Background: The new class of "CFTR modulator" drugs restore function to mutant channels in cystic fibrosis (CF) patients and lead to improvements in body-mass index and lung function especially in younger patients. While it is anticipated that early treatment with CFTR modulators will significantly delay the onset of advanced lung disease, lung neutrophils and inflammatory cytokines remain high in modulator-treated CF patients with established lung disease, underscoring the need to identify and ultimately target the source of this inflammation in CF lung. Methods: Stem cell cloning technology developed to resolve pathogenic stem cell heterogeneity in COPD lung was applied to lungs of cystic fibrosis patients (3 homozygous CFTR-F508D; 1 CFTR-F508D/L1254X; FEV1/FCV 14-30%) undergoing therapeutic lung transplantation. Single cell-derived clones corresponding to the six stem cell variants resolved by single cell RNA sequencing of these libraries were assessed by RNA-sequencing and xenografting to monitor inflammation, fibrosis, and mucin secretion. The impact of CFTR activity on these variants following gene complementation or exposure to CFTR modulators was assessed by molecular and functional studies.Findings: CF lungs display a stem cell heterogeneity marked by six predominant variants of which three are proinflammatory at the level of gene expression and their ability to drive neutrophil inflammation in xenografts in immunodeficient mice. The proinflammatory functions of these three variants were not reversible by genetic or pharmacological restoration of CFTR activity.Interpretation: The emergence of three proinflammatory stem cell variants in CF lung may explain the persistence of lung inflammation in CF patients undergoing CFTR modulator therapy.Funding: National Heart Lung and Blood Institute (NHLBI) of the NIH, Cancer Prevention and Research Institute of Texas (CPRIT), Cancer Research United Kingdom (CRUK).Declaration of Interest: W.X., F.D.M., M.D. and M.V. have filed patents related to technologies used in the present work. W.X, F.D.M., and M.V. have financial interests in MultiClonal Therapeutics, Inc. and Tract Pharmaceuticals, Inc.Ethical Approval: The lung tissues used in this study were from resected lobes, lung transplants, or fetal demise cases that were obtained under informed consent as de-identified material under approved institutional review board protocols at the University of Connecticut Health Sciences (IRB# 08-310-1), the University of Iowa Carver College of Medicine (IRB# 199507432), the University of Texas Health Sciences Center, Houston (HSC-MS-08- 0354/HSC-MS-15-1049), and the Brigham and Women’s Hospital, Boston, MA (2009P002281), respectively.
The plurality of clonogenic cells derived from human lung includes a spectrum of diverse p63+ stem cells responsible for the regeneration of normal epithelial tissue and disease-associated metaplastic lesions. Here, we report protocols for the cloning, expansion, and characterization of these stem cell variants, which in general assist in analyses of stem cell heterogeneity, genome editing, drug screening, and regenerative medicine. For complete details on the use and execution of this protocol, please refer to Kumar et al. (2011), Zuo et al. (2015), and Rao et al. (2020).
Rationale: "CFTR modulator" drugs restore function to mutant channels in cystic fibrosis (CF) patients and lead to improvements in body-mass index and lung function especially in younger patients. While it is anticipated that early treatment with CFTR modulators will significantly delay the onset of advanced lung disease, lung neutrophils and inflammatory cytokines remain high in modulator-treated CF patients with established lung disease, underscoring the need to identify and ultimately target the source of this inflammation in CF lung. Objectives: To examine the stem cell heterogeneity of CF lung to identify stem cell variants that might underlie the chronic lung inflammation in CF and the impact of CFTR genetic complementation or CFTR modulators on the inflammatory properties of the stem cell variants identified herein. Methods: Stem cell cloning technology was applied to CF lungs. Single cell-derived clones were assessed by RNA-sequencing and xenografting to monitor inflammation, fibrosis, and mucin secretion. The impact of CFTR activity on these variants following gene complementation or exposure to CFTR modulators was assessed by molecular and functional studies. Measurements and Main Results: CF lungs display a stem cell heterogeneity marked by six predominant variants of which three are proinflammatory both at the level of gene expression and their ability to drive neutrophil inflammation in xenografts. The proinflammatory functions of these variants were unallayed by genetic or pharmacological restoration of CFTR activity. Conclusions: The emergence of proinflammatory stem cell variants in CF lung may explain the persistence of lung inflammation in CF patients undergoing CFTR modulator therapy. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Abstract 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.
Abstract 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.
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)