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.