Patient-derived Orthotopic Xenograft Tumor Research Articles

In vivo loss of tumorigenicity in a patient-derived orthotopic xenograft mouse model of ependymoma.

Ependymomas (EPN) are the third most common malignant brain cancer in children. Treatment strategies for pediatric EPN have remained unchanged over recent decades, with 10-year survival rates stagnating at just 67% for children aged 0-14 years. Moreover, a proportion of patients who survive treatment often suffer long-term neurological side effects as a result of therapy. It is evident that there is a need for safer, more effective treatments for pediatric EPN patients. There are ten distinct subgroups of EPN, each with their own molecular and prognostic features. To identify and facilitate the testing of new treatments for EPN, in vivo laboratory models representative of the diverse molecular subtypes are required. Here, we describe the establishment of a patient-derived orthotopic xenograft (PDOX) model of posterior fossa A (PFA) EPN, derived from a metastatic cranial lesion. Patient and PDOX tumors were analyzed using immunohistochemistry, DNA methylation profiling, whole genome sequencing (WGS) and RNA sequencing. Both patient and PDOX tumors classified as PFA EPN by methylation profiling, and shared similar histological features consistent with this molecular subgroup. RNA sequencing revealed that gene expression patterns were maintained across the primary and metastatic tumors, as well as the PDOX. Copy number profiling revealed gains of chromosomes 7, 8 and 19, and loss of chromosomes 2q and 6q in the PDOX and matched patient tumor. No clinically significant single nucleotide variants were identified, consistent with the low mutation rates observed in PFA EPN. Overexpression of EZHIP RNA and protein, a common feature of PFA EPN, was also observed. Despite the aggressive nature of the tumor in the patient, this PDOX was unable to be maintained past two passages in vivo. Others who have successfully developed PDOX models report some of the lowest success rates for EPN compared to other pediatric brain cancer types attempted, with loss of tumorigenicity not uncommon, highlighting the challenges of propagating these tumors in the laboratory. Here, we discuss our collective experiences with PFA EPN PDOX model generation and propose potential approaches to improve future success in establishing preclinical EPN models.

EXTH-56. TARGETING GBM WITH AN ONCOLYTIC PICORNAVIRUS SVV-001 ALONE AND IN COMBINATION WITH FRACTIONATED RADIATION IN A NOVEL PANEL OF ORTHOTOPIC PDX MODELS

Abstract BACKGROUND Large panels of clinically relevant animal models are needed for the development of new therapies for GBM. SVV-001, a replication-competent oncolytic virus that can pass through the blood brain barrier, represents an attractive novel approach. Material and METHODS Surgical tissues from 23 patients were implanted into the brains of NOD/SCID mice (1x105 cells/mouse) to develop patient-derived orthotopic xenograft (PDOX) models. Analysis of histology, gene expression (RNAseq), tumor growth was performed and compared with the patient tumors during subtransplantations (up to 5 passages). Anti-tumor activities of SVV-001 were examined in vitro in primary monolayer cultures, 3D neurospheres and purified glioma stem cells derived from 13 PDOX models; and its therapeutic efficacy validated in vivo in PDOX models through single i.v. injection (1x1011 viral particle) alone and in combination with fractionated (2 Gy/day x 5 days) radiation. Changes of animal survival times were analyzed together with viral infection, cell proliferation and DNA damage. RESULTS PDOX formation was confirmed in 17/23 (73.9%) GBMs. They replicated key histopathological features and diffuse invasion of the patient tumors. RNAseq identified the differentially expressed genes and subclassified PDOX models into proneural, classic and mesenchymal groups. SVV-001 killed primary monolayer cultures (4/13 models) and neurosphere (7/13 models) and putative glioma stem cells (CD133+ and/or CD15+ cells) in vitro, infected PDOX tumors without harming normal brain cells in vivo and significantly prolonged survival times in 2/2 models. Combination with radiation showed a trend in further prolonging animal survival times through enhanced DNA damages by SVV-001. CONCLUSION A panel of 17 clinically relevant and molecularly annotated PDOX modes of GBM is developed. The strong anti-tumor activities of SVV-001 in vitro and in vivo in GBM models provided experimental rational that support the initiation of clinical trials in GBM patients.

EPCO-39. ADAPTIVE CONVERGENCE OF METHYLOMES REVEALS EPIGENETIC DRIVERS AND BOOSTERS OF REPEATED RELAPSES IN PATIENT-MATCHED CHILDHOOD EPENDYMOMAS AND IDENTIFIES TARGETS FOR ANTI-RECURRENCE THERAPIES

Abstract Ependymoma (EPN) is the third most common brain tumor in children and frequently recurs. Here, we report an integrated longitudinal analysis of epigenetic, genetic and tumorigenic changes in 30 patient-matched repeated relapses obtained from 10 pediatric patients to understand the mechanism of recurrences. Genome-wide DNA methylation analysis revealed stable molecular subtypes and convergent epigenetic reprogramming during serial relapses of the 5 RELA and 5 PFA EPNs that paralleled with elevated patient-derived orthotopic xenograft (PDOX) (13/27) formation in the late relapses. Differentially methylated CpGs (DMCs) preexisted in the primary tumors and persisted in the relapses (driver DMCs) were detected, ranging from 51 hypo-methylated in RELA to 148 hyper-methylated DMCs in PFA tumors; while newly acquired DMCs sustained in all the relapses but was absent in the primary tumors (booster DMCs) ranged from 38- 323 hyper-methylated DMCs in RELA and PFA EPNs, respectively. Integrated analysis of these DMC associated DNA methylation regions (DMRs) and RNAseq in both patient and PDOX tumors identified a small fraction of the differentially expressed genes (4.6±4.4% in RELA and 4.5±1.1% in PFA) as regulated by driver DMRs (e.g., up-regulated CACNA1H, SLC12A7, RARA in RELA and HSPB8, GMPR, ITGB4 in PFA) and booster DMRs (including the sole upregulated PLEKHG1 in RELA and NOTCH, EPHA2, SUFU, FOXJ1 in PFA tumors). Most these genes were novel to EPN relapses. Seven DMCs in RELA and 22 in PFA tumors were also identified as potential relapse predictors. Finally, integrating DNA methylation with histone modification identified LSD1 as a relapse driver gene. Combined treatment of a novel inhibitor SYC-836 with radiation significantly prolonged survival times in two PDOX models of recurrent PFA. This high-resolution epigenetic and genetic roadmap of EPN relapse and our 13 new PDOX models should significantly facilitate biological and preclinical studies of pediatric EPN recurrences.

EPEN-02. Adaptive Convergence of Methylomes Reveals Epigenetic Drivers and Boosters of Repeated Relapses in Patient-matched Childhood Ependymomas and Identifies Targets for Anti-Recurrence Therapies

Ependymoma (EPN) is the third most common brain tumor in children and frequently recurs. Here, we report an integrated longitudinal analysis of epigenetic, genetic and tumorigenic changes in 30 patient-matched repeated relapses obtained from 10 pediatric patients to understand the mechanism of recurrences. Genome-wide DNA methylation analysis revealed stable molecular subtypes and convergent epigenetic reprogramming during serial relapses of the 5 RELA and 5 PFA EPNs that paralleled with elevated patient-derived orthotopic xenograft (PDOX) (13/27) formation in the late relapses. Differentially methylated CpGs (DMCs) preexisted in the primary tumors and persisted in the relapses (driver DMCs) were detected, ranging from 51 hypo-methylated in RELA to 148 hyper-methylated DMCs in PFA tumors; while newly acquired DMCs sustained in all the relapses but was absent in the primary tumors (booster DMCs) ranged from 38- 323 hyper-methylated DMCs in RELA and PFA EPNs, respectively. Integrated analysis of these DMC associated DNA methylation regions (DMRs) and RNAseq in both patient and PDOX tumors identified a small fraction of the differentially expressed genes (4.6±4.4% in RELA and 4.5±1.1% in PFA) as regulated by driver DMRs (e.g., up-regulated CACNA1H, SLC12A7, RARA in RELA and HSPB8, GMPR, ITGB4 in PFA) and booster DMRs (including the sole upregulated PLEKHG1 in RELA and NOTCH, EPHA2, SUFU, FOXJ1 in PFA tumors). Most these genes were novel to EPN relapses. Seven DMCs in RELA and 22 in PFA tumors were also identified as potential relapse predictors. Finally, integrating DNA methylation with histone modification identified LSD1 as a relapse driver gene. Combined treatment of a novel inhibitor SYC-836 with radiation significantly prolonged survival times in two PDOX models of recurrent PFA. This high-resolution epigenetic and genetic roadmap of EPN relapse and our 13 new PDOX models should significantly facilitate biological and preclinical studies of pediatric EPN recurrences.

Fluorescent Anti-MUC5AC Brightly Targets Pancreatic Cancer in a Patient-derived Orthotopic Xenograft.

Overexpression of mucin-5AC (MUC5AC) makes it a targetable biomarker in pancreatic cancer. The present study evaluated tumor targeting with a MUC5AC antibody conjugated to a near-infrared dye in a patient-derived orthotopic xenograft (PDOX) mouse model. MUC5AC monoclonal antibody was conjugated to the near-infrared dye IRDye800CW to synthesize MUC5AC-IR800. PDOX models were established by implanting a high-MUC5AC-expressing patient-derived pancreatic tumor on the pancreas of nude mice. After 4 weeks of PDOX tumor growth, mice were imaged after receiving MUC5AC-IR800 (75 μg) intravenously. In the PDOX models, MUC5AC-IR800 selectively and brightly targeted the pancreatic tumor (tumor to background ratio: 2.46±0.465). MUC5AC-IR800 provides distinct visualization of pancreatic tumors. MUC5AC-IR800 may be used clinically in the future to improve pancreatic cancer resection. This novel fluorescent probe is also promising for targeting of pre-malignant pancreatic lesions with subsequent resection under fluorescence guidance.

Abstract 634: Targeting metastatic brain germinoma using PDOX tumor models with sequential metastases

Abstract Introduction. Intracranial germ cell tumor is an Asian-centric tumor, showing great variation in geographical distribution, with an incidence 5-8 fold greater in Asia/East Asia, than Western countries. Germinoma is one histological subtype of intracranial germ cell tumors. Current standard of care for germinoma, is chemoradiotherapy. Metastatic/recurrent germinoma have poor prognosis. There is currently no targeted therapy. Purpose: To create patient-derived orthotopic xenograft (PDOX) models of distant metastases in brain germinoma beyond brain boundaries to recapitulate sequential metastases of original patient tumors. These tumor models will serve as clinically accurate metastatic models for preclinical testing, replicating patient's pattern of tumor progression/metastases. There is a lack of such metastatic brain tumor models in the field. c-KIT is the most common mutation in germinoma. We evaluated preclinical efficacy of a c-KIT inhibitor. Experimental Procedures. Most PDOX brain tumor models are created by implanting patient-derived tumor cells in cerebrum/cerebellum. Germinoma originates from pineal gland/suprasellar region. We implanted germinoma cells nearer location of tumor origin. We were successful and achieved consistent tumor implantation into this location (100% post-surgery survival, 0% surgical mortality, 0% post-surgical neurological deficits). Summary. We successfully established two models with sequential metastases. Model 1 was created from metastatic cells from spinal recurrence (Patient 1) post-chemo-radiotherapy. This model faithfully created skull base metastases in 100% of murine replicas (n=9). Both tumor cells from brain and skull base metastases (n=7) were serially transplantable. Our findings were replicated (n=5) and consistent. Model 2 was created from metastatic cells of cerebrospinal fluid dissemination (Patient 2). Brain surface metastases were formed recapitulating patient tumor. Both models recapitulated a similar path of metastases in craniospinal axis, corresponding to respective patients. c-KIT inhibitor, was effective in significantly prolonging animal survival in Model 2 (p=0.0046) but not in Model 1 (p=0.0541). In Model 2, c-kit inhibition in-vivo was efficacious and resulted in 3-fold effect, (1) Significantly improved animal survival, (2) Macroscopically reduced frequency, severity of brain surface metastases (Controls=50%, Treated=14%, lesser in severity), (3) Microscopically reduced tumor burden to minimal residual disease limited to cerebrospinal fluid outline. Control mice brains were largely replaced by tumor bulk, while treated mice had minimal residual tumor burden. Conclusions. Understanding metastatic patterns in such aggressive models is relevant in biologic targeting for preclinical studies and clinical decision to extend radiotherapy field in recurrent disease. Citation Format: Shiying Huang, Sekar Karthik, Jack M Su, Qi Lin, YuChen Du, Ching C Lau, Adesina Adekunle, Angela Major, Kam-Man Hui, M. Tarek Elghetany, Xiaonan Li, Wan-Yee Teo. Targeting metastatic brain germinoma using PDOX tumor models with sequential metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 634.

Color-Coded Imaging of Cancer and Stromal-Cell Interaction in the Pancreatic-Cancer Tumor Microenvironment (TME).

The tumor microenvironment (TME) contains stromal cells in a complex interaction with cancer cells. This relationship has become better understood with the use of fluorescent proteins for in vivo imaging, originally developed by our laboratories. Spectrally-distinct fluorescent proteins can be used for color-coded imaging of the complex interaction of the tumor microenvironment in the living state using cancer cells expressing a fluorescent protein of one color and host mice expressing another-color fluorescent protein. Cancer cells engineered in vitro to express a fluorescent protein were orthotopically implanted into transgenic mice expressing a fluorescent protein of a different color. Confocal microscopy was then used for color-coded imaging of the TME. Color-coded imaging of the TME has enabled us to discover that stromal cells are necessary for metastasis. Patient-derived orthotopic xenograft (PDOX) tumors were labeled by first passaging them orthotopically through transgenic nude mice expressing either green, red, or cyan fluorescent protein in order to label the stromal cells of the tumor (Yang et al., Cancer Res 64:8651-8656, 2004; Yang et al. J Cell Biochem 106: 279-284, 2009). The colored stromal cells become stably associated with the PDOX tumors through multiple passages in transgenic colored nude mice or non-colored nude mice. The fluorescent protein-expressing stromal cells included cancer-associated fibroblasts and tumor-associated macrophages. Color-coded imaging enabled the visualization of apparent fusion of cancer and stromal cells. Color-coded imaging is a powerful tool visualizing the interaction of cancer and stromal cells during cancer progression and treatment.

Color-Coded Imaging of the Tumor Microenvironment (TME) in Human Patient-Derived Orthotopic Xenograft (PDOX) Mouse Models.

The tumor microenvironment (TME) contains stromal cells in a complex interaction with cancer cells. This relationship has become better understood with the use of fluorescent proteins for in vivo imaging, originally developed by our laboratories. Spectrally distinct fluorescent proteins can be used for color-coded imaging of the complex interaction of the tumor microenvironment in the living state using cancer cells expressing a fluorescent protein of one color and host mice expressing another color fluorescent protein. Cancer cells engineered in vitro to express a fluorescent protein were orthotopically implanted into transgenic mice expressing a fluorescent protein of a different color. Confocal microscopy was then used for color-coded imaging of the TME. Color-coded imaging of the TME has enabled us to discover that stromal cells are necessary for metastasis. Patient-derived orthotopic xenograft (PDOX) tumors were labeled by first passaging them orthotopically through transgenic nude mice expressing either green, red, or cyan fluorescent protein in order to label the stromal cells of the tumor. The colored stromal cells become stably associated with the PDOX tumors through multiple passages in transgenic colored nude mice or noncolored nude mice. The fluorescent protein-expressing stromal cells included cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). Using this model, specific cancer cell or stromal cell targeting by potential therapeutics can be visualized. Color-coded imaging enabled the visualization of apparent fusion of cancer and stromal cells. Color-coded imaging is a powerful tool visualizing the interaction of cancer and stromal cells during cancer progression and treatment.

The impact of initial tumor microenvironment on imaging phenotype

Models of human cancer, to be useful, must replicate human disease with high fidelity. Our focus in this study is rat xenograft brain tumors as a model of human embedded cerebral tumors. A distinguishing signature of such tumors in humans, that of contrast-enhancement on imaging, is often not present when the human cells grow in rodents, despite the xenografts having nearly identical DNA signatures to the original tumor specimen. Although contrast enhancement was uniformly evident in all the human tumors from which the xenografts’ cells were derived, we show that long-term contrast enhancement in the model tumors may be determined conditionally by the tumor microenvironment at the time of cell implantation. We demonstrate this phenomenon in one of two patient-derived orthotopic xenograft (PDOX) models using cancer stem-like cell (CSC)-enriched neurospheres from human tumor resection specimens, transplanted to groups of immune-compromised rats in the presence or absence of a collagen/fibrin scaffolding matrix, Matrigel. The rats were imaged by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and their brains were examined by histopathology. Targeted proteomics of the PDOX tumor specimens grown from CSC implanted with and without Matrigel showed that while the levels of the majority of proteins and post-translational modifications were comparable between contrast-enhancing and non-enhancing tumors, phosphorylation of Fox038 showed a differential expression. The results suggest key proteins determine contrast enhancement and suggest a path toward the development of better animal models of human glioma. Future work is needed to elucidate fully the molecular determinants of contrast-enhancement.

Abstract 2791: mTOR inhibitor and VEGFR inhibitor combination therapy regress a doxorubicin resistant osteosarcoma in a patient-derived orthotopic xenograft model and in vivo angiogenesis assay model

Abstract Introduction: The efficacy of the combination therapy of mTOR inhibitor and VEGFR inhibitor was reported in advanced renal cell cancer with anti-angiogenesis. However, none of the reports have been published in osteosarcoma (OS). A tumor from 16-year-old patient who failed doxorubicin (DOX) was previously implanted orthotopically into the lateral condyle of distal femur of nude mice to establish a patient-derived orthotopic xenograft (PDOX) model in our laboratory at AntiCancer Inc. We previously found that the OS PDOX model was resistant to DOX matching the clinical setting. We also previously established in vivo angiogenesis assay model using OS cell line with nestin green fluorescent protein (GFP) mouse and reported that OS cells have highly angiogenesis. In the present study, we evaluated the efficacy of everolimus (EVE, a mTOR inhibitor), pazopanib (PAZ, a VEGFR inhibitor) and the combination of EVE and PAZ on the DOX resistant OS PDOX model and in vivo angiogenesis assay model using OS patient derived cells (PDCs). Methods: The OS PDOX model was randomized into five groups of seven mice. An OS PDC was established from a patient-derived OS tumor fragment. After several passages, OS-PDCs were established and were transplanted into the back of nestin GFP mice to make in vivo angiogenesis assay model. For the angiogenesis study, In vivo angiogenesis assay models were randomized into five groups of eight fields: Group 1, Control (Ctrl) treated with PBS, i.p.; Group 2, DOX, 2.4 mg/kg, i.p., weekly; Group 3, EVE, 5 mg/kg, oral gavage, daily; Group 4, PAZ, 50 mg/kg, oral gavage, daily; Group 5,the combination of EVE and PAZ. Treatment was performed for 2 weeks in each model. In the OS PDOX model, treatment efficacy was evaluated on tumor volume ratio and histopathology. Adverse event was evaluated on body weight ratio. In the in vivo angiogenesis assay model, treatment efficacy was evaluated on vascular length ratio. Treatment efficacy and adverse event were evaluated after 2 weeks treatment. Results: Tumor volume ratios in OS PDOX model were the Ctrl group: 4.70 ± 0.58, DOX group: 3.97 ± 0.53, EVE group: 3.89 ± 0.80, PAZ group: 4.16 ± 1.01 and combination group: 1.70 ± 0.30, respectively. Vascular length ratios in vivo angiogenesis assay model were the Ctrl group: 1.44 ± 0.50, DOX group: 1.22 ± 0.30, EVE group: 0.97 ± 0.31, PAZ group: 0.80 ± 0.12 and combination group: 0.58 ± 0.08, respectively. EVE and PAZ combination group regress tumor volume ratio and vascular length ratio with significant difference compared to all groups. In H&E-staining, partial necrosis was detected only in OS PDOX tumors treated with combination of EVE and PAZ. Body weight ratio was no significant difference among all groups. Conclusion: The combination therapy of EVE and PAZ was the most effective in the OS PDOX mouse model and in vivo angiogenesis assay model due to suppress angiogenesis strongly. This study demonstrates that the combination therapy of mTOR inhibitor and VEGFR inhibitor has possibility to be therapeutic strategy for DOX resistant osteosarcoma. Citation Format: Hiromichi Oshiro, Yasunori Tome, Robert M. Hoffman, Kotaro Nishida. mTOR inhibitor and VEGFR inhibitor combination therapy regress a doxorubicin resistant osteosarcoma in a patient-derived orthotopic xenograft model and in vivo angiogenesis assay model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2791.

Abstract 1661: Temozolomide suppresses a doxorubicin resistant follicular dendritic cell sarcoma in a patient derived orthotopic xenograft mouse model

Abstract Introduction: Follicular dendritic cell sarcoma (FDCS) is a very rare and highly refractory soft tissue sarcoma. The CHOP chemotherapy has been used as systemic therapy for FDCS, which includes cyclophosphamide, doxorubicin (DOX), vincristine, and prednisolone. However, FDCS has poor clinical outcome even though CHOP therapy was administered. A FDCS patient tumor was previously implanted orthotopically in the biceps femoris of nude mice to establish a patient-derived orthotopic xenograft (PDOX) model in our laboratory at AntiCancer Inc. The FDCS patient had resistance to DOX in clinical setting. We previously found the FDCS PDOX model had resistance to DOX as well as clinical settings. In the present study, we evaluated the efficacy of temozolomide (TMZ), trabectedin (TRAB), and pazopanib (PAZ) on the DOX resistant FDCS PDOX model. Methods: The FDCS PDOX mouse model was established and randomized into five groups of eight mice, respectively. Group 1, Control (Ctrl) treated with PBS, i.p.; Group 2, DOX, 2.4 mg/kg, i.p., weekly; Group 3, PAZ, 50 mg/kg, oral gavage, daily; Group 4, TRAB, 0.15 mg/kg, i.v., weekly; Group 5, TMZ, 25 mg/kg, oral gavage, daily. Treatment term was for 2 weeks. Treatment efficacy was evaluated based on tumor volume ratio (tumor volume after treatment relative to tumor volume at the beginning of treatment) and histopathology. Adverse event was evaluated based on body weight ratio. Tumor volume and body weight were assessed 2 times per week. Results: Tumor volume ratio was the Ctrl group: 11.35±4.50, DOX: 8.24±1.87, PAZ: 9.67±4.66, TRAB: 6.97±3.23, TMZ: 4.28±1.41, respectively. TMZ significantly arrested the FDCS PDOX model compared to the control group (p<0.05). PAZ and TRAB did not have significant efficacy compared to the Ctrl group (p=0.93, p=0.41). In H&E-staining, partial necrosis was detected only in PDOX tumors treated with TMZ. There was no significant difference of body weight among all groups. Conclusion: The FDCS PDOX model was resistant to DOX as well as in the clinical settings demonstrating concordance of the FDCS model and clinic. TMZ was the most effective for the FDCS PDOX mouse model established from a patient who failed DOX treatment. There is possibility that TMZ has effective to FDCS patients who has resistant to the first line chemotherapy. Citation Format: Hiromichi Oshiro, Yasunori Tome, Robert M. Hoffman, Kotaro Nishida. Temozolomide suppresses a doxorubicin resistant follicular dendritic cell sarcoma in a patient derived orthotopic xenograft mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1661.

Patient-Derived Orthotopic Xenograft (PDOX) Mouse Models of Primary and Recurrent Meningioma.

Background. Meningiomas constitute one-third of all primary brain tumors. Although typically benign, about 20% of these tumors recur despite surgery and radiation, and may ultimately prove fatal. There are currently no effective chemotherapies for meningioma. We, therefore, set out to develop patient-derived orthotopic xenograft (PDOX) mouse models of human meningioma using tumor. Method. Of nine patients, four had World Health Organization (WHO) grade I tumors, five had WHO grade II tumors, and in this second group two patients also had recurrent (WHO grade III) meningioma. We also classified the tumors according to our recently developed molecular classification system (Types A, B, and C, with C being the most aggressive). We transplanted all 11 surgical samples into the skull base of immunodeficient (SCID) mice. Only the primary and recurrent tumor cells from one patient—both molecular Type C, despite being WHO grades II and III, respectively—led to the formation of meningioma in the resulting mouse models. We characterized the xenografts by histopathology and RNA-seq and compared them with the original tumors. We performed an in vitro drug screen using 60 anti-cancer drugs followed by in vivo validation. Results. The PDOX models established from the primary and recurrent tumors from patient K29 (K29P-PDOX and K29R-PDOX, respectively) replicated the histopathology and key gene expression profiles of the original samples. Although these xenografts could not be subtransplanted, the cryopreserved primary tumor cells were able to reliably generate PDOX tumors. Drug screening in K29P and K29R tumor cell lines revealed eight compounds that were active on both tumors, including three histone deacetylase (HDAC) inhibitors. We tested the HDAC inhibitor Panobinostat in K29R-PDOX mice, and it significantly prolonged mouse survival (p < 0.05) by inducing histone H3 acetylation and apoptosis. Conclusion. Meningiomas are not very amenable to PDOX modeling, for reasons that remain unclear. Yet at least some of the most malignant tumors can be modeled, and cryopreserved primary tumor cells can create large panels of tumors that can be used for preclinical drug testing.

Recombinant Methioninase Combined With Tumor-targeting Salmonella typhimurium A1-R Induced Regression in a PDOX Mouse Model of Doxorubicin-resistant Dedifferentiated Liposarcoma.

Dedifferentiated liposarcoma (DDLPS) is associated with a poor survival rate even with multi-modality treatment. In the present study, we evaluated the efficacy of recombinant methioninase (rMETase) combined with tumor-targeting Salmonella typhimurium (S. typhimurium) A1-R against a doxorubicin-resistant DDLPS in a patient-derived orthotopic xenograft (PDOX) mouse model. A recurrent high-grade DDLPS from the right retroperitoneum of a patient was grown orthotopically in the retroperitoneum of nude mice to establish a PDOX model. The PDOX models were randomly divided into the following groups: Control, no treatment; doxorubicin monotherapy; rMETase monotherapy; S. typhimurium A1-R monotherapy; S. typhimurium A1-R and rMETase combination therapy. Tumor length and width were measured before and after treatment. On day 14 after treatment, all treatments significantly inhibited DDLPS PDOX tumor growth compared to the untreated control except for doxorubicin monotherapy. rMETase combined with S. typhimurium A1-R was significantly more effective and regressed tumor volume compared to either rMETase or S. typhimurium A1-R alone. The relative body weight did not significantly differ between days 0 and 14 for individual groups. The combination of rMETase and S. typhimurium A1-R has important clinical potential for this recalcitrant sarcoma.

Tumor shedding and metastatic progression after tumor excision in patient-derived orthotopic xenograft models of triple-negative breast cancer.

Patient-derived orthotopic xenograft (PDOX) models have been verified as a useful method for studying human cancers in mice. Previous studies on the extent of metastases in these models have been limited by the necessity of welfare euthanasia (primary tumors reaching threshold size), at which point metastases may only be micrometers in diameter, few in number, and solely identified by step-sectioning of formalin-fixed paraffin-embedded tissue. These small micro-metastases are less suitable for many downstream molecular analyses than macro-metastases. Resection of the primary tumor by survival surgery has been proven to allow further time for metastases to grow. Although PDOX models of triple-negative breast cancer (TNBC) shed circulating tumor cells (CTCs) into the bloodstream and metastasize, similar to human TNBC, little data has been collected in these TNBC PDOX models regarding the association between CTC characteristics and distant metastasis following excision of the primary tumor xenograft. This study assembles a timeline of PDOX tumor shedding and metastatic tumor progression before and after tumor excision surgery. We report the ability to use tumorectomies to increase the lifespan of TNBC PDOX models with the potential to obtain larger metastases. CTC clusters and CTCs expressing a mesenchymal marker (vimentin) were associated with metastatic burden in lung and liver. The data collected through these experiments will guide the further use of PDOX models in studying metastatic TNBC.

Eribulin Regresses a Doxorubicin-resistant Dedifferentiated Liposarcoma in a Patient-derived Orthotopic Xenograft Mouse Model.

Dedifferentiated liposarcoma (DDLPS) is recalcitrant type of sarcoma. DDLPS has a low survival rate with high recurrence and metastasis. In the present study, we evaluated the efficacy of several drugs against doxorubicin-resistant DDLPS in a patient-derived orthotopic xenograft (PDOX) model for precision oncology. To establish the PDOX model, a tumor from a patient who had recurrent high-grade DDLPS from the retroperitoneum was previously grown orthotopically in the retroperitoneum of nude mice. We randomized DDLPS PDOX models into 8 treatment groups when tumor volume became approximately 100 mm3: control, no treatment; G2, doxorubicin (DOX); G3, pazopanib (PAZ); G4, gemcitabine (GEM) combined with docetaxel (DOC); G5, trabectedin (YON); G6, temozolomide (TEM); G7, palbociclib (PAL); G8, eribulin (ERB). Tumor length and width were measured both at the beginning and at the end of treatment. At the end of treatment (day 14), all treatments significantly inhibited DDLPS PDOX tumor growth compared to the untreated control, except DOX. ERB was significantly more effective and regressed tumor volume compared to other treatments on day 14 after initiation of treatment. No significant differences were found in the relative body weight on day 14 compared to day 0 in any group. The clinical potential of ERB against DDLPS is herein presented in a PDOX model.

Combination of oral recombinant methioninase and decitabine arrests a chemotherapy-resistant undifferentiated soft-tissue sarcoma patient-derived orthotopic xenograft mouse model

Cancer cells are methionine (MET) and methylation addicted and are highly sensitive to MET restriction. The present study determined the efficacy of oral-recombinant methioninase (o-rMETase) and the DNA methylation inhibitor, decitabine (DAC) on restricting MET in an undifferentiated-soft tissue sarcoma (USTS) patient-derived orthotopic xenograft (PDOX) nude-mouse model. The USTS PDOX models were randomized into five treatment groups of six mice: Control; doxorubicin (DOX) alone; DAC alone; o-rMETase alone; and o-rMETase-DAC combination. Tumor size and body weight were measured during the 14 days of treatment. Tumor growth was arrested only in the o-rMETase-DAC condition. Tumors treated with the o-rMETase-DAC combination exhibited tumor necrosis with degenerative changes. This study demonstrates that the o-rMETase-DAC combination could arrest the USTS PDOX tumor suggesting clinical promise.

Investigating circulating tumor cells and distant metastases in patient-derived orthotopic xenograft models of triple-negative breast cancer

BackgroundCirculating tumor cells (CTCs) represent a temporal “snapshot” of a patient’s cancer and changes that occur during disease evolution. There is an extensive literature studying CTCs in breast cancer patients, and particularly in those with metastatic disease. In parallel, there is an increasing use of patient-derived models in preclinical investigations of human cancers. Yet studies are still limited demonstrating CTC shedding and metastasis formation in patient-derived models of breast cancer.MethodsWe used seven patient-derived orthotopic xenograft (PDOX) models generated from triple-negative breast cancer (TNBC) patients to study CTCs and distant metastases. Tumor fragments from PDOX tissue from each of the seven models were implanted into 57 NOD scid gamma (NSG) mice, and tumor growth and volume were monitored. Human CTC capture from mouse blood was first optimized on the marker-agnostic Vortex CTC isolation platform, and whole blood was processed from 37 PDOX tumor-bearing mice.ResultsStaining and imaging revealed the presence of CTCs in 32/37 (86%). The total number of CTCs varied between different PDOX tumor models and between individual mice bearing the same PDOX tumors. CTCs were heterogeneous and showed cytokeratin (CK) positive, vimentin (VIM) positive, and mixed CK/VIM phenotypes. Metastases were detected in the lung (20/57, 35%), liver (7/57, 12%), and brain (1/57, less than 2%). The seven different PDOX tumor models displayed varying degrees of metastatic potential, including one TNBC PDOX tumor model that failed to generate any detectable metastases (0/8 mice) despite having CTCs present in the blood of 5/5 tested, suggesting that CTCs from this particular PDOX tumor model may typify metastatic inefficiency.ConclusionPDOX tumor models that shed CTCs and develop distant metastases represent an important tool for investigating TNBC.

Abstract LB-320: PPTC testing of an OLIG2 inhibitor CT-179 in patient-derived orthotopic xenograft mouse models of pediatric GBM

Abstract Background: Oligodendrocyte Lineage Transcription Factor 2 (OLIG2) is a pro-mitotic transcription factor highly expressed in glioma stem cells (GSCs) that has been shown to be essential for tumorigenesis and a significant driver of tumor growth, GSC motility, and resistance to radiation. We evaluated the efficacy of the OLIG2 inhibitor CT-179 on survival times in pediatric glioblastoma (pGBM) patient-derived orthotopic xenograft (PDOX) models. Methods: OLIG2 mRNA expression levels were examined by gene expression profiling. In vitro activity was analyzed in matched monolayer cells and 3D neurospheres from 8 PDOX models treated with CT-179 (0-10 µM) alone as well as in combination with radiation (XRT) (0-8 Gy). For in vivo drug testing, 105 pGBM cells were implanted into the brains of SCID mice. Brain penetration of CT-179 was evaluated in PDOX tumors and normal mouse brain tissues using blood concentration as reference. Therapeutic efficacy was examined in 4 PDOX models. Vehicle or CT-179 was administered by oral gavage starting one week post tumor implantation. CT-179 was dosed at 200 mg/kg every 4 days (q4d). In one model, a dose of 240 mg/kg every 3 days (q3d) was also used. XRT (2 Gy/day x 5 days) was given alone and in combination with CT-179. Median survival was determined using the Kaplan-Meier method, with the significance level for combination testing experiments set at 0.01 to account for multiple comparisons. Results: High level OLIG2 mRNA expression was detected in all PDOX tumors. CT-179 inhibited cell viability in a time- and dose-dependent manner in all 8 pGBM cell lines (IC50 0.03-10 µM). XRT potentiated the effects of CT-179 at low/moderate drug doses (0.03-1 µM). Compared with a mean serum concentration of 361.3 ± 1.5 ng/mL, oral gavage of CT-179 led to high tissue concentrations in the cerebrum (3,232.7 ± 569.2 ng/g), cerebellum (1563.3 ± 269.6 ng/g), brain stem (1685.3 ±309 ng/g) and PDOX tumors (1,814 ± 110.3 ng/g). In the PDOX models, the ratios of the median survival time for single agent CT-179 (200 mg/kg q4d) groups to their respective control groups (T/C) ranged from 0.61 to 1.19; no comparisons reached statistical significance. In one of the models, when a higher dose (240 mg/kg q3d) was used a longer T/C ratio (1.06 vs 1.02) was observed. In 1 of 4 PDOX models, combining CT-179 and XRT significantly improved survival times when compared with the control and single agent CT-179 groups, and for another PDOX there was a trend for improved survival for the combination compared to control. Conclusion: The molecular target of CT-179, OLIG2, was over-expressed in multiple pGBM xenografts. CT-179 inhibited cell viability of pGBM cells in vitro, and effectively penetrated and accumulated in CNS tissues and PDOX tumors. Single agent CT-179 showed limited impact on survival for the pGBM xenografts that were studied. The combination of CT-179 and XRT increased survival times in some pGBM PDOX models. Citation Format: Holly Lindsay, Yuchen Du, Lin Qi, Huiyuan Zhang, Sibo Zhao, Frank Braun, Mari Kogiso, Sarah Injac, Mary Sobieski, Clifford Stephan, Gordon Alton, Gregory Stein, Graham Beaton, Stephen Erickson, Beverly Teicher, Malcolm A. Smith, Xiao-Nan Li. PPTC testing of an OLIG2 inhibitor CT-179 in patient-derived orthotopic xenograft mouse models of pediatric GBM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-320.

Efficacy of Tumor-Targeting Salmonella typhimurium A1-R against Malignancies in Patient-Derived Orthotopic Xenograft (PDOX) Murine Models

We developed tumor-targeting Salmonella typhimurium (S. typhimurium) A1-R, a facultative anaerobe that is an auxotroph of leucine and arginine. The tumor-targeting efficacy of S. typhimurium A1-R was demonstrated in vivo and vitro using several malignant cell lines including melanoma, sarcoma, glioma, breast, pancreatic, colon, cervical, prostate, and ovarian cancers. Our laboratory also developed a patient-derived orthotopic xenograft (PDOX) model by implanting patient-derived malignant tumor fragments into orthotopic sites in mice. We reviewed studies of S. typhimurium A1-R against recalcitrant cancers. S. typhimurium A1-R was effective against all PDOX tumor models tested and showed stronger efficacies than chemotherapy or molecular-targeting therapy against some tumors. Furthermore, the synergistic efficacy of S. typhimurium A1-R when combined with chemotherapeutic agents, molecular-targeting agents, or recombinant methioninase was also demonstrated. We suggest potential clinical uses of this S. typhimurium A1-R treatment.

Temozolomide targets and arrests a doxorubicin-resistant follicular dendritic-cell sarcoma patient-derived orthotopic xenograft mouse model

Follicular dendritic cell sarcoma (FDCS) is a very rare and highly recalcitrant disease. A patient’s doxorubicin-resistant FDCS was previously established orthotopically on the right high thigh into the biceps femoris of mice to establish a patient-derived orthotopic xenograft (PDOX) model. The aim of the present manuscript was to identify an effective drug for this recalcitrant tumor. Here, we evaluated the efficacy of temozolomide (TMZ), trabectedin (TRAB) and pazopanib (PAZ) on the FDCS PDOX model. PDOX mouse models were randomized into five groups of eight to nine mice, respectively. Group 1, untreated control with PBS, i.p.; Group 2, treated with doxorubicin (DOX), 2.4 mg/kg, i.p., weekly for 3 weeks; Group 3, treated with PAZ, 50 mg/kg, oral gavage, daily for 3 weeks; Group 4, treated with TMZ, 25 mg/kg, oral gavage, daily for 3 weeks; Group 5, treated with TRAB, 0.15 mg/kg, i.v., weekly for 3 weeks. Body weight and tumor volume were assessed 2 times per week. TMZ arrested the FDCS PDOX model compared to the control group (p < 0.05). PAZ and TRAB did not have significant efficacy compared to the control group (p = 0.99, p = 0.69 respectively). The PDOX tumor was resistant to DOX (p= 0.99). as was the patient. The present study demonstrates that TMZ is effective for a PDOX model of FDCS established from a patient who failed DOX treatment, further demonstrating the power of PDOX to identify effective therapy including for tumors that failed first line therapy.