4. Discussion
Connexin 43 (Cx43) is a member of transmembrane proteins that are responsible in part for intercellular communication via gap junctions. Recent studies have indicated that Cx43 controls the response of GBM cells to TMZ through various mechanisms, such as modulating mitochondrial apoptosis, and activating P13K signaling (Gielen et al., 2013; Munoz et al., 2014; Pridham et al., 2022). Pridham et al.have also shown that expression of Cx43 protein in high-grade glioma is higher than other connexins (Pridham et al., 2022). They also show that high levels of Cx43 mRNA were associated with poor prognosis of GBM patients (Pridham et al., 2022). αCT1 is a mimetic peptide of the Cx43 C-terminal and can inhibit Cx43 hemichannel functions (Montgomery et al., 2021). Therefore, we hypothesized that combinatorial treatment that consists of αCT1 peptide, which inhibits Cx43 hemichannel function, and TMZ could be used to sensitize GBM to TMZ.
To test this hypothesis, we chose to use a 3-dimensional in vitroculture system to model the GBM microenvironment. Traditional 2D cell culture is commonly used in research; however, it has limitations due to inaccurately representing tissue cells in vitro (Costa et al., 2016). The brain’s extracellular matrix (ECM) is a macromolecular network of proteins and polysaccharides that acts as “scaffolding” in which neurons, glia, and other cells of the brain reside. ECM provides cells structural support and has crucial biomechanical and biochemical functions that regulate cell behaviors. The composition of the ECM is specific for each tissue type (Frantz et al., 2010). The brain’s ECM is primarily composed of glycosaminoglycans (e.g., hyaluronan), proteoglycans, and glycoproteins, and contains low levels of fibrous proteins (e.g., collagen, fibronectin, and vitronectin) (Simsa et al., 2021). Hydrogels can be used to mimic the mechanical and biochemical properties of tumor ECM and could serve as a better model than conventional 2D tumor models (Hoarau-Véchot et al., 2018). Thus, in this study, to investigate how GBM responds to the drug candidate treatment, a 3D organoid model was used to mimic brain ECM. Our lab uses a UV-crosslinked hydrogel composed of hyaluronic acid and gelatin to mimic the high HA content in brain ECM and simulate mechanical properties of the native brain tissue. We have previously shown that this hydrogel supports the growth of GBM cells (Maloney et al., 2020; Sivakumar et al., 2017).
Initial experiments conducted in this study were done using several GBM cell lines. While cell lines are not always the best models of native tumors, the cell lines chosen represent distinct GBM tumor populations and allowed us to easily test this novel treatment methodology before working with valuable patient tumor samples. We chose to use 3 cell lines (A172, U87 MG, and BT169) because they represent slightly different populations of GBM tumor cells. The U87 MG cell line was isolated from malignant gliomas from human brain tissue (ATCC HTB-14™). U87 MG cells have an epithelial-like morphology. The A172 cell line was isolated from glioblastoma from human brain tissue (ATCC® CRL-1620TM). While not perfect representations of previously defined clinical GBM subtypes, we have used U87 MG and A172 cell lines previously to represent different populations of GBM cells based on their different genetic profiles (Sivakumar et al., 2020). GSCs have the capacity to self-renew and differentiate into heterogeneous cell populations. This has been indicated as a possible driver of tumor recurrence and chemo-resistance. The BT169 cell line was used to represent a GSC GBM subpopulation. The cells form neutrosphere structures in tissue culture, which is an experimentally defined property of GSC (Ahmed et al., 2013). Thus, therapeutic methods that effectively target this GSC-like population are of utmost interest in our peptide-TMZ study. BT169 is MGMT promoter methylated, EGFR wild-type, PTEN heterozygous mutant, TP53 wild-type, IDH wild-type (ATCC® CRL-3413 ). Previous studies have shown that MGMT is associated with GBM resistance to alkylating agents such as TMZ, and methylation of the MGMT promotor is suggested to sensitize GBM to TMZ (Binabaj et al., 2018; Donson et al., 2007). Cristofano et al., demonstrated that PTEN heterozygous mice display hyperplastic features as well as high tumor incidence, which indicates that PTEN mutation may promote tumor progression (Di Cristofano et al., 1999). Nevertheless, Shen et al., demonstrated that wild-type IDH promotes primary GBM progression. Based on these indicators, we would predict a mixed response to TMZ (Shen et al., 2020).
Structural studies have shown that αCT1 directly interacts with a short α‐helical sequence along the Cx43 C-terminal called H2 domain of Cx43 (Jiang et al., 2019). αCT11 is a 9-mer peptide variant of αCT1. Because αCT11 lacks the same cell penetration sequence as αCT1, it’s likely that αCT11 isn’t taken up by cells (Jiang et al., 2019). We have shown that the 3 GBM cell lines tested do not respond to treatment with αCT11 and TMZ (Figure 2 ). However, some cell lines, A172 and BT169, do respond to combination treatment with αCT11 and TMZ at certain concentrations (Figure 3 and 4 ). Our patient derived cells show decreased viability in αCT1+TMZ combination treatments (Figure 6 ). These results indicate that the combination treatment is effective on certain populations of cells. It is hypothesized that the GSC population present in the BT169 cells and patient derived cell population are sensitive to the combination. Specific population response will be investigated in future studies.
We used high resolution confocal imaging to begin investigating the possible mechanism of action of αCT1 and TMZ in combination. It was observed that in A172 and BT169s that αCT1 without TMZ induced significant changes in cell viability so we were interested to see if any changes would also be observed in this condition. Immunofluorescent imaging shows that the Cx43 aggregates increased in A172 and BT169 cell lines significantly only after the combinatorial treatment, whereas the Cx43 aggregates number in the U87 cell line only increased slightly (Figure 5 ), which could explain why the combinatorial treatment in A172 and BT169 cell lines works better. These imaging studies will serve as a springboard for future super resolution studies to further evaluate the mechanism of action in order to design an effective treatment regimen.
Much of this study utilized tumor cell lines, and these cell lines do not accurately mimic the cellular heterogeneity of GBM. However, they serve as useful tools with which to begin exploring the utility of Cx43 inhibition in GBM. Importantly, we did also include experiments utilized a patient-derived tumor population, that does retain the heterogeneity of GBM, including preservation of glioma stem cell populations, which often contribute to the cell populations that are resistant to TMZ. In future work, we wish to expand our drug studies to patient-derived tumor organoids derived from multiple GBM patients. This will provide us with a better sense of the clinical utility of this treatment approach. In addition, we plan to embed GBM organoids within an in vitromicrofluidic blood-brain barrier model, testing systemic versus local delivery of both TMZ and αCT1. This system will provide a more complex, and physiologically accurate model system of GBM, while retaining the use of human cells.
In conclusion, the studies presented here demonstrate potential to treat GBM using a combinatorial treatment with Cx43 mimetic peptide αCT1 and TMZ. Studies were conducted using 3D tumor organoids which accurately mimic the in vivo tumor microenvironment. The platform allows for high throughput screening of various treatment concentrations and high-resolution imaging to study mechanisms of action. Further studies are under way to optimize the treatment and design effective delivery vehicles.