Western blots reveal expression of CD97 in various GBM cell lines

Tatiana Slepak et al. from the Sylvester Comprehensive Cancer Center at the University of Miami used the Azure 600 imager in their study of CD97's influence in glioblastoma (GBM)^1. This was the first study to analyze the impact of CD97 knockdown, overexpression, and functional mutations in mouse brain xenografts. GBM is highly invasive and known to have high levels of CD97. The new insights will be important for evaluating CD97 as a potential drug target in the treatment of GBM. Due to this correlation, the researchers examined the effects of CD97 on in vitro and in vivo tumor growth and invasion. The teamgenetically modified CD97 levels in patient-derived GBM stem cells and used in vitro assays and xenografts to assess effects of changing CD97 levels on the tumor cells. Slepak et al. discovered a dual-role for CD97 in GBM where the two functional domains of CD97 regulate activity causing either 1) increased cell adhesion or 2) stimulation of tumor cell invasion and proliferation.

What is Glioblastoma? GBM Invasion

GBM is the most prevalent form of brain cancer, accounting for over 47% of all cases. It remains a formidable clinical challenge with no known cure.

GBM invasion characteristics

A defining characteristic of GBM is its aggressive invasion into neighboring brain tissues. This is a trait largely attributed to glioma stem cells, which are highly adaptable cancer cells associated with invasiveness. Although numerous studies have explored the molecular targets impacting GBM invasion, effective pharmacological inhibitors remain elusive.

The potential target: G-protein coupled receptor protein CD97

An intriguing potential target is the adhesion G-protein coupled receptor (GPCR) CD97. CD97 has been found to be increased in other cancers and is associated with increased invasiveness, metastasis, and poor prognosis. Notably, in GBM, CD97 is expressed at high levels compared to normal brain tissue and low-grade gliomas.

After protein folding, CD9 undergoes self-cleavage, resulting in two fragments that remain noncovalently linked: the extracellular N-terminal fragment (NTF) and the intracellular C-terminal fragment (CTF). This unique structure allows CD97 to perform independent functions mediated by the NTF and CTF domains. For instance,? NTF interacts with other cell surface receptors and affects cell adhesion, while ?CTF is involved in G-alpha 12/13 signaling involving the Rho pathway. The Rho pathway is important in cell motility and invasion. Because of the varied functions of CD97, it is involved in many processes: cell adhesion, migration, and invasion.

Previous in vitro studies on CD97

In a 2013 study, CD97 was knocked down in a GBM cell line resulting in a reduction in invasion but no effect on proliferation in vitro. On the other hand, a more recent study from 2022 performed transcriptomic analysis of GBM tumors and found a correlation between high levels of CD97 and activation of the mitogenic pathway. Since both studies were done in vitro, Slepak et al. wanted to examine the effects of CD97 on GBM growth and invasion in vivo. To accomplish this, the research team genetically modified either CD97 levels or function in primary patient-derived GBM stem cells (pdGSCs), performed mouse xenografts, and evaluated the effects.

CD97 and tumorigenesis

Cancer cells migrate either as a single cell or as a collective group. Collective migration requires adhesion molecules to keep the cells together. In GBM, collective migration resembles a finger-like protrusion from a tumor mass or detached cell clusters. In contrast, after adopting a fibroblast-like appearance, single-cell invasion involves individual tumor cells penetrating surrounding tissues. The transformed, single GBM cells are resistant to treatment and detection, which makes eliminating them a challenging feat.

Tumorigenesis: A pathologic process that involves the transformation of normal cells to a neoplastic state and resulting in polyclonal or monoclonal neoplastic cell proliferation. [from NCI] (definition from NIH National Library of Medicine)

Changes in CD97 expression affect invasion in vitro

The researchers prepared for the in vivo studies by doing in vitro studies to first determine the best cells and conditions that modeled GBM invasion for use in the xenografts. Because previous studies had used human derived GBM cell lines. Slepak et al. were curious if changing CD97 levels in primary pdGSCs would have the same effect on their invasion.

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To overexpress CD97 and mimic what is seen in GBM tumors, the team first assessed endogenous CD97 levels in the GBM cell line U87 and three primary pdGSCs from their collection (GBM1, GBM12, and GBM22) via Western blot analysis (Figure 1A). They continued using Western blots to determine which CD97 shRNA most effectively reduced endogenous CD97 protein levels (Figure 1B). The Western blots showed their shRNA-2 was the most effective. Therefore, shRNA-2 would be used for all subsequent experiments.

>> Read more about Western Blotting: Quantitative Westerns: What is the Best Way to Normalize Your Western Blot?

After selecting for successfully transduced cells with puromycin, the effects of reduced CD97 levels on invasion through Matrigel to mimic the extracellular matrix (ECM) in vitro were evaluated. Reduced CD97 levels usually serves as a barrier to cell movement. Remarkably, the CD97-deficient cells displayed enhanced invasive properties in vitro. This difference from the previous study could be due to the longer selection time resulting in a purer population of cells.

To mimic the situation in vivo, where cells need to disrupt both the ECM and cell-to-cell junctions, a human umbilical vein endothelial cell (HUVEC) lawn was used. The team observed the same phenomenon with increased migration (Figure 1D,E). The researchers hypothesized that the cells secreted specific cytokines to migrate through the HUVEC lawn. This process was potentially regulated by CD97 levels and/or the activation state of the CD97 receptor.

To test the theory about the activation state of CD97, Slepak et al. created two functional CD97 mutants for expression in pdGSCs: a constitutively active ΔNTF mutant (activates downstream Gα12/13 signaling) and dominant-negative H436A mutant (inactivates downstream Gα12/13.signaling). The effects of secretions from the CD97 mutants on HUVEC cell-to-cell junctions via confocal microscopy were examined. The research team assessed the type of adherens junctions (AJs) present, noting the active remodeling and reticular types of AJs which favor cancer cell permeability.

A knockdown of CD97 or inhibition of CD97 downstream activity in pdGSCs (represented by the H436A mutant) promotes chemokine secretion that disrupts cell-cell adhesion was revealed. This environment is favorable for tumor invasion and provides a potential mechanism explaining the reason behind how decreased CD97 results in an increase in cell migration.

CD97 on tumor xenograft size in vivo

Having established promising in vitro evidence, the researchers proceeded to assess the relevance of these findings in vivo. They implanted modified pdGSCs into the right frontal lobe of the brains of mice. The pdGSCs had either knocked down CD97, overexpressed CD97, or expressed the CD97 ΔNTF mutant or the CD97 H436A mutant. After six weeks, the tumors were harvested and analyzed via microscopy. It was found that tumors derived from CD97-deficient cells were significantly smaller than wild-type (WT) controls, whereas overexpression of CD97 or its mutants resulted in larger tumors.

To see if the size differences were due to changes in proliferation, the Ki67 index was examined. Expression of the ΔNTF mutant (constitutively active) resulted in increased proliferation, while overexpression of the noncleavable H436A mutant produced large tumors but did not have an increase in proliferation, as measured by Ki67. The researchers hypothesize the results from low basal activity from the receptor that promotes cell survival could be sufficient to drive tumor growth.

Individual tumor cell migration was then measured. GBM tumors can be highly invasive within the brain, but they rarely metastasize outside of the central nervous system. These tumors commonly invade along the white matter tracts and blood vessels. Individual tumor cells have also been known to disseminate into the brain. CD97 knockdown cells were found much farther from the tumor edge than WT cells, which correlated with the in vitro data, indicating primary pdGSCs with reduced CD97 maintain their invasive properties in vivo.

Uncovering CD97s Role in GBM Invasion

To explore the effects of CD97 on GBM invasion, CD97 was knocked down in U87 cells and implanted into the mouse brains. The GBM cell line expresses high levels of CD97 and is known to produce fast-growing, non-invasive tumors. Using GBM cells, the effects of CD97 on invasion could be better determined. While tumor size remained unaffected, CD97 knockdown led to the presence of U87 cells with elongated bodies beyond the original tumor border, which are characteristic of invading cells. These observations underscored that reducing CD97 promoted an invasive phenotype in a subset of cells.

The proposed model for CD97 in GBM

The researchers proposed a model wherein the linking of the NTF with the transmembrane CD97 GPCR domain enhanced cell-to-cell adhesion, facilitating GBM tumor invasion as a collective unit while suppressing individual cell motility and infiltration. Conversely, NTF dissociation activates Rho-mediated growth pathways, promoting tumor cell proliferation and, consequently, tumor growth. While lower CD97 levels can reduce cell viability, because the cancer cells adapt to the changes in CD97 levels, the lower levels can also promote the transformation of the cells that do survive to be highly invasive.

Conclusion

This research done by Slepak et al. sheds valuable light on the role of CD97 in the context of GBM and deepens our understanding of its contributions to tumor invasion and progression. This study proposes a model wherein CD97 plays a pivotal role in regulating collective invasion and individual cell motility, making it a potential drug target for GBM treatment.

The Azure 600 Imager played a crucial role in evaluating CD97 levels within cells during this research through the imaging of Western blots (Figure 1). If you are in search of a reliable imaging tool capable of collecting high-quality images, schedule a demo today to explore the capabilities of our Azure Imagers.