RNA sequencing of immune cells infiltrating cerebrospinal fluid enables the characterization of the tumor microenvironment in brain metastases

Immune cells accessing cerebrospinal fluid faithfully recapitulate the characteristics of cells identified in brain metastasis, and could therefore constitute novel biomarkers of response to immune-based therapies.

Brain metastases are the most frequent tumor of the brain, with a dismal prognosis. While a fraction of patients benefit from treatment with immune checkpoint inhibitors, the majority do not. To predict response to these therapies necessitates the characterization of tumor specimens. Due to the anatomical location of brain tumors and the risk of surgical procedures, accessing samples from brain malignancies is challenging.

Results from previous studies led by Joan Seoane, Director of Preclinical and Translational Research co-program at VHIO and ICREA Professor as well as those of other groups, have evidenced that cerebrospinal fluid can provide vital insights into the genomic characteristics of brain tumors and therefore be used as a minimally invasive liquid biopsy. Spurred by these findings, the investigators conducted this present research to establish whether they could effectively characterize the immunological phenotype through the analysis of cerebrospinal fluid.

To test this hypothesis, Joan Seoane’s team analyzed samples from 48 patients with brain metastasis. These samples were obtained thanks to the generosity of patients receiving treatment at our Vall d’Hebron University Hospital (HUVH), as well as the Hospital Clínic in Barcelona, who gave their full consent to use their samples. The collection of samples was possible thanks to the dedication and expertise of these hospitals’ Neurosurgery Services.

The researchers assessed the immune cells present in the brain metastases, and in parallel, performed immune cell profiling of the cerebrospinal fluid. They sought to identify which cell types were present in the cerebrospinal fluid and compare them with those obtained from the metastatic lesions.

“By establishing similarities between the two, we have identified a novel and minimally invasive method that can allow us to predict response to immunotherapy in these patients. This pioneering approach could more precisely guide clinical decision making in treating these patients with immune-based therapeutic strategies,” adds Joan Seoane.

By analyzing cerebrospinal fluid, Joan Seoane’s team have been able to identify the T-cells that recognize the tumor, and those that are active in treatment.

“Each immune T-cell has a unique sequence that recognizes a particular tumor antigen. When their tracing and targeting commence, these cells are activated and begin to proliferate. Through this study, we have been able to characterize the individual sequences of immune cells and, in this way, identify which immune cells are fighting the tumor and discern how they evolve over time,” continues Joan Seoane.

The study was also carried out in collaboration with colleagues at the National Centre for Genomic Analysis-Centre for Genomic Regulation (CNAG-CRG), Barcelona. Directed by Holger Heyn, Head of CNAG-CRG’s Single Cell Genomics Team, they performed single cell transcriptome sequencing of around 6000 cells by scRNA-seq technology.

Identification of tumor immune infiltration patterns in brain metastases

Fig. 1

a Schematic representation of patient sample collection and the experimental procedures performed. Immunohistochemistry (IHC), whole exome squencing (WES), cerebrospinal fluid (CSF). b Stacked bar plot showing the patient distribution of primary tumor of origin of the BrM c Heatmap representing the enrichment scores of seven immune cell types (rows) in 48 BrM patients (columns). d Boxplot representing the IFNγ enrichment score distribution across the three clusters of tumor immune infiltration, as shown in d. Statistically significant differences are shown with an asterisk (P value < 0.05, T-test). All boxplots indicate median (center line), 25th and 75th percentiles (bounds of box), and minimum and maximum (whiskers). e Representative images of low, intermediate, and high CD8 staining by IHC (n = 42 patients). Scale bar, 55 µm. f Regression plots representing the Pearson correlation between CLym (left)/IFNγ signature (middle) enrichment score and CD8 IHC cell density (log2 transformed) (n = 42 patients).

Single cell transcriptome sequencing provides the highest resolution for the detection and monitoring of several different diseases. The identification of clonal T-cells in both metastasis and liquid biopsy is of particular interest. We have shown that the sequencing of T-cell receptors provides a cellular barcode that can be assessed outside of the tumor. Importantly, this approach opens up new avenues for the detection of systemic disease,” concludes Holger Heyn, co-Corresponding Author of this present study.

SourceVall d’Hebron Institute of Oncology

Rubio-Perez C, Planas-Rigol E, Trincado JL, Bonfill-Teixidor E, Arias A, Marchese D, Moutinho C, Serna G, Pedrosa L, Iurlaro R, Martínez-Ricarte F, Escudero L, Cordero E, Cicuendez M, Ruiz S, Parra G, Nuciforo P, Gonzalez J, Pineda E, Sahuquillo J, Tabernero J, Heyn H, Seoane J. (2021) Immune cell profiling of the cerebrospinal fluid enables the characterization of the brain metastasis microenvironment. Nat Commun 12(1):1503. [article].
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