While melanoma, a form of skin cancer that begins in the pigmented cells of the skin (melanocytes), is less common than other types of skin cancer, its seriousness lies in its ability to spread to other organs if not treated at an early stage. In nearly 75% patients, autopsy reveals the metastasis of melanoma to the central nervous system (CNS).

Of these, patients with the worst prognosis and rapid disease progression are those in whom melanoma spreads to the leptomeninges, the two innermost layers of the meninges that cover and protect the brain (leptomeningeal melanoma metastases, LMM).

Whereas patients with melanoma brain metastases (MBM) can respond favorably to targeted therapy and immunotherapy, patients with LMM frequently respond poorly, indicating differences between the immune microenvironments of CNS compartments.

A new study published in Clinical Cancer Research, explores the biological differences of the LMM and MBM immune landscapes through single-cell RNA sequencing (scRNA-Seq) in 43 melanoma samples from 26 patients taken from the skin, brain, leptomeninges or cerebrospinal fluid (CSF). The authors validate the key results through immunohistochemistry and flow cytometry and identify immune cell subsets that correlate with overall survival.

The findings, reported in the article, “Single cell characterization of the immune microenvironment of melanoma brain and leptomeningeal metastases” provide critical new insights that may result in the development of improved therapies.

The study led by Keiran Smalley, PhD, Director of the Melanoma and Skin Cancers Center of Excellence at the Moffitt Cancer Center focuses on identifying differences in the MBM and LMM metastatic microenvironments.

“We were somewhat surprised to find such prominent differences at these two sites and to see a lot more overlap in the immune composition of the brain and the skin metastases. It’s somewhat counter-intuitive, but also could explain why the responses at the leptomeninges are so different than those in the brain or extra-cranial sites,” says Inna Smalley, PhD, Assistant Member at the Department of Cancer Physiology at the Moffitt Cancer Center.

Inna Smalley, PhD, Assistant Member, Department of Cancer Physiology, H. Lee Moffitt Cancer Center

The researchers report that the LMM microenvironment is radically different from that of brain and skin metastases. LMMs have higher proportions of CD4 T helper cells and lower proportions of the CD8 T effector cells, indicating significant differences in the lymphoid compartment of the metastatic microenvironments.

“We observed the highest proportions of exhausted or apoptotic T cells in the LMM and lowest number of proliferating T cells, truly highlighting how ‘cold’ the immune environment is at this site compared to others,” says Inna Smalley.  ‘Cold’ tumors are characterized by the lack of T cell infiltration.

Moreover, the authors show that an LMM patient with long-term survival had an immune landscape that differed from from that of poor survivors and was similar to normal CSF.

PD-1 therapy, a popular checkpoint blockade approach used in cancer immunotherapy, increased levels of T-cells and dendritic cells in the CSF of the patient with long-term survival, whereas poor survivors did not show increases in immune cell levels. In MBM patients, PD-1 therapy increased immune infiltration with comparable T-cell transcriptional diversity as seen in skin metastases.

“We do not know how chemotherapy would affect the tumor microenvironment at these sites but it appears that systemic targeted and immune therapy do have profound effects on the tumor microenvironment of both the MBM and LMM,” says Inna Smalley. “Pre-treatment specimens tended to have very few immune cell infiltrates overall and both targeted therapy and immunotherapy was associated with increased numbers of infiltrating immune cells. Treatment with targeted therapy was associated with an increased ratio of CD8 T cells to CD4 T cells, which was less frequently seen following immunotherapy. Meanwhile, immune-therapy stimulated a more diverse lymphocyte landscape, including higher numbers of B cells and plasma cells.”

The team identified a rare population of dendritic cells (DC3s) that was associated with increased overall survival.

“DC3s positively regulate the immune environment through activation of the T cells, leading to more robust anti-tumor immune responses,” says Inna Smalley.

The use of a single-cell approach in this study allowed the research team to ask questions that would not have otherwise occurred to them.

“It allowed us to not only focus on the cells we thought were going to be important but rather to see everything that is present in the tumor microenvironment. For instance, we would have never known to look for DC3 cells,” says Inna Smalley. “In addition, patient specimens such as those harvested from CSF are incredibly difficult to analyze due to the relatively low number of cells present. Single cell analysis approaches really gives us the power and technical sensitivity to interrogate these rare cells and learn about the biology of this under-studied disease from CSF, which is much easier to obtain than surgical resections.”

Leptomeningeal metastasis is a devastating terminal complication for late-stage patients, with few therapeutic options and little actionable knowledge on the tumor and its microenvironment due to its inaccessibility.

“We hope our data will provide critical insights into the biology of these tumors and propel the clinical translation of novel therapeutic approaches that are so desperately needed for these patients,” says Inna Smalley. “We would just also like to mention how thankful we are to the patients and their families who had agreed to participate in this study and donate their tissues to research. It is truly an invaluable resource and we greatly appreciate and applaud their dedication to eradicating cancer.”

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