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，它就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，标准治疗只对很小一部分患者有效。近年来，新的药物被批准用于治疗转移性黑色素瘤。这些药物抑制PD-1和CTLA-4分子，这两种分子存在于被称为T淋巴细胞的白细胞亚群中。抑制PD-1和CTLA-4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，但联合抑制PD-1和CTLA-4后的完全缓解仅在11.5%的患者中出现。因此，重要的是要更好地了解这些药物如何起作用，以便能够开发进一步提高其疗效的方法。值得注意的是，免疫系统在身体的不同器官中以不同的方式工作。因此，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起作用是很重要的。我们的目标是了解PD-1和CTLA-4阻断剂如何在大脑中提高疗效，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），这种肿瘤特别难以治疗。与黑色素瘤相比，我们对BrM知之甚少；这是因为尽管BrM的发病率很高，但BrM患者大多被排除在临床试验之外，而且BrM在实验上的研究不足。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。忽视大脑的这些特征会带来一种危险——尽管身体其他部位的黑色素瘤治疗取得了进展——BrM的治疗再次落后，BrM成为患者生存的一个限制因素。因此，及时确定靶向PD-1和CTLA-4的药物在BrM中的作用机制至关重要。迄今为止，还没有实验研究调查靶向PD-1和CTLA-4的药物如何在BrM中起作用。为了研究后者，我们建立了黑色素瘤BrM的体内模型，证明CTLA-4和PD-1联合靶向可显著抑制BrM的生长，延长生存期。这主要是由称为细胞毒性T淋巴细胞（ctl）的T淋巴细胞亚群和另一种称为自然杀伤细胞的白细胞介导的。治疗后ctl在肿瘤中积累。因此，我们的目标是了解ctl如何移动到BrM，并确定它们如何在这种治疗的背景下杀死癌细胞。我们还观察到肿瘤中所谓髓系白细胞的积累增加。因此，我们的目标是确定这些细胞是否也是大脑中靶向PD-1和CTLA-4的药物活性所必需的。了解CTL如何移动到BrM将有助于开发能够增强大脑肿瘤内CTL积累的策略，因此有望增强针对PD-1和CTLA-4的治疗效果。如果我们的研究确定髓系白细胞在靶向PD-1和CTLA-4后参与了BrM的抑制，这将为PD-1/CTLA-4和髓系细胞联合靶向治疗的改进提供合理的依据。至少有一部分新获得的知识预计将适用于除大脑以外的其他部位的黑色素瘤。因此，从拟议的研究中获得的知识有可能有助于改善BrM患者和转移性黑色素瘤患者的预后。

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.