αCT1 peptide sensitizes glioma cells to temozolomide in a glioblastoma organoid platform

Glioblastoma (GBM) is the most common form of brain cancer. Even with aggressive treatment, tumor recurrence is almost universal and patient prognosis is poor because many GBM cell subpopulations, especially the mesenchymal and glioma stem cell populations, are resistant to temozolomide (TMZ), the most commonly used chemotherapeutic in GBM. For this reason, there is an urgent need for the development of new therapies that can more effectively treat GBM. Several recent studies have indicated that high expression of connexin 43 (Cx43) in GBM is associated with poor patient outcomes. It has been hypothesized that inhibition of the Cx43 hemichannels could prevent TMZ efflux and sensitize otherwise resistance cells to the treatment. In this study, we use a three‐dimensional organoid model of GBM to demonstrate that combinatorial treatment with TMZ and αCT1, a Cx43 mimetic peptide, significantly improves treatment efficacy in certain populations of GBM. Confocal imaging was used to visualize changes in Cx43 expression in response to combinatorial treatment. These results indicate that Cx43 inhibition should be pursued further as an improved treatment for GBM.


| INTRODUCTION
Glioblastoma (GBM) is an aggressive, terminal cancer in the brain. It is a lethal and heterogeneous disease, and even with maximally aggressive surgery and chemoradiotherapy, median survival for GBM patients is 14.5 months (Stupp et al., 2005). These tumors infiltrate the brain, are surgically incurable, and universally recur.
Upon recurrence, response rates to standard treatment are less than 5%, leading to a median survival of 8 months (Taal et al., 2014).
Despite advances in managing other types of cancer, only four new treatments have been Food and Drug Administration-approved for GBM in the last three decades. A major challenge in translating successful therapies into the clinic is modeling the genetic, epigenetic, and micro-environmental heterogeneity of GBMs, as well as accounting for the blood-brain barrier (BBB) (Rybinski & Yun, 2016). GBMs evolve spontaneously and in response to treatment, making selection of patient-specific therapies a challenge (Malkki, 2016;Sottoriva et al., 2013). Seminal studies employing multiple biopsies of single patients' tumors have shown that multiple distinct GBM molecular subtypes (i.e., classical, proneural, and mesenchymal) exist within the same tumor, although these designations are now questioned due to little clinical improvements based on subtyping (Sottoriva et al., 2013;Tang et al., 2015). Alternatively, glioma stem cell (GSC) subpopulations have arisen as a key cell type of interest due to their ability to evade therapy and drive tumor recurrence (Auffinger et al., 2015;Heimberger et al., 2003;Swartz et al., 2014).
Often, recurring tumors, while still heterogeneous, display increased presence of the mesenchymal and GSC subpopulations which correlate with poor prognosis, heightened inflammation, matrix metalloproteinase expression, and extracellular matrix (ECM) remodeling-factors that further drive tumor progression (Fedele et al., 2019;Segerman et al., 2016).
Precision medicine has largely failed to improve clinical outcomes in GBM. Precision medicine utilizes genomic and molecular profiling of tumors to identify drugs for treatment of patients' tumors. In neuro-oncology, this approach fails to incorporate important contributions of the BBB. The current approach in neuro-oncology is to limit drug selection to the few agents that are known to cross the BBB. However, this may be overly restrictive, particularly given historical examples of agents that have been thought not to be able to penetrate BBB (i.e., rituximab) that are known to be effective against GBM tumor cells (Rubenstein et al., 2013). Thus, precision medicine in neuro-oncology will require additional understanding of how the tumor and surrounding microenvironment influence BBB integrity, BBB permeability, and drug delivery. Yet, due to the inability to assess BBB-tumor interplay more effectively, temozolomide (TMZ), a BBB-permeable alkylating agent that damages DNA and induces tumor cell apoptosis, remains the ubiquitous chemotherapy for GBM patients. Clinically-and recapitulated by our bioengineered tumor organoids-there is often a correlation between clinical biomarkers such as MGMT (O 6 -methylguanine-DNA methyltransferase) promoter methylation and IDH (isocitrate dehydrogenase) mutational status and patient (or organoid) TMZ response (Verhaak et al., 2010). In patients that do not respond to TMZ, regardless of MGMT or IDH status, alternative treatments, including strategies involving TMZ sensitization, need to be investigated.
As noted above, TMZ is the most widely used GBM chemotherapy agent due to its unique ability to pass the BBB.
However, some tumor cells are able to evade TMZ via drug efflux mechanisms that expel TMZ from the cell before DNA is damaged.
Mounting evidence implicates connexin 43 (Cx43) hemichannels in drug efflux underlying chemotherapy-resistance, and Cx43 hemichannel inhibition has been shown to sensitize GSCs to TMZ (Grek et al., 2018;Murphy et al., 2016). However, available evidence comes from simple two-dimensional (2D) cell line cultures lacking the heterogeneity and complexity of tumor and brain physiology.
Another key barrier to clinical translation is that Cx43 hemichannel inhibitors, such as the Cx43-mimetic peptides αCT1 and αCT11, cannot cross the BBB, necessitating local delivery at the tumor site sustained over extended periods (months) (Grek et al., 2018;Murphy et al., 2016). Previous nanoparticle-based delivery approaches have been limited to 2-3 weeks, but sustained release technologies have great potential to increase local sustained delivery of these peptides (Roberts et al., 2020). New model systems that recapitulate three-dimensional (3D) in vivo human brain physiology, support the heterogeneity of GBM, and provide direct experimental manipulation and observation-such as tumor organoids-could be used to evaluate and optimize combinatorial therapies such as this.
Here we evaluate the potential of two Cx43-mimetic peptides, αCT11 and αCT1, to inhibit Cx43 hemichannels and sensitize GSCs and other GBM cell populations to TMZ in a 3D hyaluronic acid (HA) hydrogel-based tumor organoid system. We have previously deployed this system across a variety of tumor types, including mesothelioma, melanoma, lung adenocarcinoma, colorectal carcinoma, sarcoma, appendiceal, adrenocortical carcinoma, and gliomas Maloney et al., 2020;Mazzocchi et al., 2018;Mazzocchi et al., 2019;. The results of the current study demonstrated that αCT11 had no statically significant effect on the viability of TMZtreated organoids. However, combinatorial treatment with TMZ and αCT1 shows increased efficacy in certain GBM cell populations compared to the TMZ-only treatment. Particularly, the GSCs that are often responsible for clinical chemotherapy-resistance, showed a drastic decrease in viability after the combinatorial treatment.
Immunofluorescence microscopy indicated that Cx43 is expressed in all tested cell lines, and αCT1 increases the number of Cx43 aggregates in GBM cell lines that responded to the combinatorial treatment, which may indicate that αCT1 affects Cx43 production as well as hemichannel function. These studies provide an early indication that Cx43 hemichannel inhibition may be an effective therapy to increase TMZ efficacy, sensitizing GBM populations to TMZ with αCT1 could enable remission in patients with lower chance of tumor recurrence. Medium with proliferation supplement, 20 ng/ml epidermal growth factor, 20 ng/ml beta fibroblast growth factor, and 2 µg/ml heparin sulfate in a tissue culture incubator at 37°C with 5% CO 2 .

| Combinatorial TMZ and peptide treatment
TMZ is an anticancer drug that is commonly used in chemotherapy to treat brain cancer. TMZ is an alkylating agent prodrug, delivering a methyl group to purine bases of DNA (Zhang et al., 2012). TMZ was purchased from SelleckChem. Two Cx43 mimetic peptides, ⍺CT1 and ⍺CT11 (LifeTein) were evaluated in this experiment.
Different combinations of TMZ and αCT1 or αCT11 concentrations were tested in each cell line to determine the optimal combination.  Briefly, z-stacks were converted to binary images. Objects were identified using the regionprops3 function. These objects were then filtered by size to remove any background noise and the dead cell count was recorded.

| Organoid viability analysis
2.6 | αCT1 peptide-Cx43 interaction αCT1 peptide conjugated with fluorescein isothiocyanate (FITC-αCT1; LifeTein) was used to visualize the distribution of αCT1 peptide inside the cells during treatment. Four experimental groups were used: control (no drug or peptide), TMZ (100 μM), FITC-αCT1 (100 μM), and combination of 100 μM TMZ and 100 μM FITC-αCT1. Treatment was added 24 h after seeding the organoids. Old media was removed and replaced with fresh media supplemented with TMZ and/or FITC-αCT1 on Day 4. The organoids were fixed with 4% paraformaldehyde (PFA) for 1 h on Day 7. Constructs were stained as described in Section 2.7 for Cx43.

| Immunostaining and confocal imaging
Organoids were fixed for 1 h at room temperature with 4% PFA.
Samples were then blocked for 1 h at room temperature using goat blocking solution (5% normal goat serum with 0.1% w/v Sodium Azide) with 0.1% Triton-X. Primary antibodies (anti-Connexin 43 N-terminal antibody purchased from Sigma-Aldrich), were diluted in goat-blocking solution and samples are incubated overnight at 4°C. Samples were then washed with PBS three times for 5 min each. Secondary antibodies were also diluted in goat-blocking solution and incubated with samples overnight at 4°C. Samples were then washed with PBS three times for 5 min and stained with 4′,6-diamidino-2-phenylindole (DAPI) (NucBlue; Invitrogen) and Allexa-647 conjugated Phalloidin (Invitrogen) per the manufacturer instruction. Imaging was conducted using Nikon A1R HD Confocal Microscope. Expression of Cx43 was quantified using ImageJ.
Confocal images of the red channel, in which the fluorescent signal represents Cx43 expression, were converted to 8-bit greyscale. Background noises and artificial signals were excluded by thresholding pixels from 10 to 200. The threshold areas were then analyzed using Measure Particles. Pixel count, total area, average size, and percentage area of highlighted pixels were compared among all treatment groups.

| Statistical analysis
ATP luminescence data (n = 4) was averaged after subtracting blank values and analyzed in GraphPad Prism. Two-way analysis of variance with confidence intervals of 95% followed by a multiple comparisons test (Tukey) was used to analyze the combinatorial treatment studies.  . For that reason, we have chosen to test the effect of the combinatorial treatments on cells grown in 3D hydrogel constructs instead of on traditional flat 2D cell culture plastic. GBM tumor cells are encapsulated within the hydrogel solution, which is comprised of thiolated-HA, thiolated-gelatin, and polyethylene glycol diacrylate (PEGDA) in a 2:2:1 ratio. Six organoids were generated for each treatment group, including four organoids used for ATP assay and two for LIVE/DEAD imaging. Drug treatment proceeds for 7 days at which point, cell viability is assessed, or the samples are fixed for immunostaining. The hydrogel is also optically transparent which allows for analysis via confocal microscopy. See Figure 1 for a detailed outline of the workflow used in the studies presented here.

| Combinatorial treatment with TMZ and αCT11 does not increase GBM cell killing
To test efficacy of Cx43 targeting in combination with TMZ, we first investigated the effect of αCT11 peptide on drug efficacy. Organoids were treated with combinations of TMZ (0, 10, 100, and 1000 µM) and αCT11 (0, 4, 40, and 400 µM). This media was removed and replaced with fresh media supplemented with drugs on Day 4. Seven days after initiating drug treatment, cell viability was assessed using the CellTiter Glo 3D (Promega) assay. The assay is used to quantify ATP levels in cells and has been previously verified for studying the drug efficacy of 3D cancer organoids (Dominijanni et al., 2021). Live/dead staining was used to qualitatively confirm results from the viability assay. These images can be found in Supporting Information: Figures S1-3. The

| αCT1 successfully sensitizes GBM organoids to TMZ
Like αCT11, αCT1 is a Cx43 mimetic peptide. However, unlike αCT11, it contains a cell penetration sequence that should allow it to better permeate the cells (Jiang et al., 2019). We hypothesized that because of the presence of this additional sequence, it would significantly increase GBM cell sensitivity to TMZ compared to αCT11. We found that when the αCT1 peptide is present at high concentrations (αCT1 = 10 and 100 μM) in A172 and BT169 organoids, viability is considerably decreased compared to the control groups (αCT1 = 0) for several TMZ concentrations CHE ET AL. | 1111 (Figure 3a,c). Surprisingly, αCT1 peptide at a concentration of 10 μM is more effective in the A172 organoids than the highest concentration (100 μM). In the U87 MG organoids, however, the αCT1 had It's also worth noting that even when the TMZ concentration is zero, αCT1 shows a significant decrease in viability in the A172 organoids.
This could indicate that αCT1 is impacting several important cell pathways, not just those related to TMZ efficacy in GBM cells.

| Confocal imaging shows that combination treatment induces changes in Cx43 expression and intracellular distribution
To better understand the mechanism of αCT1 enhanced cell killing, we repeated the treatment in A172, U87, and BT169 cell lines using a fluorescence conjugated αCT1 (FITC-αCT1) combined with TMZ. The interaction between FITC-αCT1 and Cx43 was observed using confocal microscopy. The complete procedure can be found in Sections 2.6 and 2.7.
As shown in Figure 5, Cx43 level did not show significant changes after the TMZ (100 μM), or FITC-αCT1 (100 μM) treatment alone in any of the three cell lines. However, in A172 and BT169 cell lines (Figure 5a,b), the combination of 100 μM TMZ and 100 μM FITC-αCT1 treatment group increased the number of Cx43 aggregates as well as the general amount of Cx43 compared to the U87 F I G U R E 1 Schematic describing the overall workflow of the study. Tumor cells are expanded on culture plastic and passage with Trypsin (1). The cells are then suspended in hydrogel solution. Ten microliters of droplets are pipetted into a well plate and crosslinked with UV light for 2 s (2). After 24 h, drug treatment is started (3). Constructs are then analyzed for viability and Cx43 protein expression (4). cell line, which only exhibits a smaller in the Cx43 aggregates number within the cell bodies (Figure 5c). This observation indicates that FITC-αCT1 combined with TMZ may induce changes in TMZ-related signaling pathways and affect Cx43 protein functions and activities in GBM cells. Quantification of intracellular and secreted Cx43 level in these confocal images also confirms that addition of αCT1 induces increased Cx43 level in A172 and BT169 organoids (Supporting Information: Figure S9).

| Validation of αCT1-TMZ treatment in patient-derived GBM organoids
It has been established that studies using cell lines are limited (Fusenig et al., 2017). Cell lines often consist of a homogeneous population of cells that only represents a subset of the cells found in the heterogenous tumor in the patient. It has also been confirmed that some cell lines, namely the U87 cell line, are genetically and phenotypically different from the original cells obtained from patients many years ago (Allen et al., 2016). To partially address this issue, we used the BT169 cell line which is not immortalized and is cultured in conditions that enhance the formation of a heterogeneous population. Even so, we sought to validate that the combinatorial treatment of αCT1 and TMZ is effective in a patient-derived GBM cell population.
The highest concentration of TMZ (1000 μM) killed most of the cells in our patient-derived cell population even without αCT1 ( Figure 6). However, TMZ only was ineffective at lower concentrations (10 and 100 μM). Combinatorial treatment with αCT1 and TMZ resulted in significant decreases in cell viability in the TMZ 100 μM group. Interestingly, there was also a significant decrease of viability in the αCT1 = 100 μM, TMZ = 0 condition, again indicating that αCT1 also induces other cellular changes in some subsets of GBM cells. 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. (2022) have shown that expression of Cx43 protein in high-grade glioma is higher than other connexins. They also show that high levels of Cx43 messenger RNA 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 consists of αCT1 peptide, which inhibits Cx43 hemichannel function, and TMZ could be used to sensitize GBM to TMZ.

| DISCUSSION
To test this hypothesis, we used a 3-dimensional in vitro culture 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 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 UVcrosslinked hydrogel composed of HA 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 which allowed us to test this novel treatment methodology before working with valuable patient tumor samples. We chose to use three cell lines (A172, U87 MG, and BT169) because they  . 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 chemoresistance. 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). 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. (1999) demonstrated that PTEN heterozygous mice F I G U R E 6 ATP assay data and LIVE/DEAD images of patient-derived GBM cells. (a) patient-derived GBM cell viability after treatment with TMZ at 0, 10, 100, or 1000 μM alone or in combination with αCT1 at concentrations of 0, 1, 10, and 100 μM. (b) Patient-derived GBM cell ATP data responding to TMZ at 1000 μM alone or in combination with αCT1 at concentrations of 0, 1, 10, and 100 μM. (c) LIVE/DEAD images of patient-derived cells after the treatment. Green, Calcein AM-stained viable cells; Red, Ethidium homodimer-1-stained dead cell nuclei. Treatment with lower concentrations of αCT1 (1 and 10 µM) can be found in Supporting Information: Figure S7. Scale bar: 250 μm. ATP, adenosine triphosphate; GBM, glioblastoma; TMZ, temozolomide. display hyperplastic features as well as high tumor incidence, which indicates that PTEN mutation may promote tumor progression.
Nevertheless, Shen et al. (2020) demonstrated that wild-type IDH promotes primary GBM progression. Based on these indicators, we would predict a mixed response to TMZ.
Structural studies have shown that αCT1 directly interacts with a short α-helical sequence along the Cx43 C-terminus called H2 domain (Jiang et al., 2019). αCT11 is a 9-mer peptide variant of αCT1. Because αCT11 lacks the same cell penetration sequence as αCT1, it is likely that αCT11 is not taken up by cells (Jiang et al., 2019). We have shown that the three 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 αCT1 and TMZ at certain concentrations (Figures 3 and 4). Our patient-derived cells also show decreased viability in αCT1 + TMZ combination treatments ( Figure 6). These results indicate that the αCT1 + TMZ combination treatment is effective on certain populations of cells. It is hypothesized that the GSC population present in the BT169 cells and patientderived 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 in A172 and BT169 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 expression increased in A172 and BT169 cell lines significantly only after the combinatorial treatment, whereas the Cx43 expression in the U87 cell line only increased slightly ( Figure 5 and Supporting Information: Figure S9), which could explain why the combinatorial treatment in A172 and BT169 cell lines works better in cell killing. These imaging studies will serve as a springboard for future super-resolution imaging studies to further evaluate the mechanism of action.
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 also tested this combinatorial treatment on a patient-derived tumor population that retains the heterogeneity of GBM, including preservation of GSC 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.
Validation of the combinatorial treatment requires correlation of in vitro responses to in vivo and clinical outcomes. While these initial in vitro studies have shown increased cell death and decreased proliferation, the translation of in vitro dosing scheme to in vivo delivery remains challenging. Similar in vitro GBM studies showed that high concentrations of TMZ ranging from 100 to 4000 µM are used to induce cell apoptosis, however, these high concentrations are hard to achieve in clinical settings due to TMZ toxicity (Hirose et al., 2001a(Hirose et al., , 2001bJohannessen et al., 2013;Ostermann et al., 2004;Roos et al., 2007). Further pharmacokinetic studies are necessary to determine the TMZ dosing scheme to increase in vivo and clinical relevance. Even though TMZ can cross the BBR, the amount of TMZ that successfully reaches the tumor site is likely limited. To address these issues, we plan to use more complex in vitro models to investigate transport across the BBR of both TMZ and αCT1.
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.