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.

The human neocortex is the seat of many of the higher cognitive functions that make us human. These include our memory, speech and advanced learning. Our neocortex has greatly expanded during evolution, resulting in an increase in the number of nerve cells (neurons) within it, and an increase in overall size. This expansion was accompanied by folding of the cortical surface, which gives the brain its wrinkled appearance and allowed the expansion of the cortical surface area within the confinement of the developing skull. Despite their functional importance, we know relatively little about how these folds are formed in development. What is known, is that these cortical folds are very similar between individuals, so much so, that the largest folds are almost identical. This suggests that the formation of the correct number of folds in the correct location is important for their function. In fact, cognitive defects are observed in various neurodevelopmental disorders that result in abnormal cortical folding, i.e. disorders with too much folding, such as polymicrogyria, and not enough folding, such as lissencephaly, suggesting that the regulation of folding during development is crucial. My work has shown that the extracellular matrix (ECM), the proteins that surround cells in a tissue, has a key role in regulating how cortical folds form. Manipulating the ECM in tissue slices of human fetal neocortex kept in culture induced folding of the cortical plate. This ECM-induced folding was reduced or delayed in neocortex samples with specific neurodevelopmental defects, such as Down syndrome and prenatal methamphetamine exposure. Both of these disorders are known to have developmental delays or defects in the neocortex, which are currently not fully understood. The ECM-induced folding of human neocortex explants can now be used to probe these disorders and others. This is particularly important for the disorders that currently lack suitable animal models. Of these, the most relevant are the disorders that alter cortical folding, as many of the model systems used to study neocortex development naturally lack folding (such as mice). I predict that there are many more functions of ECM in human neocortex development, folding and related neurodevelopmental disorders. Therefore, the overall aim of my proposal is to investigate how ECM regulates human neocortex development and its related disorders, using the novel human neocortex explant systems I have developed. The first aim of my proposal is to use my ECM-folding assay to investigate the function of ECM genes that have already been linked to specific neurodevelopmental disorders in patients. Two candidates are the ECM protein perlecan, and the ECM receptor dystrogylcan. Both of these genes are linked to the folding disorder lissencehpaly. The second aim is to examine the exact composition of the ECM within the developing human neocortex. There is currently very little data on what ECM is expressed in the human neocortex, when it is expressed and where it is located. This is vital information that will help us understand not only the normal function of the ECM in human neocortex development, but also help understand and predict how mutations in ECM genes led to neurodevelopmental defects. The third aim is to test the function of ECM proteins in human neocortex development, using the human neocortex explant culture system and ECM-folding assay. This will increase our understanding of how ECM regulates the development and folding of the cortex, and its dysregulation. This fellowship funding would allow me to set up my independent research group, addressing these fundamental questions of how the human neocortex develops and how this development goes awry in developmental disorders. It will uncover the role of the ECM in both of these processes, and increase our knowledge and understanding of the normal neocortex development and folding, and the dysregulation seen in related disorders.

Melanoma is the most aggressive 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 used to be frequently excluded from clinical trials and BrM are experimentally strongly understudied. Notably, brain has a very distinct cellular composition, with the blood-brain barrier hindering access of drugs and molecules, and it lacks lymphatic vessels that play an important role in initiation of immune responses. 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 only very few studies investigating how the drugs targeting PD-1 and CTLA-4 work in BrM. To study this, we previously 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). For T lymphocytes to develop into CTLs that can kill cancer cells, they need help from another population of white blood cells called dendritic cells (DCs). DCs take up molecules derived from cancer cells and present these to T lymphocytes, which induces their activation into CTLs. There are different types of DCs. Our studies demonstrated that type 1 conventional dendritic cells (cDC1s) are required for the control of tumour growth in BrM. We therefore aim to determine how exactly are cDC1s involved in the control of BrM growth following therapy targeting PD-1 and CTLA-4, and how cDC1s in the brain differ from those outside the brain. Understanding how cDC1s support immune responses against BrM will enable the development of strategies that can enhance the ability of cDC1s to support CTLs in their attack against cancer and are therefore expected to potentiate the efficacy of therapy targeting PD-1 and CTLA-4. 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, as well as other cancers.