Background

Chemoresistance is described as the ability of cancer cells to circumvent or soldier through therapeutic interventions. Studies have established the existence of a certain population of tumor cells amongst an otherwise heterogeneous milieu of cells that harbor intrinsic or gradually acquire a chemoresistance phenotype. This population of cells is considered to be a potent challenge in oncology research (Zheng, 2017). Furthermore, cases of relapse, cancer dissemination, and subsequent death can directly or indirectly be linked to chemoresistance (Yeldag et al., 2018). Even though ongoing research has improved our knowledge regarding chemoresistance, better systemic and integrative models need to be developed, to tackle this complication head-on.

Considered the deadliest amongst gynecological malignancies, identifying novel targets to combat chemoresistance is currently a significant hurdle in ovarian cancer treatment. Many molecular mechanisms have been attributed to developing resistance against the cytotoxic effects of common chemotherapeutic drugs, such as cisplatin and paclitaxel. These mechanisms comprise a diverse set of complex and often intermingling cell signaling machinery such as the PI3K/Akt/mTOR pathway (Deng et al., 2019) or Wnt/β-catenin signaling (Nguyen et al., 2019), which are deemed essential for acquiring resistance. These pathways are multifactorial and poorly understood, making their targeting ineffective. Cell lines established from EOC patients are routinely utilized to investigate the alteration in these molecular pathways post drug treatments, as well as during development of resistance.

In a clinical setting, the cells gradually begin acquiring resistance to the ongoing course of treatment by expressing specific signaling molecules. These signaling molecules allow the cells to better adapt to the external environment, thereby allowing them to survive and proliferate further. Hence, developing appropriate drug-resistant cellular models to mimic this scenario is essential to understand and discover the signaling cascades associated with chemoresistance regulation (Yan et al., 2007). Drug-resistant cell models are broadly classified into conventional and clinically relevant models. The conventional method involves incremental administration of drugs at low doses with every cycle, whereas the clinically relevant model employs a pulsed treatment, wherein the cells are allowed to grow after every treatment cycle. However, both these traditionally accepted methods have a fair share of advantages and disadvantages. The conventional method creates highly resistant cells, but the heterogeneity observed in these models makes it difficult to identify the underlying molecular players responsible for chemoresistance. Moreover, it is not clinically relevant. The clinically relevant model successfully mimics the treatment cycles administered to a patient in the clinic, but it creates low-level resistance with significantly few molecular changes, thereby making it hard to detect and study. Establishing clinically relevant drug-resistant cell lines involves treating the cells with a fixed drug dosage consistently. This is called the pulse method, and it better encapsulates the biological transition in patients of an otherwise chemo-sensitive cell into gaining chemoresistance. Additionally, the repurposing of existing non-oncology drugs has also emerged as an effective adjuvant in complementing conventional chemotherapeutic regimens (Zhang et al., 2020).

Our laboratory employs a modified version of the pulse method, wherein the parent cells are treated with multiple cycles of incremental drug doses. This leads to the development of a set of isogenic ovarian cancer cell lines resistant to cisplatin, paclitaxel, or both. Furthermore, we also checked the effect of metformin, used for type 2 diabetes treatment, on acquiring chemoresistance in combination with platinum-taxol. An increase in drug concentration after every few treatment cycles is instrumental in the selection and proliferation of only the surviving cells. The isogenic chemoresistant cells obtained from the modified pulse method reflect the clinical scenario and show high resistance indices. Additionally, they also allow the investigator to accurately identify the key molecular players responsible for attaining this chemoresistant phenotype. Though we majorly narrated our methods for A2780 and OAW42 cell lines, these methods can be adapted for any cell line of various cancer origins.