RESUMO
In vitro cancer cultures, including three-dimensional organoids, typically contain exclusively neoplastic epithelium but require artificial reconstitution to recapitulate the tumor microenvironment (TME). The co-culture of primary tumor epithelia with endogenous, syngeneic tumor-infiltrating lymphocytes (TILs) as a cohesive unit has been particularly elusive. Here, an air-liquid interface (ALI) method propagated patient-derived organoids (PDOs) from >100 human biopsies or mouse tumors in syngeneic immunocompetent hosts as tumor epithelia with native embedded immune cells (T, B, NK, macrophages). Robust droplet-based, single-cell simultaneous determination of gene expression and immune repertoire indicated that PDO TILs accurately preserved the original tumor T cell receptor (TCR) spectrum. Crucially, human and murine PDOs successfully modeled immune checkpoint blockade (ICB) with anti-PD-1- and/or anti-PD-L1 expanding and activating tumor antigen-specific TILs and eliciting tumor cytotoxicity. Organoid-based propagation of primary tumor epithelium en bloc with endogenous immune stroma should enable immuno-oncology investigations within the TME and facilitate personalized immunotherapy testing.
Assuntos
Modelos Imunológicos , Neoplasias Experimentais/imunologia , Organoides/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Microambiente Tumoral/imunologia , Animais , Antígeno B7-H1/imunologia , Técnicas de Cocultura , Feminino , Humanos , Imunoterapia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias/imunologia , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Organoides/patologiaRESUMO
ABSTRACT: Chimeric antigen receptor (CAR) T cells hold promise as a therapy for B-cell-derived malignancies, and despite their impressive initial response rates, a significant proportion of patients ultimately experience relapse. Although recent studies have explored the mechanisms of in vivo CAR T-cell function, little is understood about the activation of surrounding CARneg bystander T cells and their potential to enhance tumor responses. We performed single-cell RNA sequencing on nonhuman primate (NHP) and patient-derived T cells to identify the phenotypic and transcriptomic hallmarks of bystander activation of CARneg T cells following B-cell-targeted CAR T-cell therapy. Using a highly translatable CD20 CAR NHP model, we observed a distinct population of activated CD8+ CARneg T cells emerging during CAR T-cell expansion. These bystander CD8+ CARneg T cells exhibited a unique transcriptional signature with upregulation of natural killer-cell markers (KIR3DL2, CD160, and KLRD1), chemokines, and chemokine receptors (CCL5, XCL1, and CCR9), and downregulation of naïve T-cell-associated genes (SELL and CD28). A transcriptionally similar population was identified in patients after a tisagenlecleucel infusion. Mechanistic studies revealed that interleukin-2 (IL-2) and IL-15 exposure induced bystander-like CD8+ T cells in a dose-dependent manner. In vitro activated and patient-derived T cells with a bystander phenotype efficiently killed leukemic cells through a T-cell receptor-independent mechanism. Collectively, to our knowledge, these data provide the first comprehensive identification and profiling of CARneg bystander CD8+ T cells following B-cell-targeting CAR T-cell therapy and suggest a novel mechanism through which CAR T-cell infusion might trigger enhanced antileukemic responses. Patient samples were obtained from the trial #NCT03369353, registered at www.ClinicalTrials.gov.
Assuntos
Efeito Espectador , Linfócitos T CD8-Positivos , Imunoterapia Adotiva , Animais , Humanos , Imunoterapia Adotiva/métodos , Linfócitos T CD8-Positivos/imunologia , Efeito Espectador/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos B/imunologia , Ativação Linfocitária/imunologia , Macaca mulatta , Linfócitos T Citotóxicos/imunologiaRESUMO
Synthetic lethality-an interaction between two genetic events through which the co-occurrence of these two genetic events leads to cell death, but each event alone does not-can be exploited for cancer therapeutics1. DNA repair processes represent attractive synthetic lethal targets, because many cancers exhibit an impairment of a DNA repair pathway, which can lead to dependence on specific repair proteins2. The success of poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors in cancers with deficiencies in homologous recombination highlights the potential of this approach3. Hypothesizing that other DNA repair defects would give rise to synthetic lethal relationships, we queried dependencies in cancers with microsatellite instability (MSI), which results from deficient DNA mismatch repair. Here we analysed data from large-scale silencing screens using CRISPR-Cas9-mediated knockout and RNA interference, and found that the RecQ DNA helicase WRN was selectively essential in MSI models in vitro and in vivo, yet dispensable in models of cancers that are microsatellite stable. Depletion of WRN induced double-stranded DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI models. MSI cancer models required the helicase activity of WRN, but not its exonuclease activity. These findings show that WRN is a synthetic lethal vulnerability and promising drug target for MSI cancers.
Assuntos
Instabilidade de Microssatélites , Repetições de Microssatélites/genética , Neoplasias/genética , Mutações Sintéticas Letais/genética , Helicase da Síndrome de Werner/genética , Apoptose/genética , Sistemas CRISPR-Cas/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Humanos , Modelos Genéticos , Neoplasias/patologia , Interferência de RNA , Proteína Supressora de Tumor p53/metabolismo , Helicase da Síndrome de Werner/deficiênciaRESUMO
BACKGROUND: Primary and metastatic prostate cancers have low mutation rates and recurrent alterations in a small set of genes, enabling targeted sequencing of prostate cancer-associated genes as an efficient approach to characterizing patient samples (compared to whole-exome and whole-genome sequencing). For example, targeted sequencing provides a flexible, rapid, and cost-effective method for genomic assessment of patient-derived cell lines to evaluate fidelity to initial patient tumor samples. METHODS: We developed a prostate cancer-specific targeted next-generation sequencing (NGS) panel to detect alterations in 62 prostate cancer-associated genes as well as recurring gene fusions with ETS family members, representing the majority of common alterations in prostate cancer. We tested this panel on primary prostate cancer tissues and blood biopsies from patients with metastatic prostate cancer. We generated patient-derived cell lines from primary prostate cancers using conditional reprogramming methods and applied targeted sequencing to evaluate the fidelity of these cell lines to the original patient tumors. RESULTS: The prostate cancer-specific panel identified biologically and clinically relevant alterations, including point mutations in driver oncogenes and ETS family fusion genes, in tumor tissues from 29 radical prostatectomy samples. The targeted panel also identified genomic alterations in cell-free DNA and circulating tumor cells (CTCs) from patients with metastatic prostate cancer, and in standard prostate cancer cell lines. We used the targeted panel to sequence our set of patient-derived cell lines; however, no prostate cancer-specific mutations were identified in the tumor-derived cell lines, suggesting preferential outgrowth of normal prostate epithelial cells. CONCLUSIONS: We evaluated a prostate cancer-specific targeted NGS panel to detect common and clinically relevant alterations (including ETS family gene fusions) in prostate cancer. The panel detected driver mutations in a diverse set of clinical samples of prostate cancer, including fresh-frozen tumors, cell-free DNA, CTCs, and cell lines. Targeted sequencing of patient-derived cell lines highlights the challenge of deriving cell lines from primary prostate cancers and the importance of genomic characterization to credential candidate cell lines. Our study supports that a prostate cancer-specific targeted sequencing panel provides an efficient, clinically feasible approach to identify genetic alterations across a spectrum of prostate cancer samples and cell lines.
Assuntos
Ácidos Nucleicos Livres , Neoplasias da Próstata , Linhagem Celular , Credenciamento , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Mutação , Neoplasias da Próstata/genéticaRESUMO
Wilms tumor (WT) is the most common renal malignancy of childhood. Despite improvements in the overall survival, relapse occurs in ~15% of patients with favorable histology WT (FHWT). Half of these patients will succumb to their disease. Identifying novel targeted therapies remains challenging in part due to the lack of faithful preclinical in vitro models. Here we establish twelve patient-derived WT cell lines and demonstrate that these models faithfully recapitulate WT biology using genomic and transcriptomic techniques. We then perform loss-of-function screens to identify the nuclear export gene, XPO1, as a vulnerability. We find that the FDA approved XPO1 inhibitor, KPT-330, suppresses TRIP13 expression, which is required for survival. We further identify synergy between KPT-330 and doxorubicin, a chemotherapy used in high-risk FHWT. Taken together, we identify XPO1 inhibition with KPT-330 as a potential therapeutic option to treat FHWTs and in combination with doxorubicin, leads to durable remissions in vivo.
Assuntos
Hidrazinas , Neoplasias Renais , Triazóis , Tumor de Wilms , Humanos , Proteína Exportina 1 , Transporte Ativo do Núcleo Celular , Carioferinas/genética , Carioferinas/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Linhagem Celular Tumoral , Apoptose , Recidiva Local de Neoplasia , Doxorrubicina/farmacologia , Tumor de Wilms/tratamento farmacológico , Tumor de Wilms/genética , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Ciclo Celular/metabolismoRESUMO
T cell receptor (TCR) clonotype tracking is a powerful tool for interrogating T cell mediated immune processes. New methods to pair a single cell's transcriptional program with its TCR identity allow monitoring of T cell clonotype-specific transcriptional dynamics. While these technologies have been available for human and mouse T cells studies, they have not been developed for Rhesus Macaques (RM), a critical translational organism for autoimmune diseases, vaccine development and transplantation. We describe a new pipeline, 'RM-scTCR-Seq', which, for the first time, enables RM specific single cell TCR amplification, reconstruction and pairing of RM TCR's with their transcriptional profiles. We apply this method to a RM model of GVHD, and identify and track in vitro detected alloreactive clonotypes in GVHD target organs and explore their GVHD driven cytotoxic T cell signature. This novel, state-of-the-art platform fundamentally advances the utility of RM to study protective and pathogenic T cell responses.
Assuntos
Rastreamento de Células , Receptores de Antígenos de Linfócitos T/genética , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Rastreamento de Células/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Teste de Cultura Mista de Linfócitos , Macaca mulatta , Receptores de Antígenos de Linfócitos T/metabolismo , TranscriptomaRESUMO
Despite considerable efforts to identify cancer metabolic alterations that might unveil druggable vulnerabilities, systematic characterizations of metabolism as it relates to functional genomic features and associated dependencies remain uncommon. To further understand the metabolic diversity of cancer, we profiled 225 metabolites in 928 cell lines from more than 20 cancer types in the Cancer Cell Line Encyclopedia (CCLE) using liquid chromatography-mass spectrometry (LC-MS). This resource enables unbiased association analysis linking the cancer metabolome to genetic alterations, epigenetic features and gene dependencies. Additionally, by screening barcoded cell lines, we demonstrated that aberrant ASNS hypermethylation sensitizes subsets of gastric and hepatic cancers to asparaginase therapy. Finally, our analysis revealed distinct synthesis and secretion patterns of kynurenine, an immune-suppressive metabolite, in model cancer cell lines. Together, these findings and related methodology provide comprehensive resources that will help clarify the landscape of cancer metabolism.
Assuntos
Neoplasias/metabolismo , Animais , Asparaginase/uso terapêutico , Asparagina/metabolismo , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/antagonistas & inibidores , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/metabolismo , Linhagem Celular Tumoral , Metilação de DNA , Feminino , Técnicas de Silenciamento de Genes , Humanos , Cinurenina/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/terapia , Metaboloma , Camundongos , Camundongos Nus , Neoplasias/genética , Neoplasias/terapia , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/terapiaRESUMO
Renal medullary carcinoma (RMC) is a rare and deadly kidney cancer in patients of African descent with sickle cell trait. We have developed faithful patient-derived RMC models and using whole-genome sequencing, we identified loss-of-function intronic fusion events in one SMARCB1 allele with concurrent loss of the other allele. Biochemical and functional characterization of these models revealed that RMC requires the loss of SMARCB1 for survival. Through integration of RNAi and CRISPR-Cas9 loss-of-function genetic screens and a small-molecule screen, we found that the ubiquitin-proteasome system (UPS) was essential in RMC. Inhibition of the UPS caused a G2/M arrest due to constitutive accumulation of cyclin B1. These observations extend across cancers that harbor SMARCB1 loss, which also require expression of the E2 ubiquitin-conjugating enzyme, UBE2C. Our studies identify a synthetic lethal relationship between SMARCB1-deficient cancers and reliance on the UPS which provides the foundation for a mechanism-informed clinical trial with proteasome inhibitors.
Assuntos
Carcinoma Medular/genética , Neoplasias Renais/genética , Complexo de Endopeptidases do Proteassoma/genética , Inibidores de Proteassoma/farmacologia , Proteína SMARCB1/genética , Alelos , Animais , Sistemas CRISPR-Cas , Carcinoma Medular/tratamento farmacológico , Ciclo Celular , Linhagem Celular Tumoral , Exoma , Feminino , Humanos , Hibridização in Situ Fluorescente , Rim/metabolismo , Neoplasias Renais/tratamento farmacológico , Camundongos , Camundongos Nus , Mutação , Transplante de Neoplasias , Interferência de RNA , Análise de Sequência de RNA , Ubiquitina/química , Sequenciamento Completo do GenomaRESUMO
Identifying therapeutic targets in rare cancers remains challenging due to the paucity of established models to perform preclinical studies. As a proof-of-concept, we developed a patient-derived cancer cell line, CLF-PED-015-T, from a paediatric patient with a rare undifferentiated sarcoma. Here, we confirm that this cell line recapitulates the histology and harbours the majority of the somatic genetic alterations found in a metastatic lesion isolated at first relapse. We then perform pooled CRISPR-Cas9 and RNAi loss-of-function screens and a small-molecule screen focused on druggable cancer targets. Integrating these three complementary and orthogonal methods, we identify CDK4 and XPO1 as potential therapeutic targets in this cancer, which has no known alterations in these genes. These observations establish an approach that integrates new patient-derived models, functional genomics and chemical screens to facilitate the discovery of targets in rare cancers.