Mechanisms underlying inhibition of melanoma brain metastases upon immune checkpoint targeting

黑色素瘤是最致命的皮肤癌。一旦黑色素瘤扩散到全身，它被称为转移性黑色素瘤。在这个阶段，黑色素瘤变得非常难以治疗，标准治疗仅在非常小比例的患者中有效。近年来，新的药物已被批准用于治疗转移性黑色素瘤。这些药物抑制称为PD-1和CTLA-4的分子，这些分子存在于称为T淋巴细胞的白色血细胞亚群上。PD-1和CTLA-4的抑制有助于免疫系统攻击癌症。尽管这些药物显著延长了黑色素瘤患者的生命，但仅在11.5%的患者中观察到PD-1和CTLA-4联合抑制后的完全反应。因此，更好地了解这些药物的作用机制非常重要，以便能够开发出进一步提高其疗效的方法。值得注意的是，免疫系统在体内不同器官中以不同的方式发挥作用。因此，重要的是要了解靶向PD-1和CTLA-4的药物如何在黑色素瘤最常扩散的器官内发挥作用。我们的目标是了解PD-1和CTLA-4阻断剂在大脑中的疗效如何提高，高达60%的转移性黑色素瘤患者的癌症扩散到大脑。由此产生的肿瘤被称为脑转移瘤（BrM），它们特别难以治疗。与一般的黑色素瘤相比，我们对BrM知之甚少;这是因为尽管发病率很高，但BrM患者大多被排除在临床试验之外，并且BrM在实验上被严重低估。值得注意的是，大脑具有非常独特的细胞组成，血脑屏障的存在限制了药物和免疫细胞进入肿瘤。忽视大脑的这些细节构成了一种危险，即尽管身体其他部位的黑色素瘤治疗取得了进展，但BrM的治疗再次缺乏落后，BrM成为患者生存的限制因素。因此，及时确定PD-1和CTLA-4靶向药物在BrM中的作用机制至关重要。迄今为止，还没有研究PD-1和CTLA-4靶向药物在BrM中如何发挥作用的实验研究。为了研究后者，我们建立了黑色素瘤BrM的体内模型，并证明CTLA-4和PD-1的组合靶向显著抑制BrM的生长并延长存活。这主要由称为细胞毒性T淋巴细胞（CTL）的T淋巴细胞亚群和称为自然杀伤细胞的另一种类型的白色血细胞介导。治疗后CTL在肿瘤中积累。因此，我们的目标是了解CTL如何到达BrM，并确定它们如何在这种疗法的背景下杀死癌细胞。我们还观察到肿瘤中所谓的髓系白色血细胞的积累增加。因此，我们的目标是确定这些细胞是否也需要针对PD-1和CTLA-4的药物在brain.Understanding如何CTL旅行到BrM的活动将使战略的发展，可以提高CTL的积累在脑肿瘤内，因此，预计将加强针对PD-1和CTLA-4的治疗效果。如果我们的研究确定髓系的白色血细胞参与PD-1和CTLA-4靶向后的BrM抑制，这将为PD-1/CTLA-4和髓系细胞靶向组合的改进疗法提供合理性。预计至少部分新获得的知识适用于脑以外部位的黑素瘤。因此，从拟议的研究中获得的知识有可能有助于改善BrM患者和一般转移性黑色素瘤患者的结局。

Melanoma is the deadliest form of skin cancer. Once melanoma has spread throughout the body, it is known as metastatic melanoma. At this stage melanoma becomes very difficult to treat and the standard treatment is effective only in a very small proportion of patients. In recent years new drugs have been approved for the treatment of metastatic melanoma. These drugs inhibit the molecules called PD-1 and CTLA-4 that are present on a subpopulation of white blood cells called T lymphocytes. Inhibition of PD-1 and CTLA-4 helps the immune system to attack the cancer. Although these drugs significantly extend lives of melanoma patients, complete responses upon combined inhibition of PD-1 and CTLA-4 are seen only in 11.5 % of the patients. It is therefore important to gain a better understanding of how these drugs work in order to be able to develop approaches that further improve their efficacy.Notably, the immune system works in different ways within different organs in the body. It is therefore important to understand how the drugs targeting PD-1 and CTLA-4 work within the organs to which melanoma most commonly spreads. Our goal is to understand how the efficacy of PD-1 and CTLA-4 blockade could be improved in the brain, to which cancer spreads in up to 60% of metastatic melanoma patients. The resulting tumours are called brain metastases (BrM) and they are particularly difficult to treat. In comparison to the melanoma in general, we know very little about BrM; this is because - despite their high incidence - patients with BrM are mostly excluded from clinical trials and BrM are experimentally strongly understudied. Notably, brain has a very distinct cellular composition and the presence of the blood-brain barrier restricts access of drugs and immune cells into the tumour. Ignoring these specifics of the brain poses a danger that - despite a progress in the treatment of melanoma in other parts of the body - treatment of BrM once again lacks behind and BrM become a limiting factor in patient survival. It is therefore critical to identify the mechanisms involved in the action of drugs targeting PD-1 and CTLA-4 in BrM in a timely manner.There are to date no experimental studies investigating how the drugs targeting PD-1 and CTLA-4 work in BrM. To study the latter, we established an in vivo model of melanoma BrM and demonstrated that a combined targeting of CTLA-4 and PD-1 significantly inhibits growth of BrM and prolongs the survival. This was mainly mediated by a subpopulation of T lymphocytes called Cytotoxic T lymphocytes (CTLs) and by another type of white blood cells called natural killer cells. CTLs accumulated in tumours following therapy. Therefore our goal is to understand how CTLs travel to BrM and to determine how they kill cancer cells in the context of this therapy. We also observed increased accumulation of white blood cells of so-called myeloid lineage in tumours. We therefore aim to determine whether these cells are also required for activity of drugs targeting PD-1 and CTLA-4 in the brain.Understanding how CTLs travel to BrM will enable the development of strategies that can enhance CTL accumulation within the tumour in the brain and are therefore expected to potentiate the efficacy of therapy targeting PD-1 and CTLA-4. If our study determines that white blood cells of myeloid lineage are involved in inhibition of BrM following targeting of PD-1 and CTLA-4, this will provide a rational for improved therapies combining targeting of PD-1/CTLA-4 and myeloid cells. At least part of the newly gained knowledge is expected to be applicable to melanoma at sites other than the brain. Thus, the knowledge emerging from the proposed research has a potential to contribute towards improved outcomes of patients with BrM and those with metastatic melanoma in general.