Evolutionary trajectories of small cell lung cancer under therapy.

The evolutionary processes that underlie the marked sensitivity of small cell lung cancer (SCLC) to chemotherapy and rapid relapse are unknown1-3. Here we determined tumour phylogenies at diagnosis and throughout chemotherapy and immunotherapy by multiregion sequencing of 160 tumours from 65 patients. Treatment-naive SCLC exhibited clonal homogeneity at distinct tumour sites, whereas first-line platinum-based chemotherapy led to a burst in genomic intratumour heterogeneity and spatial clonal diversity. We observed branched evolution and a shift to ancestral clones underlying tumour relapse. Effective radio- or immunotherapy induced a re-expansion of founder clones with acquired genomic damage from first-line chemotherapy. Whereas TP53 and RB1 alterations were exclusively part of the common ancestor, MYC family amplifications were frequently not constituents of the founder clone. At relapse, emerging subclonal mutations affected key genes associated with SCLC biology, and tumours harbouring clonal CREBBP/EP300 alterations underwent genome duplications. Gene-damaging TP53 alterations and co-alterations of TP53 missense mutations with TP73, CREBBP/EP300 or FMN2 were significantly associated with shorter disease relapse following chemotherapy. In summary, we uncover key processes of the genomic evolution of SCLC under therapy, identify the common ancestor as the source of clonal diversity at relapse and show central genomic patterns associated with sensitivity and resistance to chemotherapy.

Diverse clonal fates emerge upon drug treatment of homogeneous cancer cells.

Even among genetically identical cancer cells, resistance to therapy frequently emerges from a small subset of those cells1-7. Molecular differences in rare individual cells in the initial population enable certain cells to become resistant to therapy7-9; however, comparatively little is known about the variability in the resistance outcomes. Here we develop and apply FateMap, a framework that combines DNA barcoding with single-cell RNA sequencing, to reveal the fates of hundreds of thousands of clones exposed to anti-cancer therapies. We show that resistant clones emerging from single-cell-derived cancer cells adopt molecularly, morphologically and functionally distinct resistant types. These resistant types are largely predetermined by molecular differences between cells before drug addition and not by extrinsic factors. Changes in the dose and type of drug can switch the resistant type of an initial cell, resulting in the generation and elimination of certain resistant types. Samples from patients show evidence for the existence of these resistant types in a clinical context. We observed diversity in resistant types across several single-cell-derived cancer cell lines and cell types treated with a variety of drugs. The diversity of resistant types as a result of the variability in intrinsic cell states may be a generic feature of responses to external cues.

Chemically defined cytokine-free expansion of human haematopoietic stem cells.

Haematopoietic stem cells (HSCs) are a rare cell type that reconstitute the entire blood and immune systems after transplantation and can be used as a curative cell therapy for a variety of haematological diseases1,2. However, the low number of HSCs in the body makes both biological analyses and clinical application difficult, and the limited extent to which human HSCs can be expanded ex vivo remains a substantial barrier to the wider and safer therapeutic use of HSC transplantation3. Although various reagents have been tested in attempts to stimulate the expansion of human HSCs, cytokines have long been thought to be essential for supporting HSCs ex vivo4. Here we report the establishment of a culture system that allows the long-term ex vivo expansion of human HSCs, achieved through the complete replacement of exogenous cytokines and albumin with chemical agonists and a caprolactam-based polymer. A phosphoinositide 3-kinase activator, in combination with a thrombopoietin-receptor agonist and the pyrimidoindole derivative UM171, were sufficient to stimulate the expansion of umbilical cord blood HSCs that are capable of serial engraftment in xenotransplantation assays. Ex vivo HSC expansion was further supported by split-clone transplantation assays and single-cell RNA-sequencing analysis. Our chemically defined expansion culture system will help to advance clinical HSC therapies.

Non-genetic determinants of malignant clonal fitness at single-cell resolution.

All cancers emerge after a period of clonal selection and subsequent clonal expansion. Although the evolutionary principles imparted by genetic intratumour heterogeneity are becoming increasingly clear1, little is known about the non-genetic mechanisms that contribute to intratumour heterogeneity and malignant clonal fitness2. Here, using single-cell profiling and lineage tracing (SPLINTR)-an expressed barcoding strategy-we trace isogenic clones in three clinically relevant mouse models of acute myeloid leukaemia. We find that malignant clonal dominance is a cell-intrinsic and heritable property that is facilitated by the repression of antigen presentation and increased expression of the secretory leukocyte peptidase inhibitor gene (Slpi), which we genetically validate as a regulator of acute myeloid leukaemia. Increased transcriptional heterogeneity is a feature that enables clonal fitness in diverse tissues and immune microenvironments and in the context of clonal competition between genetically distinct clones. Similar to haematopoietic stem cells3, leukaemia stem cells (LSCs) display heritable clone-intrinsic properties of high, and low clonal output that contribute to the overall tumour mass. We demonstrate that LSC clonal output dictates sensitivity to chemotherapy and, although high- and low-output clones adapt differently to therapeutic pressure, they coordinately emerge from minimal residual disease with increased expression of the LSC program. Together, these data provide fundamental insights into the non-genetic transcriptional processes that underpin malignant clonal fitness and may inform future therapeutic strategies.

Butyrate and Metformin Affect Energy Metabolism Independently of the Metabolic Phenotype in the Tumor Therapy Model.

The BALB/c cell transformation assay (BALB-CTA) considers inter- and intra-tumor heterogeneities and affords the possibility of a direct comparison between untransformed and malignant cells. In the present study, we established monoclonal cell lines that originate from the BALB-CTA and mimic heterogeneous tumor cell populations, in order to investigate phenotype-specific effects of the anti-diabetic drug metformin and the short-chain fatty acid butyrate. Growth inhibitory effects were measured with a ViCell XR cell counter. The BALB/c tumor therapy model (BALB-TTM) was performed, and the extracellular glucose level was measured in the medium supernatant. Using a Seahorse Analyzer, the metabolic phenotypes of four selected clones were characterized, and effects on energy metabolism were investigated. Anti-carcinogenic effects and reduced glucose uptake after butyrate application were observed in the BALB-TTM. Metabolic characterization of the cell clones revealed three different phenotypes. Surprisingly, treatment with metformin or butyrate induced opposite metabolic shifts with similar patterns in all cell clones tested. In conclusion, the BALB-TTM is a relevant model for mechanistic cancer research, and the generation of monoclonal cell lines offers a novel possibility to investigate specific drug effects in a heterogeneous tumor cell population. The results indicate that induced alterations in energy metabolism seem to be independent of the original metabolic phenotype.

The evolution of hematopoietic cells under cancer therapy.

Chemotherapies may increase mutagenesis of healthy cells and change the selective pressures in tissues, thus influencing their evolution. However, their contributions to the mutation burden and clonal expansions of healthy somatic tissues are not clear. Here, exploiting the mutational footprint of some chemotherapies, we explore their influence on the evolution of hematopoietic cells. Cells of Acute Myeloid Leukemia (AML) secondary to treatment with platinum-based drugs show the mutational footprint of these drugs, indicating that non-malignant blood cells receive chemotherapy mutations. No trace of the 5-fluorouracil (5FU) mutational signature is found in AMLs secondary to exposure to 5FU, suggesting that cells establishing the leukemia could be quiescent during treatment. Using the platinum-based mutational signature as a barcode, we determine that the clonal expansion originating the secondary AMLs begins after the start of the cytotoxic treatment. Its absence in clonal hematopoiesis cases is consistent with the start of the clonal expansion predating the exposure to platinum-based drugs.

Cycling cancer persister cells arise from lineages with distinct programs.

Non-genetic mechanisms have recently emerged as important drivers of cancer therapy failure1, where some cancer cells can enter a reversible drug-tolerant persister state in response to treatment2. Although most cancer persisters remain arrested in the presence of the drug, a rare subset can re-enter the cell cycle under constitutive drug treatment. Little is known about the non-genetic mechanisms that enable cancer persisters to maintain proliferative capacity in the presence of drugs. To study this rare, transiently resistant, proliferative persister population, we developed Watermelon, a high-complexity expressed barcode lentiviral library for simultaneous tracing of each cell's clonal origin and proliferative and transcriptional states. Here we show that cycling and non-cycling persisters arise from different cell lineages with distinct transcriptional and metabolic programs. Upregulation of antioxidant gene programs and a metabolic shift to fatty acid oxidation are associated with persister proliferative capacity across multiple cancer types. Impeding oxidative stress or metabolic reprogramming alters the fraction of cycling persisters. In human tumours, programs associated with cycling persisters are induced in minimal residual disease in response to multiple targeted therapies. The Watermelon system enabled the identification of rare persister lineages that are preferentially poised to proliferate under drug pressure, thus exposing new vulnerabilities that can be targeted to delay or even prevent disease recurrence.

Alternative strategies for optimizing treatment of chronic lymphocytic leukemia with complex clonal architecture.

In silico simulation of pre-clinical and clinical data may accelerate pre-clinical and clinical trial advances, leading to benefits for therapeutic outcomes, toxicity and cost savings. Combining this with clonal architecture data may permit truly personalized therapy. Chronic lymphocytic leukemia (CLL) exhibits clonal diversity, evolution and selection, spontaneously and under treatment pressure. We apply a dynamic simulation model to published CLL clonal architecture data to explore alternative therapeutic strategies, focusing on BTK inhibition. By deriving parameters of clonal growth and death behavior we model continuous vs time-limited ibrutinib therapy, and find that, despite persistence of disease, time to clinical progression may not differ. This is a testable hypothesis. We model IgVH-mutated CLL vs unmutated CLL by varying proliferation and find, based on the limited available data about clonal dynamics after such therapy, that there are differences predicted in response to anti-CD20 efficacy. These models can suggest potential clinical trials, and also indicate what additional data are needed to improve predictions. Ongoing work will expand modeling to agents such as venetoclax and to T cell therapies.

Response to Hypomethylating Agents in Myelodysplastic Syndrome Is Associated With Emergence of Novel TCR Clonotypes.

Aberrant T-cell function is implicated in the pathogenesis of myelodysplastic syndrome (MDS). Monitoring the T-cell receptor (TCR) repertoire can provide insights into T-cell adaptive immunity. Previous studies found skewed TCR repertoires in MDS compared to healthy patients; however these studies that leverage mRNA-based spectratyping have limitations. Furthermore, evaluating the TCR repertoire in context of hypomethylating agents (HMAs) treatment can provide insights into the dynamics of T-cell mediated responses in MDS. We conducted immunosequencing of the CDR3 regions of TCRß chains in bone marrows of 11 MDS patients prior to treatment (n=11 bone marrows prior to treatment), and in at least 2 timepoints for each patient following treatment (n=26 bone marrow aspirates post-treatment) with (HMA), alongside analyzing bone marrows from 4 healthy donors as controls. TCR repertoires in MDS patients were more clonal and less diverse than healthy donors. However, unlike previous reports, we did not observe significant skewness in CDR3 length or spectratyping. The global metrics of TCR profiling including richness, clonality, overlaps were not significantly changed in responders or non-responders following treatment with HMAs. However, we found an emergence of novel clonotypes in MDS patients who responded to treatment, while non-responders had a higher frequency of contracted clonotypes following treatment. By applying GLIPH2 for antigen prediction, we found rare TCR specificity clusters shared by TCR clonotypes from different patients at pre- or following treatment. Our data show clear differences in TCR repertoires of MDS compared with healthy patients and that novel TCR clonotype emergence in response to HMA therapy was correlated with response. This suggests that response to HMA therapy may be partially driven by T-cell mediated immunity and that the immune-based therapies, which target the adaptive immune system, may play a significant role in select patients with MDS.

The Flavonol Isoquercitrin Promotes Mitochondrial-Dependent Apoptosis in SK-Mel-2 Melanoma Cell via the PI3K/AKT/mTOR Pathway.

Isoquercitrin (IQ), a major flavonol present in Prunus mume fruit, has gained much attention in recent studies because of its superior bioavailability and physiological effects. In this study, the anti-cancer mechanism of IQ against human melanoma, particularly its effect on the mitochondria-mediated apoptosis, was investigated. Treatment with IQ at 25 µM concentration effectively inhibited the proliferation of SK-MEL-2 skin cancer cells while the same concentration did not exhibit cytotoxicity against human keratinocytes HaCaT. Morphological analysis and clonogenic assay also showed that IQ can alter the growth and long-term survival of SK-MEL-2 cells. IQ also induced apoptosis in the melanoma cells as manifested in the nuclear morphology changes, DNA fragmentation, increase in the apoptosis rate (17.69% at 25 µM) and accumulation of sub-G1 cell cycle phase population (19.55% at 25 µM). Western blot analysis revealed the involvement of the mitochondrial apoptosis signaling pathway in the anti-cancer property of IQ. Treatment with IQ resulted in the decrease in the levels of procaspase-8 and -9, and Bcl-2 protein, and an increase in the expression of cleaved PARP and Bax. Moreover, AIF and Endo G protein expression increased, indicating a caspase-independent mitochondrial-mediated apoptosis. The anti-proliferative activity of IQ against SK-MEL-2 can also be attributed to the downregulation of the PI3K/AktmTOR signaling pathway. These findings showed that IQ can be developed into a chemopreventive therapeutic agent against the melanoma cells.

Detection of chemotherapy-resistant patient-derived acute lymphoblastic leukemia clones in murine xenografts using cellular barcodes.

Clonal heterogeneity fuels leukemia evolution, therapeutic resistance, and relapse. Upfront detection of therapy-resistant leukemia clones at diagnosis may allow adaptation of treatment and prevention of relapse, but this is hampered by a paucity of methods to identify and trace single leukemia-propagating cells and their clonal offspring. Here, we tested methods of cellular barcoding analysis, to trace the in vivo competitive dynamics of hundreds of patient-derived leukemia clones upon chemotherapy-mediated selective pressure. We transplanted Nod/Scid/Il2Rγ-/- (NSG) mice with barcoded patient-derived or SupB15 acute lymphoblastic leukemia (ALL) cells and assessed clonal responses to dexamethasone, methotrexate, and vincristine, longitudinally and across nine anatomic locations. We illustrate that chemotherapy reduces clonal diversity in a drug-dependent manner. At end-stage disease, methotrexate-treated patient-derived xenografts had significantly fewer clones compared with placebo-treated mice (100 ± 10 vs. 160 ± 15 clones, p = 0.0005), while clonal complexity in vincristine- and dexamethasone-treated xenografts was unaffected (115 ± 33 and 150 ± 7 clones, p = NS). Using tools developed to assess differential gene expression, we determined whether these clonal patterns resulted from random clonal drift or selection. We identified 5 clones that were reproducibly enriched in methotrexate-treated patient-derived xenografts, suggestive of pre-existent resistance. Finally, we found that chemotherapy-mediated selection resulted in a more asymmetric distribution of leukemia clones across anatomic sites. We found that cellular barcoding is a powerful method to trace the clonal dynamics of human patient-derived leukemia cells in response to chemotherapy. In the future, integration of cellular barcoding with single-cell sequencing technology may allow in-depth characterization of therapy-resistant leukemia clones and identify novel targets to prevent relapse.

Mechanisms of stretch-mediated skin expansion at single-cell resolution.

The ability of the skin to grow in response to stretching has been exploited in reconstructive surgery1. Although the response of epidermal cells to stretching has been studied in vitro2,3, it remains unclear how mechanical forces affect their behaviour in vivo. Here we develop a mouse model in which the consequences of stretching on skin epidermis can be studied at single-cell resolution. Using a multidisciplinary approach that combines clonal analysis with quantitative modelling and single-cell RNA sequencing, we show that stretching induces skin expansion by creating a transient bias in the renewal activity of epidermal stem cells, while a second subpopulation of basal progenitors remains committed to differentiation. Transcriptional and chromatin profiling identifies how cell states and gene-regulatory networks are modulated by stretching. Using pharmacological inhibitors and mouse mutants, we define the step-by-step mechanisms that control stretch-mediated tissue expansion at single-cell resolution in vivo.

Secondary myelodysplastic syndrome and leukemia in acquired aplastic anemia and paroxysmal nocturnal hemoglobinuria.

Acquired aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) are pathogenically related nonmalignant bone marrow failure disorders linked to T-cell-mediated autoimmunity; they are associated with an increased risk of secondary myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Approximately 15% to 20% of AA patients and 2% to 6% of PNH patients go on to develop secondary MDS/AML by 10 years of follow-up. Factors determining an individual patient's risk of malignant transformation remain poorly defined. Recent studies identified nearly ubiquitous clonal hematopoiesis (CH) in AA patients. Similarly, CH with additional, non-PIGA, somatic alterations occurs in the majority of patients with PNH. Factors associated with progression to secondary MDS/AML include longer duration of disease, increased telomere attrition, presence of adverse prognostic mutations, and multiple mutations, particularly when occurring early in the disease course and at a high allelic burden. Here, we will review the prevalence and characteristics of somatic alterations in AA and PNH and will explore their prognostic significance and mechanisms of clonal selection. We will then discuss the available data on post-AA and post-PNH progression to secondary MDS/AML and provide practical guidance for approaching patients with PNH and AA who have CH.

Functional and genomic characterization of three novel cell lines derived from a metastatic gallbladder cancer tumor.

BACKGROUND: Gallbladder cancer (GBC) is the most common tumor of the biliary tract. The incidence of GBC shows a large geographic variability, being particularly frequent in Native American populations. In Chile, GBC represents the second cause of cancer-related death among women. We describe here the establishment of three novel cell lines derived from the ascitic fluid of a Chilean GBC patient, who presented 46% European, 36% Mapuche, 12% Aymara and 6% African ancestry. RESULTS: After immunocytochemical staining of the primary cell culture, we isolated and comprehensively characterized three independent clones (PUC-GBC1, PUC-GBC2 and PUC-GBC3) by short tandem repeat DNA profiling and RNA sequencing as well as karyotype, doubling time, chemosensitivity, in vitro migration capability and in vivo tumorigenicity assay. Primary culture cells showed high expression of CK7, CK19, CA 19-9, MUC1 and MUC16, and negative expression of mesothelial markers. The three isolated clones displayed an epithelial phenotype and an abnormal structure and number of chromosomes. RNA sequencing confirmed the increased expression of cytokeratin and mucin genes, and also of TP53 and ERBB2 with some differences among the three cells lines, and revealed a novel exonic mutation in NF1. The PUC-GBC3 clone was the most aggressive according to histopathological features and the tumorigenic capacity in NSG mice. CONCLUSIONS: The first cell lines established from a Chilean GBC patient represent a new model for studying GBC in patients of Native American descent.

Mutations in long-lived epithelial stem cells and their clonal progeny in pre-malignant lesions and in oral squamous cell carcinoma.

Oral squamous cell carcinomas (OSCCs) are the most common cancers of the oral cavity, but the molecular mechanisms driving OSCC carcinogenesis remain unclear. Our group previously established a murine OSCC model based on a 10-week carcinogen [4-nitroquinoline 1-oxide (4-NQO)] treatment. Here we used K14CreERTAM;Rosa26LacZ mice to perform lineage tracing to delineate the mutational profiles in clonal cell populations resulting from single, long-lived epithelial stem cells, here called LacZ+ stem cell clones (LSCCs). Using laser-capture microdissection, we examined mutational changes in LSCCs immediately after the 10-week 4-NQO treatment and >17 weeks after 4-NQO treatment. We found a 1.8-fold ±0.4 (P = 0.009) increase in single-nucleotide variants and insertions/deletions (indels) in tumor compared with pre-neoplastic LSCCs. The percentages of indels and of loss of heterozygosity events were 1.3-fold±0.3 (P = 0.02) and 2.2-fold±0.7 (P = 0.08) higher in pre-neoplastic compared with tumor LSCCs. Mutations in cell adhesion- and development-associated genes occurred in 83% of the tumor LSCCs. Frequently mutated genes in tumor LSCCs were involved in planar cell polarity (Celsr1, Fat4) or development (Notch1). Chromosomal amplifications in 50% of the tumor LSCCs occurred in epidermal growth factor receptor, phosphoinositide 3-kinase and cell adhesion pathways. All pre-neoplastic and tumor LSCCs were characterized by key smoking-associated changes also observed in human OSCC, C>A and G>T. DeconstructSigs analysis identified smoking and head and neck cancer as the most frequent mutational signatures in pre-neoplastic and tumor LSCCs. Thus, this model recapitulates a smoking-associated mutational profile also observed in humans and illustrates the role of LSCCs in early carcinogenesis and OSCCs.

A Multistage Murine Breast Cancer Model Reveals Long-Lived Premalignant Clones Refractory to Parity-Induced Protection.

Breast cancers evolve in a multistage process that can span decades after a carcinogenic exposure. It follows that long-lived precursor breast lesions persist in a subclinical state prior to completing malignant transformation, yet widely used breast cancer models lack an experimental framework for targeting premalignant disease. Inspired by classic multistage skin carcinogenesis protocols, we combined chemical carcinogenesis with transgenic mouse modeling to resolve mouse mammary carcinogenesis into discrete initiation and progression stages. At the initiation stage, exposure to the carcinogen 7,12-dimethylbenzanthracene (DMBA) generated "initiated mammary epithelial cells" (iMEC) by introducing a stereotyped HrasQ61L driver mutation. Whether DMBA exposure occurred during puberty or adulthood, mice efficiently acquired iMEC clones that eluded detection by conventional histology, yet were long lived, persisting in a clinically silent state for months in the absence of a cooperating event. At the progression stage, inducible activation of oncogenic Wnt signaling drove rapid and synchronous transformation of latent iMECs into overt mammary carcinomas, while Wnt activation in neighboring normal mammary epithelium yielded only benign hyperplasia over this same time period. Although early parity (completion of a full-term pregnancy) reduces breast cancer risk in some contexts, standard parity-induced protection schemes failed to eliminate iMECs in our multistage model, suggesting Wnt-responsive iMECs are maintained by hormone-independent mechanisms. Variations on our multistage modeling strategy may help to identify and validate cellular and molecular targets for breast cancer chemoprevention.

Prexasertib (LY2606368) reduces clonogenic survival by inducing apoptosis in primary patient-derived osteosarcoma cells and synergizes with cisplatin and talazoparib.

Progress in the systemic control of osteosarcoma has been limited over the past decades thus indicating the urgent clinical need for the development of novel treatment strategies. Therefore, we have recently developed new preclinical models to study promising novel agents for the treatment of pediatric osteosarcoma. The checkpoint kinase (chk) inhibitor prexasertib (LY2606368) and its salt form (LSN2940930) have recently been shown to be active in adult and pediatric malignancies, including sarcoma. We have now tested the potency of prexasertib in clonogenic survival assays in two new lines of primary patient-derived osteosarcoma cells and in two established osteosarcoma cell lines as a single agent and in combination with cisplatin and the poly ADP-ribose polymerase (PARP) inhibitor talazoparib. Prexasertib alone results in strongly reduced clonogenic survival at low nanomolar concentrations and acts by affecting cell cycle progression, induction of apoptosis and induction of double-stranded DNA breakage at concentrations that are well below clinically tolerable and safe plasma concentrations. In combination with cisplatin and talazoparib, prexasertib acts in a synergistic fashion. Chk1 inhibition by prexasertib and its combination with the DNA damaging agent cisplatin and the PARP-inhibitor talazoparib thus emerges as a potential new treatment option for pediatric osteosarcoma which will now have to be tested in preclinical primary patient derived in vivo models and clinical studies.

Functional and genomic characterization of three novel cell lines derived from a metastatic gallbladder cancer tumor

BACKGROUND: Gallbladder cancer (GBC) is the most common tumor of the biliary tract. The incidence of GBC shows a large geographic variability, being particularly frequent in Native American populations. In Chile, GBC represents the second cause of cancer-related death among women. We describe here the establishment of three novel cell lines derived from the ascitic fluid of a Chilean GBC patient, who presented 46% European, 36% Mapuche, 12% Aymara and 6% African ancestry. RESULTS: After immunocytochemical staining of the primary cell culture, we isolated and comprehensively characterized three independent clones (PUC-GBC1, PUC-GBC2 and PUC-GBC3) by short tandem repeat DNA profiling and RNA sequencing as well as karyotype, doubling time, chemosensitivity, in vitro migration capability and in vivo tumorigenicity assay. Primary culture cells showed high expression of CK7, CK19, CA 19-9, MUC1 and MUC16, and negative expression of mesothelial markers. The three isolated clones displayed an epithelial phenotype and an abnormal structure and number of chromosomes. RNA sequencing confirmed the increased expression of cytokeratin and mucin genes, and also of TP53 and ERBB2 with some differences among the three cells lines, and revealed a novel exonic mutation in NF1. The PUC-GBC3 clone was the most aggressive according to histopathological features and the tumorigenic capacity in NSG mice. CONCLUSIONS: The first cell lines established from a Chilean GBC patient represent a new model for studying GBC in patients of Native American descent.