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\begin{document}

\title{\selectlanguage{greek}α\selectlanguage{english}CT1 Peptide Sensitizes Glioma Cells to Temozolomide in a Glioblastoma
Organoid Platform\selectlanguage{english}}



\author[1]{Jingru Che}%
\author[1]{Thomas J. DePalma}%
\author[1]{Hemamylammal Sivakumar}%
\author[1]{Louisa S. Mezache}%
\author[2]{Miranda M. Tallman}%
\author[3]{Monica Venere}%
\author[1]{Katelyn Swindle-Reilly}%
\author[1]{Rengasayee Veeraraghavan}%
\author[1]{Aleksander Skardal}%
\affil[1]{The Ohio State University}%
\affil[2]{The Ohio State University Wexner Medical Center}%
\affil[3]{The Ohio State University Comprehensive Cancer Center}%


\vspace{-1em}



  
  \date{May 26, 2024}


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\selectlanguage{english}
\begin{abstract}
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 3-dimensional organoid model of GBM to demonstrate that
combinatorial treatment with TMZ and \selectlanguage{greek}α\selectlanguage{english}CT1, a Cx43 mimetic peptide,
significantly improves treatment efficacy in certain populations of GBM.
Confocal imaging was used to analyze 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.%
\end{abstract}%



\sloppy


\textbf{1. 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 4 new treatments have been FDA-approved for
GBM in the last 3 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 (MMP) expression, and 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\textsuperscript{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 \selectlanguage{greek}α\selectlanguage{english}CT1 and \selectlanguage{greek}α\selectlanguage{english}CT11, 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 3D \emph{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, \selectlanguage{greek}α\selectlanguage{english}CT11 and
\selectlanguage{greek}α\selectlanguage{english}CT1, to inhibit Cx43 hemichannels and sensitize GSCs and other GBM
cells populations to TMZ in a 3D hyaluronic acid (HA) and collagen
hydrogel-based tumor organoid system that we have deployed across a
variety of tumor types, including mesothelioma, melanoma, lung
adenocarcinoma, colorectal carcinoma, sarcoma, appendiceal,
adrenocortical carcinoma, and gliomas (Forsythe et al., 2019; Maloney et
al., 2020; Mazzocchi et al., 2019; Mazzocchi et al., 2018; Votanopoulos,
Forsythe, et al., 2019; Votanopoulos, Mazzocchi, et al., 2019;
Votanopoulos \& Skardal, 2020). The results of the current study
demonstrated that \selectlanguage{greek}α\selectlanguage{english}CT11 had no statically significant effect on the
viability of TMZ treated organoids. However, combinatorial treatment
with TMZ and \selectlanguage{greek}α\selectlanguage{english}CT1 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 \selectlanguage{greek}α\selectlanguage{english}CT1 increases the number of Cx43 aggregates in
GBM cell lines that responded to the combinatorial treatment, which may
indicate that \selectlanguage{greek}α\selectlanguage{english}CT1 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 \selectlanguage{greek}α\selectlanguage{english}CT1 could enable
remission in patients with lower chance of tumor recurrence.

\textbf{2. Materials and Methods}

\emph{2.1. Cell Culture-GBM Cell Lines}

There are multiple distinct subpopulations that have been shown to
respond differently to radiation and treatment with TMZ (Lee, 2016).
Three GBM cell lines were employed in order to compare how different
populations of cells respond to the treatment in GBM organoids. GBM cell
lines U-87 MG (ATCC\selectlanguage{ngerman}\textsuperscript{®} HTB-14\textsuperscript{TM}), A172
(ATCC\textsuperscript{®} CRL-1620\textsuperscript{TM}) (obtained from
ATCC, Manassas, VA), and BT169
(ATCC\textsuperscript{®}CRL-3413\selectlanguage{english}\textsuperscript{\textbf{}} ) were
employed. Each cell line was cultured separately. U-87 MG and A172
cancer cell lines were cultured in high glucose Dulbecco's Modified
Eagle Medium (DMEM) with 10\% fetal bovine serum (FBS), 1\% L-glutamine,
and 1\% penicillin/streptomycin in a tissue culture incubator at 37degC
with 5\% CO\textsubscript{2}. B169 cell line was cultured in NeuroCult
Basal Medium with proliferation Supplement, 20 ng/mL EGF, 20 ng/mL \selectlanguage{greek}β-\selectlanguage{english}FGF
and 2 \selectlanguage{greek}µ\selectlanguage{english}g/mL heparin sulfate in a tissue culture incubator at 37\selectlanguage{ngerman}°C with
5\% CO\textsubscript{2}.

\emph{2.2. Patient-Derived GBM Cells}

GBM 3691 cells were obtained as de-identified specimens that were
initially acquired as primary human brain tumor patient specimens in
accordance with appropriate, approved Institutional Review Board (IRB)
protocols. GBM 3691 was a kind gift from Dr. Jeremy Rich (University of
Pittsburgh; IRBs from Duke University and The Cleveland Clinic;
transferred via MTAs). Cells were cultured in suspension at 37°C at 5\%
CO\textsubscript{2} in Neurobasal media (minus phenol red; Gibco) with
added B27 (minus Vitamin A; Gibco), human fibroblast growth factor-2 (10
ng/mL; Miltenyi Biotec), human epidermal growth factor (10 ng/mL;
Miltenyi Biotec), L-glutamine (2 mM; Gibco), sodium pyruvate (1 mM;
Gibco), and penicillin/streptomycin (100 I.U./ml/100 ug/ml; Gibco).
Prior to seeding within hydrogels, cell aggregates are dissociated into
a single cell suspension using TrypLE (Gibco).

\emph{2.3. GBM Organoid Fabrication}

Bioengineered hydrogels are a widely used tool in the tissue engineering
field. They serve as a biomimetic scaffold that supports cells and can
deliver a wide range of bioactive agents to the embedded cells. The
hydrogel used in these studies is composed of thiolated-hyaluronic acid
(HA), thiolated-gelatin, and polyethylene glycol diacrylate (PEGDA)
dissolved in water containing 0.1\% w/v photoinitiator (Irgacure 2959)
to a final concentration of 1\%w/v. The 3 components are combined in a
2:2:1 ratio (HA:Gelatin:PEGDA) by volume. Cells are then suspended
within the solution at a concentration of 5 million cells/ml. A 10 \selectlanguage{greek}μ\selectlanguage{english}L
droplet of hydrogel solution is pipetted onto each well of a PDMS coated
48-well resulting in organoid constructs containing 50,000 cells per
organoid. The organoids are then cured with 365nm wavelength UV light
for 2 seconds.

\emph{2.4. Combinatorial Temozolomide and Peptide Treatment}

Temozolomide (TMZ) is an anti-cancer 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 (Houston, TX). Two Cx43 mimetic
peptides, [?]CT1 and [?]CT11 (LifeTein, Somerset, NJ) were evaluated in this
experiment. Different combinations of TMZ and \selectlanguage{greek}α\selectlanguage{english}CT1 or \selectlanguage{greek}α\selectlanguage{english}CT11
concentrations were tested in each cell line to determine the optimal
combination. Organoids are treated with combinations of TMZ (0, 10, 100,
or 1000 \selectlanguage{greek}μ\selectlanguage{english}M) and \selectlanguage{greek}α\selectlanguage{english}CT11 (0, 4, 40, 400 \selectlanguage{greek}μ\selectlanguage{english}M) or \selectlanguage{greek}α\selectlanguage{english}CT1 peptide (0, 1, 10, 100
\selectlanguage{greek}μ\selectlanguage{english}M) for 7 days, with the drugs and peptide replenished on day 4. TMZ and
peptide were dissolved in media and added directly to the organoids.

\emph{2.5. Organoid Viability Analysis}

After the organoids were treated for 7 days, the viability was assessed
by Live/Dead imaging and quantitatively validated with ATP Luminescent
assay (Cell Titer-Glo 3D\selectlanguage{ngerman}\textsuperscript{®}, Promega). Luminescence
level is proportional to the number of viable cells, thus reflects the
level of activity in an organoid. The assay was conducted per the vendor
protocol. Briefly, media was removed from the well and replaced with 200
\selectlanguage{greek}μ\selectlanguage{english}L of assay solution (100 \selectlanguage{greek}μ\selectlanguage{english}L of CellTiter-Glo\selectlanguage{ngerman}® 3D Reagent mixed with 100
\selectlanguage{greek}μ\selectlanguage{english}L DMEM). The plate was then mixed vigorously on a plate shaker for 5
minutes at 100 rpm followed by incubation at room temperature for an
additional 25 minutes. 180 \selectlanguage{greek}μ\selectlanguage{english}L of the mixture from each well was
transferred to a white flat bottom Polystyrene 96 well plate.
Luminescence was measured using a microplate reader (Varioskan LUX,
ThermoFisher).

LIVE/DEAD staining was performed on day 7 of organoid culture. Calcein
AM (excitation and emission \textasciitilde{}495/515 nm, 1:2000
dilution) and Ethidium Homodimer-1 (EthD-1) (excitation/emission
~528/617 nm, 1:500 dilution) were dissolved in 200 \selectlanguage{greek}μ\selectlanguage{english}L of a PBS and DMEM
mixture (1:1), and introduced to each organoid with a 30-minute culture.
Fluorescent imaging was performed using Nikon A1R Confocal live cell
confocal microscope (CMIF, OSUCCC). Z-Stacks were obtained for each
organoid and presented as maximum intensity projections.

\selectlanguage{greek}\emph{2.6. αCT1 Peptide-Cx43 Interaction}\selectlanguage{english}

\selectlanguage{greek}α\selectlanguage{english}CT1 peptide conjugated with Fluorescein isothiocyanate (FITC- \selectlanguage{greek}α\selectlanguage{english}CT1;
LifeTein) was used to visualize the distribution of \selectlanguage{greek}α\selectlanguage{english}CT1 peptide inside
the cells during treatment. Four experimental groups were used: control
(no drug or peptide), TMZ (100 \selectlanguage{greek}μ\selectlanguage{english}M), FITC-\selectlanguage{greek}α\selectlanguage{english}CT1 (100 \selectlanguage{greek}μ\selectlanguage{english}M), and combination
of 100 \selectlanguage{greek}μ\selectlanguage{english}M TMZ and 100 \selectlanguage{greek}μ\selectlanguage{english}M FITC-\selectlanguage{greek}α\selectlanguage{english}CT1. Treatment was added 24 hours after
seeding the organoids and replenished on day 4, the organoids were fixed
with 4\% PFA for 1 hour on day 7. Constructs were stained as described
in section 2.7 for Cx43.

\emph{2.7. Immunostaining and Confocal Imaging}

Organoids were fixed for 1 hour at room temperature with 4\% PFA.
Samples were then blocked for 1 hour 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\selectlanguage{ngerman}°C. Samples were then
washed with PBS 3 times for 5 minutes each. Secondary antibodies were
also diluted in goat blocking solution and incubated with samples
overnight at 4°C. Samples were then washed with PBS 3 times for 5
minutes and stained with DAPI (NucBlue, Invitrogen) and Allexa-647
conjugated Phalloidin (Invitrogen) per the manufacturer instruction.
Imaging was conducted using Nikon A1R HD Confocal Microscope.

\emph{2.8. Statistical Analysis}

ATP luminescence data (n=4) was averaged after subtracting blank values
and analyzed in GraphPad Prism. Two-Way ANOVA with confidence intervals
of 95\% followed by a multiple comparisons test (Tukey) was used to
analyze the combinatorial treatment studies.

\textbf{3. Results}

\emph{3.1. 3D GBM Hydrogel Organoid Fabrication and Drug Treatment
Workflow}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 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. 10mL
droplets are pipetted into a well plate and crosslinked with UV light
for 2 seconds (2). After 24 hours, drug treatment is started (3).
Constructs are then analyzed for viability and Cx43 protein expression
(4).

Previous work from our lab and others has shown that cancer cells grown
in 3D hydrogels retain more properties of \emph{in vivo} tumor cells
(Devarasetty et al., 2020; Forsythe et al., 2019; Mazzocchi et al.,
2018; Votanopoulos, Forsythe, et al., 2019; Votanopoulos, Mazzocchi, et
al., 2019). 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 2-dimension 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 \textbf{Figure 1} for a
detailed outline of the workflow used in the studies presented here.

\selectlanguage{greek}\emph{3.2. Combinatorial treatment with TMZ and αCT11 does not increase
GBM cell killing.}\selectlanguage{english}

To test efficacy of Cx43 targeting in combination with TMZ, we first
investigated the effect of \selectlanguage{greek}α\selectlanguage{english}CT11 peptide on drug
efficacy\emph{.}Organoids were treated with combinations of TMZ (0, 10,
100, 1000 \selectlanguage{greek}µ\selectlanguage{english}M) and \selectlanguage{greek}α\selectlanguage{english}CT11 (0, 4, 40, 400 \selectlanguage{greek}µ\selectlanguage{english}M) with the drug and media
replenished after 4 days. After combinatorial treatment of GBM organoids
for 7 days. 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\textbf{Supplemental Figures 1-3} . The GBM organoids were
grouped by three concentrations of TMZ (10, 100, or 1000 \selectlanguage{greek}µ\selectlanguage{english}M) and a
control group (TMZ = 0), and the organoid viability is compared across
three concentrations of \selectlanguage{greek}α\selectlanguage{english}CT11 (4, 40, or 400 \selectlanguage{greek}µ\selectlanguage{english}M) and a control group
({[}\selectlanguage{greek}α\selectlanguage{english}CT11{]} = 0) in each TMZ subgroup.

The viability of all 3 GBM cell lines decreased as TMZ concentration
increased (\textbf{Figure 2} ). In A172 and BT169 organoids, there is no
statistically significant difference between cells treated with TMZ
alone and \selectlanguage{greek}α\selectlanguage{english}CT11 (\textbf{Figure 2A and 2C} ). The U87 organoids showed
significantly decreased viability in the TMZ 10 \selectlanguage{greek}µ\selectlanguage{english}M + \selectlanguage{greek}α\selectlanguage{english}CT11 4 \selectlanguage{greek}µ\selectlanguage{english}M
condition, but in no other condition (\textbf{Figure 2B} ).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\selectlanguage{greek}\textbf{Figure 2. ATP assay results of cell lines treated with αCT11 and
TMZ}\selectlanguage{english} .\textbf{A} , A172 cell line viability after treatment with TMZ at
0, 10, 100, or 1000 \selectlanguage{greek}μ\selectlanguage{english}M alone or in combination with \selectlanguage{greek}α\selectlanguage{english}CT11 at
concentrations of 0, 4, 40, 400 \selectlanguage{greek}μ\selectlanguage{english}M .\textbf{B} , U87 cell line ATP data
viability after treatment with TMZ at 0, 10, 100, 1000 \selectlanguage{greek}μ\selectlanguage{english}M alone or in
combination with \selectlanguage{greek}α\selectlanguage{english}CT11 at concentrations of 0, 4, 40, 400 \selectlanguage{greek}μ\selectlanguage{english}M .\textbf{C}
, BT169 cell line viability after treatment with TMZ at 0, 10, 100, 1000
\selectlanguage{greek}μ\selectlanguage{english}M alone or in combination with \selectlanguage{greek}α\selectlanguage{english}CT11 at concentrations of 0, 1, 10, 100
\selectlanguage{greek}μ\selectlanguage{english}M .

\selectlanguage{greek}\emph{3.3. αCT1 successfully sensitizes GBM organoids to temozolomide}\selectlanguage{english}

Like \selectlanguage{greek}α\selectlanguage{english}CT11, \selectlanguage{greek}α\selectlanguage{english}CT1 is a Cx43 mimetic peptide. However, unlike \selectlanguage{greek}α\selectlanguage{english}CT11, 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 \selectlanguage{greek}α\selectlanguage{english}CT11. Using an ATP
assay to quantify cell viability, we found that when the \selectlanguage{greek}α\selectlanguage{english}CT1 peptide is
present at high concentrations (\selectlanguage{greek}α\selectlanguage{english}CT1 = 10 and 100 \selectlanguage{greek}μ\selectlanguage{english}M) in A172 and BT169
organoids, viability is considerably decreased compared to the control
groups (\selectlanguage{greek}α\selectlanguage{english}CT1 = 0) for several TMZ concentrations (\textbf{Figure 3A and
3C} ). Surprisingly, \selectlanguage{greek}α\selectlanguage{english}CT1 peptide at a concentration of 10 \selectlanguage{greek}μ\selectlanguage{english}M is more
effective in the A172 organoids than the highest concentration (100 \selectlanguage{greek}μ\selectlanguage{english}M).
In the U87 MG organoids, however, the \selectlanguage{greek}α\selectlanguage{english}CT1 had little influence on cell
growth (\textbf{Figure 3B} ).

The LIVE/DEAD images (\textbf{Figure 4} ) show comparable results as the
ATP assay. The highest concentration of TMZ (1000 \selectlanguage{greek}µ\selectlanguage{english}M) combined with
highest concentration of \selectlanguage{greek}α\selectlanguage{english}CT1 (100 \selectlanguage{greek}µ\selectlanguage{english}M) indicates the most significant
cell killing in A172 and BT169 cell lines (\textbf{Figure 4A and 4C} ).
This combination of TMZ and \selectlanguage{greek}α\selectlanguage{english}CT1 treatment group, however, did not
increase cell killing efficacy in the U87 cell line, which is consistent
with the results of the ATP assay (\textbf{Figure 4B} ). LIVE/DEAD
images of combinatorial treatment with TMZ and lower concentrations of
\selectlanguage{greek}α\selectlanguage{english}CT1 (1 and 10 \selectlanguage{greek}µ\selectlanguage{english}M) are shown in \textbf{Supplemental Figures 4-6} .

This finding indicates that the combinatorial treatment with TMZ and
\selectlanguage{greek}α\selectlanguage{english}CT1 may only be effective in certain GBM tumor populations. It's also
worth noting that even when the TMZ concentration is zero, \selectlanguage{greek}α\selectlanguage{english}CT1 shows a
significant decrease in viability in the A172 organoids. This could
indicate that \selectlanguage{greek}α\selectlanguage{english}CT1 is impacting several important cell pathways, not
just those related to TMZ efficacy in GBM cells.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\selectlanguage{greek}\textbf{Figure 3. ATP assay results of cell lines treated with αCT1 and
TMZ.}\selectlanguage{english} \textbf{A} , A172 cell line viability after treatment with TMZ at
0, 10, 100, or 1000 \selectlanguage{greek}μ\selectlanguage{english}M alone or in combination with \selectlanguage{greek}α\selectlanguage{english}CT11 at
concentrations of 0, 1, 10, 100 \selectlanguage{greek}μ\selectlanguage{english}M. \textbf{B} , U87 cell line ATP data
responding t viability after treatment with o TMZ at 0, 10, 100, 1000 \selectlanguage{greek}μ\selectlanguage{english}M
alone or in combination with \selectlanguage{greek}α\selectlanguage{english}CT1 at concentrations of 0, 1, 10, 100
\selectlanguage{greek}μ\selectlanguage{english}M.\textbf{C} , BT169 cell line viability after treatment with TMZ at 0,
10, 100, 1000 \selectlanguage{greek}μ\selectlanguage{english}M alone or in combination with \selectlanguage{greek}α\selectlanguage{english}CT1 at concentrations of
0, 1, 10, 100 \selectlanguage{greek}μ\selectlanguage{english}M.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\selectlanguage{greek}\textbf{Figure 4. LIVE/DEAD images of three tested cell lines with
different combinations of αCT1 (0 and 100 μM) and TMZ (0, 10, 100, 1000
μM). A,}\selectlanguage{english} LIVE/DEAD images of A172. \textbf{B} , LIVE/DEAD images of
U87.\textbf{C} , LIVE/DEAD images of BT169. Green -- Calcein AM-stained
viable cells; Red -- Ethidium homodimer-1-stained dead cell nuclei.
Scale bar -- 250 \selectlanguage{greek}μ\selectlanguage{english}m. LIVE/DEAD images of samples treated with 1 and 10
\selectlanguage{greek}μ\selectlanguage{english}M \selectlanguage{greek}α\selectlanguage{english}CT1 can be found in Supplemental Figures 4-6.

\emph{3.4 Confocal imaging shows that combination treatment induces
changes in Cx43 expression and intracellular distribution.}

To better understand the mechanism of \selectlanguage{greek}α\selectlanguage{english}CT1 enhanced cell killing, we
repeated the treatment in A172, U87, and BT169 cell lines using a
fluorescence conjugated \selectlanguage{greek}α\selectlanguage{english}CT1 (FITC-\selectlanguage{greek}α\selectlanguage{english}CT1) combined with TMZ. The
interaction between FITC-\selectlanguage{greek}α\selectlanguage{english}CT1 and N-terminal Cx43 was visualized under
the confocal microscope. The complete procedure can be found in sections
2.6 and 2.7.

As shown in \textbf{Figure 5} , Cx43 level did not show significant
changes after the TMZ (100 \selectlanguage{greek}μ\selectlanguage{english}M), or FITC-\selectlanguage{greek}α\selectlanguage{english}CT1 (100 \selectlanguage{greek}μ\selectlanguage{english}M) treatment in any
of the three cell lines. However, in A172 and BT169 cell lines
(\textbf{Figure 5A and 5B} ), the combination of 100 \selectlanguage{greek}μ\selectlanguage{english}M TMZ and 100 \selectlanguage{greek}μ\selectlanguage{english}M
FITC-\selectlanguage{greek}α\selectlanguage{english}CT1 treatment group significantly increased the number of Cx43
aggregates as well as the Cx43 signal intensity compared to the U87 cell
line, which only exhibits a slight increase in the Cx43 aggregates
number within the cell bodies (\textbf{Figure 5C} ). This observation
indicates that FITC-\selectlanguage{greek}α\selectlanguage{english}CT1 combined with TMZ may induce changes in TMZ-
related signaling pathways and affect Cx43 protein functions and
activities in GBM cells.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5. Immunofluorescence staining of GBM cells after
treatment. A} , A172 cells after treatment in four conditions: control
(no drug or peptide), TMZ (100 \selectlanguage{greek}μ\selectlanguage{english}M), FITC-\selectlanguage{greek}α\selectlanguage{english}CT1 (100 \selectlanguage{greek}μ\selectlanguage{english}M), and combination
of 100 \selectlanguage{greek}μ\selectlanguage{english}M TMZ and 100 \selectlanguage{greek}μ\selectlanguage{english}M FITC-\selectlanguage{greek}α\selectlanguage{english}CT1. \textbf{B} , BT169 cells after
treatment in four conditions. \textbf{C} , U87 cells after treatment in
four conditions. Blue, DAPI; Green, FITC- \selectlanguage{greek}α\selectlanguage{english}CT1; Red, N-terminal Cx43;
Gray, Phalloidin.

\selectlanguage{greek}\emph{3.5. Validation of αCT1-temozolomide treatment in patient-derived
glioblastoma organoids}\selectlanguage{english}

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 \selectlanguage{greek}α\selectlanguage{english}CT1
and TMZ is effective in a patient derived GBM cell population.

The highest concentration of TMZ (1000 \selectlanguage{greek}μ\selectlanguage{english}M) killed most of the cells in
our patient-derived cell population even without \selectlanguage{greek}α\selectlanguage{english}CT1 (\textbf{Figure 6}
). However, TMZ only was ineffective at lower concentrations (10 \selectlanguage{greek}μ\selectlanguage{english}M and
100 \selectlanguage{greek}μ\selectlanguage{english}M). Combinatorial treatment with \selectlanguage{greek}α\selectlanguage{english}CT1 and TMZ resulted in
significant decreases in cell viability in the TMZ 100 \selectlanguage{greek}μ\selectlanguage{english}M group.
Interestingly, there was also a significant decrease of viability in the
\selectlanguage{greek}α\selectlanguage{english}CT1=100 \selectlanguage{greek}μ\selectlanguage{english}M, TMZ=0 condition, again indicating that \selectlanguage{greek}α\selectlanguage{english}CT1 also induces
other cellular changes in some subsets of GBM cells.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 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 \selectlanguage{greek}μ\selectlanguage{english}M alone or in combination with \selectlanguage{greek}α\selectlanguage{english}CT1 at
concentrations of 0, 1, 10, 100 \selectlanguage{greek}μ\selectlanguage{english}M. \textbf{B} , patient derived GBM
cell ATP data responding to TMZ at 1000 \selectlanguage{greek}μ\selectlanguage{english}M alone or in combination with
\selectlanguage{greek}α\selectlanguage{english}CT1 at concentrations of 0, 1, 10, 100 \selectlanguage{greek}μ\selectlanguage{english}M. \textbf{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 \selectlanguage{greek}α\selectlanguage{english}CT1 (1 and 10 \selectlanguage{greek}µ\selectlanguage{english}M) can be
found in Supplemental Figure 7. Scale bar -- 250 \selectlanguage{greek}μ\selectlanguage{english}m.

\textbf{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 \emph{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). \selectlanguage{greek}α\selectlanguage{english}CT1 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 \selectlanguage{greek}α\selectlanguage{english}CT1 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 \emph{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 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-V\selectlanguage{ngerman}échot 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\selectlanguage{english}).
U87 MG cells have an epithelial-like morphology. The A172 cell line was
isolated from glioblastoma from human brain tissue (ATCC(r)
CRL-1620\textsuperscript{TM}).~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\textsuperscript{(r)} CRL-3413\selectlanguage{english}\textsuperscript{\textbf{}} ).
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 \selectlanguage{greek}α\selectlanguage{english}CT1 directly interacts with a short
\selectlanguage{greek}α\selectlanguage{english}-\selectlanguage{english}helical sequence along the Cx43 C-terminal called H2 domain of Cx43
(Jiang et al., 2019). \selectlanguage{greek}α\selectlanguage{english}CT11 is a 9-mer peptide variant of \selectlanguage{greek}α\selectlanguage{english}CT1. Because
\selectlanguage{greek}α\selectlanguage{english}CT11 lacks the same cell penetration sequence as \selectlanguage{greek}α\selectlanguage{english}CT1, it's likely that
\selectlanguage{greek}α\selectlanguage{english}CT11 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 \selectlanguage{greek}α\selectlanguage{english}CT11 and
TMZ (\textbf{Figure 2} ). However, some cell lines, A172 and BT169, do
respond to combination treatment with \selectlanguage{greek}α\selectlanguage{english}CT11 and TMZ at certain
concentrations (\textbf{Figure 3 and 4} ). Our patient derived cells
show decreased viability in \selectlanguage{greek}α\selectlanguage{english}CT1+TMZ combination treatments
(\textbf{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 \selectlanguage{greek}α\selectlanguage{english}CT1 and TMZ in combination. It was
observed that in A172 and BT169s that \selectlanguage{greek}α\selectlanguage{english}CT1 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
(\textbf{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 \emph{in
vitro}microfluidic blood-brain barrier model, testing systemic versus
local delivery of both TMZ and \selectlanguage{greek}α\selectlanguage{english}CT1. 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 \selectlanguage{greek}α\selectlanguage{english}CT1 and
TMZ. Studies were conducted using 3D tumor organoids which accurately
mimic the \emph{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.

\textbf{Acknowledgements:}

A.S acknowledges funding from NIH grants R21CA229027 and R21CA263137,
the Pelotonia Foundation, and the Ohio State University Comprehensive
Cancer Center. TJD and MMT acknowledge support from the Pelotonia
Graduate Fellowship Program. JC acknowledges support from the OSU
College of Engineering Undergraduate Research Distinction Program. MV
acknowledges funding from a Research Scholar Grant RSG-18-066-01-TBG
from the American Cancer Society, NIH grant R21CA250118, and funds from
The Ohio State University Comprehensive Cancer Center/Department of
Radiation Oncology The authors thank Dr. Jeremy Rich (University of
Pittsburgh) for the kind gift of the GBM 3691.

\textbf{Conflicts of Interest:}

The authors have no conflicts of interest to disclose.

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