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'Adult' or 'somatic' stem cells harbor an intrinsic ability to regenerate tissues. Heterogeneity of such stem cells along the gastrointestinal tract yields the known segmental specificity of this organ and may contribute to the pathology of certain enteric conditions. Here we detail technology for the generation of 'libraries' of clonogenic cells from 1-mm-diamter endoscopic biopsy samples from the human gastrointestinal tract. Each of the 150-300 independent clones in a typical stem cell library can be clonally expanded to billions of cells in a few weeks while maintaining genomic stability and the ability to undergo multipotent differentiation to the specific epithelia from which the sample originated. The key to this methodology is the intrinsic immortality of normal intestinal stem cells (ISCs) and culture systems that maintain them as highly immature, ground-state ISCs marked by a single-cell clonogenicity of 70% and a corresponding 250-fold proliferative advantage over spheroid technologies. Clonal approaches such as this enhance the resolution of molecular genetics, make genome editing easier, and may be useful in regenerative medicine, unravelling heterogeneity in disease, and facilitating drug discovery.

High-grade serous ovarian carcinoma (HGSOC) is the most common and lethal form of epithelial ovarian cancer. While HGSOCs respond well to platinum and taxane chemotherapy, the majority of patients eventually relapse with treatment-resistant tumors. This new and ominous resistance property of recurrent tumors is generally thought to be acquired in a process aided by the mutagenic activities of the chemotherapy itself. However, using advanced and robust technology that enables the direct cloning of cancer stem cells (CSCs) from HGSOCs, our study challenges the notion that chemotherapy resistance is acquired. In particular, patient-specific “libraries” of CSCs generated from therapy-naive tumors show that, while the majority of the CSCs are rapidly killed by platinum-taxane treatment, a discrete subset is highly resistant to these drugs to which they had no prior exposure. More telling is that these pre-existing, platinum-taxane resistant CSCs also display a profound “polyresistance” to a wide range of unrelated chemotherapeutic drugs. Importantly, gene expression profiles of polyresistant CSCs are highly uniform within a particular patient and clearly distinct from those of sensitive clones. This finding suggests that resistant clones within a patient carry the same gene signature, suggesting that resistance is a singular and knowable event in each patient. Comparing the resistance gene expression signatures across patients is revealing a very limited set of common pathways involving nuclear receptors that likely hold the key to the polyresistance phenomena. Taken together, our study supports the concept that these poly-resistant variants exist and can be cloned from most if not all HGOC patients. Further, the cloning of these polyresistant cells sets in motion the possibility that such variants can be exploited to identify both key pathways of polyresistance and drugs that specifically and pre-emptively eliminate these clones to prevent onset of recurrent disease. Citation Format: Jingzhong Xie, Yusuke Yamamoto, Audrey-Ann Liew, Bailiang Wang, Rajasekaran Mahalingam, Marcin Duleba, Wei Rao, Suchan Niroula, Kristina Goller, Yanting Zhang, Shan Wang, Amir Jazaeri, Peter Davies, Suzy Torti, Giulio Draetta, Matthew Anderson, Molly Brewer, Christopher Crum, Frank McKeon, Wa Xian. Pre-existing, poly-resistant cancer stem cells in high-grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A78.

Chronic obstructive pulmonary disease (COPD) is a progressive condition of chronic bronchitis, small airway obstruction, and emphysema, and represents a leading cause of death worldwide. While inflammation, fibrosis, mucus hypersecretion, and metaplastic epithelial lesions are hallmarks of this disease, their origins and dependent relationships remain unclear. Here we apply single-cell cloning technologies to lung tissue of patients with and without COPD. Unlike control lungs, which were dominated by normal distal airway progenitor cells, COPD lungs were inundated by three variant progenitors epigenetically committed to distinct metaplastic lesions. When transplanted to immunodeficient mice, these variant clones induced pathology akin to the mucous and squamous metaplasia, neutrophilic inflammation, and fibrosis seen in COPD. Remarkably, similar variants pre-exist as minor constituents of control and fetal lung and conceivably act in normal processes of immune surveillance. However, these same variants likely catalyze the pathologic and progressive features of COPD when expanded to high numbers.

INTRODUCTION: High-grade ovarian cancer (HGOC) shows excellent responses to standard-of-care surgery and paclitaxel/carboplatin therapy only to relapse 6-24 months later with typically resistant disease. While the origin of this recurrent, resistant disease is unclear, most believe it is acquired by the action of chemotherapeutics. Using novel stem cell technology that enables the cloning of cancer stem cells (CSCs) from epithelial cancers, we have generated large libraries of CSCs from multiple cases of HGOC. And while the vast majority of these CSC clones are killed by standard-of-care chemotherapeutic drugs, a minor fraction shows profound resistance not only to paclitaxel/carboplatin but to a wide range of structurally unrelated chemotherapeutic drugs to which these cells had no prior exposure. We describe screens for drugs that selectively target this resistant CSC population. METHODS: Libraries of 10- to 100,000 CSC clones were generated from individual, therapy naïve, HGOC resections using technology we developed for cloning so-called “adult” stem cells from normal columnar epithelia (Wang et al., 2015, Nature, 522, 173). RESULTS: Paclitaxel/carboplatin resistant CSCs were identified in CSC libraries derived from therapy naïve tumors at ratios of 1:50 to 1:300. By copy number variation, these resistant variant clones proved distinct from the bulk of CSCs, and by gene expression analysis varied from sensitive clones by more than 700 differentially expressed genes. Independent resistant clones from the same library clustered with other resistant clones by both copy number variation and gene expression profiles, suggesting the possibility that resistance within a single tumor is dominated by a single type of resistant CSCs. Clones resistant to paclitaxel/carboplatin were screened in a 384-well format against a wide range of experimental drug-like molecules. These pre-existing resistant clones also proved to be profoundly resistant to a large number of structurally unrelated chemotherapeutic drugs. This same screening program identified drugs that act alone or in combination with paclitaxel to eliminate these resistant clones, suggesting a route to personalized medicine for addressing the problem of recurrent disease in HGOC. CONCLUSIONS: Tumors from patients with HGOC possess clonogenic CSCs including variants that are resistant to a broad spectrum of chemotherapeutics to which they have not been exposed. It is likely that such CSCs would survive standard-of-care chemotherapy and contribute to the recurrent disease seen in HGOC. We have identified known and experimental drugs that specifically eliminate these resistant variants and the overall platform represents a potential strategy to addressing the problem of recurrent disease in these patients. Citation Format: Jingzhong Xie, Yusuke Yamamoto, Marcin Duleba, Bailiang Wang, Rajasekaran Mahalingam, Shan Wang, Wei Rao, Suchan Niroula, Clifford Stephan, Peter Davies, Amir Jazaeri, Giulio Draetta, Molly Brewer, Matt L. Anderson, Christopher P. Crum, Frank McKeon, and Wa Xian. PRE-EXISTENCE OF POLY-RESISTANT CANCER STEM CELLS IN HIGH-GRADE OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-115.