Orthotopic Engraftment Research Articles

Abstract 6945: Dissecting the interplay of cell identity and extrachromosomal DNA in glioblastoma

Abstract The five-year survival rate of glioblastoma multiforme (GBM) is 5.8%, and its standard of care has not changed since 2005. This stagnation in therapeutic development is due, in part, to its characteristic heterogeneity–multiforme–highlighting a critical need to understand the biological processes underlying this GBM hallmark. Despite the well-documented individual relationships between extrachromosomal DNA (ecDNA), GBM, and heterogeneity, the interplay between the three is not well understood. The Furnari lab has generated a library of cancer avatars, or human induced pluripotent stem cells (hiPSCs) engineered to harbor cancer driving mutations. Single cell RNA sequencing (scRNA-seq) was used to characterize the longitudinal evolution of the first avatars engineered to have TP53−/−;PDGFRAΔ8-9 and PTEN−/−;NF1−/− genotypes. The TP53−/−;PDGFRAΔ8-9 avatar acquired ecDNA, while the PTEN−/−; NF1−/− avatar did not. Re-analysis of the scRNA-seq data identified a subpopulation of cells in the TP53−/−;PDGFRAΔ8-9 avatar that had high expression of CDK4 and PDGFRA, two genes that are commonly co-amplified, found on ecDNA, and drive cellular states. This subpopulation of cells also had highly expressed genes near the CDK4 locus indicating a structural variant and possibly ecDNA. This population diverged transcriptionally from other cells and was associated with the OPC-like cellular state, which has been shown to be associated with high levels of PDGFRA and, at times, co-expression with CDK4. While TP53−/−;PDGFRAΔ8-9 tumors contained cells across all four GBM cellular states, PTEN−/−;NF1−/− tumors had a significantly less diverse composition of cellular states suggesting that ecDNA may facilitate cellular state diversity. With the eventual goal to directly test whether ecDNA enables transcriptional state heterogeneity compared to chromosomally inherited amplifications, CRISPR-Cas9 and CRISPR-C were implemented to engineer EGFRvIII ecDNA in U87. While inverse PCR confirmed the successful generation of ecDNA in U87, ecDNA was lost following long term in vitro cell passaging. Current follow up work aims to identify different intrinsic (oncogene amplified, genetic background, cell type, cell line) and extrinsic (in vitro conditions, orthotopic engraftment) contexts that promote the selection and amplification of engineered ecDNA, as it would be would be valuable to have genotypically identical cancer models with different classes of amplifications (i.e., engineered ecDNA vs. lentiviral overexpression, which is chromosomally inherited). Together, these results highlight the potential to use hiPSC-derived models and single cell methods to investigate gliomagenesis and cell identity. Citation Format: Brett Taylor, Brandon Jones, Daisuke Kawauchi, Yohei Miyake, Raghav Vadla, Nathan Jameson, Shunichiro Miki, Tomoyuki Koga, Bing Ren, Frank Furnari. Dissecting the interplay of cell identity and extrachromosomal DNA in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6945.

Generation of Orthotopic and Subcutaneous Patient-Derived Xenograft Models from Diverse Clinical Tissue Samples of Pediatric Extracranial Solid Tumors.

Realistic and renewable laboratory models that accurately reflect the distinct clinical features of childhood cancers have enormous potential to speed research progress. These models help us to understand disease biology, develop new research methods, advance new therapies to clinical trial, and implement personalized medicine. This chapter describes methods to generate patient-derived xenograft models of neuroblastoma and rhabdomyosarcoma, two tumor types for which children with high-risk disease have abysmal survival outcomes and survivors have lifelong-debilitating effects from treatment. Further, this protocol addresses model development from diverse clinical tumor tissue samples, subcutaneous and orthotopic engraftment, and approaches to avoid model loss.

P02.09.A THE PLASTICITY AND SURVIVAL OF GLIOBLASTOMA STEM CELLS DEPEND ON GD3, WHOSE INHIBITION IS SYNERGISTIC WITH RADIO-CHEMOTHERAPY

Abstract BACKGROUND Glioblastoma is the most frequent and aggressive primary brain tumor in adults. No curative treatment is available, and the identification of relevant therapeutic targets is an unmet medical need. Our team previously demonstrated that A2B5 positive cells have a stemness behavior, with self-renewal, multipotency ability, and tumor initiation ability in vivo. A2B5 is recognizing different gangliosides, including GT3 and its precursor GD3. However, A2B5 is an IgM, that cannot be used directly as a therapeutic strategy.Then, our aim was to demonstrate that GD3 was a relevant and actionable target for glioblastoma stem cells (GSC). MATERIAL AND METHODS We used the GCS lines we already derived from fresh patient samples. Then, we sorted GD3 positive cells from patient samples to derive new GCS lines and explore GD3 properties. We downregulated the GD3 synthase (ST8Sia1) by using Sh. The tumor cells were orthotopically engrafted in nude mice. RESULTS First, we showed that GD3 was expressed by 15 to 20% of cells from patient tumor bulks but not by normal brain. Then, we showed that 60% to 90% of GSC expressed GD3. Importantly, this fraction dropped between 20 to 40% after cell differentiation. We sorted GD3 positive cells from patient samples and showed their ability to exhibit stemness properties including the generation of neurospheres (expressing stem cell markers like SOX2 and nestin), clonogenicity, and tumorigenicity after orthotopic engraftment. We also demonstrated that GD3+ cells had plasticity properties with differentiation and dedifferentiation abilities depending on their culture medium. Then, by using shST8SIA1 in GSC lines, we showed that the reduction of ST8Sia1/GD3 was associated with a decreasing size of spheres, and reduced clonogenicity and migratory capacities. Moreover, the reduction of ST8Sia1/GD3 expression increased the temozolomide and radiotherapy sensitivity in vitro with a synergistic effect. Finally, the reduction of ST8Sia1/GD3 expression in U87 cell line was associated with an increasing mouse survival after orthotopic engraftment in nude mice. CONCLUSION Taken together, our results strongly suggest that GD3 and ST8SIA1 are essential for GSC properties. GD3 appears to be an interesting and actionable stem cell marker, opening promising therapeutic development for GBM patients.

IMMU-17. DEVELOPMENT OF AN INTRACRANIAL CANNULA IMPLANTATION SYSTEM FOR SERIAL LOCOREGIONAL CAR T CELL INFUSIONS IN MICE

Abstract Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children. As many tumors lack efficacious treatments, there is a crucial need to develop more promising therapeutic options, such as immunotherapies. Of particular interest is the use of chimeric antigen receptor (CAR) T cell therapy. Current clinical trials are ongoing targeting the cell surface molecules HER2, EGFR, B7-H3, IL13RA2, and the disialoganglioside GD2. While the majority of trials deliver CAR T cells into the cerebral spinal fluid with repeated doses, this has not been completed in preclinical models due to difficulty performing multiple stereotactic surgeries. To evaluate repeated locoregional delivery of CAR T cells in preclinical murine models, we established an indwelling catheter system that recapitulates indwelling catheters currently being used in human clinical trials. Following orthotopic injection and engraftment of tumor cells in mice, intratumoral placement of a fixed guide cannula is completed on a stereotactic apparatus and secured with screws and acrylic resin. Treatment cannulas are then inserted through the fixed guide cannula for repeated CAR T cell delivery. With this system, we tested novel CAR T cells directed against glypican 2 (GPC2), resulting in significant tumor regression in medulloblastoma model 7316-4509 (p<0.01), as well as significantly prolonged survival in thalamic diffuse midline glioma 7316-3058 (p<0.05). Stereotactic placement of the guide cannula can be adjusted to deliver CAR T cells directly into the lateral ventricle or other locations in the brain. This platform offers a reliable mechanism for preclinical testing of repeated intracranial infusions of CAR T cells and other novel therapeutics, offering a superior preclinical model for translation to clinical trials.

Neuropilin-1 Knockout and Rescue Confirms Its Role to Promote Metastasis in MDA-MB-231 Breast Cancer Cells.

Breast cancer (BC) metastasis remains a leading cause of female mortality. Neuropilin-1 (NRP-1) is a glycoprotein receptor that plays ligand-dependent roles in BC. Clinical studies indicate its correlation with metastatic disease; however, its functional role in BC metastasis remains uncertain. CRISPR-Cas9 was used to knockout the NRP-1 gene in MDA-MB-231 BC cells, and the effects on metastasis were determined using an orthotopic mouse engraftment model. NRP-1 expression in knockout cells was rescued using a recombinant cDNA with a silent mutation in the sgRNA target-adjacent PAM sequence. Differentially expressed genes between NRP-1 knockout and control cells were determined using whole-transcriptome sequencing and validated using real-time PCR. NRP-1KO cells showed a pronounced reduction in the metastasis to the lungs. KEGG pathway analysis of the transcriptome data revealed that PI3K and ECM receptor interactions were among the top altered pathways in the NRP-1KO cells. In addition, reduction in metastasis enhancers proteins, Integrin-β3 and Tenascin-C, and genes CCL20 and FN1 and upregulation of metastasis suppressor genes, ACVRL and GPX3 in NRP-1KO were detected. These findings provide evidence for a functional role for NRP-1 in BC metastasis, supporting further exploration of NRP-1 and the identified genes as targets in treating metastatic BC.

The Blockade of Mitogen-Activated Protein Kinase 14 Activation by Marine Natural Product Crassolide Triggers ICD in Tumor Cells and Stimulates Anti-Tumor Immunity

Immunogenic cell death (ICD) refers to a type of cell death that stimulates immune responses. It is characterized by the surface exposure of damage-associated molecular patterns (DAMPs), which can facilitate the uptake of antigens by dendritic cells (DCs) and stimulate DC activation, resulting in T cell immunity. The activation of immune responses through ICD has been proposed as a promising approach for cancer immunotherapy. The marine natural product crassolide, a cembranolide isolated from the Formosan soft coral Lobophytum michaelae, has been shown to have cytotoxic effects on cancer cells. In this study, we investigated the effects of crassolide on the induction of ICD, the expression of immune checkpoint molecules and cell adhesion molecules, as well as tumor growth in a murine 4T1 mammary carcinoma model. Immunofluorescence staining for DAMP ectolocalization, Western blotting for protein expression and Z'-LYTE kinase assay for kinase activity were performed. The results showed that crassolide significantly increased ICD and slightly decreased the expression level of CD24 on the surface of murine mammary carcinoma cells. An orthotopic tumor engraftment of 4T1 carcinoma cells indicated that crassolide-treated tumor cell lysates stimulate anti-tumor immunity against tumor growth. Crassolide was also found to be a blocker of mitogen-activated protein kinase 14 activation. This study highlights the immunotherapeutic effects of crassolide on the activation of anticancer immune responses and suggests the potential clinical use of crassolide as a novel treatment for breast cancer.

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice.

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy and radiotherapy. As many tumors lack efficacious treatments, there is a crucial need to develop more promising therapeutic options, such as immunotherapies; the use of chimeric antigen receptor (CAR) T cell therapydirected against CNS tumors is of particular interest. Cell surface targets such as B7-H3, IL13RA2, and the disialoganglioside GD2 are highly expressed on the surface of several pediatric and adult CNS tumors,raising the opportunity to use CAR T cell therapy against these and other surface targets. To evaluate the repeated locoregional delivery of CAR T cells in preclinical murine models, an indwelling catheter system that recapitulates indwelling catheters currently being used in human clinical trials was established. Unlike stereotactic delivery, the indwelling catheter system allows for repeated dosing without the use of multiple surgeries. This protocol describes the intratumoral placement of a fixed guide cannula that has been used to successfully test serial CAR T cell infusions in orthotopic murine models of pediatric brain tumors. Following orthotopic injection and engraftment of the tumor cells in mice, intratumoral placement of a fixed guide cannula is completed on a stereotactic apparatus and secured with screws and acrylic resin. Treatment cannulas are then inserted through the fixed guide cannula for repeated CAR T cell delivery. Stereotactic placement of the guide cannula can be adjusted to deliver CAR T cells directly into the lateral ventricle or other locations in the brain. This platform offers a reliable mechanism for the preclinical testing of repeated intracranial infusions of CAR T cells and other novel therapeutics for these devastating pediatric tumors.

Modeling Therapy-Driven Evolution of Glioblastoma with Patient-Derived Xenografts.

Adult-type diffusely infiltrating gliomas, of which glioblastoma is the most common and aggressive, almost always recur after treatment and are fatal. Improved understanding of therapy-driven tumor evolution and acquired therapy resistance in gliomas is essential for improving patient outcomes, yet the majority of the models currently used in preclinical research are of therapy-naïve tumors. Here, we describe the development of therapy-resistant IDH-wildtype glioblastoma patient-derived xenografts (PDX) through orthotopic engraftment of therapy naïve PDX in athymic nude mice, and repeated in vivo exposure to the therapeutic modalities most often used in treating glioblastoma patients: radiotherapy and temozolomide chemotherapy. Post-temozolomide PDX became enriched for C>T transition mutations, acquired inactivating mutations in DNA mismatch repair genes (especially MSH6), and developed hypermutation. Such post-temozolomide PDX were resistant to additional temozolomide (median survival decrease from 80 days in parental PDX to 42 days in a temozolomide-resistant derivative). However, temozolomide-resistant PDX were sensitive to lomustine (also known as CCNU), a nitrosourea which induces tumor cell apoptosis by a different mechanism than temozolomide. These PDX models mimic changes observed in recurrent GBM in patients, including critical features of therapy-driven tumor evolution. These models can therefore serve as valuable tools for improving our understanding and treatment of recurrent glioma.

A cell-laden hydrogel as prophylactic vaccine and anti-PD-L1 amplifier against autologous tumors.

Immune checkpoint blockade (ICB) can elicit anti-cancer response against tumors growing at normal organs while sparing adjacent tissues. However, many orthotopic tumors respond poorly to ICB therapy due to the lack of pre-existing immune effector cells. Here, we describe a vaccine strategy that induces protective immunity and benefits ICB therapy. An injectable hydrogel platform that forms scaffold subcutaneously was applied to deliver autologous cancer cells undergoing oncolysis (ACCO) as immunogenic antigen source and toll-like receptor 9 agonists (CpG) as additional adjuvant. When administered as a prophylactic, the hydrogel-based vaccine, denoted as (ACCO+CpG)@Gel, successfully built a durable and tumor antigen-specific immune memory against subsequent challenges with orthotopic engraftment of autologous tumors including melanoma, colon carcinoma, and lung carcinoma. Although the vaccination did not completely prevent tumor occurrence, tumors orthotopically established in vaccinated mice acquired significant enhancement in tumor-infiltrating CD8+ T cells and intratumoral PD-L1 expression, which ameliorated the immune status and rendered the originally irresponsive tumors responsible to anti-PD-L1 therapy. Further treatment with PD-L1 blockade therapy efficiently delayed the tumor growth and prolonged the survival of these orthotopic cancer models. Thus, without the need for precisely delivering immunoactivatory agents to tumor or locally remodeling tumor microenvironment, "priming" intractable or inaccessible tumors for subsequent ICB therapy could be achieved by prophylactic vaccination with (ACCO+CpG)@Gel. These findings highlighted (ACCO+CpG)@Gel as a generalized framework of protective vaccine strategy that could be broadly applicable to potentiate ICB therapy against multiple types of orthotopic tumors growing in different regions.

An Orthotopic Patient-Derived Xenograft (PDX) Model Allows the Analysis of Metastasis-Associated Features in Colorectal Cancer.

Metastasis is the primary cause of death in patients with colorectal cancer (CRC), urging the need for preclinical models that recapitulate the metastatic process at the individual patient level. We used an orthotopic patient-derived xenograft (PDX) obtained through the direct implantation of freshly dissociated CRC cells in the colon of immunocompromised mice to model the metastatic process. Ortho-PDX engraftment was associated to a specific set of molecular features of the parental tumor, such as epithelial-to-mesenchymal transition (EMT), TGF-β pathway activation, increased expression of stemness-associated factors and higher numbers of circulating tumor cells (CTCs) clusters expressing the metastatic marker CD44v6. A parallel analysis of orthotopic/metastatic xenografts and organoids showed that tumor cells underwent mesenchymal-to-epithelial transition at the metastatic site and that metastasis-derived organoids had increased chemotherapy resistance. These observations support the usefulness of ortho-PDX as a preclinical model to study metastasis-related features and provide preliminary evidence that EMT/stemness properties of primary colorectal tumors may be crucial for orthotopic tumor engraftment.

Abstract 264: Neutrophil elastase (NE) inhibition enhances metastasis free survival by altering regulation of tumor intrinsic cytoskeletal and cellular adhesion pathways in murine breast cancer models

Abstract Background: Neutrophil elastase (NE), a serine protease exclusively secreted in neutrophils, is a crucial mediator of chronic inflammation and tumor progression. Studies from our group reveal that NE may be a prognostic marker of metastasis. In a cohort of 192 breast cancer patients (58% ER/PR+ve, 20%-HER-2 +ve and 22% TNBC), our results show that higher infiltration of NE-positive Tumor Associated Neutrophils (TANs), is associated with a decrease in recurrence free survival (hazard ratio=3.4, 95% CI, 1.1-5.5), regardless of breast cancer subtype Further, we observe that the genetic deletion of Elane (encoding NE) inhibits lung metastasis in in vivo models of breast cancer. Yet the precise mechanism(s) by which NE promotes tumorigenesis and metastasis of breast cancer remains to be elucidated. To address this gap in knowledge, we have identified the tumor-intrinsic and -extrinsic mechanisms by which NE promotes metastasis. Methods: The role of NE in breast cancer metastasis was assessed using Elane+/+ and Elane-/- mice in FVB/NJ genetic background, bearing PyMT tumors (orthotopic and spontaneous) respectively. These mice develop lung metastasis in 80-90% of the tumor-bearing mice within 1-3 months of primary tumor initiation. To assess metastasis-free survival (MFS), mice received orthotopic engraftment of PyMT tumors, followed by resection and subsequent monitoring for metastasis. The efficacy of the NE inhibitor AZD9668 was assessed in Elane+/+ mouse models by treating the mice with 100mg/kg B.I.D treatments. Preliminary results: Genetic ablation of NE (Elane-/-) in the PyMT models, reduced lung metastasis by ~90% (Lung metastatic index =21.8 vs. 2.7, respectively; p=0.0044). Survival studies in FVB Elane-/- showed a MFS benefit of 345 days compared to controls, which succumbed to metastasis-related death in 46 days post primary tumor resection. RNA-sequencing analysis of PyMT- Elane+/+ and Elane-/- tumors showed differential regulation of tumor intrinsic actin cytoskeleton and integrin signaling pathways between the two genetic backgrounds. These NE-regulated pathways are critical for cell-to-cell contact and tissue integrity, explaining the delay in metastasis in the Elane-/- mice. 100mg/kg daily treatment of AZD9668 reduced lung metastasis in PyMT mice by 94% compared to vehicle-treated mice (0.49+/- 0.21% vs. 0.03+/-0.01%; p=0.05) and significantly reduced instances of primary tumor recurrence. Conclusions: Collectively, our studies suggest that genetic and pharmacological ablation of NE reduces metastasis and extends MFS in in vivo models of breast cancer. Our preclinical studies presented here are likely to provide the much-needed rationale for the use of this class of NE inhibitors as a viable treatment strategy for the metastatic breast cancer. Citation Format: Amriti R. Lulla, Said Akli, Lucas D. Warma, Natalie W. Fowlkes, Kelly K. Hunt, Xiayu Rao, Jing Wang, Stephanie S. Watowich, Khandan Keyomarsi. Neutrophil elastase (NE) inhibition enhances metastasis free survival by altering regulation of tumor intrinsic cytoskeletal and cellular adhesion pathways in murine breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 264.

Ortho- and Ectopic Zebrafish Xeno-Engraftment of Ocular Melanoma to Recapitulate Primary Tumor and Experimental Metastasis Development.

There are currently no animal models for metastatic ocular melanoma. The lack of metastatic disease models has greatly hampered the research and development of novel strategies for the treatment of metastatic ocular melanoma. In this protocol we delineate a quick and efficient way to generate embryonic zebrafish models for both the primary and disseminated stage of ocular melanoma, using retro-orbital orthotopic and intravascular ectopic cell engraftment, respectively. Combining these two different engraftment strategies we can recapitulate the etiology of cancer in its totality, progressing from primary, localized tumor growth under the eye to a peri-vascular metastasis formation in the tail. These models allow us to quickly and easily modify the cancer cells prior to implantation with specific labeling, genetic or chemical interference; and to treat the engrafted hosts with (small molecular) inhibitors to attenuate tumor development. Here, we describe the generation and quantification of both orthotopic and ectopic engraftment of ocular melanomas (conjunctival and uveal melanoma) using fluorescently labelled stable cell lines. This protocol is also applicable for engraftment of primary cells derived from patient biopsy and patient/PDX derived material (manuscript in preparation). Within hours post engraftment cell migration and proliferation can be visualized and quantified. Both tumor foci are readily available for imaging with both epifluorescence microscopy and confocal microscopy. Using these models, we can confirm or refute the activity of either chemical or genetic inhibition strategies within as little as 8 days after the onset of the experiment, allowing not only highly efficient screening on stable cell lines, but also enables patient directed screening for precision medicine approaches.

Ortho- and Ectopic Zebrafish Xeno-Engraftment of Ocular Melanoma to Recapitulate Primary Tumor and Experimental Metastasis Development

There are currently no animal models for metastatic ocular melanoma. The lack of metastatic disease models has greatly hampered the research and development of novel strategies for the treatment of metastatic ocular melanoma. In this protocol we delineate a quick and efficient way to generate embryonic zebrafish models for both the primary and disseminated stage of ocular melanoma, using retro-orbital orthotopic and intravascular ectopic cell engraftment, respectively. Combining these two different engraftment strategies we can recapitulate the etiology of cancer in its totality, progressing from primary, localized tumor growth under the eye to a peri-vascular metastasis formation in the tail. These models allow us to quickly and easily modify the cancer cells prior to implantation with specific labeling, genetic or chemical interference; and to treat the engrafted hosts with (small molecular) inhibitors to attenuate tumor development. Here, we describe the generation and quantification of both orthotopic and ectopic engraftment of ocular melanomas (conjunctival and uveal melanoma) using fluorescently labelled stable cell lines. This protocol is also applicable for engraftment of primary cells derived from patient biopsy and patient/PDX derived material (manuscript in preparation). Within hours post engraftment cell migration and proliferation can be visualized and quantified. Both tumor foci are readily available for imaging with both epifluorescence microscopy and confocal microscopy. Using these models, we can confirm or refute the activity of either chemical or genetic inhibition strategies within as little as 8 days after the onset of the experiment, allowing not only highly efficient screening on stable cell lines, but also enables patient directed screening for precision medicine approaches.

Abstract 3008: Characterization of a panel of orthotopic bioluminescent patient-derived models for drug discovery

Abstract Background: Cancer is a complicated disease featuring geographic, genetic and immunological diversity. The tumor microenvironment (TME) is an essential part of tumor development, influencing tumor growth, metastasis and also therapeutic response. Patient-derived xenografts (PDX) and PDX-derived organoids (PDXO) are widely-used preclinically for drug evaluation with the prominent advantage of largely maintaining the histo- and molecular- pathology of the original tumor. Over decades of studies using PDX models, we found limitations in monitoring the orthotopic and metastatic pattern of cancer. With the development of gene engineering technologies (particularly for luciferase labeling), we now present the transduction of patient-derived models with firefly luciferase, to enable real-time monitoring of tumor burden in mice following orthotopic engraftment of either PDX tumor cells or PDXO in vivo. Methods: A panel of PDX cell slurry or PDXO cultures were transduced with lentiviral vectors carrying both the firefly luciferase gene as well as GFP. Mixed pools or GFP-sorted cells were used for in vivo validation. Bioluminescent PDX or PDXO were orthotopically and subcutaneously engrafted into immunodeficient mice. Real-time tumor growth and metastasis were monitored using the IVIS® Spectrum In Vivo Imaging System. Results: We have established a series of luciferase-labeled PDXO from different cancer types, including liver, ovarian, pancreatic and lung cancer and characterized their growth following orthotopic implantation. We have also established a series of luciferase-labeled PDX models from non-epithelial origin, including brain tumors, osteosarcoma and lymphomas. All of the models were confirmed with GFP signaling in vitro and showed significant luciferase signaling and growth in vivo. A mixed pool of bioluminescent PDXs or PDXOs presented heterogenous luciferase signals in tumors, while single clones or cell sorting by GFP or selected passage in vivo improved the consistency of the bioluminescent signal. Conclusions: PDX models are widely used as preclinical tools for clinical predictivity, however their application in orthotopic/metastatic modeling has been challenging due to the limitation in labelling PDXs for real-time monitoring. With the development of gene engineering technology in vitro and bioluminescent imaging (BLI) in vivo, we have developed and characterized a large panel of bioluminescent PDX and PDXO models from a range of different cancer types for real-time monitoring of orthotopic engrafted tumors in an organ-specific location to represent the TME, and the potential metastatic spread. Citation Format: Jessie Jingjing Wang, Jun Zhou, Ling Chen, Rongyun Zhou, Xiaoxi Xu, Henry Q. X. Li, Davy Xuesong Ouyang. Characterization of a panel of orthotopic bioluminescent patient-derived models for drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3008.

Design and Synthesis of Cathepsin-K-Activated Osteoadsorptive Fluorogenic Sentinel (OFS) Probes for Detecting Early Osteoclastic Bone Resorption in a Multiple Myeloma Mouse Model.

We describe the design and synthesis of OFS-1, an Osteoadsorptive Fluorogenic Sentinel imaging probe that is adsorbed by hydroxyapatite (HAp) and bone mineral surfaces, where it generates an external fluorescent signal in response to osteoclast-secreted cathepsin K (Ctsk). The probe consists of a bone-anchoring bisphosphonate moiety connected to a Förster resonance energy transfer (FRET) internally quenched fluorescent (IQF) dye pair, linked by a Ctsk peptide substrate, GHPGGPQG. Key structural features contributing to the effectiveness of OFS-1 were defined by structure-activity relationship (SAR) and modeling studies comparing OFS-1 with two cognates, OFS-2 and OFS-3. In solution or when preadsorbed on HAp, OFS-1 exhibited strong fluorescence when exposed to Ctsk (2.5-20 nM). Time-lapse photomicrographs obtained after seeding human osteoclasts onto HAp-coated well plates containing preadsorbed OFS-1 revealed bright fluorescence at the periphery of resorbing cells. OFS-1 administered systemically detected early osteolysis colocalized with orthotopic engraftment of RPMI-8226-Luc human multiple myeloma cells at a metastatic skeletal site in a humanized mouse model. OFS-1 is thus a promising new imaging tool for detecting abnormal bone resorption.

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis.

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant transgenic mouse models of ovarian cancer exist, these mice are expensive and take a long time to develop tumors. Intraperitoneal injection xenograft models lack human stroma and do not accurately model ovarian cancer metastasis. Even patient derived xenografts (PDX) do not fully recapitulate the human stromal microenvironment as serial PDX passages demonstrate significant loss of human stroma. The ability to easily model human ovarian cancer within a physiologically relevant stromal microenvironment is an unmet need. Here, the protocol presents an orthotopic ovarian cancer mouse model using human ovarian cancer cells combined with patient-derived carcinoma-associated mesenchymal stem cells (CA-MSCs). CA-MSCs are stromal progenitor cells, which drive the formation of the stromal microenvironment and support ovarian cancer growth and metastasis. This model develops early and diffuses metastasis mimicking clinical presentation. In this model, luciferase expressing ovarian cancer cells are mixed in a 1:1 ratio with CA-MSCs and injected into the ovarian bursa of NSG mice. Tumor growth and metastasis are followed serially over time using bioluminescence imaging. The resulting tumors grow aggressively and form abdominal metastases by 14 days post injection. Mice experienced significant decreases in body weight as a marker of systemic illness and increased disease burden. By day 30 post injection, mice met endpoint criteria of >10% body weight loss and necropsy confirmed intra-abdominal metastasis in 100% of mice and 60%-80% lung and parenchymal liver metastasis. Collectively, orthotopic engraftment of ovarian cancer cells and stroma cells generates tumors that closely mimic the early and diffuse metastatic behavior of human ovarian cancer. Furthermore, this model provides a tool to study the role of ovarian cancer cell: stroma cell interactions in metastatic progression.

Abstract PS17-04: Immune checkpoint blockade reprograms tumor microenvironment and systemic immune landscape in obesity associated triple negative breast cancer

Abstract Few targeted therapies exist for triple negative breast cancer (TNBC), an aggressive and deadly subtype. Obesity exacerbates poor outcomes in TNBC due to elevated invasion and metastasis leading to increased mortality. Obesity has paradoxically been shown to improve immune checkpoint therapies in other cancers, yet the underlying mechanisms are unclear. Despite being an inflammatory state, obesity increases immune checkpoint ligands PD-1 and PD-L1 which drives immunosuppression. Thus, we tested the hypothesis that obesity-driven changes to the immune milieu will improve ICB efficacy in TNBC. Syngeneic tumors were generated via orthotopic engraftment of E0771 basal-like TNBC cell line into age-matched immune-competent C57BL/6J female littermates on obesogenic or low fat diets. Mice were treated with anti-mouse anti-PD-1 or isotype control (IgG2a) every 3rd day until sacrifice. Obese mice gained almost 2-fold great body weight and 3.6-fold greater adiposity compared to lean mice and immunotherapy did not impact weights or body composition. Obesity led to immunosuppression systemically in bone marrow and spleen in tumor-free mice, which was exacerbated in tumor-bearing mice. Obese mice had significantly greater tumor progression and fewer regressed tumors at endpoint vs. lean mice. Anti-PD-1 significantly reduced tumor progression in obese mice with 4.2-fold reduction in volume and 5.7-fold reduction in tumor weights in obese mice vs. isotype controls. Anti PD-1 significantly reduced immunosuppressive cells including M2-like tumor associated macrophages and monocytic and granulocytic myeloid derived suppressor cells (MDSC) and raised anti-tumor M1-like macrophages, cytotoxic CD8+ T cells, and dendritic cells. Last, the microbiome has potent effects on responses to anti-tumor therapies such as chemotherapy and immune checkpoint blockade. Obesity is a major regulator of the gut microbiome. We found that beneficial bacteria belonging to genus Bifidobacterium was higher in lean compared to obese mice, which could limit tumor progression and lead to greater regression through robust anti-tumor immunity. In obese mice, beneficial bacteria belonging to genus Ruminococcus, Adlercreutzia, Corpococcus that promote anti-tumor immunity increased with anti PD-1 immunotherapy. In sum, we show for the first time that obesity-induced systemic and microenvironmental immunosuppression augmented tumor incidence and tumor progression. Furthermore, anti-PD-1 immune checkpoint blockade successfully reduced tumor progression in obese mice through reprogramming not only the TME but systemic immune milieu as well. Immunosuppressive targets unique to the obese TME could be targeted in concert with checkpoint inhibitors in future interventions to enhance durable anti-tumor immunity. Citation Format: Ajeeth K Pingili, Mehdi Chaib, Laura M Sipe, Emily J Miller, Bin Teng, Rahul Sharma, Qusai AI Abdallah, Deidra Daria, Radhika Sekhri, Heejoon Jo, Mims S Tahliyah, D. Neil Hayes, Joseph F Pierre, Liza Makowski. Immune checkpoint blockade reprograms tumor microenvironment and systemic immune landscape in obesity associated triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-04.

TMOD-07. HUMAN DIFFUSE MIDLINE GLIOMA AVATARS AS A PLATFORM TO SEARCH FOR NOVEL THERAPEUTIC TARGETS

Abstract Diffuse midline glioma is the leading cause of brain tumor death among the pediatric population. Drugs that show notable promise in preclinical models inevitably fail to demonstrate efficacy in clinical trials, likely due to the inadequacy of preclinical models. We have recently proposed glioblastoma models derived from human induced pluripotent stem cells (hiPSCs) genetically engineered with different combinations of glioblastoma-associated genetic alterations as a platform to search for therapeutic targets. These glioblastoma avatars authentically recapitulated the different pathobiology of glioblastoma subtypes, depending on what genetic alterations to be introduced. To investigate the biology and to develop novel therapeutics for diffuse midline glioma with H3K27M mutation, we have established a novel model by introducing H3.3 K27M mutation together with one of the most common concurrent genetic alterations, TP53 R248Q mutation, into hiPSCs through CRISPR/Cas9 genome engineering. Orthotopic engraftment of the neural progenitor cells derived from these edited hiPSCs formed diffusely invasive brainstem tumors with histological features of the diffuse midline glioma. These tumor avatars presented a global reduction in H3K27me3 accompanied by the expression of H3K27M. Transcriptome analyses of these models revealed that these avatars with H3K27M cluster apart from the pediatric glioma samples without this particular mutation, and that they present signatures of oligodendroglial progenitor differentiation as discovered in patient samples with this mutation. Using these models faithfully recapitulating histology and pathobiology of the patient tumors, we have performed drug screening and confirmed that their sensitivity to known drugs, including an EZH2 inhibitor and histone deacetylase inhibitors. On these faithful human avatars of diffuse midline glioma with H3K27M, we have applied bioinformatics algorithms of drug sensitivity prediction aiming at developing novel therapeutics for this devastating pediatric glioma.

Establishment and characterization of a cell line and patient-derived xenograft (PDX) from peritoneal metastasis of low-grade serous ovarian carcinoma

Peritoneal spread indicates poor prognosis in patients with serous ovarian carcinoma (SOC) and is generally treated by surgical cytoreduction and chemotherapy. Novel treatment options are urgently needed to improve patient outcome. Clinically relevant cell lines and patient-derived xenograft (PDX) models are of critical importance to therapeutic regimen evaluation. Here, a PDX model was established, by orthotopic engraftment after subperitoneal tumor slurry injection of low-grade SOC, resulting in an early-stage transplantable peritoneal metastasis (PM)-PDX model. Histology confirmed the micropapillary and cribriform growth pattern with intraluminal tumor budding and positivity for PAX8 and WT1. PM-PDX dissociated cells show an epithelial morphotype with a 42 h doubling time and 40% colony forming efficiency, they are low sensitive to platinum derivatives and highly sensitive to paclitaxel (IC50: 6.3 ± 2.2 nM, mean ± SEM). The patient primary tumor, PM, PM-PDX and derived cell line all show a KRAS c.35 G > T (p.(Gly12Val)) mutation and show sensitivity to the MEK inhibitor trametinib in vitro (IC50: 7.2 ± 0.5 nM, mean ± SEM) and in the PM mouse model. These preclinical models closely reflecting patient tumors are useful to further elucidate LGSOC disease progression, therapy response and resistance mechanisms.

Longitudinal assessment of tumor development using cancer avatars derived from genetically engineered pluripotent stem cells

Many cellular models aimed at elucidating cancer biology do not recapitulate pathobiology including tumor heterogeneity, an inherent feature of cancer that underlies treatment resistance. Here we introduce a cancer modeling paradigm using genetically engineered human pluripotent stem cells (hiPSCs) that captures authentic cancer pathobiology. Orthotopic engraftment of the neural progenitor cells derived from hiPSCs that have been genome-edited to contain tumor-associated genetic driver mutations revealed by The Cancer Genome Atlas project for glioblastoma (GBM) results in formation of high-grade gliomas. Similar to patient-derived GBM, these models harbor inter-tumor heterogeneity resembling different GBM molecular subtypes, intra-tumor heterogeneity, and extrachromosomal DNA amplification. Re-engraftment of these primary tumor neurospheres generates secondary tumors with features characteristic of patient samples and present mutation-dependent patterns of tumor evolution. These cancer avatar models provide a platform for comprehensive longitudinal assessment of human tumor development as governed by molecular subtype mutations and lineage-restricted differentiation.