Breast cancer is the most common cause of cancer related deaths for women worldwide. In Europe alone, approximately 630,000 women died from breast cancer in 2018. According to current European and American guidelines for treatment, breast cancers are categorized into molecular subtypes based on the expression of hormone receptors PR and ER as well as Her2 and Ki67. The subtypes include Luminal A, Luminal B, Her2+/non-luminal, and basal-like/triple negative, and each subtype has its own course of treatment. For the luminal subtypes, treatment includes endocrine therapy and for the Her2+/non-luminal subtype treatment includes the anti-Her2 drug trastuzumab (Cardoso et al., 2019; Duffy et al., 2017; Howlader et al., 2018; Telli et al., 2019). In addition, for the luminal A subtype there are a variety of new drugs that target specific pathways such as Everolimus for mTOR, Abemaciclib, Palbociclib, Ribociclib for CDK 4/6, and Olaparib for those with BRCA1/2 mutations (Telli et al., 2019). While a variety of options of targeted drugs is promising, they are only relevant for those with the relevant molecular background. This means these solutions are relevant for only a small group, for example only 2–3% of breast cancer patients have BRCA1/2 mutations (Griguolo et al., 2018). In addition, it has been shown that intrinsically tamoxifen resistant cancers often have a large number of gene alterations, making the choice of an alternative therapy anything but straightforward (Hultsch et al., 2018; Poudel et al., 2019). Yet, for the basal-like/triple negative subtype where neither endocrine therapy or anti-Her2 therapy is appropriate, the first line of therapy is classic chemotherapy (Denkert et al., 2017; Yuan et al., 2019) and there is only one approved immunotherapy drug, Atezolizumab (Heimes and Schmidt, 2019). In recent years, it has been demonstrated that targeted therapies that were approved for one indication may be effective in treating others, this is known as drug repurposing. For example, the drug that targets the mTOR pathway, Everolimus that was originally developed as an immunosuppressant drug for transplant patients, is now approved for use in luminal A breast cancer (Neumayer et al., 1999; O’Shaughnessy et al., 2018). New targeted or immunotherapy drugs, that can treat the basal-like/triple negative may already exist.
In addition to endocrine therapy and anti-Her2 drugs, the majority of patients are treated according to a conventional chemotherapy protocol which includes anthracyclines and/or taxanes, with few known biomarkers for predicting response to a given treatment (Cardoso et al., 2019; Denkert et al., 2017; Duffy et al., 2017; Gu et al., 2016). Drug choice is also complicated for targeted therapy, as sometimes there are several drugs that target the same pathway and there are no clear biomarkers to help with the choice of which targeted drug to use. An unguided choice for first line therapy can lead to a delay in effective treatment, and thus risk progression of the disease. Furthermore, each course of treatment is accompanied by suffering due to adverse side effects of chemotherapy (Fotheringham et al., 2019; Jensen, 2006). Even when actionable biomarkers do exist, their diagnostic value is not individual, and some patients may not respond to the predicted therapy. Later, some patients who initially respond to the treatment may develop recurrence and progression of the disease (Bastien et al., 2015; Gu et al., 2016; de Melo Gagliato et al., 2016). New tools to predict drug efficacy for individual patients would extend survival and prevent treatment with ineffective drugs. In summary, new tools for treatment selection are needed to give doctors guidance where no biomarkers currently exist, to predict when patients may not respond to targeted options, to explore opportunities for drug repurposing and to assay drug efficacy where drug toxicity and the risk of side effects is high. Expanding the field in these ways will lead to more effective treatment plans, better quality of life for patients and fewer breast cancer deaths.
One approach to developing new tools to guide individualized treatment selection utilizes chemotherapy sensitivity and resistance assays (CSRAs) which use viable tissue from a tumor to provide predictive information about response to treatment (Burstein et al., 2011; Morgan et al., 2016). These techniques have the benefit of being inexpensive, quick and compatible with high-throughput screening to help guide treatment decision making (Morgan et al., 2016). Initially, assays were developed using cells from tumors in two dimensional (2D) tissue culture (Joo et al., 2009; Ochs et al., 2005). Yet, 2D culture of tumor cells does not accurately mimic the complex relationship between the cells, and the access to oxygen, nutrients and signaling molecules. Furthermore, in the 2D culturing process, there is selection of specific cell populations and the cells undergo significant changes in gene expression (Hickman et al., 2014; I, et al., 2010; Jo et al., 2018; Morgan et al., 2016; Richard et al., 2015; Weiswald et al., 2015). To address these limitations, a number of three dimensional (3D) models have been developed including tumor cells seeded in a matrix of extracellular proteins, multicellular tumor spheroids, organoids, tissue slices, and bioreactors and microfluidic models (Brancato et al., 2020; Grinshpun et al., 2018; Jo et al., 2018; Majumder et al., 2015; Morgan et al., 2016; Mulholland et al., 2018; Orditura et al., 2018; Tanigawa et al., 2016; Weiswald et al., 2015).
Our work focuses on one type of 3D model, namely spheroids, which are scaffold-free multicellular spheres containing cancer cells. These spheroids take into account cell-cell interactions and can facilitate the production of the endogenous extracellular matrix to provide local tumor microenvironment-like conditions. Additional environmental considerations, including the access to nutrients, oxygen, growth factors, metabolites and paracrine factors, are also recapitulated (Brancato et al., 2020; Weiswald et al., 2015). The microtissue culture system from InSphero AG, Switzerland (3D InSight™ system) has been used to create multicellular tumor spheroids from a single cell suspension using a range of tissue culture lines (Anastasov et al., 2015; Falkenberg et al., 2015, 2016; Herter et al., 2017; Rimann et al., 2014; Thoma et al., 2013) and patient derived samples from osteoblastic, chondroblastic and renal cell carcinoma (Amann et al., 2014; Bolck et al., 2019). A similar technique was used by Shuford, et al. who demonstrated that spheroid cultures could be generated from patient-derived ovarian tissue samples with a 90% success rate, with an overall accurate prediction of response to first-line accuracy in 89% of samples. This clearly demonstrates the potential of this method to facilitate treatment choice, as well as to explore non-standard therapies, both of which would prevent a delay in effective treatment, eliminate unnecessary suffering, and ultimately improve prognosis. Still, in the study cited above, 11% of results were falsely negative, with the spheroids indicating no response and the actual patient showing a response. Clearly, this method requires further improvement (Shuford et al., 2019). There are currently no clinically recommended CSRAs by the American Society of Clinical Oncology but their potential, following further development, is acknowledged (Burstein et al., 2011). As more studies show the utility and efficacy of such models, we are confident that they will be integrated into the medical decision-making pipeline.
Here, we present a successful method to grow in vitro spheroids from patient-derived tumor tissue. Our model is demonstrated on breast cancer, and will be further expanded to include additional histotypes in future pro- and retrospective studies.