Development of patient-derived tumor models toward understanding disease biology and drug screening

Abstract

Cancer is a complex disease of uncontrolled cell proliferation, which cripples the normal functioning of tissues. According to GLOBOCAN 2020, an estimated 19.3 million new cancer cases and around 10 million cancer deaths have been reported globally, indicative of the increasing cancer burden worldwide. Decades of research have gone into establishing various model systems that can serve as a platform not only for understanding the fundamental molecular basis of cancer but also for translational aspects. Selecting an ideal model system that faithfully reflects the given tumor system is one of the critical challenges faced by researchers. In this regard, model systems derived from patient biopsies serve as a powerful and robust tool for understanding the disease mechanism, preclinical drug testing, and predicting patient response. Moreover, they can be used to study the existing variations in terms of etiology, molecular aspects, and biological responses of cancers in different populations. However, there is a dearth of such tumor models, particularly derived from Indian patients. Therefore, this thesis aimed to develop two different culture-based model systems using liquid (blood) and surgical biopsies (tumor tissue) from advanced-stage breast and oral cancer patients of Indian origin, respectively. In the first part of the thesis, we present a biodegradable polymer—polycaprolactone (PCL)-based novel ex vivo 3D culture system for the expansion of circulating tumor cells (CTCs) derived from breast cancer patients. The second part of the study has dealt with the establishment and detailed characterization of a novel oral cancer cell line derived from the tissue biopsy of an advanced-stage Indian patient with a long-term tobacco chewing habit. Development of ex-vivo 3D culture system for breast cancer patient-derived circulating tumor cells (CTCs) exhibiting differential epithelial-mesenchymal (EM) phenotypes Breast cancer is the most prevalent cancer in women, with 2.3 million new cases and ~6.85 lakh deaths in 2020 worldwide. It is reported that 90% of cancer-associated deaths are related to metastasis —a multi-step process facilitating the spread of tumor cells from the primary site to secondary vital organs. Circulating tumor cells (CTCs) that are shed from the primary tumor into the circulation are the precursors of the metastatic cascade. In this regard, liquid biopsy, which contains CTCs and other tumor-derived markers, has gained much attention in the recent past as a promising clinical tool (both for prognostic and diagnostic purposes). However, the pathophysiological role of CTCs in cancer metastasis is poorly explored due to their rarity in blood, thus making it challenging to establish suitable model systems. Ex vivo culture of CTCs allows the expansion of these rare populations, thus enabling detailed characterization, drug screening, and real-time monitoring during personalized treatment. Nevertheless, the current methods to isolate CTCs are majorly based on pre-enrichment for specific markers (majorly epithelial markers), thereby causing the loss of detection of heterogeneous CTCs (epithelial, mesenchymal, and other types). Furthermore, several of these techniques demand cell fixation, which essentially destroys the scope for further biological/functional characterization of CTCs. Prior studies from our laboratory have established a polymer-based 3D scaffold system in which breast cancer cells form tumoroids and show enhanced metastatic potential mimicking breast cancer progression in vivo. This in-house engineered 3D scaffold system was exploited to develop a newer 3D ex vivo model system to culture CTCs and subsequently to understand their biology, including their heterogeneity and dynamicity. As opposed to the available CTC culturing methods that rely on pre-enrichment for EpCAM+ cells, in this work, we isolated RBC-depleted nucleated cells from the blood of advanced-stage breast cancer patients without any prior enrichment. After that, we cultured them in 3D PCL scaffolds for 14 days. We first confirmed the presence of cells in 3D culture by F-actin and nucleus staining, followed by the identification of CTCs as CK+ and CD45– cells. Using scanning electron microscopy (SEM) and immunophenotyping of pan-laminin, we demonstrated the deposition of extracellular matrix (ECM) on the scaffolds, which potentially aids the attachment of cells to the scaffolds. Detection of Ki67 and 5-bromo-2'-deoxyuridine (BrdU) positive cells revealed active cell proliferation in the 3D scaffolds. Furthermore, we demonstrated that CTCs exhibit intra- and inter-patient heterogeneity in epithelial (E) and mesenchymal (M) phenotypes that are otherwise missed out on marker-based prior enrichment approaches. Thus, the strategy of culturing whole-blood derived CTCs in PCL scaffolds offers a pathophysiologically relevant model for studying CTC biology, the interaction of CTCs with other immune cells, and individualized drug screening. Moreover, this system can be explored further to culture CTCs obtained from other carcinomas. Establishment and characterization of a novel oral cancer cell line of Indian origin exhibiting cancer stem cell-like properties Over the last decade, oral squamous cell carcinoma of the gingivobuccal complex (OSCC-GB) has been the leading cause of mortality in the Indian population amongst all other types of head and neck carcinomas. Interestingly, Indian oral cancer cases show dramatic etiological differences compared to that of the Western countries, primarily due to tobacco chewing habits, tobacco-associated inflammation, and fibrosis. However, given the dearth of available oral cancer cell lines, particularly of Indian origin, there is a dire need for well-characterized oral cancer cell lines to understand the disease mechanism and identify novel drug targets. Thus, we aimed to establish a novel oral cancer cell line from Indian patient origin. In this study, we have established a cell line derived from an Indian patient with a long-term tobacco chewing habit. The cell line is named as IIOC019 after the ethnic origin, institute, cancer site, and sample number —Indian Institute of Science Oral Cancer (IIOC019). It is a spontaneously immortalized novel cell line that has been sub-cultured for over 60 passages. IIOC019 cells exhibit polygonal, cobblestone morphology along with cytokeratin 8 (CK8) expression indicating their squamous epithelial origin. The short tandem repeat (STR) profiling of IIOC019 did not match with any previously reported cell lines, thereby confirming the novelty of this cell line. Karyotype and DNA ploidy analysis revealed aneuploidy with complex chromosomal aberrations. Notably, this cell line formed aggressive tumors when injected into immunocompromised nude mice recapitulating the original patient tumor histopathology. Given that a small subpopulation of cancer stem cells (CSCs) in tumors contributes to cancer maintenance, drug resistance, and disease relapse/recurrence, we next sought to explore the cancer stemness properties exhibited by the newly derived IIOC019 cell line. Using orosphere, side-population, and Aldefluor assays, we identified the presence of a CSC subpopulation within the established cell line. Further, this cell line could be passaged in vivo in serial xenotransplantation assays, suggesting the presence of long-term repopulating stem-like cells within the cell line. These traits collectively make it a suitable model system to study the role of cancer stemness in oral cancer pathology relevant to the Indian cohort. Taken together, the present study has focused on developing two novel solid tumor model systems that can be utilized for understanding the molecular mechanisms of disease progression and preclinical cancer therapeutics.