Improvement of NK cell-infusion therapy for metastatic breast cancer by targeting tumour-associated macrophages

乳腺癌是女性癌症死亡的主要原因之一。虽然大多数乳腺癌(原发肿瘤)可以通过手术切除，但它经常扩散到身体的不同部位，如骨和肺(转移性肿瘤)，这是导致这种疾病高死亡率的原因。由于现有的治疗方法不足以延长转移性乳腺癌患者的生存期，因此需要新的方法来改善预后。一种新的治疗策略是注入被称为自然杀伤(NK)细胞的免疫细胞，这种细胞可以直接杀死某些类型的癌细胞。临床和临床前研究表明，NK细胞输注对乳腺癌患者的治疗效果不大，且无不良反应。然而，一些报道表明，NK细胞在肿瘤环境中不能表现出完全的抗肿瘤能力，这表明需要消除这种抑制环境才能将NK细胞用作治疗工具。最近的研究表明，其他类型的免疫细胞，即肿瘤相关巨噬细胞(TAMs)，可以抑制原发肿瘤中的NK细胞功能。然而，它们在转移性肿瘤中的作用在很大程度上是未知的。利用转移性乳腺癌的动物模型，我曾报道TAMs是转移肿瘤环境中最丰富的细胞类型之一，它们的积累有助于癌细胞建立致命的转移肿瘤。此外，我最近的研究表明，转移性肿瘤中的TAMs在体外可以抑制NK细胞诱导的肿瘤细胞死亡。我们还发现，当巨噬细胞受到条件培养液的刺激时，它们获得了抑制NK细胞的功能。条件培养液是从培养的癌细胞中获得的，因此包含了癌细胞分泌的几种因子。因此，我预测，转移肿瘤环境中的TAM受到癌细胞的教育，以防止输注的NK细胞对转移性乳腺癌的治疗效果。我的项目的主要目标是通过阻断TAMs的功能来提高以NK细胞为基础的治疗转移性乳腺癌的疗效。为了实现这一最终目标，这项提议有三个具体目标。第一个目标是确定TAMs如何抑制NK细胞的功能。我们将从小鼠转移性肿瘤中分离TAMs，并利用我们建立的体外实验方法研究其对NK细胞运动、激活、杀伤因子表达和肿瘤杀伤活性的影响。第二个目标是确定可以增强人类NK细胞疗效的治疗靶点。我们将确定在癌细胞培养的人巨噬细胞中高表达的候选因子，并评估这些因子的消融是否能阻止巨噬细胞介导的NK细胞功能抑制。我们还将定义培养人巨噬细胞成为NK抑制细胞的癌细胞衍生因子。第三个目标是开发与临床相关的模型，用于验证治疗靶点和进一步的药物发现。我们已经建立了一种将人诱导多能干细胞(IPSC)分化为成熟巨噬细胞和NK细胞的方案。使用这些细胞，我们将把我们的体外试验发展成更具临床相关性的模型。我们还将建立一个小鼠模型，通过该模型我们可以评估人NK细胞输注联合阻断已确定的治疗靶点对人乳腺癌细胞转移形成的治疗效果。我相信这项研究将向我们揭示TAMs抑制NK细胞功能的基本因素，这将引导我们开发新的方法来提高以NK细胞为基础的治疗转移性疾病的疗效。来自该奖项的资金将使我能够扩大我的研究小组，增强我的独立性，发展进一步的合作，从而确定可能延长转移性乳腺癌患者生存的新策略。

Breast cancer is one of the leading causes of cancer death in women. Although most cancer in the breast (primary tumours) can be resected surgically, it often spreads to the different part of the body such as the bone and lung (metastatic tumours) and this is responsible for the high mortality of the disease. Since existing therapies are not sufficient to prolong survival of patients with metastatic breast cancer, novel approaches are required to improve outcome. An emerging therapeutic strategy is the infusion of immune cells called natural killer (NK) cell that can directly kill certain types of cancer cells. Clinical and pre-clinical studies have shown that infusion of NK cells exerts modest therapeutic effects in breast cancer patients without adverse side effects. However, several reports show that NK cells cannot exhibit full anti-tumour ability in the environment of the tumour, suggesting that elimination of such a suppressive environment is needed to use the NK cells as a therapeutic tool. Recent studies suggest that other types of immune cells called tumour-associated macrophages (TAMs) can suppress NK cell functions in the primary tumours. However, their roles in the metastatic tumours are largely unknown. Using animal models of metastatic breast cancer, I have reported that TAMs are one of the most abundant cell types in the environment of metastatic tumours, and their accumulation helps cancer cells to establish the lethal metastatic tumours. Furthermore, my recent studies suggest that the TAMs in the metastatic tumours can suppress NK cell-induced tumour cell death in vitro. We have also found that macrophages acquire NK cell suppressive functions when they are stimulated with conditioned media that are harvested from cultured cancer cells and thus include several factors secreted by cancer cells. I thus predict that the TAMs in the metastatic tumor environment are educated by cancer cells to prevent therapeutic effects of the infused NK cells on the metastatic breast cancer. The overarching ambition of my project is to improve efficacy of the NK cell-based therapy against metastatic breast cancer by blocking functions of TAMs. To achieve this ultimate goal, this proposal has three specific aims. The first aim is to define how TAMs suppress NK cell functions. We will isolate TAMs from murine metastatic tumors, and investigate their effects on NK cell movement, activation, killing factor expression, and tumor killing activity using in vitro assays established by us. The second aim is to identify therapeutic targets that can potentiate human NK cell efficacy. We will identify candidate factors that are highly expressed in human macrophages educated by cancer cells, and assess whether ablation of these factors prevent the macrophage mediated suppression of the NK cell functions. We will also define the cancer cell-derived factors that educate human macrophages to be NK suppressive cells. The third aim is to develop clinically relevant models for validation of the therapeutic targets and further drug discovery. We have established a protocol to differentiate human induced pluripotent stem cell (iPSC) into mature macrophages and NK cells. Using these cells, we will develop our in vitro assay into a more clinically relevant model. We will also establish a mouse model by which we can assess therapeutic effects of human NK cell infusion combined with blockade of identified therapeutic targets on the metastasis formation of human breast cancer cells. I believe that this research will show us the essential factors for TAMs to suppress NK cell functions, which will lead us to develop novel approaches to improve efficacy of NK cell-based therapy for metastatic diseases. The funding from this award would allow me to expand my research group, enhance my independence, develop further collaborations, and thereby identify new strategies that will potentially prolong survival of metastatic breast cancer patients.