Patient-derived Xenograft Lines Research Articles

DRES-12. QUANTIFYING INDIVIDUALIZED ABT-414 SENSITIVITY AND BLOOD-BRAIN BARRIER PENETRANCE FROM SERIAL IMAGING OF PATIENT-DERIVED XENOGRAFTS MODELS OF GLIOBLASTOMA

Abstract INTRODUCTION Glioblastoma (GBM) is an aggressive primary brain tumor, known for its poor prognosis. Due to its diffuse invasiveness into normal-appearing brain, localized treatments such as surgical resection and radiotherapy are typically supplemented with chemotherapy. However, to reach invading tumor cells, such antineoplastic drugs must cross the blood-brain barrier (BBB). That is, while angiogenesis induces BBB breakdown in dense tumor regions, the BBB remains rather intact for invading GBM cells. As a result, it is unclear whether BBB-impermeable drugs are delivered at a sufficient level to be effective. METHODS In order to study heterogeneity in BBB breakdown, experiments were conducted using both flank and intracranial patient-derived xenografts (PDXs) treated with the EGFR-targeted monoclonal antibody drug conjugate, depatuxizumab mafodotin (ABT-414). Time-series bioluminescence imaging (BLI) data was used to develop a differential equation model of tumor growth for three PDX cell lines. Data from untreated PDXs, both in flank and intracranially, were used to parameterize tumor proliferation rates. Flank PDX data were used to parameterize individual sensitivity to ABT-414, whereas intracranial PDX data were used to determine the proportion of drug exposed to the tumor. RESULTS Each PDX line differed in response to the study drug ABT-414. As expected, such heterogeneous responses can primarily be attributed to differences in both drug sensitivity and the proportion of drug that reached the tumor. Notably, the estimated proportion of drug that reached the tumor was highest in the PDX line with the longest survival times, despite also having higher estimates of resistance. This suggests that PDXs with greater overall BBB breakdown may respond better to this agent. CONCLUSIONS Although more cell lines are needed to validate our approach, parameterizing this model for PDXs gives valuable insight into the extent of BBB breakdown in patient GBMs and may aid in determining optimal therapies for individual patients.

GENE-55. GENE EXPRESSION SIGNATURE ASSOCIATED WITH AGGRESSIVE GLIOBLASTOMA GROWTH IS ENRICHED IN CHROMATIN MODIFICATION AND STEMNESS TRANSCRIPTIONAL REGULATION PROGRAMS

Abstract Prognosis of patients diagnosed with IDHwt glioblastoma is influenced by known clinical and demographic factors, and likely by physiological characteristics. Our goal is to determine tumor-intrinsic gene expression signatures associated with aggressive tumor growth in patient-derived xenografts (PDXs). Cancer stem cell (CSC) lines established from 10 IDHwt glioblastoma tumors were implanted orthotopically in cohorts of 10 to 15 nude mice (3x10E5 viable cells/mouse), for development of PDX under uniform conditions. Mice were monitored and sacrificed when symptomatic. Five PDX lines, presenting median survival of 29 to 59 days were classified as short (S) survivors, and 5 lines with median survival between 111 and 134 days as long (L) survivors. RNA was isolated from terminal PDX tumors (n=3/line) and sequenced using Illumina HiSeq 2000. Differential gene expression analysis between tumors in S and L survival groups was conducted using the lmFit and eBayes, and genes were ranked by Benjamini-Hochberg adjusted P-values, set to adj.p< 0.05, resulting in 1663 genes upregulated and 1539 genes downregulated in the aggressive S group. Gene ontology analysis was performed using Metacore (Clarivate Analytics) and Metascape (http://metascape.org). Chromatin modification was significantly enriched in the aggressive tumor group (Metacore, p= 1x 10–12). Remarkably, 40% (654/1663) of the genes upregulated in the aggressive PDX tumors were co-expressed with an epigenetic master regulator in the TCGA glioblastoma RNAseq dataset (cBio Portal, q< 10E-15), and this subset was also highly enriched in chromatin modification, stemness transcriptional regulation and DNA repair (q=10E-45 to q=10E-13). These results indicate that novel host-independent prognostic gene expression signatures can be derived from the PDX models and underline the potential of epigenetic regulators as therapeutic target for aggressive glioblastomas. Our results further indicate that these models are suitable for testing a new generation of epigenetic drugs currently in pre-clinical and clinical development.

ANGI-10. CHEMOTHERAPEUTIC STRESS INDUCES TRANSDIFFERENTIATION OF GLIOBLASTOMA CELLS TO PROMOTE VASCULAR MIMICRY

Abstract Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor affecting adults, with a median survival of approximately 21 months after diagnosis. A key factor underlying the limited efficacy of current treatment modalities is the remarkable plasticity exhibited by GBM cells, which allows them to effectively adapt to changes induced by our current treatment strategies. In addition to their plasticity, GBM tumors are also highly vascularized with aberrant vessels that further promote its aggressiveness. Recent research has demonstrated that GBM cells have the ability to transdifferentiate into endothelial cells (ECs), which suggests that GBM cells may use their properties of plasticity and vascularity in concert, leading to the creation of tumor-derived blood vessels. The mechanism behind this transdifferentiation remains unclear. Here, we show that treatment with temozolamide (TMZ)-based chemotherapy (the current standard of care) induces time-dependent increases in expression of markers for glioma stem cells (GSCs) and immature and mature ECs over 8 days of treatment (p < .001) in multiple patient-derived xenograft (PDX) lines. In addition, GBM tumors growing as orthotopic xenografts in nude mice showed significantly increased expression of GSC markers (CD15 and CD133) and EC markers (CD105 and CD144) after 8 days of TMZ treatment (p < .01). Ex-vivo FACS analysis of these orthotopic xenografts showed the presence of immature and mature EC populations in addition to GSC populations. To assess the functionality of these increased EC populations, a tube forming assay was performed. Results showed that the tube forming capacity of PDX lines was significantly increased (p < .01) after therapy. Furthermore, immunofluorescence analysis revealed increased tumor-derived vessels in TMZ-recurrent tumors. Overall, this study identifies chemotherapeutic stress as a new driver of transdifferentiation of tumor cells to endothelial cells and highlights cellular plasticity as a key player in therapeutic resistance and tumor recurrence.

MiR-221 Targets QKI to Enhance the Tumorigenic Capacity of Human Colorectal Cancer Stem Cells.

miRNAs are key players in the integrated regulation of cellular processes and shape many of the functional properties that define the "cancer stem cell" (CSC) phenotype. Little is known, however, about miRNAs that regulate such properties in human colorectal carcinoma. In this study, we compared the expression levels of 754 miRNAs between paired samples of EpCAM+/CD44+ cancer cells (enriched in CSCs) and EpCAM+/CD44neg cancer cells (with CSC depletion) sorted in parallel from human primary colorectal carcinomas and identified miR-221 as the miRNA that displayed the highest level of preferential expression in EpCAM+/CD44+ cancer cells. High levels of miR-221 expression were associated with Lgr5+ cells in mouse colon crypts and reduced survival in patients with colorectal carcinoma. Constitutive overexpression of miR-221 enhanced organoid-forming capacity of both conventional colorectal carcinoma cell lines and patient-derived xenografts (PDX) in vitro. Importantly, constitutive downregulation of miR-221 suppressed organoid-forming capacity in vitro and substantially reduced the tumorigenic capacity of CSC populations from PDX lines in vivo. Finally, the most abundant splicing isoform of the human Quaking (QKI) gene, QKI-5, was identified as a functional target of miR-221; overexpression of miR-221-reduced QKI-5 protein levels in human colorectal carcinoma cells. As expected, overexpression of QKI-5 suppressed organoid-forming capacity in vitro and tumorigenic capacity of colorectal carcinoma PDX cells in vivo. Our study reveals a mechanistic link between miR-221 and QKI and highlights their key role in regulating CSC properties in human colorectal cancer. SIGNIFICANCE: These findings uncover molecular mechanisms underlying the maintenance of cancer stem cell properties in colon cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/20/5151/F1.large.jpg.

The CHK1 Inhibitor Prexasertib Exhibits Monotherapy Activity in High-Grade Serous Ovarian Cancer Models and Sensitizes to PARP Inhibition.

PARP inhibitors are approved for the treatment of high-grade serous ovarian cancers (HGSOC). Therapeutic resistance, resulting from restoration of homologous recombination (HR) repair or replication fork stabilization, is a pressing clinical problem. We assessed the activity of prexasertib, a checkpoint kinase 1 (CHK1) inhibitor known to cause replication catastrophe, as monotherapy and in combination with the PARP inhibitor olaparib in preclinical models of HGSOC, including those with acquired PARP inhibitor resistance. Prexasertib was tested as a single agent or in combination with olaparib in 14 clinically annotated and molecularly characterized luciferized HGSOC patient-derived xenograft (PDX) models and in a panel of ovarian cancer cell lines. The ability of prexasertib to impair HR repair and replication fork stability was also assessed. Prexasertib monotherapy demonstrated antitumor activity across the 14 PDX models. Thirteen models were resistant to olaparib monotherapy, including 4 carrying BRCA1 mutation. The combination of olaparib with prexasertib was synergistic and produced significant tumor growth inhibition in an olaparib-resistant model and further augmented the degree and durability of response in the olaparib-sensitive model. HGSOC cell lines, including those with acquired PARP inhibitor resistance, were also sensitive to prexasertib, associated with induction of DNA damage and replication stress. Prexasertib also sensitized these cell lines to PARP inhibition and compromised both HR repair and replication fork stability. Prexasertib exhibits monotherapy activity in PARP inhibitor-resistant HGSOC PDX and cell line models, reverses restored HR and replication fork stability, and synergizes with PARP inhibition.

Acetylcholine Receptor Activation as a Modulator of Glioblastoma Invasion.

Grade IV astrocytomas, or glioblastomas (GBMs), are the most common malignant primary brain tumor in adults. The median GBM patient survival of 12–15 months has remained stagnant, in spite of treatment strategies, making GBMs a tremendous challenge clinically. This is at least in part due to the complex interaction of GBM cells with the brain microenvironment and their tendency to aggressively infiltrate normal brain tissue. GBMs frequently invade supratentorial brain regions that are richly innervated by neurotransmitter projections, most notably acetylcholine (ACh). Here, we asked whether ACh signaling influences the biology of GBMs. We examined the expression and function of known ACh receptors (AChRs) in large GBM datasets, as well as, human GBM cell lines and patient-derived xenograft lines. Using RNA-Seq data from the “The Cancer Genome Atlas” (TCGA), we confirmed the expression of AChRs and demonstrated the functionality of these receptors in GBM cells with time-lapse calcium imaging. AChR activation did not alter cell proliferation or migration, however, it significantly increased cell invasion through complex extracellular matrices. This was due to the enhanced activity of matrix metalloproteinase-9 (MMP-9) from GBM cells, which we found to be dependent on an intracellular calcium-dependent mechanism. Consistent with these findings, AChRs were significantly upregulated in regions of GBM infiltration in situ (Ivy Glioblastoma Atlas Project) and elevated expression of muscarinic AChR M3 correlated with reduced patient survival (TCGA). Data from the Repository for Molecular Brain Neoplasia Data (REMBRANDT) dataset also showed the co-expression of choline transporters, choline acetyltransferase, and vesicular acetylcholine transporters, suggesting that GBMs express all the proteins required for ACh synthesis and release. These findings identify ACh as a modulator of GBM behavior and posit that GBMs may utilize ACh as an autocrine signaling molecule.

P2.06-10 ABT-806 Derived Antibody Drug Conjugates (ADCs) Inhibit Growth of Malignant Mesothelioma In-Vivo

Malignant mesothelioma (MM) is an aggressive malignancy of the pleura with limited therapeutic options, and is associated with a poor prognosis. EGFR is known to be highly over-expressed in mesothelioma with reported EGFR overexpression between 44 to 97%. We have investigated an anti-EGFR antibody (ABT-806), which is tumour specific and robustly inhibits EGFR-expressing tumors. We have previously shown that ABT-806 ADCs demonstrate potent anti-tumor activity in 806 immunohistochemistry (IHC) positive MSTO-211H MM cell line xenograft model. We present data in MM using ABT-806 novel ADCs [ABT-414 (ABT-806- monomethyl auristatin F), ABBV-221 (ABT-806- monomethyl auristatin E), ABBV-322 (ABT-806- pyrrolobenzodiazepine)] and ABBV-321 (Affinity-matured ABT-806- pyrrolobenzodiazepine) in MM patient derived xenografts (PDX). We evaluated expression of EGFR and mAb 806 IHC in MM cell lines and PDXs. PDXs were implanted into 5 to 10 NOD-Scid mice per group and treated with control ADC, saline, cisplatin or ABT-806 ADCs and followed longitudinally with caliper measurements. Comparative statistics were performed in Graphpad prism. Quantitative biodistribution and imaging of mAb806 uptake (89Zr-ch806) were performed to allow correlation of mAb806 concentration in MM tumours. Three PDX models were selected according to their 806 IHC statuses (2 epithelioid 806 IHC positive, 1 biphasic histology 806 IHC negative). In one epithelioid PDX model, ABBV-322 resulted in significantly reduced tumor growth on day 27 post therapy with median tumour volumes of 180 mm3 (ADC control) compared with 78mm3 (ABBV-322; p=0.0159 two-sided). Moreover, the median survival was also significantly longer in ABBV-322 treated models (p=0.018). In the other epithelioid PDX model, ABBV-321 also resulted in significant responses (median 428mm3 (ADC control) vs 167mm3 (ABBV-321, p= 0.0201) [Figure 1]. In the 806 IHC negative PDX model, the differences in tumor volumes between all groups were found to be non-statistically significant (ADC control vs ABT-414, ADC control vs ABBV-221, ADC-control vs ABBV-321 groups) with p=0.0597 for one-way ANOVA. MSTO211H cell line xenograft model also demonstrated significant anti-tumour response to both ABT-414 and ABBV-221 (p<0.01). Whole-body PET/MR images also confirmed localization of 89Zr-Ch806 to the established MSTO-211H xenograft tumours. In a disease with limited therapies, ABT-806 targeting ADCs in MM demonstrated significant responses in 806+ PDX and cell lines. These data support clinical expansion of these compounds in 806+ MM patients.

Detection and targeting insulin growth factor receptor type 2 (IGF2R) in osteosarcoma PDX in mouse models and in canine osteosarcoma tumors

Osteosarcoma (OS) represents 3.4% of all childhood cancers with overall survival of 70% not improving in 30 years. The consistent surface overexpression of insulin-like growth factor-2 receptor (IGF2R) has been reported in commercial and patient-derived xenograft (PDX) OS cell lines. We aimed to assess efficacy and safety of treating PDX and commercial OS tumors in mice with radiolabeled antibody to IGF2R and to investigate IGF2R expression on canine OS tumors. IGF2R expression on human commercial lines 143B and SaOS2 and PDX lines OS-17, OS-33 and OS-31 was evaluated by FACS. The biodistribution and microSPECT/CT imaging with 111Indium-2G11 mAb was performed in 143B and OS-17 tumor-bearing SCID mice and followed by radioimmunotherapy (RIT) with 177Lutetium-2G11 and safety evaluation. IGF2R expression in randomly selected canine OS tumors was measured by immunohistochemistry. All OS cell lines expressed IGF2R. Biodistribution and microSPECT/CT revealed selective uptake of 2G11 mAb in 143B and OS-17 xenografts. RIT significantly slowed down the growth of OS-17 and 143B tumors without local and systemic toxicity. Canine OS tumors expressed IGF2R. This study demonstrates the feasibility of targeting IGF2R on OS in PDX and spontaneous canine tumors and sets the stage for further development of RIT of OS using comparative oncology.

Induction of Acquired Resistance towards EGFR Inhibitor Gefitinib in a Patient-Derived Xenograft Model of Non-Small Cell Lung Cancer and Subsequent Molecular Characterization.

In up to 30% of non-small cell lung cancer (NSCLC) patients, the oncogenic driver of tumor growth is a constitutively activated epidermal growth factor receptor (EGFR). Although these patients gain great benefit from treatment with EGFR tyrosine kinase inhibitors, the development of resistance is inevitable. To model the emergence of drug resistance, an EGFR-driven, patient-derived xenograft (PDX) NSCLC model was treated continuously with Gefitinib in vivo. Over a period of more than three months, three separate clones developed and were subsequently analyzed: Whole exome sequencing and reverse phase protein arrays (RPPAs) were performed to identify the mechanism of resistance. In total, 13 genes were identified, which were mutated in all three resistant lines. Amongst them the mutations in NOMO2, ARHGEF5 and SMTNL2 were predicted as deleterious. The 53 mutated genes specific for at least two of the resistant lines were mainly involved in cell cycle activities or the Fanconi anemia pathway. On a protein level, total EGFR, total Axl, phospho-NFκB, and phospho-Stat1 were upregulated. Stat1, Stat3, MEK1/2, and NFκB displayed enhanced activation in the resistant clones determined by the phosphorylated vs. total protein ratio. In summary, we developed an NSCLC PDX line modelling possible escape mechanism under EGFR treatment. We identified three genes that have not been described before to be involved in an acquired EGFR resistance. Further functional studies are needed to decipher the underlying pathway regulation.

Abstract 81: ARL13B interacts with IMPDH2 to modulate purine synthesis and temozolomide resistance in glioblastoma

Abstract Glioblastoma, a universally lethal primary brain tumor, harnesses cellular plasticity to drive therapeutic adaptation. Critical factors in developing this plasticity are histone modifiers such as Polycomb Repressor Complex 2 protein EZH2. In order to examine tumor cell plasticity in depth, we conducted multiple ChIP Sequencing runs and demonstrate that EZH2 binds within an enhancer region of ARL13B during temozolomide (TMZ) therapy and induces an H3K4 mono-methylation mark. Concurrently, we observed an increase in H3K27ac at the transcription start site of ARL13B as well as a lack of H3K27 tri-methylation, EZH2’s canonical histone mark. Based on this we hypothesize that EZH2 could be non-canonically regulating ARL13B to allow for cellular plasticity and ultimately drive therapeutic adaptation. Delving further into this regulation we demonstrate that knockdown of ARL13B in patient derived xenograft cells significantly increased survival of mice in an orthotopic GBM model when compared to controls (p-value &amp;lt;0.0001). The Cancer Genome Atlas (TCGA) patient dataset demonstrates time to recurrence in patients with downregulated ARL13B is substantially increased as compared to ARL13B upregulated patients (log-rank p-value=0.0012). Searching for a mechanism behind this survival benefit, we preformed mass spectrometry on an ARL13B pulldown in a patient derived xenograft line during TMZ therapy and identified inosine monophosphate dehydrogenase 2 (IMPDH2), the rate-limiting enzyme in de-novo guanine nucleotide biosynthesis, as a significant binding partner of ARL13B during TMZ chemotherapy (p-value &amp;lt;0.0001). Probing this novel interaction further we examined the de-novo and salvage purine biosynthesis pathways using radiolabeled carbon tracing experiments. In ARL13B knockdown cells, purine salvage pathway usage is upregulated 7-fold (p-value &amp;lt;0.0001) while de-novo pathway usage was decreased about 50% (p-value=0.004) in a TMZ specific manner. Examination of IMPDH2 enzymatic activity using a formazin reduction assay demonstrated a decrease in activity over 8 days of TMZ exposure (p&amp;lt;.001). Moreover, ARL13B knockdown GBM cells treated with TMZ show a robust increase in DNA double-strand breaks compared to control cells exposed to TMZ, demonstrated by γH2X staining. Finally, a potent inhibitor of IMPDH2 (Mycophenolate Mofetil) significantly extended median survival in an orthotopic PDX mouse model only when in combination with TMZ (p&amp;lt;.01). Based on these data we hypothesize that EZH2 regulates a novel ARL13B and IMPDH2 interaction which when lost forces cells into salvage synthesis exclusively. This synthesis shift forces cells to uptake and incorporate purines that have been alkylated by TMZ therapy which increases DNA double strand breaks and ultimately impairs therapeutic adaptation. Citation Format: Jack Shireman, Eunus Ali, Miranda Saathoff, Cheol Park, Issam Ben-Sahra, Atique U. Ahmed. ARL13B interacts with IMPDH2 to modulate purine synthesis and temozolomide resistance in glioblastoma [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 81.

Abstract 52: PDX-derived 3D InSightTM tumor microtissues as ex-vivo human experimental models for evaluating therapeutic responses

Abstract Background The selection of appropriate preclinical models comes always with the major question on how accurately and robustly they can represent the complexity of human disease. Patient-derived xenograft (PDX) models faithfully preserve the biological features and the genetic expression profile of human tumor specimens. However, one limiting aspect of patient-derived models is the replacement of the human host microenvironment by murine stroma within the tumor. Lack of cross-species compatibility compromises the induction of a broad range of signaling pathways that cannot be entirely recapitulated. With our in vitro 3D InSightTMTumor Microtissues derived from PDX lines, we provide a relevant physiological environment and a strategy to assess candidate drugs for novel therapeutic approaches. Aim Development of in vitro 3D InSightTM Tumor Microtissues from PDX lines aimed to retain the cellular heterogeneity found in the original human tumor tissue. Material &amp; Methods and Results PDX cell suspensions of lung, breast and melanoma origin were successfully used to assess 3D aggregation in 96-well format and characterized over 10 days in culture. After careful removal of mouse cell contaminants in each PDX sample, 3D PDX cell cultures were supplied with exogenous normal human dermal fibroblasts (nHDF). Furthermore, to provide a more physiological cancer niche, PDX cells were also co-cultured with tumor-matched cancer-associated fibroblasts (CAFs). 3D in vitro tumors were analyzed histologically and cancer phenotypic alterations were evaluated through the analysis of epithelial-to-mesenchymal transition (EMT) markers. The morphology, viability and growth rate of PDX-derived microtumors were assessed by size analysis (cell scanner) and ATP assay. To assess the distribution of various cell populations within the tumor, 3D PDX samples were screened for standard stromal vs. epithelial-tumor cells markers (e.g. FAP, pan-CK, E-Cadherin), and diagnostic cancer type-specific biomarkers. 3D PDX samples were also employed to investigate the efficacy of specific targeted therapies based on distinct molecular signatures of PDX tumor models. Immunohistochemistry assessment of 3D microtumors validated the resemblance with their respective PDX tumor models. 3D tumor growth rate and cell behavior observations reflected the diversity of disease progression in vivo. Conclusion Further efforts will focus on employing this platform to establish more complex co-cultures with integration of additional relevant stromal and immune cells, to enable a reliable preclinical translational research of tumor/immune cell interactions. We suggest that in vitro 3D PDX models offer a more suitable and robust approach to expedite faithful efficacy assessment of immunomodulators and approval of optimal drug candidates. Citation Format: Francesca Chiovaro, Irina Agarkova, Nicole Buschmann, Chloe' Pichon, Teresa Langova, Armin Maier, Julia Schueler, Patrick Guye. PDX-derived 3D InSightTM tumor microtissues as ex-vivo human experimental models for evaluating therapeutic responses [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 52.

Abstract 4001: Unraveling the molecular mechanism of racial disparity in hepatocellular carcinoma (HCC): A link between type I interferon (IFN-I) signaling and HCC disparity

Abstract Among HCC patients, there is a statistically significant increase in incidence and mortality as well as a decrease in 5-year survival rates in African-American (hereafter referred to as black) patients compared to non-Hispanic white (hereafter referred to as white) patients. Currently, there is a gap in our understanding of the molecular mechanism underlying this racial disparity. The purpose of this study is to unravel this molecular mechanism. Gene expression profiles were analyzed by RNA-sequencing (RNA-Seq). Canonical pathway analysis of the differentially expressed genes was performed by Ingenuity Pathway Analysis (IPA). Differential expression of the genes was validated by qRT-PCR and immunohistochemistry. The role of the identified genes in regulating proliferation was determined by transient knockdown by siRNA in a patient-derived xenograft (PDX) cell line established from a black HCC patient followed by MTT assay. In TCGA database, RNA-Seq data from 377 HCC patients were available, of which 17 were black and 184 were white. 279 genes were identified, with a p-value&amp;lt;0.01, that are differentially regulated between black and white patients. IPA analysis identified Type I Interferon (IFN-I) signaling as the only pathway that showed statistically significant activation in black patients. RNA-Seq on HCC samples from 14 white and 18 black patients from the tissue bank at VCU Medical Center identified 283 differentially regulated genes and IPA analysis revealed that the only pathway that showed statistically significant activation in black patients is the IFN-I signaling pathway. Activation of the IFN-I signaling pathway in black patients was identified even after correction of the differential gene expression data for HCV status, indicating that the observed findings are independent of HCV infection. Four interferon stimulated genes (ISGs), ISG15, IFI6, MX1 and OAS1, showing significantly increased expression in black HCC patients in both the TCGA database and our own analysis, were chosen for further analysis by qRT-PCR in HCC patient-derived xenografts (PDX) samples from black and white patients, both HCV positive and negative. The levels of the four ISGs were very low in two HCV-negative white HCC PDX lines. In three HCV-negative black HCC PDX lines, their expression levels were significantly and robustly higher. In a HCV-positive black HCC PDX line, their expression was further higher. The results for OAS1, ISG15 and MX1 were validated at the protein level using immunohistochemistry. Transient knock down of IFI6 and ISG15 significantly decreased proliferation of a PDX line established from a black HCC patient. Persistent activation of the IFN-I signaling pathway might be a key determinant of racial disparity in black HCC patients. This pathway might be exploited to develop targeted treatment that work best for black HCC patients. Citation Format: Saranya Chidambaranathan-Reghupaty, Mikhail Dozmorov, Rachel Mendoza, Zhao Lai, Paul B. Fisher, Trevor Reichman, Devanand Sarkar. Unraveling the molecular mechanism of racial disparity in hepatocellular carcinoma (HCC): A link between type I interferon (IFN-I) signaling and HCC disparity [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 4001.

Abstract 2844: In vitro recapitulation of in vivo obesity-promoted colorectal cancer growth using a patient-derived xenograft engineered tumor model

Abstract Strong epidemiological evidence links obesity to an increased risk of colorectal cancer (CRC). However, the precise molecular mechanisms underlying such an association have not been fully elucidated, partly due to the lack of physiologically relevant models. Here, we established a novel PEG-fibrinogen-based engineered tumor model using patient-derived xenograft (PDX) CRC co-cultured with 3T3-L1 adipocytes and an orthotopically implanted PDX CRC model of obesity. The PDX CRC cells were isolated from PDXs propagated subcutaneously in NOD-SCID mice and encapsulated within a biomimetic polymer, PEG-fibrinogen, to create 3D engineered PDX CRC tumors (3DePCCTs) or cultured in a standard 2D manner. We compared cell viability, colony area, and cell subpopulations within the 3DePCCTs and stiffness of the 3DePCCTs to the in vivo propagated tumors. A stage IV CRC tumor was employed in vivo and in vitro to study obesity-promoted tumor growth. Responsiveness of the 3DePCCTs to the growth promoting effects of obesity was investigated through continuous co-culture with insulin sensitive (IS) and resistant (IR) (treated with TNFα and 1% hypoxia) 3T3-L1 adipocytes (adipocytes replaced every 72 hrs). The responsiveness of the stage IV CRC PDX line was validated using an orthotopically implanted CRC model of obesity in which Rag1tm1Mom mice were fed a high fat Western diet + 4% sugar water (HFWD+S) to induce obesity or a chow diet to maintain a lean phenotype. The cells within the 3DePCCTs remained viable and the PDX-line dependent differential growth of tumor colony area within the 3DePPCTs recapitulated line-dependent difference in in vivo tumor growth. Based on flow cytometry, human (70±3%) and Cytokeratin 20+ (31±1) cell subpopulations within the 3DePCCTs were similar to the original in vivo tumor tissue and maintained over time, whereas supporting mouse stromal cells took over 2D cultured cells (n=3 batches). Stiffness of the 3DePCCTs was within the range of the in vivo tumor tissue stiffness (0.3 to3.6 KPa, n=3 tissues). In vitro adipocytes maintained IR for at least 72 hrs based on significantly higher MCP1 (at least 10 folds) and lower GLUT4 (maximally 0.1 fold) (n=3 batches). The in vitro coculture model revealed a significantly (p &amp;lt; 0.05) greater number of cancer cell colonies within 3DePCCTs after 8 days of co-culture with IR adipocytes compared to IS adipocytes and this difference increased through day 29. In the in vivo model, a significant (p &amp;lt; 0.05) greater than 2-fold increase in weight of PDX CRC tumors grown in mice fed the HFWD+S diet was observed. We have established the ability to maintain PDX CRC cells in 3D culture long-term (29 days) and generated a novel in vitro obesity-mimetic engineered tumor model that recapitulated the growth promoting effects of the in vivo orthotopic, IR tumor microenvironment and could be used to examine mechanistic questions and therapeutic targets. Citation Format: Iman Hassani, Benjamin Anbiah, Bulbul Ahmed, Nicole L. Habbit, Michael W. Greene, Elizabeth A. Lipke. In vitro recapitulation of in vivo obesity-promoted colorectal cancer growth using a patient-derived xenograft engineered tumor model [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 2844.

Abstract 2505: Comprehensive molecular and genomic characterization of pancreatic tropism in metastatic renal cell carcinoma

Abstract Introduction &amp; Objectives: Patients with metastatic renal cell carcinoma (mRCC) involving the pancreas have been shown to exhibit a relatively indolent course, yet the biologic explanation is unclear. We sought to characterize the genomic landscape of patients with mRCC harboring pancreatic metastases (PM) to identify molecular drivers of pancreatic tropism. Materials &amp; Methods: mRCC patients harboring PM from UTSW and Cleveland Clinic were identified. Clinicopathologic data and oncologic outcomes were analyzed. Samples were obtained from primary tumors, metastatic sites (including pancreatic or other distant metastases), and matched normal tissue. Whole exome (WES) and RNA sequencing of tumors was conducted. Patient-derived xenograft (PDX) models were generated from a subset of patients, and the engrafted tumors were analyzed. Results: 31 mRCC patients with PM were included with 54 tumor samples derived from the primary tumor or thrombus (24), PM (21), or other metastatic sites (9). Median follow-up was 101 months. Clinicopathologic characteristics were similar between the two institutional cohorts, and all but one patient were favorable or intermediate IMDC risk. All patients had clear cell histology. 8 patients (26%) were metastatic at diagnosis, and median time to metastasis in the remaining patients was 74 months (IQR 32-120). Overall (OS) and cancer-specific (CSS) survival did not vary by IMDC risk group. OS was strikingly superior for mRCC patients with PM than a historic control of mRCC patients without PM (p&amp;lt;0.001), even after controlling for IMDC risk score. Morphologically, tumors largely displayed low-grade acinar patterns. WES with matched normal tissue and RNAseq were completed with adequate quality for 48 and 30 samples, respectively. 14 PDX lines were generated, of which 5 (36%) engrafted stably (≥2 passages). WES from 2 tumorgraft specimens revealed preservation of specific mutations in the corresponding human samples. Conclusions: mRCC patients with PM exhibit remarkably favorable survival outcomes. The relatively indolent biology of these tumors is reflected histologically and genomically and can be recapitulated in PDX models. Understanding tumor heterogeneity may help refine prognostic models for mRCC and hold implications for improved personalization of therapy. Citation Format: Nirmish Singla, Jacob Choi, Oreoluwa Onabolu, Layton Woolford, Christina Stevens, Vanina Tcheuyap, Tiffani McKenzie, Zhiqun Xie, Tao Wang, Renee McKay, Alana Christie, Payal Kapur, Brian Rini, James Brugarolas. Comprehensive molecular and genomic characterization of pancreatic tropism in metastatic renal cell carcinoma [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 2505.

Abstract LB-317: Use of in vivo microdialysis to evaluate biomarker levels in the tumor and tumor free flank of freely-moving PDX mice

Abstract The tumor microenvironment (TME) constitutes unique surroundings where cancer cells communicate with one another and with the host immune system by releasing a multitude of factors. Information about the physiological state of the TME can help in gaining a broader understanding of cancer biology and developing novel cancer therapeutics. Previous work has demonstrated the ability to use in vivo microdialysis to monitor the release of adenosine and its metabolites from murine cancer models. To further these findings, we used in vivo microdialysis to measure biomarkers in the patient-derived xenograft (PDX) cancer model. By using human tumor tissue models, we aim to bridge the gap between rodent models and human tumors to help develop better cancer therapeutics. The current set of studies used microdialysis in different PDX lines representing human tumor models (provided by CRL DRS Freiburg). Xenograft growth was monitored and upon a tumor volume &amp;gt; 400 mm3, the animals underwent surgical implantation of microdialysis probes in the tumor and the tumor-free flank. The implanted probes were perfused with dialysate fluid and samples from the both flanks were continuously collected over several hours. Levels of ATP, inosine and kynurenine metabolites were quantified by EIA or LC-MS/MS analysis. Analysis of microdialysate samples from tumor and tumor-free flank demonstrated differences in analyte levels between the two sampling sites within one animal indicating that the levels of ATP and metabolites measured in PDX tumors is unique to the tumor microenvironment. In addition, the various PDX models showed differences in biomarker levels between the different tumor types. There is a wide range of biomarkers which are important indicators of energy consumption and provide an insight in the differences in metabolic activity between the different xenografts. Moreover, these data demonstrate that microdialysis can be used to test targeted therapies and follow relevant biomarker levels over time. Thereby, providing better insight in effects of new therapeutic treatments within the patient specific tumor tissue itself. Citation Format: Mariette Heins, Loreen Weichert, Sabine Gorynia, Gunnar Flik, Arash Rassoulpour. Use of in vivo microdialysis to evaluate biomarker levels in the tumor and tumor free flank of freely-moving PDX mice [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-317.

Abstract 3753: Patient derived xenograft colorectal cancer in a micro-vascularized tumor on a microfluidic chip

Abstract Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in the United States. Cellular and biochemical cues from the tumor microenvironment (TME) modulate tumor aggressiveness including metastasis and treatment failure with cancer therapeutics. Conventional tumor models exhibit several shortcomings including lack of cell heterogeneity which limits their potential to examine more complex phenomena associated with drug testing applications. Patient-derived xenografts (PDX) represent an alternative model which maintains tumor heterogeneity. Physiological correlation of current preclinical models is limited by the degree of endothelial vascularization of tumor and incorporation of the tumor cell heterogeneity with an appropriate extracellular matrix to form a three-dimensional (3D) tumor. Herein, we have developed a micro-vascularized tumor on a chip model to examine the dynamics of tumor cell metastasis using CRC PDX lines. PDX tumors established from stage II, III-B, IV adenocarcinomas were propagated subcutaneously in SCID-NOD mice. Two days prior to tumor dissociation, the tumor chip containing an intricate vascular network for the growth of endothelial cells was primed and coated with 200 µg/ml fibronectin. EA.hy926 endothelial cells were seeded in the vascular channel and were allowed to form a lumen over 48 hours. The excised PDX tumor was dissociated and the cells (20 x 106 cells/ml) were mixed with a polymer precursor containing Poly(ethylene glycol)-fibrinogen and the photoinitiator eosin Y, then seeded into the primary tumor chamber and crosslinked under visible light. Serum containing media was perfused through the vascular channel at a constant flow rate of 1µl/minute. Metastasis was observed by maintaining the chip long-term. Significant differences (p = 0.034) in tumor cell migration in the chip (intravasation, tumor cell circulation through the vascular channel, invasion and extravasation) over time were observed between the PDX CRC stages. Stage II tumor cells intravasated to the vascular channel by day 8 followed by circulation and adhered to the endothelium as single circular cells or clusters by day 15. Stage III-B tumor cells were observed to intravasate, circulate, and adhere to the vascular channel by day 8, and the endothelium was overtaken by the cancer cells by day 8. Stage IV tumor cells began intravasation from the primary chamber to the vascular channel and circulated through the endothelium by day 8, while invasion to the secondary chamber adjacent to the primary chamber was observed by day 15. The Stage IV tumor cells also formed clusters in the vascular channel at a distant site and extravasated to the secondary chamber which is consistent with the staging of the tumor. Finally, this chip can be used for screening anti-cancer drugs for CRC patients. Citation Format: Benjamin Anbiah, Iman Hassani, Bulbul Ahmed, Nicole Habbit, Michael Greene, Balabhaskar Prabhakarpandian, Elizabeth Lipke. Patient derived xenograft colorectal cancer in a micro-vascularized tumor on a microfluidic chip [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 3753.

Establishment of Highly Transplantable Cholangiocarcinoma Cell Lines from a Patient-Derived Xenograft Mouse Model.

Cholangiocarcinoma (CCA) is a deadly malignant tumor of the liver. It is a significant health problem in Thailand. The critical obstacles of CCA diagnosis and treatment are the high heterogeneity of disease and considerable resistance to treatment. Recent multi-omics studies revealed the promising targets for CCA treatment; however, limited models for drug discovery are available. This study aimed to develop a patient-derived xenograft (PDX) model as well as PDX-derived cell lines of CCA for future drug screening. From a total of 16 CCA frozen tissues, 75% (eight intrahepatic and four extrahepatic subtypes) were successfully grown and subpassaged in Balb/c Rag-2-/-/Jak3-/- mice. A shorter duration of PDX growth was observed during F0 to F2 transplantation; concomitantly, increased Oct-3/4 and Sox2 were evidenced in 50% and 33%, respectively, of serial PDXs. Only four cell lines were established. The cell lines exhibited either bile duct (KKK-D049 and KKK-D068) or combined hepatobiliary origin (KKK-D131 and KKK-D138). These cell lines acquired high transplantation efficiency in both subcutaneous (100%) and intrasplenic (88%) transplantation models. The subcutaneously transplanted xenograft retained the histological architecture as in the patient tissues. Our models of CCA PDX and PDX-derived cell lines would be a useful platform for CCA precision medicine.

Leveraging a robust patient-derived xenograft platform to characterize predictors for engraftment and oncologic outcomes in renal cell carcinoma patients.

e16100 Background: Patient-derived xenograft (PDX) models of renal cell carcinoma (RCC) preserve the biological features of patient tumors, providing a platform for biomarker identification and preclinical drug testing. We sought to identify predictors of successful tumor engraftment and evaluate the prognostic value of engraftment in patients with RCC using a robust murine PDX platform. Methods: 1,200 specimens derived from nephrectomy, thrombectomy, metastasectomy, or biopsy were orthotopically (renally) implanted into NOD/SCID mice between 2008-2018. Non-RCC pathology was excluded. Stable engraftment was defined by successful passage of tumor tissue at least twice with histologic confirmation. Clinicopathologic characteristics were stratified by engraftment status, and multivariate (MVA) logistic regression was used to identify predictors of engraftment. Kaplan-Meier and Cox regression analyses were used to assess the prognostic value of engraftment on patient overall (OS) and disease-free (DFS) survival. Results: 1,003 independent PDX lines derived from 770 RCC patients were included. 157 (15.6%) lines successfully engrafted and exhibited higher tumor grade, stage, size, and presence of sarcomatoid or rhabdoid components. 79.3% of all tumors were of clear cell histology, and histologic distribution did not vary by engraftment status. We have completed whole exome sequencing and RNAseq on 197 and 213 PDX lines, respectively, and downstream analyses will be reported. On MVA, sarcomatoid (OR 5.71, p &lt; 0.001), rhabdoid (OR 2.79, p = 0.046), and advanced stage (OR 1.72, p = 0.049) were significant predictors for engraftment, while high grade and metastatic tumor source were significant only on UVA. Engraftment was associated with poor OS (HR 2.11, p &lt; 0.001) and DFS (HR 1.85, p = 0.020) in patients after controlling for sarcomatoid, rhabdoid, grade, stage, and age on MVA. Conclusions: Aggressive RCC biology correlates with successful engraftment in PDX models. Engraftment remains independently predictive of OS and DFS even after controlling for adverse pathologic features. Engraftment in mice may illuminate aspects of tumor biology not captured by clinicopathologic variables and provide insight into novel determinants of tumor aggressiveness and metastasis.

ABT-806 derived antibody drug conjugates (ADCs) inhibit growth of malignant mesothelioma in-vivo.

e14677 Background: Malignant mesothelioma (MM) is an aggressive malignancy of the pleura with limited therapeutic options, and is associated with a poor prognosis. EGFR is known to be highly over-expressed in mesothelioma with reported EGFR overexpression between 44 to 97%. We have investigated an anti-EGFR antibody (ABT-806), which is tumor specific and robustly inhibits EGFR-expressing tumors. We have previously shown that ABT-806 ADCs demonstrate potent anti-tumor activity in 806 immunohistochemistry (IHC) positive MSTO-211H MM cell line xenograft model. We present data in MM using ABT-806 novel ADCs [ABT-414 (ABT-806- monomethyl auristatin F), ABBV-221 (ABT-806- monomethyl auristatin E) and ABBV-321 (ABT-806- pyrrolobenzodiazepine)] in MM patient derived xenografts (PDX). Methods: We evaluated expression of EGFR and mAb 806 IHC in MM cell lines and PDXs. PDXs were implanted into 5 to 10 NOD-Scid mice per group and treated with control ADC, saline, cisplatin or ABT-806 ADCs and followed longitudinally with caliper measurements. Comparative statistics were performed in Graphpad prism. Results: Three PDX models were selected according to their 806 IHC statuses (2 epithelioid 806 IHC positive, 1 biphasic histology 806 IHC negative). In one epithelioid PDX model, ABBV-321 resulted in significantly reduced tumor growth on day 27 post therapy with median tumor volumes of 180 mm3 (ADC control) compared with 78mm3 (ABBV-321; p = 0.0159 two-sided). Moreover, the median survival was also significantly longer in ABBV-321 treated models (p = 0.018). In the other epithelioid PDX model, ABBV-321 also resulted in significant responses (median 428mm3 (ADC control) vs 167mm3 (ABBV-321, p = 0.0201). In the 806 IHC negative PDX model, the differences in tumor volumes between all groups were found to be non-statistically significant (ADC control vs ABT-414, ADC control vs ABBV-221, ADC-control vs ABBV-321 groups) with p = 0.0597 for one-way ANOVA. MSTO211H cell line xenograft model also demonstrated significant anti-tumor response to both ABT-414 and ABBV-221 (p &lt; 0.01). Conclusions: In a disease with limited therapies, ABT-806 targeting ADCs in MM demonstrated significant responses in 806+ PDX and cell lines. These data support clinical expansion of these compounds in 806+ MM patients.

Targeting the IGF1R/PI3K/AKT Pathway Sensitizes Ewing Sarcoma to BET Bromodomain Inhibitors.

Inhibitors of the bromodomain and extra-terminal domain (BET) family proteins modulate EWS-FLI1 activities in Ewing sarcoma. However, the efficacy of BET inhibitors as a monotherapy was moderate and transient in preclinical models. The objective of this study was to identify the mechanisms mediating intrinsic resistance to BET inhibitors and develop more effective combination treatments for Ewing sarcoma. Using a panel of Ewing sarcoma cell lines and patient-derived xenograft lines (PDX), we demonstrated that IGF1R inhibitors synergistically increased sensitivities to BET inhibitors and induced potent apoptosis when combined with BET inhibitors. Constitutively activated AKT significantly protected Ewing sarcoma cells against BET inhibitors, suggesting that IGF1R regulates responsiveness to BET inhibitors mainly through the PI3K/AKT pathway. Although two Ewing sarcoma cell lines were resistant to IGF1R inhibitors, they retained synergistic response to a combination of BET inhibitors and mTOR inhibitors, suggesting that BET proteins, when IGF1R is not functional, cross-talk with its downstream molecules. Furthermore, the combination of a BET inhibitor and an IGF1R inhibitor induced potent and durable response in xenograft tumors, whereas either agent alone was less effective. Taken together, our results suggest that IGF1R and the downstream PI3K/AKT/mTOR kinase cascade mediate intrinsic resistance to BET inhibitors in Ewing sarcoma. These results provide the proof-of-concept for combining BET inhibitors with agents targeting the IGF1R pathway for treating advanced Ewing sarcoma.