Parental Tumor Tissue Research Articles

In vitro drug testing using patient-derived ovarian cancer organoids

BackgroundOvarian cancer is the most lethal gynecological cancer. As the primary treatment, chemotherapy has a response rate of only 60–70% in advanced stages, and even lower as a second-line treatment. Despite guideline recommendations, which drugs will be most effective remains unclear. Thus, a strategy to prioritize chemotherapy options is urgently needed. Cancer organoids have recently emerged as a method for in vitro drug testing. However, limited clinical correlations have been assessed with test results from cancer organoids, particularly in gynecological cancers. We therefore aimed to generate patient-derived organoids (PDOs) of ovarian cancer, to assess their drug sensitivities and correlations with patient clinical outcomes.MethodsPDOs were generated from fresh tumors obtained during surgical resection, which was then cultured under matrix gel and appropriate growth factors. Morphological and molecular characterization of PDOs were assessed by phase contrast microscopy and paraffin-embedded histopathology. Expressions of PAX8, TP53, WT1, CK7, and CK20 were tested by immunohistochemical staining and compared with parental tumor tissues and the human protein atlas database. PDOs were subjected to in vitro drug testing to determine drug sensitivity using Titer-Glo® 3D Cell Viability Assay. PDO viability was measured, and area under the curve calculated, to compare responses to various compounds. Correlations were calculated between selected patients’ clinical outcomes and in vitro drug testing results.ResultsWe established 31 PDOs. Among them, 28 PDOs can be expanded, including 15, 11, and 2 from ovarian, endometrial, and cervical cancers, respectively. The PDOs preserved the histopathological profiles of their originating tumors. In vitro drug testing of 10 ovarian cancer PDOs revealed individual differential responses to recommended drugs, and interpersonal heterogeneity in drug sensitivity, even with the same histology type. Among four patients who were platinum sensitive, resistant, or refractory, PDO drug responses correlated well with their clinical courses.ConclusionIn vitro drug testing using ovarian cancer organoids is feasible and correlates well with patient clinical responses. These results may facilitate development of precision chemotherapy and personalized screening for repurposed or new drugs.

Patient-Derived Tumor Organoids Combined with Function-Associated ScRNA-Seq for Dissecting the Local Immune Response of Lung Cancer.

In vitro models coupled with multimodal approaches are needed to dissect the dynamic response of local tumor immune microenvironment (TIME) to immunotherapy. Here the patient-derived primary lung cancer organoids (pLCOs) are generated by isolating tumor cell clusters, including the infiltrated immune cells. A function-associated single-cell RNA sequencing (FascRNA-seq) platform allowing both phenotypic evaluation and scRNA-seq at single-organoid level is developed to dissect the TIME of individual pLCOs. The analysis of 171 individual pLCOs derived from seven patients reveals that pLCOs retain the TIME heterogeneity in the parenchyma of parental tumor tissues, providing models with identical genetic background but various TIME. Linking the scRNA-seq data of individual pLCOs with their responses to anti-PD-1 (αPD-1) immune checkpoint blockade (ICB) allows to confirm the central role of CD8+ T cells in anti-tumor immunity, to identify potential tumor-reactive T cells with a set of 10 genes, and to unravel the factors regulating T cell activity, including CD99 gene. In summary, the study constructs a joint phenotypic and transcriptomic FascRNA-seq platform to dissect the dynamic response of local TIME under ICB treatment, providing a promising approach to evaluate novel immunotherapies and to understand the underlying molecular mechanisms.

The establishment of kidney cancer organoid line in drug testing.

Kidney cancer is a common urological malignancy worldwide with an increasing incidence in recent years. Among all subtypes, renal cell carcinoma (RCC) represents the most predominant malignancy in kidney. Clinicians faced a major challenge to select the most effective and suitable treatment regime for patients from a wide range of modalities, despite improved understanding and diagnosis of RCC. Recently, organoid culture gained more interest as the 3D model is shown to be highly patient specific which is hypothetically beneficial to the investigation of precision medicine. Nonetheless, the development and application of organotypic culture in RCC is still immature, therefore, the primary objective of this study was to establish an organoid model for RCC. Patients diagnosed with renal tumor and underwent surgical intervention were recruited. RCC specimen was collected and derived into organoids. Derived organoids were validated by histological examminations, sequencing and xenograft. Drug response of organoids were compared with resistance cell line and patients' clinical outcomes. Our results demonstrated that organoids could be successfully derived from renal tumor and they exhibited high concordance in terms of immunoexpressional patterns. Sequencing results also depicted concordant mutations of driver genes in both organoids and parental tumor tissues. Critical and novel growth factors were discovered during the establishment of organoid model. Besides, organoids derived from renal tumor exhibited tumorigenic properties in vivo. In addition, organoids recapitulated patient's in vivo drug resistance and served as a platform to predict responsiveness of other therapeutic agents. Our RCC organoid model recaptiluated histological and genetic features observed in primary tumors. It also served as a potential platform in drug screening for RCC patients, though future studies are necessary before translating the outcomes into clinical practices.

Establishment of patient-derived organoids for guiding personalized therapies in breast cancer patients.

Breast cancer has become the most commonly diagnosed cancer. The intra- and interpatient heterogeneity induced a considerable variation in treatment efficacy. There is an urgent requirement for preclinical models to anticipate the effectiveness of individualized drug responses. Patient-derived organoids (PDOs) can accurately recapitulate the architecture and biological characteristics of the origin tumor, making them a promising model that can overtake many limitations of cell lines and PDXs. However, it is still unclear whether PDOs-based drug testing can benefit breast cancer patients, particularly those with tumor recurrence or treatment resistance. Fresh tumor samples were surgically resected for organoid culture. Primary tumor samples and PDOs were subsequently subjected to H&E staining, immunohistochemical (IHC) analysis, and whole-exome sequencing (WES) to make comparisons. Drug sensitivity tests were performed to evaluate the feasibility of this model for predicting patient drug response in clinical practice. We established 75 patient-derived breast cancer organoid models. The results of H&E staining, IHC, and WES revealed that PDOs inherited the histologic and genetic characteristics of their parental tumor tissues. The PDOs successfully predicted the patient's drug response, and most cases exhibited consistency between PDOs' drug susceptibility test results and the clinical response of the matched patient. We conclude that the breast cancer organoids platform can be a potential preclinical tool used for the selection of effective drugs and guided personalized therapies for patients with advanced breast cancer.

Leveraging Patient-Derived Organoids for Personalized Liver Cancer Treatment.

Primary liver cancer (PLC) is a primary cause of cancer-related death worldwide, and novel treatments are needed due to the limited options available for treatment and tumor heterogeneity. 66 surgically removed PLC samples were cultured using the self-developed 2:2 method, and the final success rate for organoid culture was 40.9%. Organoid performance has been evaluated using comprehensive molecular measurements, such as whole-exome and RNA sequencing, as well as anticancer drug testing. Multiple organoids and their corresponding tumor tissues contained several of the same mutations, with all pairs sharing conventional TP53 mutations. Regarding copy number variations and gene expression, significant correlations were observed between the organoids and their corresponding parental tumor tissues. Comparisons at the molecular level provided us with an assessment of organoid-to-tumor concordance, which, in combination with drug sensitivity testing provided direct guidance for treatment selection. Finally, we were able to determine an appropriate pharmacological regimen for a patient with ICC, demonstrating the clinical practicality in tailoring patient-specific drug regimens. Our study provides an organoid culture technology that can cultivate models that retain most of the molecular characteristics of tumors and can be used for drug sensitivity testing, demonstrating the broad potential application of organoid technology in precision medicine for liver cancer treatment.

Innovative Approach to Isolate and Characterize Glioblastoma Circulating Tumor Cells and Correlation with Tumor Mutational Status.

Circulating tumor cells (CTCs) are one of the most important causes of tumor recurrence and distant metastases. Glioblastoma (GBM) has been considered restricted to the brain for many years. Nevertheless, in the past years, several pieces of evidence indicate that hematogenous dissemination is a reality, and this is also in the caseof GBM. Our aim was to optimize CTCs' detection in GBM and define the genetic background of single CTCs compared to the primary GBM tumor and its recurrence to demonstrate that CTCs are indeed derived from the parental tumor. We collected blood samples from a recurrent IDH wt GBM patient. We genotyped the parental recurrent tumor tissue and the respective primary GBM tissue. CTCs were analyzed using the DEPArray system. CTCs Copy Number Alterations (CNAs) and sequencing analyses were performed to compare CTCs' genetic background with the same patient's primary and recurrent GBM tissues. We identified 210 common mutations in the primary and recurrent tumors. Among these, three somatic high-frequency mutations (in PRKCB, TBX1, and COG5 genes) were selected to investigate their presence in CTCs. Almost all sorted CTCs (9/13) had at least one of the mutations tested. The presence of TERT promoter mutations was also investigated and C228T variation was found in parental tumors and CTCs (C228T heterozygous and homozygous, respectively). We were able to isolate and genotype CTCs from a patient with GBM. We found common mutations but also exclusive molecular characteristics.

Alteration in molecular properties during establishment and passaging of endometrial carcinoma patient-derived xenografts

Patient-derived xenograft (PDX) tumor models are known to maintain the genomic and phenotypic profiles, including the histopathological structures, of the parental tumors. On the other hand, unique enrichment of single-nucleotide variants or copy number aberrations has been reported in several types of tumors. However, an understanding of endometrial carcinoma PDXs is limited. The purpose of the present study was to clarify the presence or absence of the molecular properties of endometrial carcinomas in PDXs passaged up to eight times. Established PDXs of endometrioid carcinomas maintained their histopathological characteristics, but those of carcinosarcomas predominantly consisted of sarcomatous components when compared to the parental tumors. Alterations in the proportion of cells with positive/negative immunohistochemical staining for estrogen receptor, PTEN, PAX8, and PAX2 were observed, whereas the proportions of cells with AE1/AE3, TP53, ARID1A, PMS2, and MSH6 staining were unchanged. Variants of cancer-associated genes were compared between PDXs and parental tumors. Mutations in POLE and a frameshift deletion in BRCA1 were observed in the parental tumor tissue in each of the six cases, and additional genomic alterations, which were not apparently related to histopathological and immunohistochemical alterations, were found in the PDXs of these cases. The genomic and phenotypic alterations observed between endometrial carcinoma PDXs and parental tumors were partly associated with endometrial cancer-specific characteristics related to cellular differentiation and gene mutations.

Abstract 170: Glioblastoma patient-derived organoids as a model for discovering novel markers of therapy resistance in the context of tumor microenvironment: potential role of melanoma antigens

Abstract Introduction: Glioblastoma is resistant to standard treatment, leading to tumor recurrence and early death of patients. Using organoid models, we aim to explore the mechanisms of resistance to cancer therapies and identify novel therapeutic vulnerabilities, including stress protecting melanoma-associated antigens (MAGEs). MAGEs are normally restricted to expression in testis but are abnormally activated in cancer and are associated with therapy resistance. Methods: Organoids have been established from fresh tumor biopsies and are stored in Gliobank, a Slovenian collection of patient tumor samples with corresponding clinical data. Organoids were first characterized and compared with the corresponding parental tumor tissue by immunofluorescence and qPCR of selected markers. Organoids were then treated with a combination of irradiation, the chemotherapeutic agent temozolomide and/or a chemokine receptor antagonist, and response to therapy was assessed by immunofluorescence, qPCR, cell viability and invasion assays, multiplex ELISA and proximity ligation assay. Results: Organoids recapitulated the gene expression profile of parental glioblastoma tissues, including expression of genes related to cancer stem cells, DNA damage response, cell cycle progression, epithelial- to- mesenchymal transition, and cytokine signaling. In addition, the organoids preserved the cellular composition of the parental glioblastoma tissues, consisting of glioblastoma (stem) cells, macrophages, microglia, lymphocytes, and endothelial cells. Standard treatment decreased viability and invasion of organoids in only 2 of 6 patients, suggesting that organoids recapitulate tumor therapy resistance. The increased expression of MDM2 and CDKN1A in organoids after treatment with irradiation and temozolomide suggests that the p53 pathway and DNA damage response mechanisms may contribute to the therapy resistance. Next, we detected high abundance of secreted cytokines in the culture medium of the organoids, including CXCL12. However, treatment of the organoids with the CXCR4 antagonist Plerixafor, which blocked the interactions between CXCL12 and CXCR4 in the organoids, had no effect on organoid viability and invasion, suggesting resistance of glioblastoma to this immunotherapy. Since high expression of several type I MAGEs correlates with poor prognosis of glioma patients based on TCGA database analysis, we investigate the role of MAGEs in therapy resistance of glioblastoma organoids. Conclusions: Patient-derived tumor organoids provide a valuable tool to 1) identify novel therapeutic vulnerabilities in the context of the tumor microenvironment, 2) evaluate the effect of novel candidate treatments including immunotherapy, and 3) discover novel markers of therapy resistance. Citation Format: Barbara Breznik, Bernarda Majc, Anamarija Habič, Gloria Krapež, Andrej Porčnik, Jernej Mlakar, Tamara Lah Turnšek, Metka Novak, Klementina Fon Tacer. Glioblastoma patient-derived organoids as a model for discovering novel markers of therapy resistance in the context of tumor microenvironment: potential role of melanoma antigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 170.

Abstract 1058: Establishment of patient-derived organoid models of primary liver cancer and enable clinical personalized oncology

Abstract Background: Primary liver cancer (PLC) is the second cause of cancer-related mortality worldwide and includes mainly hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). Due to the limited treatment options and diversity of subtypes there is an unmet need to develop novel clinical approach to capture tumor heterogeneity and to guide personalized therapy in PLC. Methods: 33 surgically resected PLC patient tissues were cultured for organoid models. Patient-derived organoids (PDOs) and matched primary tumors were molecularly characterized by whole exome sequencing (WES) and RNA sequencing (RNA-seq). Subset of PDOs were screened for sensitivity to clinical medication recommended by National Comprehensive National network (NCCN) guideline to evaluate the feasibility of PLC-derived organoids in drug testing. Results:11 HCC, 1 ICC and 2 neuroendocrine tumors from patients were successfully established for organoid models. The success rate for generation of PLC organoids was 42.4% (14/33). To investigate whether organoids preserved the molecular characteristics of the originating tumors, 14 matched tumor-organoid pairs of genomic profile concordance and 3 pairs of gene expression profiles were analyzed based on WES and RNA-seq, respectively. Of the total somatic mutations found in the tumor tissues, a median of 55.5% (range 12.5% - 92.9%) mutations was observed in the corresponding PLC organoids. TP53 was the most common mutated gene either in tumors or in the organoids, with the same mutational frequency of 71.4% (10/14). Gene expression of organoids were highly correlated with their matched parental tumor tissue (median Spearman's correlation coefficient r = 0.81, range 0.76 - 0.85). To investigate the utility of PLC organoids for drug screening, multiple anti-cancer compounds (n=6-9) in standard clinical care were tested in 5 organoids which were showed high genomic concordance with their originating tumors. A good consistency between the drug screening and validation results was observed on patient NO.43 with ICC. The organoid of NO.43 was most sensitive to combined chemotherapy of irinotecan plus cisplatin. Together with drug testing result and clinical practice guidelines, this ICC patient was treated with irinotecan plus cisplatin after surgery and demonstrated durable response in clinical. In one-year follow-up no sign of recurrence was observed. Conclusion: PLC organoids intermediately to highly recapitulates the molecular and biological features of tumors. Drug sensitivity testing on PLC organoid models have the potential to guide personalized treatment in the clinical setting. Citation Format: Jianhua Rao, Feng Cheng, Long Zhang, Xiaohu Sun, Chao Song, Xuejiao Ma, Lei Ye, Wanglong Deng, Yansong Li, Zaozao Chen. Establishment of patient-derived organoid models of primary liver cancer and enable clinical personalized oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1058.

Patient-derived renal cell carcinoma organoids for personalized cancer therapy.

BackgroundKidney cancer is one of the most common solid tumors. The advancement of human kidney cancer research and treatment has been hindered by a lack of research models that faithfully recapitulate the diversity of the disease.MethodsWe established an effective three‐dimensional culture system for generating kidney cancer organoids from clinical renal cell carcinoma samples. Renal cell carcinoma (RCC) organoids were characterized by H&E staining, immunofluorescence, whole‐exome sequencing, RNA sequencing and single‐cell RNA sequencing. The use of RCC organoids in personalized cancer therapy was assessed by testing their responses to treatment drugs and chimeric antigen receptor T cells.ResultsUsing this organoid culture system, 33 kidney cancer organoid lines from common kidney cancer subtypes, including clear cell renal cell carcinoma (ccRCC), papillary renal cell carcinoma (pRCC), and chromophobe renal cell carcinoma (chRCC), were generated. RCC organoids preserved the histological architectures, mutational landscapes, and transcriptional profile of the parental tumor tissues. Single‐cell RNA‐sequencing revealed inter‐ and intra‐tumoral heterogeneity in RCC organoids. RCC organoids allowed for in vitro drug screening and provided a tool for assessing the efficacy of chimeric antigen receptor T cells.ConclusionsPatient‐derived RCC organoids are valuable pre‐clinical models for academic research and personalized medicine.

Abstract 3111: Establishment of patient-derived xenografts in penile cancer

Abstract Background: Penile Cancer (PeCa) is a rare cancer with over 90% squamous cell carcinoma (SCC)histology. Risk factors include human papillomavirus (HPV) infection and phimosis. Molecular oncogenic pathways between HPV-Positive and HPV-Negative PeCa are difficult to validate because there are few preclinical models of PeCa and no models of HPV-Positive PeCa. Our goal was to establish PeCA patient-derived xenograft (PDX) models to elucidate the molecular signature of HPV-Positive and HPV-Negative PeCa for therapeutic benefit. Methods: PeCa specimens from penectomies and inguinal lymph node dissections (ILND) were collected from 2019-2021. Tumor specimens were dissected and tissue coated in Matrigel and transplanted subcutaneously into NOD SCID gamma (NSG™) mice. Tumor growth was monitored and was harvested at ≈ 1500m3 for subsequent propagation. Short tandem repeat fingerprinting (STR) analysis was performed on genomic DNA from PDX tissue and parental patient tumor tissue. Immunohistochemistry was performed on all PDX models and original patient primary tumors to validate the expression of p16 protein, a surrogate marker for HPV-Positive disease. p16 positivity was determined using a 0-3 scoring where a score of 3 was assessed as positive and a score of ≤2 as negative. For HPV genotyping: PCR was performed using DNA ELISA kit HPV, followed by RHA Kit HPV SPF10-LiPA25. Results: 8 PDXs were established from tissue from 7 patients. Age, race, and type of surgery were determined. SCC histology was confirmed in all the patients. Basaloid variant was present in 3 patients, and Verrucous variant was present in 1 patient. For pathological staging, 2 patients were staged as pT3N3Mx, 2 patients as pT2N1M0, 1 patient as pT3N0 and 3 patients as pT3Nx. One ILND had no evidence of disease. 2 PDX models (MDAPe3 and MDAPe7) were derived from two specimens from the same patient (1 pretreatment, 1 post-treatment). All models were matched to the patient's primary tumor by STR fingerprinting analysis. Squamous cell carcinoma histology was confirmed in all PDXs. 6 PDX models are currently in passage 3 and 6 PDXs were p16 positive. HPV genotyping in p16 positive PDXs detected high-risk HPV 16 and p16 negative xenografts were also HPV negative. Conclusion: In our study, we established PeCa PDX models that recapitulated the patients' histology. We report to our knowledge the first HPV-positive penile cancer xenografts. Plans for comprehensive characterization and future targeted therapy experiments are in development. Citation Format: Luis A. Segarra, Niki M. Zacharias, Alberto Pieretti, Angelita Alaniz, Tapati Maity, Sue Martinez, Priya Rao, Natalie Fowlkes, Jad Chahoud, Xin Lu, Magaly Martinez Ferrer, Christopher Wood, Curtis Pettaway. Establishment of patient-derived xenografts in penile cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3111.

BIOM-39. METHYLATION AND MUTATION PROFILES IN MENINGIOMA CELL-DERIVED EXTRACELLULAR VESICLE DNA REFLECT EPIGENETIC AND GENOMIC ALTERATIONS IN ORIGINAL TUMORS

Abstract The majority of meningiomas are benign but approximately 20% of display an aggressive behavior, resulting in significant patient morbidity and mortality. Standard monitoring after meningioma resection relies on serial MRI examinations, which are time-consuming, expensive and provide no information on molecular alterations that may indicate progression towards a more aggressive tumor. Extracellular vesicles (EVs) are released by tumor cells and contain high molecular weight DNA, rendering circulating EVs a potential biomarker source for non-invasive disease monitoring and for obtaining information on genetic and epigenetic alterations. We quantified EVs in plasma of 46 meningioma patients (n = 29 M1, 12 M2, 5 M3) by nanoparticle tracking analysis and detected significantly higher levels compared to age-matched healthy donors (n = 18). EV concentrations correlated with malignancy grade (p = 0.0049) and with the extent of peritumoral edema (p = 0.0031). Comparisons between paired pre- and postoperative samples revealed that EV levels counts dropped significantly the day after tumor resection and were reduced to normal levels after about one week. Completely resected patients (Simpson grade I) displayed a greater reduction of postoperative EV concentrations than incompletely resected patients. DNA methylation profiling was performed on EVs secreted by cultured meningioma cells, as well as matched cells and original tumors using 850k arrays (n = 7 M1, 5 M2, 3 M3). All EV samples were correctly identified as meningiomas by the Heidelberg classifier, and methylation subclasses were also correctly assigned in almost all cases. t-SNE analysis showed that EVs mapped in close proximity to their corresponding parental cells and tumor tissue. Tumor specific mutations and copy number variations were detected in EV-DNA with high accuracy. Differential quantitative proteomic analysis of EVs, cells and tumors identified shared proteins that could potentially be useful for enriching tumor-derived circulating EVs from biofluids.

Establishment and characterization of prostate organoids from treatment-naïve patients with prostate cancer.

Three-dimensional (3D) organoid culture systems are emerging as potential reliable tools to investigate basic developmental processes of human disease, especially cancer. The present study used established and modified culture conditions to report successful generation and characterization of patient-derived organoids from fresh primary tissue specimens of patients with treatment-naïve prostate cancer (PCa). Fresh tissue specimens were collected, digested enzymatically and the resulting cell suspensions were plated in a 3D environment using Matrigel as an extracellular matrix. Previously established 12-factor medium for organoid culturing was modified to create a minimal 5-factor medium. Organoids and corresponding tissue specimens were characterized using transcriptomic analysis, immunofluorescent analysis, and immunohistochemistry. Furthermore, patient-derived organoids were used to assess the drug response. Treatment-naïve patient-derived PCa organoids were obtained from fresh radical prostatectomy specimens. These PCa organoids mimicked the heterogeneity of corresponding parental tumor tissue. Histopathological analysis demonstrated similar tissue architecture and cellular morphology, as well as consistent immunohistochemical marker expression. Also, the results confirmed the potential of organoids as an in vitro model to assess potential personalized treatment responses as there was a differential drug response between different patient samples. In conclusion, the present study investigated patient-derived organoids from a cohort of treatment-naïve patients. Derived organoids mimicked the histological features and prostate lineage profiles of their corresponding parental tissue and may present a potential model to predict patient-specific treatment response in a pre-clinical setting.

Establishment and genetic characterization of cell lines derived from proliferating nasal polyps and sinonasal inverted papillomas

To better understand the pathogenesis of nasal polyps (NPs) and sinonasal inverted papillomas (SIPs), we aimed to establish cell lines from fresh tissues of NPs and SIPs and characterize them. Primary cell cultures were obtained from two NP tissues (NP2 and NP3) and one SIP tissue (IP4). All the cells were polygonal in shape, expressed cytokeratin 14, and had normal diploid chromosome status. HPV58 DNA was detected in NP3. To obtain immortal primary cells, NP2 and IP4 cells were transduced with a combination of mutant CDK4, cyclinD1 and TERT. These cells were thereafter named NP2/K4DT and IP4/K4DT, respectively. HPV58-positive NP3 cells were transduced with TERT alone, the resulting cells named NP3/T. Phenotypic and genotypic identity of original tissues and derived cells was investigated. All the cell cultures with transgenes were confirmed to be derived from their parental cells and primary tumor tissues by analysis of short tandem repeats (STR) and maintained in vitro growth, genetic profiles and gene expression characteristics of the primary cells. These virtually immortalized cells, as well as the primary cells, have potential as in vitro models for studying the pathogenesis of NPs and SIPs and for preclinical study to develop new therapeutic agents.

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.

Patient-derived organoids of bladder cancer recapitulate antigen expression profiles and serve as a personal evaluation model for CAR-T cells in vitro.

ObjectivesRecent advances in patient‐derived cancer organoids have opened a new avenue for personalised medicine. We aimed to establish an in vitro technological platform to evaluate chimeric antigen receptor (CAR)‐T cell‐mediated cytotoxicity against bladder cancer.MethodsPatient‐derived bladder cancer organoids (BCOs) were derived using classic medium containing R‐spondin 1 and noggin. The features of BCOs were characterised via H&E, whole‐exome sequencing and immunofluorescence of specific markers. Surface antigen expression profiles of the recently identified CAR‐recognisable targets were determined with a panel of antibodies via immunohistochemistry. A co‐cultivation system consisting of BCOs and engineered T cells targeting a specific antigen was utilised to test its efficacy to model immunotherapy by cytotoxic assays and ELISA.ResultsBladder cancer organoid lines of basal and luminal subtypes were established. The histopathological morphology, genomic alteration, and specific marker expression profiles showed that the BCO lines retained the characteristics of the original tumors. Among all tested CAR‐recognisable antigens in other solid tumors, MUC1 was simultaneously expressed in organoids and parental tumor tissues. Given the surface antigen profiles, second‐generation CAR‐T cells targeting MUC1 were prepared for modelling in vitro immunotherapy responses in BCOs. Specific immune cytotoxicity occurred only in the MUC1+ organoids but not in the MUC1‐ organoids or control CAR‐T cells.ConclusionPatient‐derived BCOs recapitulate the heterogeneity and key features of parental cancer tissues, and these BCOs could be useful for preclinical testing of CAR‐T cells in vitro.

Human Lung Adenocarcinoma-Derived Organoid Models for Drug Screening

SummaryLung cancer is an extremely heterogeneous disease, and its treatment remains one of the most challenging tasks in medicine. Few existing laboratory lung cancer models can faithfully recapitulate the diversity of the disease and predict therapy response. Here, we establish 12 patient-derived organoids from the most common lung cancer subtype, lung adenocarcinoma (LADC). Extensive gene and histopathology profiling show that the tumor organoids retain the histological architectures, genomic landscapes, and gene expression profiles of their parental tumors. Patient-derived lung cancer organoids are amenable for biomarker identification and high-throughput drug screening in vitro. This study should enable the generation of patient-derived lung cancer organoid lines, which can be used to further the understanding of lung cancer pathophysiology and to assess drug response in personalized medicine.

Abstract IA27: Patient-derived organoids in pediatric cancer research

Abstract Recent advances in in vitro culture technologies, such as adult stem cell-derived organoids, have opened up new avenues for the development of novel, more physiologic human cancer models. Such preclinical models are essential for efficient translation of basic cancer research into novel treatment regimens. We succeeded in growing organoids from a range of pediatric solid tumors, including Wilms’ tumors, renal cell carcinomas, and different types of rhabdoid tumors (i.e., AT/RT, MRT). Tumor organoids retain many characteristics of parental tumor tissue. For instance, Wilms’ tumor organoids retain the cellular heterogeneity of tumors, as they are composed of an intricate network of different cell types. Moreover, we demonstrate that tumor organoids are amenable to gene editing and high-throughput drug screens. In conclusion, our pediatric cancer organoids capture disease and tissue heterogeneity and provide a platform for basic cancer research, drug screening, and personalized medicine. Citation Format: Camilla Calandrini, Frans Schutgens, Rurika Oka, Thanasis Margaritis, Tito Candelli, Luka Mathijsen, Carola Ammerlaan, Ravian van Ineveld, Sepideh Derakhshan, Lars Custers, Philip Lijnzaad, Harry Begthel, Hinri Kerstens, Maarten Rookmaker, Marianne Verhaar, Patrick Kemmeren, Ronald de Krijger, Kathy Pritchard-Jones, Anne Rios, Marry van den Heuvel-Eibrink, Frank Holstege, Ruben van Boxtel, Hans Clevers, Jarno Drost. Patient-derived organoids in pediatric cancer research [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr IA27.

Organoid Cultures as Preclinical Models of Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related deaths worldwide. There is an unmet need to develop novel clinically relevant models of NSCLC to accelerate identification of drug targets and our understanding of the disease. Thirty surgically resected NSCLC primary patient tissue and 35 previously established patient-derived xenograft (PDX) models were processed for organoid culture establishment. Organoids were histologically and molecularly characterized by cytology and histology, exome sequencing, and RNA-sequencing analysis. Tumorigenicity was assessed through subcutaneous injection of organoids in NOD/SCID mice. Organoids were subjected to drug testing using EGFR, FGFR, and MEK-targeted therapies. We have identified cell culture conditions favoring the establishment of short-term and long-term expansion of NSCLC organoids derived from primary lung patient and PDX tumor tissue. The NSCLC organoids recapitulated the histology of the patient and PDX tumor. They also retained tumorigenicity, as evidenced by cytologic features of malignancy, xenograft formation, preservation of mutations, copy number aberrations, and gene expression profiles between the organoid and matched parental tumor tissue by whole-exome and RNA sequencing. NSCLC organoid models also preserved the sensitivity of the matched parental tumor to targeted therapeutics, and could be used to validate or discover biomarker-drug combinations. Our panel of NSCLC organoids closely recapitulates the genomics and biology of patient tumors, and is a potential platform for drug testing and biomarker validation.

Feasibility of primary human cell cultures as a model for adamantinomatous craniopharyngioma research: Evidence from RNA-Seq analysis.

Adamantinomatous craniopharyngioma (ACP) is a benign epithelial tumor of the sellar region. Whether primary human cell cultures can be used as a stable research model has yet to be determined. The characteristics of three cultured craniopharyngioma primary cell (CPC) lines were identified using immunofluorescence. The culture duration for each CPC line was 10, 20 and 30 days. Cell lines and paired parental tumor tissues were subsequently analyzed using transcriptome sequencing (RNA-Seq). Transcriptomic differences between ACP tissues and CPC lines were compared. CPCs maintained the original epithelial lineage markers, including pan-cytokeratin and epithelial cell adhesion molecule. However, the Pearson's correlation coefficient of transcriptomes between each pair of CPC lines and ACP tissues decreased from 0.657 (cultured for 10 days) to 0.61 (cultured for 20 days) and further to 0.547 (cultured for 30 days). The number of differentially expressed genes between ACP tissues and CPCs was increased from 1,247 (cultured for 10 days) to 1,643 (cultured for 20 days) and then to 1,949 (cultured for 30 days). The results of Gene Set Enrichment Analysis demonstrated that the diversity of gene sets increased with longer culture time. Significant differences in the majority of signature gene sets were not observed between ACP tissues and CPCs, with the exception of keratinization phenotype [normalized enrichment score (NES)=−2.02, false discovery rate (FDR)=0.0038] and epithelial cell phenotype (NES=−1.82, FDR=0.032). Cell proliferation (NES=1.78, FDR=0.028) and mitosis (NES=1.93, FDR=0.012) were enhanced in CPCs. Therefore, primary human cell cultures can be used as a suitable research platform for ACP, however further experiments are required.