存在與更長的存活時間相關(HR=0.97，p<0.01)。

Results:In univariate Cox regression analysis, type M2 pro-tumorigenic macrophages (CD163+CMAF+HLA-DRA1-) were associated with shorter survival time (HR=9.54, p=0.03), whereas type M1 anti-tumorigenic macrophages (CD68+pSTAT1+HLA-DRA1+) were associated with longer survival time (HR=0.87, p=0.03). Furthermore, the presence of pSTAT1 expressing immunogenic tumor cells (CK5+pSTAT1+CD68-CD163-HLA-DRA1-) was associated with longer survival time (HR=0.97, p<0.01).

結論：M1型巨噬細胞和表達pSTAT1的腫瘤細胞的表達與較長的存活率相關，M2型巨噬細胞的表達與較短的存活率相關。這些可能提供新的免疫治療靶點。

Conclusion:The expression of type M1 macrophages and pSTAT1 expressing tumor cells were associated with longer survival and the expression of type M2 macrophages was associated with shorter survival. These may provide new immunotherapeutic targets.