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1.
Microsyst Nanoeng ; 8: 130, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36561926

RESUMEN

Cancer patients with advanced disease are characterized by intrinsic challenges in predicting drug response patterns, often leading to ineffective treatment. Current clinical practice for treatment decision-making is commonly based on primary or secondary tumour biopsies, yet when disease progression accelerates, tissue biopsies are not performed on a regular basis. It is in this context that liquid biopsies may offer a unique window to uncover key vulnerabilities, providing valuable information about previously underappreciated treatment opportunities. Here, we present MyCTC chip, a novel microfluidic device enabling the isolation, culture and drug susceptibility testing of cancer cells derived from liquid biopsies. Cancer cell capture is achieved through a label-free, antigen-agnostic enrichment method, and it is followed by cultivation in dedicated conditions, allowing on-chip expansion of captured cells. Upon growth, cancer cells are then transferred to drug screen chambers located within the same device, where multiple compounds can be tested simultaneously. We demonstrate MyCTC chip performance by means of spike-in experiments with patient-derived breast circulating tumour cells, enabling >95% capture rates, as well as prospective processing of blood from breast cancer patients and ascites fluid from patients with ovarian, tubal and endometrial cancer, where sensitivity to specific chemotherapeutic agents was identified. Together, we provide evidence that MyCTC chip may be used to identify personalized drug response patterns in patients with advanced metastatic disease and with limited treatment opportunities.

2.
Cancer Res ; 82(4): 681-694, 2022 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-34916221

RESUMEN

Blood-borne metastasis of breast cancer involves a series of tightly regulated sequential steps, including the growth of a primary tumor lesion, intravasation of circulating tumor cells (CTC), and adaptation in various distant metastatic sites. The genes orchestrating each of these steps are poorly understood in physiologically relevant contexts, owing to the rarity of experimental models that faithfully recapitulate the biology, growth kinetics, and tropism of human breast cancer. Here, we conducted an in vivo loss-of-function CRISPR screen in newly derived CTC xenografts, unique in their ability to spontaneously mirror the human disease, and identified specific genetic dependencies for each step of the metastatic process. Validation experiments revealed sensitivities to inhibitors that are already available, such as PLK1 inhibitors, to prevent CTC intravasation. Together, these findings present a new tool to reclassify driver genes involved in the spread of human cancer, providing insights into the biology of metastasis and paving the way to test targeted treatment approaches. SIGNIFICANCE: A loss-of-function CRISPR screen in human CTC-derived xenografts identifies genes critical for individual steps of the metastatic cascade, suggesting novel drivers and treatment opportunities for metastatic breast cancers.


Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Células Neoplásicas Circulantes/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/sangre , Neoplasias de la Mama/patología , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Metástasis de la Neoplasia , Células Neoplásicas Circulantes/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , RNA-Seq/métodos , Análisis de Supervivencia , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Quinasa Tipo Polo 1
3.
J Vis Exp ; (147)2019 05 14.
Artículo en Inglés | MEDLINE | ID: mdl-31157780

RESUMEN

Blood-borne metastasis accounts for most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.


Asunto(s)
Metástasis de la Neoplasia/diagnóstico , Células Neoplásicas Circulantes , Animales , Humanos , Ratones , Micromanipulación , Metástasis de la Neoplasia/patología
4.
Nature ; 566(7745): 553-557, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30728496

RESUMEN

A better understanding of the features that define the interaction between cancer cells and immune cells is important for the development of new cancer therapies1. However, focus is often given to interactions that occur within the primary tumour and its microenvironment, whereas the role of immune cells during cancer dissemination in patients remains largely uncharacterized2,3. Circulating tumour cells (CTCs) are precursors of metastasis in several types of cancer4-6, and are occasionally found within the bloodstream in association with non-malignant cells such as white blood cells (WBCs)7,8. The identity and function of these CTC-associated WBCs, as well as the molecular features that define the interaction between WBCs and CTCs, are unknown. Here we isolate and characterize individual CTC-associated WBCs, as well as corresponding cancer cells within each CTC-WBC cluster, from patients with breast cancer and from mouse models. We use single-cell RNA sequencing to show that in the majority of these cases, CTCs were associated with neutrophils. When comparing the transcriptome profiles of CTCs associated with neutrophils against those of CTCs alone, we detect a number of differentially expressed genes that outline cell cycle progression, leading to more efficient metastasis formation. Further, we identify cell-cell junction and cytokine-receptor pairs that define CTC-neutrophil clusters, representing key vulnerabilities of the metastatic process. Thus, the association between neutrophils and CTCs drives cell cycle progression within the bloodstream and expands the metastatic potential of CTCs, providing a rationale for targeting this interaction in treatment of breast cancer.


Asunto(s)
Neoplasias de la Mama/patología , Ciclo Celular , Metástasis de la Neoplasia/patología , Células Neoplásicas Circulantes/patología , Neutrófilos/patología , Animales , Neoplasias de la Mama/terapia , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular , Exones/genética , Femenino , Perfilación de la Expresión Génica , Humanos , Uniones Intercelulares , Ratones , Mutación/genética , Metástasis de la Neoplasia/genética , Células Neoplásicas Circulantes/metabolismo , Neutrófilos/metabolismo , Análisis de Secuencia de ARN , Secuenciación del Exoma
5.
Cell ; 176(1-2): 98-112.e14, 2019 01 10.
Artículo en Inglés | MEDLINE | ID: mdl-30633912

RESUMEN

The ability of circulating tumor cells (CTCs) to form clusters has been linked to increased metastatic potential. Yet biological features and vulnerabilities of CTC clusters remain largely unknown. Here, we profile the DNA methylation landscape of single CTCs and CTC clusters from breast cancer patients and mouse models on a genome-wide scale. We find that binding sites for stemness- and proliferation-associated transcription factors are specifically hypomethylated in CTC clusters, including binding sites for OCT4, NANOG, SOX2, and SIN3A, paralleling embryonic stem cell biology. Among 2,486 FDA-approved compounds, we identify Na+/K+ ATPase inhibitors that enable the dissociation of CTC clusters into single cells, leading to DNA methylation remodeling at critical sites and metastasis suppression. Thus, our results link CTC clustering to specific changes in DNA methylation that promote stemness and metastasis and point to cluster-targeting compounds to suppress the spread of cancer.


Asunto(s)
Neoplasias de la Mama/genética , Metástasis de la Neoplasia/genética , Células Neoplásicas Circulantes/patología , Animales , Neoplasias de la Mama/patología , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Metilación de ADN/fisiología , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Proteína Homeótica Nanog/metabolismo , Metástasis de la Neoplasia/fisiopatología , Células Neoplásicas Circulantes/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción SOXB1/metabolismo , Complejo Correpresor Histona Desacetilasa y Sin3
6.
Front Med (Lausanne) ; 5: 34, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29520361

RESUMEN

Circulating tumor cells (CTCs) are defined as those cells that detach from a cancerous lesion and enter the bloodstream. While generally most CTCs are subjected to high shear stress, anoikis signals, and immune attack in the circulatory system, few are able to survive and reach a distant organ in a viable state, possibly leading to metastasis formation. A large number of studies, both prospective and retrospective, have highlighted the association between CTC abundance and bad prognosis in patients with various cancer types. Yet, beyond CTC enumeration, much less is known about the distinction between metastatic and nonmetastatic CTCs, namely those features that enable only some CTCs to survive and seed a cancerous lesion at a distant site. In addition, critical aspects such as CTC heterogeneity, mechanisms that trigger CTC intravasation and extravasation, as well as vulnerabilities of metastatic CTCs subpopulations are poorly understood. In this short review, we highlight recent studies that successfully adopted functional and computational analysis to gain insights into CTC biology. We also discuss approaches to overcome challenges that are associated with CTC isolation, molecular and computational analysis, and speculate regarding few open questions that currently frame the CTC research field.

7.
ACS Synth Biol ; 3(12): 986-9, 2014 Dec 19.
Artículo en Inglés | MEDLINE | ID: mdl-25524106

RESUMEN

The ability to control mammalian genes in a synergistic mode using synthetic transcription factors is highly desirable in fields of tissue engineering, stem cell reprogramming and fundamental research. In this study, we developed a standardized toolkit utilizing an engineered CRISPR/Cas9 system that enables customizable gene regulation in mammalian cells. The RNA-guided dCas9 protein was implemented as a programmable transcriptional activator or repressor device, including targeting of endogenous loci. For facile assembly of single or multiple CRISPR RNAs, our toolkit comprises a modular RNAimer plasmid, which encodes the required noncoding RNA components.


Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Regulación de la Expresión Génica/genética , Ingeniería Genética/métodos , Células HEK293 , Humanos , Plásmidos/genética
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