Emerging Advances of Detection Strategies for Tumor-Derived Exosomes

Exosomes derived from tumor cells contain various molecular components, such as proteins, RNA, DNA, lipids, and carbohydrates. These components play a crucial role in all stages of tumorigenesis and development. Moreover, they reflect the physiological and pathological status of parental tumor cells. Recently, tumor-derived exosomes have become popular biomarkers for non-invasive liquid biopsy and the diagnosis of numerous cancers. The interdisciplinary significance of exosomes research has also attracted growing enthusiasm. However, the intrinsic nature of tumor-derived exosomes requires advanced methods to detect and evaluate the complex biofluid. This review analyzes the relationship between exosomes and tumors. It also summarizes the exosomal biological origin, composition, and application of molecular markers in clinical cancer diagnosis. Remarkably, this paper constitutes a comprehensive summary of the innovative research on numerous detection strategies for tumor-derived exosomes with the intent of providing a theoretical basis and reference for early diagnosis and clinical treatment of cancer.

Patient-Derived Organoids in Precision Medicine: Drug Screening, Organoid-on-a-Chip and Living Organoid Biobank

Organoids are in vitro self-assembling, organ-like, three-dimensional cellular structures that stably retain key characteristics of the respective organs. Organoids can be generated from healthy or pathological tissues derived from patients. Cancer organoid culture platforms have several advantages, including conservation of the cellular composition that captures the heterogeneity and pharmacotypic signatures of the parental tumor. This platform has provided new opportunities to fill the gap between cancer research and clinical outcomes. Clinical trials have been performed using patient-derived organoids (PDO) as a tool for personalized medical decisions to predict patients’ responses to therapeutic regimens and potentially improve treatment outcomes. Living organoid biobanks encompassing several cancer types have been established, providing a representative collection of well-characterized models that will facilitate drug development. In this review, we highlight recent developments in the generation of organoid cultures and PDO biobanks, in preclinical drug discovery, and methods to design a functional organoid-on-a-chip combined with microfluidic. In addition, we discuss the advantages as well as limitations of human organoids in patient-specific therapy and highlight possible future directions.

Urothelial cancer organoids: a tool for bladder cancer research

Background Bladder cancer ranks among the top ten most common tumor types worldwide and represents a growing healthcare problem, accounting for a large part of total healthcare costs. Chemotherapy is effective in a subset of patients, while causing severe side effects. Tumor pathogenesis and drug resistance mechanisms are largely unknown. Precision medicine is failing in bladder cancer, as bladder tumors are genetically and molecularly very heterogeneous. Currently, therapeutic decision-making depends on assessing a single fragment of surgically acquired tumor tissue. Objective New preclinical model systems for bladder cancer are indispensable for developing therapeutic strategies tailored to individual patient and tumor characteristics. Organoids are small 3D tissue cultures that simulate small-size organs “in a dish” and tumoroids are a special type of cancer organoid (i.e., malignant tissue). Materials and methods Since 2016, we have collaborated with the renowned Hubrecht Institute to provide proof of concept of tissue-based bladder tumoroids mimicking parental tumors. We have developed a living biobank containing bladder organoids and tumoroids grown from over 50 patient samples, which reflect crucial aspects of bladder cancer pathogenesis. Results Histological and immunofluorescence analysis indicated that the heterogeneity and subclassification of tumoroids mimicked those of corresponding parental tumor samples. Thus, urothelial tumoroids mimic crucial aspects of bladder cancer pathogenesis. Conclusion Research with urothelial tumoroids will open up new avenues for bladder cancer pathogenesis and drug-resistance research as well as for precision medicine approaches.

Patient-Derived Cancer Organoids for Precision Oncology Treatment

The emergence of three-dimensional human organoids has opened the door for the development of patient-derived cancer organoid (PDO) models, which closely recapitulate parental tumor tissue. The mainstays of preclinical cancer modeling include in vitro cell lines and patient-derived xenografts, but these models lack the cellular heterogeneity seen in human tumors. Moreover, xenograft establishment is resource and time intensive, rendering these models difficult to use to inform clinical trials and decisions. PDOs, however, can be created efficiently and retain tumor-specific properties such as cellular heterogeneity, cell–cell and cell–stroma interactions, the tumor microenvironment, and therapeutic responsiveness. PDO models and drug-screening protocols have been described for several solid tumors and, more recently, for gliomas. Since PDOs can be developed in clinically relevant time frames and share many characteristics of parent tumors, they may enhance the ability to provide precision oncologic care for patients. This review explores the current literature on cancer organoids, highlighting the history of PDO development, organoid models of glioma, and potential clinical applications of PDOs.

Exosomes from Plasma of Neuroblastoma Patients Contain Doublestranded DNA Reflecting the Mutational Status of Parental Tumor Cells

Neuroblastoma (NB) is an aggressive infancy tumor, leading cause of death among preschool age diseases. Here we focused on characterization of exosomal DNA (exo-DNA) isolated from plasma cell-derived exosomes of neuroblastoma patients, and its potential use for detection of somatic mutations present in the parental tumor cells. Exosomes are small extracellular membrane vesicles secreted by most cells, playing an important role in intercellular communications. Using an enzymatic method, we provided evidence for the presence of double-stranded DNA in the NB exosomes. Moreover, by whole exome sequencing, we demonstrated that NB exo-DNA represents the entire exome and that it carries tumor-specific genetic mutations, including those occurring on known oncogenes and tumor suppressor genes in neuroblastoma (ALK, CHD5, SHANK2, PHOX2B, TERT, FGFR1, and BRAF). NB exo-DNA can be useful to identify variants responsible for acquired resistance, such as mutations of ALK, TP53, and RAS/MAPK genes that appear in relapsed patients. The possibility to isolate and to enrich NB derived exosomes from plasma using surface markers, and the quick and easy extraction of exo-DNA, gives this methodology a translational potential in the clinic. Exo-DNA can be an attractive non-invasive biomarker for NB molecular diagnostic, especially when tissue biopsy cannot be easily available.

Patient-Derived Cancer Organoids for Precision Oncology Treatment

The emergence of three-dimensional human organoids has opened the door for development of patient-derived cancer organoid (PDO) models, which closely recapitulate parental tumor tissue. Mainstays of preclinical cancer modeling include in vitro cell lines and patient-derived xenografts, but these models lack the cellular heterogeneity seen in human tumors. Moreover, xenograft establishment is resource- and time-intensive, rendering these models difficult to use to inform clinical trials and decisions. PDOs, however, can be created efficiently and retain tumor-specific properties such as cellular heterogeneity, cell-cell and cell-stromal interactions, tumor microenvironment, and therapeutic responsiveness. PDO models and drug screening protocols have been described for several solid tumors and, more recently, for gliomas. Since PDOs can be developed in clinically relevant timeframes and share many characteristics of parent tumors, they may enhance the ability to provide precision oncologic care for patients. This review explores the current literature on cancer organoids, highlighting the history of PDO development, organoid models of glioma, and potential clinical applications of PDOs.

A bladder cancer patient-derived xenograft displays aggressive growth dynamics in vivo and in organoid culture

Bladder cancer is among the most prevalent cancers worldwide. Currently, few bladder cancer models have undergone thorough characterization to assess their fidelity to patient tumors, especially upon propagation in the laboratory. Here, we establish and molecularly characterize CoCaB 1, an aggressive cisplatin-resistant muscle-invasive bladder cancer patient-derived xenograft (PDX) and companion organoid system. CoCaB 1 was a subcutaneous PDX model reliably transplanted in vivo and demonstrated an acceleration in growth upon serial transplantation, which was reflected in organoid and 2D cell culture systems. Transcriptome analysis revealed progression towards an increasingly proliferative and stem-like expression profile. Gene expression differences between organoid and PDX models reflected expected differences in cellular composition, with organoids enriched in lipid biosynthesis and metabolism genes and deprived of extracellular components observed in PDXs. Both PDX and organoid models maintained the histological fidelity and mutational heterogeneity of their parental tumor. This study establishes the CoCaB 1 PDX and organoid system as companion representative tumor models for the development of novel bladder cancer therapies.

EPCO-28. SPHEROID STUDY OF A GLIOBLASTOMA TUMOR IDENTIFIED A SUBCLONAL MUTATION OF EGFR T790M

BACKGROUND Glioblastoma (GBM) is the most common and the most devastating primary brain cancer in adults. Precision genetic medicine approach does not provide benefit to majority of GBM patients. A major reason for this is tumor heterogeneity harboring potential resistance mechanisms. Here, we describe an approach to grow spheroids of GBM, containing tumor, stroma and vascular tissues. We show that this approach can identify undetected subclonal driver mutations that can potentially cause drug resistance later in the disease course. METHODS We grew spheroids of a GBM patient in a multi-well array and monitored them visually. Exome sequencing of the parental tumor (coverage of 557X) and of spheroids after 10 days (n=3) and 20 days (n=5) of growth was performed. In addition, we sequenced six spheroids that were grown for 10 days and treated by Temozolomide for the next 10 days. RESULTS 70 somatic mutations were detected in the parental tumor. 42/70 of the tumor’s somatic mutations were also detected in the spheroids. 192 somatic mutations were detected in the spheroids only and not in the parental tumor despite high coverage sequencing of the parental tumor. In cancer genes: six somatic mutations were identified in the parental tumor, 5/6 were also detected in the spheroids and 13 mutations were detected only in the spheroids. EGFR V774M mutation was detected in both tumor and spheroids. However – EGFR T790M was independently detected in 7/11 of the spheroids sequenced after 20 days or growth but not in the parental tumor. CONCLUSION Analysis of GBM spheroids enables detection of rare subclonal mutations not detected in parental tumor. We hypothesize that this is due to positive selection of the ex vivo growth conditions. This analysis enabled detection of EGFR T790M – an important targetable resistance mutation in lung cancer not previously described in primary GBM.

Newly established gastrointestinal cancer cell lines retain the genomic and immunophenotypic landscape of their parental cancers

Human cancer cell lines are frequently used as model systems to study molecular mechanisms and genetic changes in cancer. However, the model is repeatedly criticized for its lack of proximity to original patient tumors. Therefore, understanding to what extent cell lines cultured under artificial conditions reflect the phenotypic and genomic profiles of their corresponding parental tumors is crucial when analyzing their biological properties. To directly compare molecular alterations between patient tumors and derived cell lines, we have established new cancer cell lines from four patients with gastrointestinal tumors. Tumor entities comprised esophageal cancer, colon cancer, rectal cancer and pancreatic cancer. Phenotype and genotype of both patient tumors and derived low-passage cell lines were characterized by immunohistochemistry (22 different antibodies), array-based comparative genomic hybridization and targeted next generation sequencing (48-gene panel). The immunophenotype was highly consistent between patient tumors and derived cell lines; the expression of most markers in cell lines was concordant with the respective parental tumor and characteristic for the respective tumor entities in general. The chromosomal aberration patterns of the parental tumors were largely maintained in the cell lines and the distribution of gains and losses was typical for the respective cancer entity, despite a few distinct differences. Cancer gene mutations (e.g., KRAS, TP53) and microsatellite status were also preserved in the respective cell line derivates. In conclusion, the four examined newly established cell lines exhibited a phenotype and genotype closely recapitulating their parental tumor. Hence, newly established cancer cell lines may be useful models for further pharmacogenomic studies.

Elucidation of tumor-stromal heterogeneity and the ligand-receptor interactome by single cell transcriptomics in real-world pancreatic cancer biopsies

Precision medicine approaches in pancreatic ductal adenocarcinoma (PDAC) are imperative for improving disease outcomes. However, the long-term fidelity of recently deployed ex vivo preclinical platforms, such as patient-derived organoids (PDOs), remains unknown. Through single-cell RNA sequencing (scRNA-seq), we identify substantial transcriptomic evolution of PDOs propagated from the parental tumor, which may alter predicted drug sensitivity. In contrast, scRNA-seq is readily applicable to limited biopsies from human primary and metastatic PDAC and identifies most cancers as being an admixture of previously described epithelial transcriptomic subtypes. Integrative analyses of our data provide an in-depth characterization of the heterogeneity within the tumor microenvironment, including cancer-associated fibroblast (CAF) subclasses, and predict a multitude of ligand-receptor interactions, revealing potential targets for immunotherapy approaches. While PDOs continue to enable prospective therapeutic prediction, our analysis also demonstrates the complementarity of using orthogonal de novo biopsies from PDAC patients paired with scRNA-seq to inform clinical decision-making.