Vaccine and Drug Combination Therapy for Human Cancers

某些化疗方案会触发癌细胞死亡，同时诱导树突状细胞成熟和随后的免疫反应。然而，到目前为止，化疗诱导的免疫原性细胞死亡(ICD)仅限于某些药物。相比之下，几种化疗药物可以调节抗肿瘤免疫反应，尽管不会引发经典的ICD。此外，在许多情况下，肿瘤细胞在治疗后不会死亡。在这里，使用最广泛使用的癌症化疗药物之一多西紫杉醇作为模型，我们检查了不死于免疫原性细胞死亡的肿瘤细胞的表型和功能后果。多西紫杉醇处理的肿瘤细胞没有诱导ATP或HMGB1的分泌，也没有诱导细胞死亡。然而，在化疗后检查的所有细胞系中都观察到了钙网织蛋白的暴露。经多西他赛治疗后，CEA、MUC-1或PSA特异性CD8+CTL的杀伤作用显著增强。这种杀伤与抗原处理机制组件的增加有关，并主要由钙网蛋白膜转位介导，这是由钙网蛋白、PERK或钙网蛋白阻断肽的功能性敲除所确定的。通过连续作用于多西紫杉醇，筛选出一株多西紫杉醇耐药细胞系(MDR-1+，CD133+)。这些细胞虽然抵抗多西紫杉醇的直接细胞抑制作用，但不抵抗导致CTL杀伤增强的化学调节作用。我们提供了“免疫原性调节”的操作定义，即肿瘤细胞暴露在非致命性/亚致死性剂量的化疗中，改变肿瘤表型，使肿瘤对CTL杀伤更加敏感。这些观察结果与免疫原性细胞死亡是不同的，也是互补的，并突出了化疗与免疫治疗相结合的机制。非小细胞肺癌(NSCLC)的标准治疗方案是铂类化疗，包括顺铂加长春瑞滨。亚致死性接触某些化疗药物已被证明改变了人类肿瘤细胞的表型或生物学，使它们更容易受到细胞毒性T淋巴细胞(CTL)介导的溶解。然而，顺铂/长春瑞滨对肿瘤T细胞毒敏感性的影响及其分子机制尚未完全阐明。在这里，我们检测了这种化疗对五种不同的人肺癌细胞系的生长、细胞表面表型和CTL介导的体外裂解的影响，并探讨了与增强CTL敏感性相关的分子机制。这些研究表明，人肺肿瘤细胞亚致死性暴露于铂对偶可调节肿瘤细胞的表型，并增加对MHC限制性穿孔素/颗粒酶介导的CTL杀伤的敏感性。这些研究还表明，暴露于化疗环境中可显著降低转化生长因子-β/白介素8的蛋白分泌比率。我们检测了两个肺肿瘤细胞系的基因表达谱，以确定亚致死性顺铂/长春瑞滨反应的共同基因特征，发现只有16个转录本协同表达，包括细胞因子/趋化因子表达和细胞凋亡，如肿瘤坏死因子-α、白介素8、CXCL5和bcl2样基因。总体而言，这些结果表明，顺铂/长春瑞滨的亚致死性暴露通过调节a)肿瘤表型、b)细胞因子/趋化因子环境和c)促凋亡/抗凋亡基因比率增加了对穿孔素/颗粒酶介导的CTL杀伤的敏感性。这里提供的数据提出了一种与免疫原性细胞死亡截然不同并与之互补的复杂机制。这一分子标记可能有助于预测免疫治疗的反应，并为联合使用疫苗和顺铂/长春瑞滨方案的潜在临床益处提供理论依据。

Certain chemotherapeutic regimens trigger cancer cell death while inducing dendritic cell maturation and subsequent immune responses. However, chemotherapy-induced immunogenic cell death (ICD) has thus far been restricted to select agents. In contrast, several chemotherapeutic drugs modulate antitumor immune responses, despite not inducing classic ICD. In addition, in many cases tumor cells do not die after treatment. Here, using docetaxel, one of the most widely used cancer chemotherapeutic agents, as a model, we examined phenotypic and functional consequences of tumor cells that do not die from immunogenic cell death. Docetaxel treatment of tumor cells did not induce ATP or HMGB1 secretion, or cell death. However, calreticulin exposure was observed in all cell lines examined after chemotherapy treatment. Killing by CEA, MUC-1, or PSA-specific CD8+ CTLs was significantly enhanced after docetaxel treatment. This killing was associated with increases in components of antigen-processing machinery, and mediated largely by calreticulin membrane translocation, as determined by functional knockdown of calreticulin, PERK, or calreticulin-blocking peptide. A docetaxel-resistant cell line was selected (MDR-1+, CD133+) by continuous exposure to docetaxel. These cells, while resistant to direct cytostatic effects of docetaxel, were not resistant to the chemomodulatory effects that resulted in enhancement of CTL killing. We provided an operational definition of "immunogenic modulation," where exposure of tumor cells to nonlethal/sublethal doses of chemotherapy alters tumor phenotype to render the tumor more sensitive to CTL killing. These observations are distinct and complementary to immunogenic cell death and highlight a mechanism whereby chemotherapy can be used in combination with immunotherapy. Chemotherapy with platinum doublets, including cisplatin plus vinorelbine, is standard of care for non-small cell lung cancer (NSCLC). Sublethal exposure to certain chemotherapeutic agents has been demonstrated to alter the phenotype or biology of human tumor cells, rendering them more susceptible to cytotoxic T lymphocyte (CTL)-mediated lysis. However, the effects of cisplatin/vinorelbine on tumor sensitivity to T-cell cytotoxicity and its molecular mechanisms have not been fully elucidated. Here, we examined the effect of this chemotherapy on growth, cell-surface phenotype, and CTL-mediated lysis of five distinct human lung carcinoma cell lines in vitro, and examined the molecular mechanisms associated with enhanced CTL sensitivity. These studies demonstrate that sublethal exposure of human lung tumor cells to the platinum doublet modulates tumor cell phenotype, and increases sensitivity to MHC-restricted perforin/granzyme-mediated CTL killing. These studies also demonstrate that exposure to chemotherapy markedly decreased the protein secretion ratio of TGF-beta/IL-8. We examined the gene expression profile of two lung tumor cell lines in order to identify a shared gene signature in response to sublethal cisplatin/vinorelbine and found coordinate expression of only 16 transcripts, including those for cytokine/chemokine expression and apoptosis such as TNF-alpha, IL8, CXCL5, and BCL-2 like genes. Overall, these results suggest that sublethal exposure to cisplatin/vinorelbine increases sensitivity to perforin/granzyme-mediated CTL killing by modulation of a) tumor phenotype, b) cytokine/chemokine milieu, and c) the pro-apoptotic/anti-apoptotic gene ratio. The data presented here propose a complex mechanism that is distinct from and complementary to that of immunogenic cell death. This molecular signature may be useful in predicting responses to immunotherapy as well as provide the rationale for the potential clinical benefit of the combined use of vaccine with cisplatin/vinorelbine regimens.