Development Of Patient-derived Xenograft Models Research Articles

Abstract 5475: Development of high-throughput 3D bioprinted pediatric models for precision medicine

Abstract Childhood cancer patients with high-risk disease have poor prognosis. Therapies given to treat these patients are often highly toxic and determined empirically, exposing children to damaging and ineffective therapies. Precision medicine is a promising strategy for these patients. However, tumor biopsies often lack sufficient sample for phenotypic drug screening, requiring cell expansion via primary cell culture or development of patient-derived xenograft models - a slow process with variable success rates. There is an unmet need for sensitive, predictive, and timely drug testing models. Dynamic interactions between cells and the extracellular matrix (ECM) impact cellular signaling and response to cancer therapy. The development of patient-derived tumor models that are reflective of the patient sample and achieved in a clinically relevant timeframe will be game changing. In-silico analysis of 265 ECM gene expression from a high-risk neuroblastoma and sarcoma patient cohort (n=145) identified collagens and fibronectin to be the most abundantly expressed ECM genes in the tumor samples. To reflect this environment, we developed tuneable tissue ECM-like bioinks where cells are embedded within the bioink that mimics growth constraints within a tumor [1]. Specifically, we functionalized the bioinks with peptides of collagen I, fibronectin, and laminin to mimic the ECM environment and combined this with HTP 3D bioprinting technology to create patient-derived tumoroids. In this proof-of-concept study, we identified conditions that enable the growth and expansion of the neuroblastoma and sarcoma cells in a 3D ECM-like environment. The tumor samples proliferate in the bioinks and HTP drug screening was used to determine drug sensitivity. Importantly, the bioprinted cells reflect the genetic and phenotypic characteristics of the original patient tumors and retained their tumorigenic capacity in vivo. Collectively, we have successfully generated preclinical models that reflect patient tumors and are directly compatible with preclinical drug testing. Importantly, our 3D bioprinting platform has the potential to advance cancer precision medicine in a clinically relevant timeframe. These findings have broader applications for a range of cancer types for preclinical testing, drug discovery and cancer biology.1. Utama, RH, et al. Macromolecular Bioscience, 2021. 21(9):e2100125 Citation Format: Valentina Poltavets, MoonSun Jung, Joanna Skhinas, Kathleen Kimpton, Alvin Kamili, Gabe Tax, Jie Mao, Louise Cui, Marie Wong, Mark J. Cowley, Loretta Lau, Emmy ME Dolman, John J. Gooding, Maria Kavallaris. Development of high-throughput 3D bioprinted pediatric models for precision medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5475.

Abstract PO-129: Using the castration resistant prostate cancer patient-derived xenograft (PDX) tumor model to identify mRNA/miRNA biomarkers which distinguish abiraterone treatment responders and non-responders

Abstract Prostate cancer (PCa) is the most frequent cancer occurring in men in the United States and is the second leading cause of cancer deaths in men. Median survival for patients with metastatic hormone refractory PCa (HRPC/CRPC) is around 18 months. Abiraterone (Abi) is a next-generation androgen receptor targeting agent which has significant effects on PCa patients' survival, and has been approved for treatment of metastatic and CRPC. However, it is clear that there is development of Abi resistance (Abi-R). Abi inhibits the steroidogenic enzyme CYP17A1, a critical enzyme in androgen biosynthesis in the adrenals, testis as well as tumor tissues. To screen and identify biomarkers (genes and microRNA (miRNA)) for Abi-R in a heterogenous tumor, we used patient derived PCa xenograft animal model (PCa-PDX mice). Recent advances in the development of PDX models and their increasing sophistication have led to their escalating use for anticancer drug research and development, and as predictive clinical models. Hence, for the correct identification of Abi-R markers, we proposed using PCa-PDX animal model (PDX mice). We hypothesized that changes in miRNA expression underlie Abi resistance (Abi-R) mechanisms. Each tumor bearing mouse was castrated resulting in tumor regression followed by regrowth/relapse phase. Mice were then treated with Abi. The tumors were collected either once they regressed (called CRABS), or reinoculated into castrated host if they showed continued growth. The new host was treated with Abi once tumors reached 50-300 mm3. Tumors were harvested when they reached a volume of ~600-800 mm3 (CRABR2). The RNA was extracted from tumors using the miRvana kits, and RNA/miRNA-seq performed. We compared the Abi sensitive vs the Abi resistant (CRABR2) groups, to identify 316 genes and 55 miRNA whose expression was significantly changed between these two groups. We used Venn diagrams to overlap the validated gene targets for the miRNA (5,557 genes) with our 316 gene dataset. 85 common genes were identified, of which 63 genes were validated targets of hsa-miR-335 (1.7 fold in CRABS vs CRABR2), and 8 genes were validated targets of hsa-miR-574 (0.4 fold in CRABS vs CRABR2). Hsa-miR-335 has been previously identified to function as a candidate tumor suppressor in PCa. Reduced expression of miR-335 was found in human PCa tissues comparing with paired adjacent benign prostate tissues (P < 0.05). Moreover, the increased expression of miR-335 suppressed cell proliferation, invasion and migration of PCa cell lines in vitro. It has been found to suppress bone metastasis in PCa via targeting endothelial nitric oxide synthase. MicroRNA-574-5p promotes metastasis of non-small cell lung cancer, triple negative breast cancer and colorectal cancer to name a few. However, their function in Abi resistance has not been explored. In conclusion, we have identified two promising miRNA for distinguishing Abi sensitive from resistant tumors whose targeting should have profound effects on sensitizing castration resistant tumors to Abi. Citation Format: Bin Ouyang, Dan Song, Jacek Biesiada, Yan Ren, Mario Medvedovic, Pheruza Tarapore. Using the castration resistant prostate cancer patient-derived xenograft (PDX) tumor model to identify mRNA/miRNA biomarkers which distinguish abiraterone treatment responders and non-responders [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-129.

Mouse models of high-risk neuroblastoma.

Informative and realistic mouse models of high-risk neuroblastoma are central to understanding mechanisms of tumour initiation, progression, and metastasis. They also play vital roles in validating tumour drivers and drug targets, as platforms for assessment of new therapies and in the generation of drug sensitivity data that can inform treatment decisions for individual patients. This review will describe genetically engineered mouse models of specific subsets of high-risk neuroblastoma, the development of patient-derived xenograft models that more broadly represent the diversity and heterogeneity of the disease, and models of primary and metastatic disease. We discuss the research applications, advantages, and limitations of each model type, the importance of model repositories and data standards for supporting reproducible, high-quality research, and potential future directions for neuroblastoma mouse models.

Development of patient-derived xenograft models of prostate cancer for maintaining tumor heterogeneity.

Prostate cancer (Pca) is a heterogeneous disease with multiple morphological patterns. Thus, the establishment of a patient-derived xenograft (PDX) model that retains key features of the primary tumor is of great significance. This review demonstrates the characteristics and advantages of the Pca PDX model and summarizes the main factors affecting the establishment of the model. Because this model well recapitulates the diverse heterogeneity observed in the clinic, it was extensively utilized to discover new therapeutic targets, screen drugs, and explore metastatic mechanisms. In the future, clinical phenotype and different stages of the Pca patient might be faithfully reflected by PDX model, which provides tremendous potential for understanding Pca biology and achieving individualized treatment.

The IL ‐6 signaling complex is a critical driver, negative prognostic factor, and therapeutic target in diffuse large B‐cell lymphoma

Interleukin‐6 (IL‐6) is a growth factor for normal B cells and plasma cell‐derived malignancies. Here, we show that the IL‐6 signaling pathway is also active in a subset of diffuse large B‐cell lymphoma (DLBCL) patients with particularly poor prognosis. Primary DLBCL cells and DLBCL cell lines expressing IL‐6R engraft and form orthotopic lymphomas in humanized mice that ectopically produce human IL‐6, and in mice reconstituted with a human immune system. We show that a subset of DLBCL cases have evolved mechanisms that ensure constitutive activation of the IL‐6 signaling pathway, i.e., the expression of both chains of the IL‐6R, the expression of the cytokine itself, and the mutational inactivation of a negative regulator of IL‐6 signaling, SOCS1. IL‐6 signaling promotes MYC‐driven lymphomagenesis in a genetically engineered model, and treatment with the IL‐6R‐specific antibody tocilizumab reduces growth of primary DLBCL cells and of DLBCL cell lines in various therapeutic settings. The combined results uncover the IL‐6 signaling pathway as a driver and negative prognosticator in aggressive DLBCL that can be targeted with a safe and well‐tolerated biologic.

International experience in the development of patient-derived xenograft models of diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma is the most aggressive form of high grade glioma in children with no effective therapies. There have been no improvements in survival in part due poor understanding of underlying biology, and lack of representative in vitro and in vivo models. Recently, it has been found feasible to use both biopsy and autopsy tumors to generate cultures and xenograft models. To further model development, we evaluated the collective international experience from 8 collaborating centers to develop DIPG pre-clinical models from patient-derived autopsies and biopsies. Univariate and multivariate analysis was performed to determine key factors associated with the success of in vitro and in vivo PDX development. In vitro cultures were successfully established from 57% of samples (84.2% of biopsies and 38.2% of autopsies). Samples transferred in DMEM media were more likely to establish successful culture than those transported in Hibernate A. In vitro cultures were more successful from biopsies (84.2%) compared with autopsies (38.2%) and as monolayer on laminin-coated plates than as neurospheres. Primary cultures successfully established from autopsy samples were more likely to engraft in animal models than cultures established from biopsies (86.7% vs. 47.4%). Collectively, tumor engraftment was more successful when DIPG samples were directly implanted in mice (68%), rather than after culturing (40.7%). This multi-center study provides valuable information on the success rate of establishing patient-derived pre-clinical models of DIPG. The results can lead to further optimization of DIPG model development and ultimately assist in the investigation of new therapies for this aggressive pediatric brain tumor.

Abstract 5013: Rapid generation of phenomic and functional profiles of patient-derived 3D cell culture models for identification of treatment vulnerabilities of breast cancer: Early results of the EFRE-PoP project

Abstract Targeted treatment for breast cancer subsets currently relies on the occurrence of estrogen, progesterone and Her2/neu receptors. For triple negative breast cancer (TNBC) there is no identified targeted therapy. The mutational landscape for breast cancer subsets has been characterized, but drug development has been limited due to the lack of appropriate preclinical models. Development of patient-derived xenograft models (PDX) has been difficult with take rates of below 30%.To establish a series of patient-derived 3D (PD3D) cell culture models as a versatile resource for ex vivo drug sensitivity screens as well as secondary establishment of PDX. We obtained breast cancer specimens either by biopsy or surgical resection. Upon arrival tumor tissue was minced and enzymatically digested. After subsequent filtering, respective tissue size fractions were seeded as Matrix-droplets into 24-well plates and incubated under standard conditions. Growth of PD3Ds was monitored daily by microscopy. PD3Ds were splitted when their diameter reached appropriate size. Organoids were than fixed and embedded. FFPE sections of donor-tissues and derived PD3D models were than used for IHC and inspected by a pathologist. In parallel, mutational profiling of snap-frozen tumor tissue and cell cultures was performed. All models were subjected to a semi-automated multi-drug response assay in a 384-well format to assess individual compound sensitivities.We established a scalable workflow for culturing and screening of PD3D cell cultures from limited quantities of breast cancer tissue from true-cut and vacuum biopsies as well as surgical specimen. Tissue fragments of ca. 3mm in diameter were sufficient to successfully establish PD3D cell cultures at a high yield. The time needed to expand PD3D cell cultures to obtain a sufficient amount of cells for subsequent molecular analyses varies from 2-6 weeks depending on amount and quality of samples as well as grade and stage of the donor tumor tissue. At the time, we can report a rate of culture establishment rate of 75% (11 models from 15 patient samples) for various clinical relevant subgroups, including TNBC, HR+, Her 2 pos. BC and one DCIS sample. The immunohistology and mutational profiles of these samples are currently being confirmed. The established workflow for culturing PD3D cell cultures offers high yield rates within a potentially clinically relevant time-frame for HTP cytotoxicity screenings. We are encouraged that this method is suitable for various molecular subtypes of breast cancer. Once adequately validated in co-clinical trials, PD3D models would make for an intriguing tool in supporting clinical decision-making for individual patients. Particularly patients with breast cancer refractory to standard of care compounds could benefit from this approach. Citation Format: Verena I. Kiver, Guido Gambara, Olga Gorea, Jens-Uwe Blohmer, Philipp Jurmeister, Carsten Denkert, Alessandra Silvestri, Caroline M. Schweiger, Maxine Silvestrov, Ulrich Keilholz, Christian R. Regenbrecht. Rapid generation of phenomic and functional profiles of patient-derived 3D cell culture models for identification of treatment vulnerabilities of breast cancer: Early results of the EFRE-PoP project [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5013.

Abstract 77: Establishment of lung cancer patient derived xenograft model from circulating tumor cells: Identification of somatic mutations associated with metastatic potentials

Abstract Carcinoma metastasis is initiated by a subpopulation of circulating tumor cells (CTCs) found in the blood of patients. However, the existence and genetic alternations of metastasis-initiating cells (MICs) among CTCs has not been experimentally demonstrated. The establishment of xenograft model using CTCs should help us to understand the biologic behaviors and genetic alternations of MICs associated the promotion of metastasis. Here, we demonstrate that xenograft model can be established by CTCs obtained from lung cancer patients and somatic mutations analyzed from xenograft model in which MICs develop metastatic tumor. After injecting mononuclear cells of lung cancer patients via tail vein, mouse was sacrificed at four weeks. After primary cultured cells were isolated from mouse lung tissues, the MICs were selected by using FASC sorting (EpCam+) and then re-injected via tail vein. And tumor formations in mouse were examined at 8 weeks. For identifying somatic mutations, target sequencing with NGS was performed in tumor DNA and cell free DNA (CF DNA) of lung cancer patients bloods. Our results indicated that nonsynonymous IDH, EGFR, KIT, ABL1, PTEN, ATM, P53, and SMAD4 were found in both tumor tissues and CF DNA of lung cancer patients’ bloods. IDH and EGFR was alternative homo. Others newly found in CF DNA. For further examination of the biologic behaviors of tumor cells which harvested from xenograft model using CTCs, we cultured primary cells from lung tissues and analyzed the anti-cancer drug resistance. Our results showed that primary cultured cells (BH-007) were resistant against cisplatin and erlotinib. In assay of migration and invasion, BH-007 cells possessed high metastatic potential when compared with A549 and H23 cell lines. These data suggested that several somatic mutations affect the anti-cancer drug resistance in lung cancer patients. Our future researches will include development of patient-derived xenograft models for preclinical testing of novel therapeutics. And we develop the possible integration of CF DNA analysis and PDX model from CTCs in the design of co-clinical studies for testing in individual lung cancer patients. Citation Format: Chansu Lee, Youngwook Kim, Eunkyung Bae, Sunghoon Cho, Sohyun Youn, Kwang-Sung Ahn, Bong-Seog Kim. Establishment of lung cancer patient derived xenograft model from circulating tumor cells: Identification of somatic mutations associated with metastatic potentials. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 77.

Distinct Properties of Leukemia Stem Cells in Primary Refractory Acute Myeloid Leukemia

Introduction: Acute Myeloid Leukemia (AML) patients who are refractory to induction chemotherapy (RF) have a dismal prognosis. Properties of AML cell populations that cause the refractory phenotype are poorly understood. It is postulated that the leukemia stem cell (LSC) pool promotes chemotherapy resistance in AML; yet whether differences exist in this pool as a function of eventual remission status, and thus may account for induction failure, is unknown. Our group has recently found that in AML patients who initially achieve complete remission (CR) and then relapse, the LSC pool evolves to become larger and more immunophenotypically diverse at the time of relapse [Blood 2013; 122(21): abstract #883]. Since relapsed AML patients are often refractory to salvage chemotherapy, we postulated that diagnostic specimens from RF AML patients would have enlarged, immunophenotypically-diverse LSC pools relative to AML patients achieving CR, implicating these properties in the refractory phenotype. Defining unique properties of LSCs in RF AML might provide greater insight into the mechanisms responsible for the treatment-resistant phenotype.Methods: RF AML patients were defined by having ≥ 6% leukemic blasts in their bone marrow or peripheral blood after 1 or 2 cycles of anthracycline/cytarabine-based induction chemotherapy. All studies were conducted with pre-treatment AML specimens on approved IRB protocols at the University of Rochester Medical Center. LSC frequency was determined by limiting dilution analysis and xenotransplantation into sublethally irradiated NOD scid gamma (NSG) mice. To determine immunophenotypically-defined cell populations harboring functional LSC activity, AML specimens were sorted into four distinct populations defined by CD34 and CD38 staining. The presence of LSC activity in the four resultant populations (CD34+/CD38-, CD34+/CD38+, CD34-/CD38+, and CD34-/CD38-) was determined by the ability of each sorted population to engraft NSG mice in primary and secondary transplantation experiments. RNA-seq analysis was conducted on LSC-enriched cell populations.Results: Specimens from RF AML patients were more likely to engraft NSG mice relative to those from AML patients achieving CR (6/6 vs. 13/29 in RF vs. CR patients, respectively; P=0.02). The LSC pool from six RF AML patients was studied in detail. LSC frequency ranged from 1/19 to 1/326,123. Four of the 6 RF specimens harbored LSC frequencies 6- to 7,700-fold higher than those previously reported in primary AML [Sarry et al. J Clin Invest 2011; 121(1384-395]. In 5/6 RF specimens, LSCs were present in more than one immunophenotypically-defined cell population. Relative to specimens from AML patients achieving CR, specimens from RF patients were more likely to harbor LSCs in both CD34+ and CD34- populations (P=0.04). Treatment of NSG mice xenografted with specimens from RF patients with anthracycline/cytarabine-based chemotherapy failed to eradicate disease from engrafted mice; thus, therapeutic outcome of NSG mice engrafted with specimens from RF patients resembles that seen in patients. To identify genes potentially driving the refractory LSC phenotype, we compared gene expression profiles of CD34+ cells from RF patients to those from AML patients achieving long-term remission and to those from normal donors. Ninety-nine genes were uniquely deregulated in RF LSCs, including numerous Homeobox transcription factors and components of the Wnt and Hedgehog signaling pathways, all implicated in the maintenance of stemness. Pathway analysis revealed that amino acid metabolic pathways critical to other treatment-resistant cancers and molecules involved in IL-1 signaling and implicated in metastasis of solid tumors are dysregulated in primary refractory LSCs.Conclusions: This study is the first to systematically analyze the LSC pool in primary refractory AML. Similar to our findings in relapsed AML, RF AML patients harbor enlarged, phenotypically diverse LSC pools relative to AML patients achieving CR. Challenge of leukemic mice with an anthracycline/cytarabine treatment regimen mimics the effect seen in patients, facilitating the development of patient-derived xenograft models of RF AML. We identify a refractory LSC gene signature, opening the door to future mechanistic investigations and novel therapeutic approaches for this AML patient population in great need. DisclosuresCalvi:Fate Therapeutics: Patents & Royalties. Becker:Millenium: Research Funding.

Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

BackgroundThere is a paucity of effective therapies for recurrent/aggressive meningiomas. Establishment of improved in vitro and in vivo meningioma models will facilitate development and testing of novel therapeutic approaches.MethodsA primary meningioma cell line was generated from a patient with an olfactory groove meningioma. The cell line was extensively characterized by performing analysis of growth kinetics, immunocytochemistry, telomerase activity, karyotype, and comparative genomic hybridization. Xenograft models using immunocompromised SCID mice were also developed.ResultsHistopathology of the patient tumor was consistent with a WHO grade I typical meningioma composed of meningothelial cells, whorls, and occasional psammoma bodies. The original tumor and the early passage primary cells shared the standard immunohistochemical profile consistent with low-grade, good prognosis meningioma. Low passage KCI-MENG1 cells were composed of two cell types with spindle and round morphologies, showed linear growth curve, had very low telomerase activity, and were composed of two distinct unrelated clones on cytogenetic analysis. In contrast, high passage cells were homogeneously round, rapidly growing, had high telomerase activity, and were composed of a single clone with a near triploid karyotype containing 64–66 chromosomes with numerous aberrations. Following subcutaneous and orthotopic transplantation of low passage cells into SCID mice, firm tumors positive for vimentin and progesterone receptor (PR) formed, while subcutaneous implant of high passage cells yielded vimentin-positive, PR-negative tumors, concordant with a high-grade meningioma.ConclusionsAlthough derived from a benign meningioma specimen, the newly-established spontaneously immortal KCI-MENG1 meningioma cell line can be utilized to generate xenograft tumor models with either low- or high-grade features, dependent on the cell passage number (likely due to the relative abundance of the round, near-triploid cells). These human meningioma mouse xenograft models will provide biologically relevant platforms from which to investigate differences in low- vs. high-grade meningioma tumor biology and disease progression as well as to develop novel therapies to improve treatment options for poor prognosis or recurrent meningiomas.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0596-8) contains supplementary material, which is available to authorized users.

Advances in patient-derived tumor xenografts: From target identification to predicting clinical response rates in oncology

Most oncology compounds entering clinical development have passed stringent preclinical pharmacology evaluation criteria. However, only a small fraction of experimental agents induce meaningful antitumor activities in the clinic. Low predictability of conventional preclinical pharmacology models is frequently cited as a main reason for the unusually high clinical attrition rates of therapeutic compounds in oncology. Therefore, improvement in the predictive values of preclinical efficacy models for clinical outcome holds great promise to reduce the clinical attrition rates of experimental compounds.Recent reports suggest that pharmacology studies conducted with patient derived xenograft (PDX) tumors are more predictive for clinical outcome compared to conventional, cell line derived xenograft (CDX) models, in particular when therapeutic compounds were tested at clinically relevant doses (CRDs). Moreover, the study of the most malignant cell types within tumors, the tumor initiating cells (TICs), relies on the availability of preclinical models that mimic the lineage hierarchy of cells within tumors. PDX models were shown to more closely recapitulate the heterogeneity of patient tumors and maintain the molecular, genetic, and histological complexity of human tumors during early stages of sequential passaging in mice, rendering them ideal tools to study the responses of TICs, tumor- and stromal cells to therapeutic intervention.In this commentary, we review the progress made in the development of PDX models in key areas of oncology research, including target identification and validation, tumor indication search and the development of a biomarker hypothesis that can be tested in the clinic to identify patients that will benefit most from therapeutic intervention.