In-Depth Immune-Checkpoint Analysis of Glioma at Transcript and Protein Level

Glioma is the most common type of malignant brain tumor and is associated with high morbidity and mortality.1 The most common and invasive type of glioma, glioblastoma, has a median survival of around 15 months.1 Over the last decade, there has been very little progress in treating glioma.1 Meanwhile, in other cancers, immunotherapy has emerged as an approach to achieve long-term remission through the targeting and manipulation of the immune system.2 In particular, immune checkpoint inhibition has created breakthroughs in the treatment of many solid tumor types.2,3 These therapies interfere with the tumor immune microenvironment (TIME), which tumor cells can manipulate to try to evade the immune system.3 However, clinical trials found no benefit of therapy targeting the immune checkpoint proteins PD-1 (or its ligand) and CTLA-4 in glioma.2

To develop effective immune checkpoint therapy for glioma, we need to know more about the function of immune checkpoints in the disease and how they interact with each other. To do so, researchers from Sichuan University in Chengdu, China, studied the relationship between RNA expression and protein expression and the colocalization of a selection of cell markers and immune checkpoints in glioma.3


Figure 1. Representative images of glioma samples stained with 7 markers: DAPI (blue), B7-H3 (yellow), PD-L1 (green), GFAP (pink), CD3 (white), IBA-1 (orange), VISTA (red) 3.

RNA Sequencing

To begin, the researchers analyzed glioma samples from two genome atlases and studied the relationship between clinical features and RNA expression of three checkpoint inhibitors (PD-L1, VISTA and B7-H3) and three cell markers (GFAP, IBA-1 and CD3, markers for glioma cells, tumor-associated macrophages (TAMs) and T-cells, respectively).3 The team found that, at the RNA level, all markers except for PD-L1 had higher levels of expression in glioma tissue than normal tissues, and increased expression of PD-L1, B7-H1, CD3 and IBA-1 were significantly associated with increasing tumor grade and predicted worse overall survival.3 Collectively, these findings confirm the importance of immune checkpoints, T cells and TAMs in the development of glioma, the researchers say.

Multiplex Immunofluorescence

To better understand the relationship between phenotype and these transcript-level changes, the team looked at protein expression in glioma samples from 168 patients.3 They performed seven-colour multiplex immunofluorescence (mIF) staining combined with multispectral tissue cytometry (TissueFAXS SPECTRA and StrataQuest), which allowed them not only to quantify levels of expression but analyze the co-expression of immune checkpoints and cell markers in different grades of glioma. They then correlated these findings with clinicopathological features like age, sex, grade and recurrence.

The team found that only B7-H3 was significantly associated with tumor grade, overall survival, recurrence status and age, while expression of CD3 decreased with increasing tumor grade.

The team states that their results highlight B7-H3 as a potential molecular target for immunotherapy and illustrate that it can serve as a prognostic biomarker in glioma by both RNA-seq and mIF.

VISTA, which is known to play a role in downplaying T cell-mediated immune responses, was primarily expressed in tumor cells and TAMs. This strong correlation between VISTA and TAMs in glioma was a novel finding.

The team found that expression of PD-L1, primarily in T cells, was not ubiquitous, and they suggest the lack of relationship between its expression and tumor grade may explain why past attempts to use anti-PD-L1 therapy in glioma have been unsuccessful.

In terms of the interrelationship between the three immune checkpoints, B7-H3 and VISTA expression was positively correlated with PD-L1 expression.

Immune Checkpoint Insights

Collectively, the researchers suggest that their findings indicate that the three immune checkpoints work cooperatively to help glioma tumor cells evade the immune system.

“The correlation of PD-L1, VISTA, and B7-H3 was perhaps consistent with a mutually exclusive expression pattern, which led to immunosuppression of glioma,”3 they write.

They suggest that the co-expression of PD-L1 with T cells, the co-expression of VISTA with TAMs and the relationship between B7-H3, VISTA and PD-L1 in glioma cells likely come together to produce an immunosuppressive environment for glioma. This insight could help inform the development of immunotherapy for glioma.

“We evaluated the clinical and expression characteristics of these immune checkpoints in the sample by mIF and transcriptome data and provided new insights into immunotherapy and the correlation discovery of immune checkpoints,”3 the team concluded in Clinical Immunology.

Multispectral Tissue Cytometry

Parts of the results were obtained by whole-slide  multispectral imaging and image analysis using the TissueFAXS SPECTRA and StrataQuest analysis software (TissueGnostics). TissueFAXS SPECTRA allows the scanning of up to 8 markers in one run and thereby is an ideal solution for in-depth phenotyping. A liquid crystal tunable filter (LCTF) is used as well as an inbuild spectral unmixing algorithm – leading to elimination of bleed through and autofluorescence.

TissueFAXS systems are modular, adaptable and upgradable, allowing you to tailor its capabilities to your needs, including high-throughput multispectral,widefield fluorescence, confocal and brightfield whole-slide imaging. Meanwhile, StrataQuest offers an advanced and powerful image-processing solution with the option of over 100 Apps to assist automation and the Advanced Mode, giving freedom to experienced users to create their own solutions. Contact us to get more details about our products today.

References and Further Reading

  1. Ghouzlani A, Kandoussi S, Tall M, et al. Immune Checkpoint Inhibitors in Human Glioma Microenvironment. Front. Immunol. 2021;12:679425. doi: 10.3389/fimmu.2021.679425.
  2. Ghouzlani A, Rafii S, Karkouri M, et al. The Promising IgSF11 Immune Checkpoint Is Highly Expressed in Advanced Human Gliomas and Associates to Poor Prognosis. Front. Oncol. 2021;10:608609. doi: 10.3389/fonc.2020.608609.
  3. Wang L, Wang Y, He B, et al. Expression and clinical significance of VISTA, B7-H3, and PD-L1 in glioma. Clin Immunol. 2022;245:109178. Doi: 10.1016/j.clim.2022.109178.

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