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
(O6-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 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
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 αCT11 had no statically significant effect on the
viability of TMZ treated 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.