Original Patient Tumor Research Articles

Abstract C121: Racial/ethnic differences of pediatric brain tumors in the development of orthotopic PDX models

Abstract Brain tumor is leading cause of cancer-related death in children. While significant advances have been made in molecularly subgrouping tumors of same pathologic diagnosis, little is known about the biologic differences among racial/ethnic populations, and there is a lack of animal models that represent different racial/ethnic patients. Here, we report our analysis of tumorigenicity of a total of 215 pediatric brain tumors in SCID mice. All surgical tumor tissues were obtained from cryo lab and directly implanted into the anatomically matched locations in mouse brains, i.e., cerebral tumors (such as GBM) into mouse right cerebra, and cerebellar tumors (such as medulloblastoma) into mouse cerebella. The animals were closely monitored following institutional-approved animal protocols. Tumor formation was validated either through the harvesting of visible tumors or via histopathologic examination of paraffin-embedded whole mouse brains. From the 215 tumors, racial/ethnic information was validated in 180 tumors. Overall tumor formation was 41.2% (52/126) in white, 26.9% (7/26) in black, 50% (8/16) in more than one race, and 20% (2/5) in Asian patients. When different tumor types were compared, children with medulloblastoma exhibited similar tumor take rate, ranging from 50% (3/6) in black, to 54% (15/28) in whites and 67% (2/3) of American Indian or Alaska Native, whereas in GBM, it was 79% (11/14) in white, and 1/1 in other racial groups, and in ependymoma, it ranged from 14% (1/7) in American Indian or Alaska to 18% (4/22) in white patients. The tumor take of atypical teratoid/rhabdoid tumor (ATRT) was 63% (5/8) in white but 0% (0/3) in black patient. Low-grade gliomas (total 26) did not form xenografts, and sample size was small (<5) for other types of tumors. Histopathologic and comprehensive molecular characterization of these models confirmed their replication of the original patient tumors. In summary, this study suggested the differences of tumorigenicity among different racial populations and supports the expansion of patient cohorts, particularly the minorities, to draw definitive conclusions. These orthotopic PDX models (7 from African American, 8 from American Indian/Alaska, and 1 each from more than one race and Asian) provided a novel panel of clinically relevant and racial-specific models to facilitate the biologic and preclinical studies on cancer disparities. Note: This abstract was not presented at the conference. Citation Format: Lin Qi, Mari Kogiso, Yuchen Du, Yulun Huang, Huiyuan Zhang, Frank Braun, Holly Lindsay, Sibo Zhao, Sarah Injac, Lazlo Perlaky, Patricia Baxter, Wan-Yee Teo, Zhigang Liu, Xiumei Zhao, Yujing Zhang, Jack M.F. Su, Xiao-Nan Li. Racial/ethnic differences of pediatric brain tumors in the development of orthotopic PDX models [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C121.

Abstract P1-03-06: Development of patient-derived xenograft tumor models and 3D spheroid culture from advanced hormone receptor-positive inflammatory breast cancer patients for evaluation of new therapeutics

Abstract Background: Breast cancer (BC) is a heterogeneous disease comprising different clinical, histopathological, and molecular subtypes. Hormone receptor-positive (HR+) advanced BC cancer, in particular the inflammatory BC (IBC) represents a significant clinical challenge because of the development of endocrine resistance during either adjuvant or metastatic treatment translating to poor outcome. We aim to develop unique PDX tumor models and 3D spheroid/organoid cultures that recapture advanced IBC using pleural effusions or/and circulating tumor cells (CTCs) as tumor resource and that can help understand the mechanism of IBC metastasis for new drug development. Methods: Five samples of pleural effusion-derived tumor cells or circulating tumor cells (CTCs) from three advanced IBC patients were obtained from Northwestern Memorial Hospital to establish IBC PDX tumor model in immunodeficient NSG female mice via subcutaneous or breast fat pad xenografting and develop the derived 3D organoid/spheroid cultures for anti-cancer drug evaluation. STR profiling against with original patient tumor DNA was conducted to confirm the tumor authentication. Results: Three developed IBC PDX tumor models (one HR+-IBC and two triple negative (TN)-IBC) were demonstrated by pathology to have highly heterogeneous characteristics and metastatic features similar to the original patient tumor. NSG mice xenografted with original TN-IBC pleural effusion, or derived spheroid cultures with varying passage numbers (<3 passages or >6 passages) manifested different inflammatory clinical symptoms. Mice xenografted with one case of original TN-IBC pleural effusion cells (~40% of tumor-associated macrophages and ~60% of tumor cells) developed palpable tumors and was shown to have the most IBC-like characteristics including reddish skin and ulcerative lesions. They were sacrificed 2 months after xenograft due to declining health. Mice xenografted with >6 passages or <3 passages of spheroid cultures manifested little to moderate skin lesions and developed tumor sizes of ~1 cm within 2-3 months while having a longer life span. Liver and lung metastases were observed in the breast fat pad of xenografted PDX tumor mice with one case of original HR+-IBC pleural effusion. 3D tumor spheroid/organoid cultures were successfully established from original IBC pleural effusion or/and IBC PDX tumor cultures. Spheroid generated from HR+-IBC pleural effusion showed phenotypic plasticity: the HR+-IBC spheroid cells display both tumor and macrophage cell characteristics and spheroid cultures can convert to adhesive cell phenotypes with addition of condition media. These spheroid/organoid cultures are used for evaluation of new drug strategies such as selectively targeting ER degraders (SERDS), CDK4/6 inhibitor, or Tyrosine kinase inhibitors for treatment of HR+-IBC or TN-IBC. Conclusions: Our results suggested 3D spheroid cultures and PDX tumor models from advanced IBC recapture aggressive features of the disease and can be an ideal model for the mechanism study of metastatic advanced IBC and preclinical testing of novel agents in IBC and endocrine resistant disease. Citation Format: Wenan Qiang, Zhangfeng Zhong, Pamela Monahan, Kristy Tse, Youbin Zhang, Qiang Zhang, Lorenzo Gerratana, Andrew Davis, Demirkan Gursel, Reiner Bleher, Jian-Jun Wei, Charles D. James, Thomas V. O’Halloran, Massimo Cristofanilli. Development of patient-derived xenograft tumor models and 3D spheroid culture from advanced hormone receptor-positive inflammatory breast cancer patients for evaluation of new therapeutics [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-03-06.

Biliary tract cancer patient-derived xenografts: Surgeon impact on individualized medicine.

Background & AimsBiliary tract tumors are uncommon but highly aggressive malignancies with poor survival outcomes. Due to their low incidence, research into effective therapeutics has been limited. Novel research platforms for pre-clinical studies are desperately needed. We sought to develop a patient-derived biliary tract cancer xenograft catalog.MethodsWith appropriate consent and approval, surplus malignant tissues were obtained from surgical resection or radiographic biopsy and implanted into immunocompromised mice. Mice were monitored for xenograft growth. Established xenografts were verified by a hepatobiliary pathologist. Xenograft characteristics were correlated with original patient/tumor characteristics and oncologic outcomes. A subset of xenografts were then genomically characterized using Mate Pair sequencing (MPseq).ResultsBetween October 2013 and January 2018, 87 patients with histologically confirmed biliary tract carcinomas were enrolled. Of the 87 patients, 47 validated PDX models were successfully generated. The majority of the PDX models were created from surgical resection specimens (n = 44, 94%), which were more likely to successfully engraft when compared to radiologic biopsies (p = 0.03). Histologic recapitulation of original patient tumor morphology was observed in all xenografts. Successful engraftment was an independent predictor for worse recurrence-free survival. MPseq showed genetically diverse tumors with frequent alterations of CDKN2A, SMAD4, NRG1, TP53. Sequencing also identified worse survival in patients with tumors containing tetraploid genomes.ConclusionsThis is the largest series of biliary tract cancer xenografts reported to date. Histologic and genomic analysis of patient-derived xenografts demonstrates accurate recapitulation of original tumor morphology with direct correlations to patient outcomes. Successful development of biliary cancer tumografts is feasible and may be used to direct subsequent therapy in high recurrence risk patients.Lay summaryPatient biliary tract tumors grown in immunocompromised mice are an invaluable resource in the treatment of biliary tract cancers. They can be used to guide individualized cancer treatment in high-risk patients.

A Novel Anionic-phosphate-platinum Complex Effectively Targets a Cisplatinum-resistant Osteosarcoma in a Patient-derived Orthotopic Xenograft Mouse Model.

We have previously developed a novel bone-targeting platinum compound, 3Pt, and showed that it has strong inhibitory activity against osteosarcoma cells and orthotopic cell-line xenograft mouse models. In the present report, we compared the efficacy of 3Pt to cisplatinum (CDDP) in a CDDP-resistant relapsed osteosarcoma patient-derived orthotopic xenograft (PDOX) mouse model. The tumor of a patient with osteosarcoma of the distal femur was treated with CDDP-based chemotherapy followed by surgery. The surgical specimen was used to establish a PDOX model. An osteosarcoma cell line was also established from the original patient tumor. Osteosarcoma cell viability was assessed with the WST-8 assay and the IC50 values were calculated. The PDOX models were randomized into three groups: untreated control, CDDP-treated group, and 3Pt-treated group. Tumor size and body weight were measured twice a week. 3Pt had a strong concentration-dependent cytocidal effect in vitro. The IC50 value of 3Pt was significantly lower than that of CDDP. On day 14 of the treatment, 3Pt caused a significantly greater tumor growth inhibition compared to the untreated control and CDDP-treated mice. 3Pt is a promising clinical candidate for the treatment of recalcitrant osteosarcoma.

Bioengineered Cancer-Fibroblast Interactions in Patient-Derived Colorectal Cancer Organoid Models

The development of patient-derived organoid (PDO) technologies has greatly expanded the toolbox for drug discovery and personalized drug screening for several cancer types. While PDO models more closely represent the molecular characteristics and heterogeneity of patient tumors than traditional immortalized cancer cell lines, they are inherently limited in their ability to reflect the tumor microenvironment in vitro as they comprise exclusively of epithelial cells. The lack of stromal cells in PDO models, such as cancer-associated fibroblasts (CAFs), poses a major problem as these tumor microenvironmental components contribute to the various hallmarks of cancer and response to therapy. Particularly for colorectal cancer, CAFs comprise the majority of the tumor microenvironment and play important roles in cancer progression and drug resistance. In this study, we addressed this problem by establishing in vitro conditions that robustly enable the co-culture of CRC PDO with patient-derived CAFs. We report the development of an engineered tumor microenvironment consisting of CRC PDO encapsulated within a well-defined three-dimensional (3D) hyaluronan-gelatin hydrogel and co-cultured with patient-derived CAFs. Basement membrane extracts conventionally used for PDO culture exhibit batch-to-batch variability. Considering that the CRC extracellular matrix is high in hyaluronan and collagen I, and that hyaluronan-based matrices have been shown to be conducive for the culture of various human cancers, we hypothesized that hyaluronan-gelatin hydrogels may serve as a suitable alternative 3D matrix to support the co-culture of CRC PDO and CAFs. Through RNA- and whole-exome sequencing, we first show that these hydrogels are capable of maintaining key molecular characteristics of the original patient tumors in CRC PDO but not support the culture of CAFs. Further, based on our findings that CRC PDO culture medium poorly supports CAF viability, we developed a co-culture strategy that maintains the viability of both CRC PDO and CAFs. We found that in the absence of growth factors added to the co-culture, CAFs were able to maintain the proliferation of the cultured CRC PDO in the hydrogels and restore distinct biological pathways absent in the PDO culture alone but present in patient tissues. Lastly, we demonstrate that these CRC PDO-CAFs models are suitable for evaluating standard-of-care drugs, making them potentially very useful for realizing personalized cancer medicine.

Abstract A006: Preclinical models from rare aggressive tumors: Establishment, drug sensitivity and genomic analyses of mesothelioma PDX as models for oncology drug development

Abstract Malignant mesothelioma is a rare but aggressive disease with few therapeutic options and a poor prognosis. The main cause of the disease is the exposition to asbestos, even after 50 years of latency. Intended therapeutic goals, such as increase of patient survival and improvement of quality of life have only been achieved to a limited extent. Curative treatment is currently still not possible. Therefore, there is a strong clinical need for translational models. For this, we have characterized patient-derived xenograft models (PDX) of mesothelioma to evaluate their suitability for preclinical research. For PDX establishment, tumor tissue from surgically resected pleura or peritoneum mesotheliomas, was implanted subcutaneously into immunodeficient mice and serially passaged for at least 3 in vivo-passages. Established PDX models were characterized towards sensitivity to standard of care drugs. PDX tissue was collected for RNA sequencing and immunohistochemistry. The generated molecular profiles (comparative gene expression and sequence variations) of the models were analyzed comprehensively. A panel of 6 mesothelioma PDX has been established. In these PDX models, characteristic histological structure of the original patient tumor was preserved. The PDX models responded individually to standard of care drugs like pemetrexed, cisplatin, and gemcitabine as well as combinations thereof. The combination of pemetrexed or gemcitabine with cisplatin showed a stronger tumor growth inhibition than the respective monotherapies. The analyzed mutational and gene expression landscape resembles the known pattern of mesotheliomas. In summary, our established mesothelioma PDX will serve as a preclinical tool to investigate cellular and molecular mechanisms of drug sensitivity and resistance. They will provide a valuable preclinical model system to identify and validate biomarkers and to develop new therapeutic approaches. Citation Format: Michael Becker, Bernadette Brzezicha, Jana Rolff, Theresia Conrad, Beate Rau, Wolfgang Walther, Jens Hoffmann. Preclinical models from rare aggressive tumors: Establishment, drug sensitivity and genomic analyses of mesothelioma PDX as models for oncology drug development [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A006. doi:10.1158/1535-7163.TARG-19-A006

Abstract GMM-021: UTILIZATION OF PATIENT-DERIVED TUMOR XENOGRAFT (PDX) MODELS IN ONCOLOGY

Abstract BACKGROUND: The 5-year survival rate of patients with recurrent epithelial ovarian cancer is only 30%, in part due to relapse and resistance to first-line platinum-based chemotherapy. Even with marked progress toward understanding ovarian cancer biology, the translation of research findings into new therapies is still an enormous barrier to progress. Recent data suggests a 90% failure rate for new oncology drugs in the clinic. Development and preclinical testing of new cancer therapies has been limited by the availability of clinically relevant models that recapitulate the molecular and phenotypic characteristics of primary ovarian cancers. To overcome this barrier, we have initiated collaborative project between Stephenson Cancer Center (SCC) and Oklahoma Medical Research Foundation (OMRF) with the goal to generate a biobank of patient-derived xenograft (PDX) models derived directly from ovarian cancer patients undergoing therapy. RESULTS: PDX tumor models are generated in the Patient-Derived Xenograft and Preclinical Therapeutics (PDX-PCT) Core at OMRF and used to improve preclinical evaluation of new drugs towards more personalized medicine. We have collected blood samples and tumor tissues from consenting patients having primary debulking surgery at Stephenson Cancer Center since 2015 (>140 unique patients). Fresh tumor tissue is minced into small tumor chunks (~2 mm) and implanted into immunocompromised mice. PDXs are expanded for 3-4 passages and characterized based on tumor type, histology and molecular characteristics. Immunohistochemical and molecular analysis showed that histology of the original patient tumors as well as the expression of commonly used markers for high-grade serous ovarian cancer such as cytokeratin, PAX8 and WT1 are perfectly maintained in the derivative PDX lines. Genotyping of patients' tumors and their corresponding PDXs using Illumina's Exome Array revealed that single nucleotide polymorphism (SNP) profile found in the original tumors is perfectly maintained in PDX lines. One of the PDX limitations is that human tumor stromal and immune cells are being replaced with mouse equivalents upon in vivo propagation of PDXs, which negatively affects quality of genomic or proteomic analyses. To overcome this challenge, we optimized a method to deplete mouse cells from PDX tumors by magnetic cells separation. Our data showed that ovarian cancer PDXs contain around 28-54% of human tumor cells, where the rest are mouse cells. We also optimized protocols to utilize pure population of human tumor cells isolated from PDXs to transduce these with lentiviral vectors expressing GFP and/or luciferase to generate luciferized PDXs. Luciferized ovarian PDXs are excellent models used for non-invasive orthotopic tumor growth and drug response monitoring. CONCLUSIONS: Collection of our PDXs illustrates heterogeneity and diversity of human ovarian tumors. However, each PDX maintains essential molecular features of the original patients' tumor. Our goal is to generate clinically faithful ovarian tumor models that will provide a platform to learn more about cancer biology and to screen these for new better therapies. Citation Format: Magdalena Cybula, Katherine Moxley, Lin Wang, Luyao Wang, Magdalena Bieniasz. UTILIZATION OF PATIENT-DERIVED TUMOR XENOGRAFT (PDX) MODELS IN ONCOLOGY [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-021.

TMOD-21. TARGETING ANAPLASTIC MENINGIOMA WITH PANOBINOSTAT IN PATIENT-DERIVED ORTHOTOPIC XENOGRAFT (PDOX) MODELS DERIVED FROM A MATCHING PAIR OF PRIMARY AND RECURRENT TUMORS

Abstract BACKGROUND Meningioma is the most common brain tumor in adults. Despite the overall benign nature of meningioma, skull base tumors can be difficult to completely resect while others exhibit progression and aggressive profiles. The lack of clinically relevant animal models is blocking the development of novel therapies. MATERIAL AND METHODS Twelve surgical specimens (1 × 105) from 11 adult meningioma patients were implanted into the frontal cranial-base of the brain of SCID mice. Mice were then followed and assessed for tumor formation. Tumor growth was confirmed by small animal MRI. Pathologic features of the PDOX models and the matched patient tumors were compared with standard H&E and immunohistochemical staining. RNAseq was performed to examine the molecular fidelity of PDOX tumors and to identify new therapeutic targets. A panel of 60 clinically-relevant drugs was developed for screening drug sensitivity. In vivo examination of therapeutic efficacy of Panobinostat was performed in two models by treating preformed PDOX tumors with i.p. injection (10 mg/kg), 5 days on, 5 days off for 2 cycles. RESULTS Intracranial xenograft formation was confirmed in two samples derived from the same patient, the first an atypical meningioma (K029MEN-P) and the second, which progressed to anaplastic meningioma at recurrence (K029MEN-R). MRI scanning revealed that the PDOX tumors grew from the skull base. These patient tumor cells can be cryopreserved for long-term maintenance of tumorigenicity. The xenograft tumors replicated histopathological features of parental tumors. Overall gene expression profiles of PDOX were similar to the original patient tumors. Using MEN primary culture cells, we screened 60 drugs and identified 12 (20%) active compounds. Panobinostat also significantly prolonged survival of mice bearing orthotopic meningiomas. CONCLUSION A set of meningioma PDOX models derived from primary and recurrent tumor was established. Our data further demonstrate that panobinostat exerts potent antitumor activity against high-grade meningioma.

Patient-derived xenografts of central nervous system metastasis reveal expansion of aggressive minor clones.

The dearth of relevant tumor models reflecting the heterogeneity of human central nervous system metastasis (CM) has hindered development of novel therapies. We established 39 CM patient-derived xenograft (PDX) models representing the histological spectrum, and performed phenotypic and multi-omic characterization of PDXs and their original patient tumors. PDX clonal evolution was also reconstructed using allele-specific copy number and somatic variants. PDXs retained their metastatic potential, with flank-implanted PDXs forming spontaneous metastases in multiple organs, including brain, and CM subsequent to intracardiac injection. PDXs also retained the histological and molecular profiles of the original patient tumors, including retention of genomic aberrations and signaling pathways. Novel modes of clonal evolution involving rapid expansion by a minor clone were identified in 2 PDXs, including CM13, which was highly aggressive in vivo forming multiple spontaneous metastases, including to brain. These PDXs had little molecular resemblance to the patient donor tumor, including reversion to a copy number neutral genome, no shared nonsynonymous mutations, and no correlation by gene expression. We generated a diverse and novel repertoire of PDXs that provides a new set of tools to enhance our knowledge of CM biology and improve preclinical testing. Furthermore, our study suggests that minor clone succession may confer tumor aggressiveness and potentiate brain metastasis.

Profiling the Stromal and Vascular Heterogeneity in Patient-derived Xenograft Models of Head and Neck Cancer: Impact on Therapeutic Response.

Head and neck squamous cell carcinomas (HNSCC) represent a group of epithelial neoplasms that exhibit considerable heterogeneity in clinical behavior. Here, we examined the stromal and vascular heterogeneity in a panel of patient-derived xenograft (PDX) models of HNSCC and the impact on therapeutic response. Tumor sections from established tumors were stained for p16 (surrogate for human papillomavirus (HPV) infection), stromal (Masson’s trichrome) and vascular (CD31) markers. All PDX models retained the HPV/p16 status of the original patient tumor. Immunohistochemical evaluation revealed the presence of multiple vessel phenotypes (tumor, stromal or mixed) in the PDX panel. Vascular phenotypes identified in the PDX models were validated in a tissue microarray of human HNSCC. Treatment with a microtubule targeted vascular disrupting agent (VDA) resulted in a heterogeneous antivascular and antitumor response in PDX models. The PDX with the tumor vessel phenotype that exhibited higher CD31+ vessel counts and leaky vasculature on magnetic resonance imaging (MRI) was sensitive to VDA treatment while the PDX with the stromal vessel phenotype was resistant to therapy. Collectively, our results demonstrate the phenotypic and functional vascular heterogeneity in HNSCC and highlight the impact of this heterogeneity on response to antivascular therapy in PDX models of HNSCC.

Hormonal Regulation of Patient-Derived Endometrial Cancer Stem-like Cells Generated by Three-Dimensional Culture.

Low-grade and early-stage endometrial cancer usually has a favorable prognosis, whereas recurrent or metastatic disease is often difficult to cure. Thus, the molecular mechanisms underlying advanced pathophysiology remain to be elucidated. From the perspective of the origin of advanced endometrial cancer, the characterization of cancer stem-like cells (CSCs) will be the first step toward the development of clinical management. We established long-term culturable patient-derived cancer cells (PDCs) from patient endometrial tumors by spheroid cell culture, which is favorable for the enrichment of CSCs. PDC-derived xenograft tumors were generated in immunodeficient NOD/Shi-scid, IL-2RγKO Jic mice. Morphologically, PDCs derived from three distinct patient samples and their xenograft tumors recapitulated the corresponding original patient tumors. Of note, CSC-related genes including ALDH1A1 were upregulated in all of these PDCs, and the therapeutic potentiality of aldehyde dehydrogenase inhibitors was demonstrated. In addition, these PDCs and their patient-derived xenograft (PDX) models exhibited distinct characteristics on the basis of their hormone responsiveness and metastatic features. Interestingly, genes associated with inflammation and tumor immunity were upregulated by 17β-estradiol in PDC lines with high estrogen receptor expression and were also overexpressed in secondary PDCs obtained from metastatic tumor models. These results suggest that PDC and PDX models from endometrial cancer specimens would be useful to elucidate CSC traits and to develop alternative diagnostic and therapeutic options for advanced disease.

Abstract 4710: Surface protein marker and single cell gene expression profiling of individual tumor cells dissociated from small cell lung cancer pdx mouse models can be correlated with in vivo sensitivity to the p70S6K/AKT1/3 inhibitor M2698

Abstract Small Cell Lung Cancer (SCLC) is characterized by rapid tumor growth and currently, there are few therapeutic options or predictive biomarkers. Patient derived xenograft (PDX) models are capable of recapitulating solid tumor growth including the intra-tumor heterogeneity (ITH) observed in the original patient tumor. The present study aimed to correlate surface marker profiles of SCLC PDX models with previously observed drug responses to M2698, a potent, selective inhibitor of p70S6K and AKT 1/3, and investigate ITH through gene expression profiling of tumor cell subpopulations. Drug response data had been established in a preclinical screen of 45 PDX models of SCLC, in which two mice were implanted subcutaneously with tumors from each model; one mouse was treated with vehicle while the other was treated with M2698 25 mg/kg QD po until the tumor in the vehicle-treated mouse reached ~1200 mm3. Tumor control (stasis or regression) was seen in 12 (27%) of the models. We showed previously that cell surface marker profiles of PDX tumor tissue demonstrated high intra-model reproducibility for many surface markers and uniquely associate with each model. Also, distinctly heterogeneous markers were identified that allowed FACS sorting of tumor cell subpopulations with similarly distinct gene expression profiles. To build on these data, a subset of models that were the most and the least sensitive to M2698 (n=7) in the above described screen were selected for implantation into a new set of mice. Tumors were profiled once they reached ~800 mm3. We evaluated 80+ markers commonly used to identify tumor initiating cells (e.g. CD44, CD90, CD133, CD166, CD184), EMT or aggressiveness (e.g. CD166, EphB2, CD324, CD325), poor outcome in SCLC (CD49b, CD221, claudin3) or drug targets (e.g. CD184, EGFR, Her2) to establish extensive marker profiles. Our data reveal that surface marker profiles in these models allow a meaningful subclassification of SCLC PDX tissue. Correlation of these profiles with efficacy of M2698 (above) suggest that surface markers may have predictive value. Our results prequalify a number of these markers for validation. Furthermore, several models harbor cell subpopulations identifiable by various surface markers. Along with their distinct gene expression profiles this suggests an equilibrium between functionally different compartments within a lesion. In one case, the ratio of two populations shifted concurrently with growth differences, raising the possibility of a dynamic relationship between this equilibrium and the growth stage. Overall, our workflow may provide tools for sample characterization, quality control and elucidation of cellular response markers to varying selective pressures, such as drug challenges. Citation Format: Warren Porter, Eileen Snowden, Friedrich Hahn, Mitchell Ferguson, Frances Tong, William S. Dillmore, Anderson Clark, Hong Zhang, Rainer Blaesius. Surface protein marker and single cell gene expression profiling of individual tumor cells dissociated from small cell lung cancer pdx mouse models can be correlated with in vivo sensitivity to the p70S6K/AKT1/3 inhibitor M2698 [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 4710.

Abstract 1941: Fluorescence lifetime imaging of treatment response in neuroendocrine tumor organoids

Abstract Gastroenteropancreatic neuroendocrine tumors (GEP-NET) are now the second most common form of gastrointestinal cancer, and account for roughly 60% of all neuroendocrine tumors. Unfortunately, no animal models or cell lines exist that reflect the in vivo characteristics of human GEP-NET’s for use as predictive models. Therefore, pre-clinical work is often not representative of the clinical outcome, so clinical trials begin in patients without rigorous pre-clinical validation of the treatment regimen. There is an established need to predict the most effective treatments prior to clinical trials for GEP-NET patients, to increase treatment efficacy and reduce toxicities. Here we show that a three-dimensional organoid model of primary patient tumors is representative of the original patient tumors. Furthermore, this study aims to use the organoid model to test a novel pre-clinical combination of ABT263, an experimental cancer drug that has been shown to enhance the effects of other chemotherapeutic agents, and the standard drug everolimus, on GEP-NET organoids. Previous studies have shown that ABT263, which targets Bcl-2, Bcl-XL and Bcl-w, induces apoptosis when used in combination with everolimus, making this a promising drug combination for GEP-NET patients. Treatment response to ABT263, everolimus, and the combination, was tested in three-dimensional organoids derived from six primary GEP-NET patients, using two-photon fluorescence lifetime imaging microscopy (FLIM). FLIM captures the fluorescence lifetime of metabolic cofactors NAD(P)H and FAD. Further, the fluorescence intensity of NAD(P)H divided by that of FAD (the redox ratio) was measured in all samples. Altogether, two-photon imaging of NAD(P)H and FAD fluorescence intensities and lifetimes (optical metabolic imaging, or OMI) in primary patient GEP-NET organoids provides a novel tool to measure metabolic changes with drug treatment. OMI measured metabolic differences induced by treatment in primary patient GEP-NETs when compared to control, at 72 hours post-treatment. All patients except one showed a decrease in redox ratio, which corresponds with a response, to the combination treatment. As metabolic imaging is performed to collect treatment response from more patients, this OMI data will be integrated into a multivariate analysis of variance to predict treatment response. Independent measures of organoid response, including changes in organoid diameter and immunohistochemistry, have been used to validate OMI predictions. We have shown that ABT263 plus everolimus is a promising combination treatment for GEP-NET’s. Overall, this work establishes the feasibility of personalized drug screens for GEP-NET’s using OMI in primary tumor organoids. Additionally, this platform can be used to identify new treatment regimens for improved GEP-NET patient outcomes, such as the ABT263 plus everolimus combination tested here. Citation Format: Amani A. Gillette, Christopher P. Babiarz, Cheri A. Pasch, Dustin A. Deming, Melissa C. Skala. Fluorescence lifetime imaging of treatment response in neuroendocrine tumor organoids [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 1941.

Abstract 3337: A racially/ethnically diverse 3D PDX model of prostate cancer

Abstract Prostate cancer (PCa) incidence and mortality rates in African American men are double that of any other race/ethnicity in the United States. Thorough understanding of the biological factors that contribute to this long-standing cancer health disparity (CHD) is required to improve the major public health concern and close this gap. However, few models exist that can compare racially-diverse specimens directly and provide a platform for dissecting the impact of ancestry-dependent factors on disease pathway selection and drug susceptibility. Both the conventional 2D culture of clonal human cell lines and the purely rodent-based in vivo models fail to reflect the heterogeneity of human tumors, often leading to inaccurate prediction of in vivo tumor response in patients, and confounding researchers’ ability to detect potentially subtle biological factors that may contribute to prostate CHD. PCa patient-derived xenografts (PDXs) offer substantially greater fidelity to original patient tumors but are non-adherent and ultimately non-viable in extended in vitro 2D culture. Therefore, a population-based PCa platform which accurately mimics the three-dimensional (3D) tumor microenvironment (TME) is urgently needed. We have employed MIMETAS’ OrganoPlate®, a high throughput microfluidic culture platform containing 40-96 individual tissue chips, for ex vivo 3D culture of multiple racially/ethnically diverse PCa PDXs (African American, Caucasian, Hispanic) developed at MD Anderson Cancer Center (the MDA PCa PDXs series). MDA PCa PDX tumors were reconstituted from single-cell digestates into multicellular clusters, suspended within HyStem® hyaluronic acid hydrogel precursor solutions, and loaded into the OrganoPlate®. PDXs were maintained in 3D either as monocultures, as cocultures with bone marrow-derived stromal fibroblasts, or as tricultures with endothelial cell blood vessel mimics under continuous perfusion. High-content fluorescence imaging identified retention of stable, viable cultures for at least 7 days. Positive immunofluorescent staining for human nuclear antigen (HNA) confirmed that nearly 100% of encapsulated PCa cells were of human origin. For each PCa model developed, appropriate expression of phenotypic prostate-specific antigen (PSA) and androgen receptor (AR) was maintained over the life of the culture. PCa cultures were treated with various chemotherapeutic drugs and viability was monitored to generate dose response curves for comparison to clinical data. This engineered “tumor-on-a-chip” will better predict patient responses and, by incorporating PCa cells from patients with diverse ancestries, support CHD research. Citation Format: Lindsey K. Sablatura, Kristin M. Bircsak, Peter Shepherd, Rick Kittles, Pamela E. Constantinou, Anthony D. Saleh, Nora M. Navone, Daniel A. Harrington. A racially/ethnically diverse 3D PDX model of prostate cancer [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 3337.

Abstract 1067: A patient-derived xenograft (PDX) platform for cancer translational, precision medicine and health disparity research

Abstract Introduction and Objective: This purpose of this program is to develop and characterize PDXs from multiple organ sites and patients of multiple ethnic backgrounds, and to make these resources available to the scientific community for translational, precision medicine, and health disparity research. Methods: PDXs were directly developed from patient cancer specimens, molecularly characterized with next-generation sequencing, and annotated with clinical information. Various aspects of its applications were tested. Results: UC Davis, working in collaboration with The Jackson Laboratory (JAX), has developed PDX models, reflecting a wide range of tumor types, from 150 of our patients. Each of these models is annotated with clinical and genomic information. The fidelity of these PDXs was validated by the retention of histopathological features and the conservation of genetic aberrations (i.e., 92-97%) of the original patient tumors in bladder cancer PDXs tested. Extensive preclinical studies showed that these PDXs could potentially be used to screen multiple therapeutic agents simultaneously to identify the most efficacious drugs or drug combinations, re-purpose FDA-approved drugs, decipher the mechanisms of primary resistance, decrypt the mechanisms of secondary resistance, guide drug development, and identify biomarkers. For drug repurposing, the EGFR/ERBB2 dual inhibitor lapatinib effectively prolonged the overall survival (OS) of mice carrying ERBB2+ bladder cancer PDXs from 18.4 days to 25.4 days (p=0.007). For screening of targeted therapy in a PDX carrying PI3K, ERBB2 and Src alterations, only a PI3K inhibitor (BEZ235) prolonged the OS (p<0.0001) while lapatinib and a Src inhibitor ponatinib had no effect. For biomarker development, DNA adduct levels correlated with cancer response to alkylating agents and this project has already been translated into a clinical trial. For determination of the mechanisms of secondary resistance, loss of tumor suppressor gene LSP1 expression was associated with secondary resistance to an inhibitor of the PI3K pathway which is commonly mutated in many cancers. With the support of the Minority PDX Development and Trial Center (M-PDTC) U54 grant, UC Davis will continue to establish over 200 PDX models from bladder, lung squamous cell, gastric and hepatocellular carcinoma, with >60% of the specimens coming from minority patients, for health disparity research. Conclusions: PDXs have great potential for cancer translational, precision medicine and health disparity research. The NCI-funded U54 center with minority PDXs is open for collaboration through the PDX Development and Trial Centers Research Network (PDXNet). Citation Format: Chong-Xian Pan, Hongyong Zhang, Ai-Hong Ma, Shuxiong Zeng, Maike Zimmermann, Clifford Tepper, Paul Henderson, Luis Carvajal-Carmona, Regina Gandour-Edwards, Moon Chen, Susan Airhart, Ralph de Vere Whtie. A patient-derived xenograft (PDX) platform for cancer translational, precision medicine and health disparity research [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 1067.

The development and characterization of patient derived xenografts from non-small cell lung cancer brain metastases

Introduction: NSCLC brain metastasis cell lines and in vivo brain metastasis models are not widely accessible. The purpose of this study was to establish and characterize a direct-from patient-derived xenograft (PDX) model of non-small cell lung cancer (NSCLC) brain metastases. Methods: Surgically obtained tissue was implanted subcutaneously and as orthotopic intracranial implants into immunodeficient mice. Histology and DNA loci were compared between original tumor and subsequent PDX passages. Tumors underwent RNA and DNA sequencing and relevant therapeutic targets were identified. Tumor growth rates were assessed following treatment with radiation, MEK inhibitor selumetinib, or MET inhibitor savolitinib. Cell lines were established. Results: Nine NSCLC brain metastases PDXs were established. Morphologically, strong retention of cytoarchitectural features was observed between original patient tumor and subcutaneous and intracranial tumors. Preservation of thyroid transcription factor 1 expression was seen in all xenografts. Short tandem repeat analysis demonstrated strong concordance between patient tumors and subsequent PDX passages. RNA sequencing analysis revealed high correlation between matched patient and PDX samples. Significant growth inhibition was shown with radiation, with selumetinib in tumors harboring KRAS G12C mutation and with savolitinib in tumors with MET exon 14 skipping mutation. Significant tumor growth delay was observed with the combination of radiation and savolitinib compared to radiation or savolitinib alone in the PDX harboring MET exon 14 skipping mutation. Early passage cell strains showed high consistency between patient and PDX tumors. Conclusion: We have established a robust human xenograft model system for investigating NSCLC brain metastases. These PDXs and cell lines show strong phenotypic and molecular correlation with the original patient tumors and provide a valuable resource for testing preclinical therapeutics.

Application of Prostate Cancer Models for Preclinical Study: Advantages and Limitations of Cell Lines, Patient-Derived Xenografts, and Three-Dimensional Culture of Patient-Derived Cells.

Various preclinical models have been developed to clarify the pathophysiology of prostate cancer (PCa). Traditional PCa cell lines from clinical metastatic lesions, as exemplified by DU-145, PC-3, and LNCaP cells, are useful tools to define mechanisms underlying tumorigenesis and drug resistance. Cell line-based experiments, however, have limitations for preclinical studies because those cells are basically adapted to 2-dimensional monolayer culture conditions, in which the majority of primary PCa cells cannot survive. Recent tissue engineering enables generation of PCa patient-derived xenografts (PDXs) from both primary and metastatic lesions. Compared with fresh PCa tissue transplantation in athymic mice, co-injection of PCa tissues with extracellular matrix in highly immunodeficient mice has remarkably improved the success rate of PDX generation. PDX models have advantages to appropriately recapitulate the molecular diversity, cellular heterogeneity, and histology of original patient tumors. In contrast to PDX models, patient-derived organoid and spheroid PCa models in 3-dimensional culture are more feasible tools for in vitro studies for retaining the characteristics of patient tumors. In this article, we review PCa preclinical model cell lines and their sublines, PDXs, and patient-derived organoid and spheroid models. These PCa models will be applied to the development of new strategies for cancer precision medicine.

Abstract LB-037: 3D organoids from milligrams of genitourinary cancer patients tissue retain key features of original tumors

Abstract 3D organoid culture, especially that generated from patient samples, is becoming prevalent as 2D cell culture models fail to provide clinical relevance. However, a relatively large amount of starting tissue is still required for this process, and the turnover rate and overall success rate vary greatly if relying solely on spontaneous aggregation. Here, we report the successful use of magnetic 3D bioprinting to drive organoid formation derived from fresh tumor samples of patients with prostate cancer, renal cell cancer, and urothelial cancer. Only milligrams to tens of milligrams of starting tissues (from fine-needle biopsies, core biopsies, or surgical excision) were used. Most organoids formed overnight, with a very high overall success rate (&gt95%, n = 25). We have demonstrated that these cultures can be maintained, passed, and cryopreserved like conventional 2D cultures. More importantly, we characterized the organoids derived from prostatic adenocarcinomas and neuroendocrine tumors by the means of immunostaining and showed that the organoids expressed the same signature proteins as the originating tumors. This clinical concordance is crucial for downstream translational and biological research. We also showed that these adenocarcinoma cells responded to enzalutamide, an androgen receptor inhibitor that is used clinically to treat prostatic adenocarcinoma. In summary, we have established an efficient and robust system to create patient-derived 3D organoids; molecular characterization shows clinical concordance with original patient tumors; and our system shows great potential in drug screening with patient-derived 3D cultures. Citation Format: Yue Wang, Glauco Souza, Robert J. Amato. 3D organoids from milligrams of genitourinary cancer patients tissue retain key features of original tumors [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 LB-037.

Abstract 1046: Developing patient-derived xenograft tumor models that recapture clinical manifestation of inflammatory breast cancer patients

Abstract Inflammatory breast cancer (IBC) is the most aggressive clinical manifestation of breast cancer (BC), with striking erythema of the overlying skin, and exhibiting features of early metastatic and treatment-refractory disease. Malignant cells and infiltrating leukocytes, e.g. tumor-associated microphages (TAMs) commonly designated "M2" macrophages, play a major part in metastasis and treatment resistance. TAMs help promote tumor growth and progression. There is a significant need for preclinical models that retain the characteristics of the original patient tumor for investigating mechanisms of IBC metastasis and for testing candidate therapies. Six samples of pleural effusion-derived tumor cells or circulating tumor cells (CTCs) from five IBC or metastatic breast cancer (MBC) patients were obtained from Northwestern Memorial Hospital. IBC cells were used for 3D tumor spheroid cultures, and for developing PDX tumor models in immunodeficient mice. Short Tandem Repeat profiling against with original patient tumor DNA was conducted to establish patient DNA identify, and to serve as a reference for authenticating derivative tumor models. The tumor genomic mutation analysis indicated the most common alterations of five IBC or MBC patients were TP53 (75%), CCND1 (75%), BRCA2 (50%), NF1 (50%), FGFR1 (50%), CDK6 (50%), EGFR (50%), MYC (50%), BRAF (50%). Examination IBC pleural effusion cellular composition indicated ~60% were tumor cells and ~40% were M2 TAMs (CD163+). In early 3D spheroid IBC culture, a symbiotic phenomenon was observed between tumor cells and attached TAMs; Anti-cancer drugs GW2580 and Pexidartinib inhibited 32% and 68% of cell proliferation, respectively, when added to spheroid cultures at 100 nanomolar. Histopathologic analysis of derivative IBC and MBC PDX revealed highly heterogeneous characteristics and metastatic features characteristic of corresponding patient tumors. Subcutaneous tumors also demonstrated metastasis to liver and lung tissue. NSG mice engrafted with original IBC pleural effusion cells or derived spheroid cultures manifested a variety of inflammatory clinical symptoms. Mice engrafted with original pleural effusion cells developed palpable tumor and manifested most IBC-characteristic including reddish and ulcerated skin lesions necessitating euthanasia within 2 months of cell engraftment. Mice engrafted with > 6 passage or <4 passages of spheroid cultures (lower % of TAMs present) manifested lesser skin inflammation and lesion, and developed tumors of ~ 1 cm within 2-3 months. This may indicate the role of TAMs in the development of IBC skin lesion. Our results indicate IBC 3D spheroid cultures and PDX tumor models recapitulate clinical characteristics observed in IBC patients, and will prove useful for studying mechanisms of IBC metastasis as well as responsiveness to investigational therapies. Citation Format: Wenan Qiang, Youbin Zhang, Demirkan Gursel Gursel, Jian-Jun Wei, Charles David James, Thomas V. O'Halloran, Massimo Cristofanilli. Developing patient-derived xenograft tumor models that recapture clinical manifestation of inflammatory breast cancer patients [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 1046.

Abstract 5020: A patient-derived organoid model of neuroendocrine prostate cancer transdifferentiation informing the role of the BAF complex component ARID1A

Abstract Metastatic castration-resistant prostate cancer (mCRPC) is a genetically and phenotypically heterogeneous disease. Resistance to contemporary androgen-deprivation therapy (ADT) is inevitable, and occurs via several different mechanisms. One of these is lineage plasticity leading to transdifferentiation of AR-positive adenocarcinoma (ACPC) to the neuroendocrine (NEPC) subtype. The modern era of second-generation ADT has seen a significant increase in the percentage of NEPC, yet little is known about the genetic and epigenetic drivers of this phenotype. Using modified organoid media conditions that we previously developed, we have established a set of mCRPC organoid lines derived from four biopsies (two samples before and two after carboplatin + etoposide treatment, 1 year apart) of a single patient tumor that capture the genetic and phenotypic heterogeneity of the original metastatic tumor. Genomic and histologic analysis show that the original patient tumor is composed of both adenocarcinoma and neuroendocrine subpopulations that are maintained in the organoids at a ratio of approximately 1:4, respectively. The two subpopulations share a common ancestor and a putative early driver event in the homozygous loss of CDKN1B, but our analysis of clonal evolution indicates that the majority NEPC population did not transdifferentiate directly from the minority ACPC population in the metastasis, but rather in the primary tumor or in a prior metastatic lesion. The ACPC subpopulation contains a high copy gain of an upstream regulatory region of the AR gene and is genetically distinct from the NEPC population that lacks this alteration, but uniquely contains a truncating mutation in the ARID1A gene. ARID1A is frequently mutated and/or deleted in several different cancer types. It has been found to be altered in ~2% of prostate cancer patients, but its role is not understood. It is a key component of the BAF complex, which is a known regulator of neural development. Immunofluourescent staining of our organoid cells shows that mutant ARID1A correlates with the NEPC population. Using single-cell RNA-seq and principle component clustering analysis, we have identified five different transcriptionally defined populations: one population that corresponds to the genetically defined ACPC population and four additional transcriptionally defined populations that represent different states of differentiation of the NEPC component, as determined by expression of lineage markers. A full understanding of this model with respect to differentiation of the NEPC population and the specific role of ARID1A in the process will inform the development of mechanism-based treatments to inhibit NEPC formation. Citation Format: Mike L. Beshiri, Caitlin M. Tice, Adam G. Sowalsky, Crystal Tran, Fatima Karzai, William Dahut, Kathy Kelly. A patient-derived organoid model of neuroendocrine prostate cancer transdifferentiation informing the role of the BAF complex component ARID1A [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 5020.