Patient-derived Xenograft Tissue Research Articles

Development and validation of an efficacy assessment platform utilizing patient-derived xenograft (PDX) models in ovarian cancer research.

e17564 Background: Ovarian cancer predominantly undergoes treatment through surgical and chemotherapeutic interventions. However, with an increasing rate of recurrence, there's a need for systemic chemotherapy. A paradigm shift towards personalized treatment is crucial with concerns escalating about chemotherapy resistance post PARP inhibitor treatment. This paper emphasizes the significance of preclinical drug evaluation platforms using Patient-Derived Xenograft (PDX) models, which resonate with the genuine characteristics of ovarian cancer tissues. Methods: The 112 epithelial ovarian patients' tumors sectioned to approximately 3 mm2 were subcutaneously transplanted into at least two female 5-week-old BALB/c nude mice. The xenograft tumors engrafted into mice from patient-derived tumors were defined as P1. After that, xenograft tumors serially engrafted into the following mice were termed P2 to P3. We collected and banked small pieces of tumor tissue from each passage of PDX tissue for performing Whole Exome Sequencing (WES) sequencing and RNA Sequencing. Results: We established 54 epithelial ovarian cancer PDX models that are stably growing. They show a ratio of 70.37% Serous, 11.11% clear cell, and 3.7% endometriod. The stage is 14.81% I/II, 61.11% III/IV, and 24.07% Recurrent. BRCA status consists of 59.26% wild type, 9.26% BRCA 1 mutation, 5.56% BRCA 2 mutation, and 11.11% BRAC VUS. We have obtained a variety of ovarian cancer PDX models and confirmed retained the histological characteristics of patients by H&E staining. We validated the similarity of genomic information in patients and PDX models via WES and RNA sequencing. Conclusions: We have a stable and growing PDX model that accurately reflects the actual tissue characteristics of ovarian cancer. In the future, we plan to characterize drug response further and build new models resistant to existing anti-cancer drugs. Based on these, we will validate the efficacy of new drugs and suggest strategies to overcome cancer resistance.

Abstract 4250: Adherent and spheroid cell models of patient-derived xenograft for drug development and translational research

Abstract Introduction: The development of new drugs in cancer therapy comprises toxicity and efficacy tests with increasing complexity. First and foremost, in vitro experiments are performed with well-established cancer cell lines, which are subsequently validated in animal experiments as prerequisite for clinical trials. Until a few years ago, there were gaps in the complexity chain between in vitro and in vivo experiments. Ethical considerations have led to use of systems like organ on a chip or mini-organ 3D in vitro models. In cancer research, cell cultures or organoids are generated from patient tumor material. However, these cultures only reflect the tumor of one patient, which is why panels of patient tumors should be used to evaluate the effectiveness of drugs on different patients. In vivo panel screens of patient derived xenografts (PDX) mouse models need high numbers of animals and takes several months. A pre-screen with in vitro models generated from in vivo PDX tissues allows large scale and faster pre-screens complementing in vivo systems for more focused in vivo analyses. Methods: Currently we have a pool of more than 600 established PDX models of 21 tumor entities. From this pool, cancer tissues of glioblastoma, mesothelioma, gastric, head/neck, lung and breast cancer were processed in single cell suspensions and cultured under defined conditions to obtain adherent cells or spheroids. The generated PDX in vitro cultures were analyzed for cellular impurities, cancer stem cell content and perpetuation of in vivo PDX characteristics. FACS analyses for tumor specific markers, chemo sensitivity assays and growth characteristics of the PDX derived cell lines (especially for glioblastoma) were analyzed. Results: From PDX tissues used, 90% grew as adherent and/or spheroid PDX derived in vitro cultures, in which mouse cells were entirely depleted. A high percentage of these cultures showed enriched cancer stem cell features and stem cell marker expression. Tumor marker expression and standard drug sensitivity data correlate to the in vivo PDX and derived in vitro cell culture models. RNAseq data were used to predict drug sensitivities in silico for untested drugs and drug combinations on our newly established PDX derived glioblastoma cell lines. Initial screens with predicted candidates were performed. Promising conditions were successfully repeated in corresponding animal PDX models. Conclusion: The newly developed technology for establishment if in vitro cell cultures from PDX efficiently generates stably growing cell lines possessing all key features of the original PDX. These cell lines can be used for initial pre-screens to optimize and improve selection of pharmacologically active drugs or drug combinations before initiating in vivo PDX studies. Citation Format: Lars Winkler, Joshua Alcaniz, Maria Stecklum, Wolfgang Walther, Jens Hoffmann. Adherent and spheroid cell models of patient-derived xenograft for drug development and translational research [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 4250.

Abstract 5006: Discovery of immunogenic neo-peptides from actionable RNA fusions for developing cancer vaccines

Abstract The global surge in cancer cases, with a staggering 20 million new instances reported in 2023 alone, underscores an urgent demand for advanced and reliable cancer therapeutics. Immunotherapies centered around neoantigens have emerged as a promising avenue for enhancing treatment efficacy and have become a focal point in cancer research. We hypothesize that the tumor cells present a rich neoantigenic repertoire and the immunogenic neoantigens arising from the junctions of proteins translated from chimeric RNAs present viable targets for peptide and mRNA vaccines, offering a promising approach to impede tumor progression. To validate our hypothesis, we acquired a dataset of RNA fusions detected through RNAseq of tissue extracted from patient-derived xenograft (PDX) models established by XenoSTART, a global non clinical oncology contract research organization. This comprehensive dataset encompassed 985 PDX models across 27 cancer types, revealing three fusions associated with the neuregulin-1 (NRG1) gene. NRG1 gene fusions have been identified as a clinically actionable target for multiple solid tumors as they result in ErbB-mediated pathway activation. CD74-NRG1 has emerged as the most frequently reported NRG1 fusion across many cancers such as pancreatic ductal adenocarcinoma, triple negative breast cancer and ovarian cancer. The CD74-NRG1 fusion was identified in two PDX models harboring lung cancer (ST2891 and ST3204) and in an ovarian cancer model (ST088). The lung cancer PDXs displayed a notable abundance of fusion junction-crossing reads. We generated the CD74 (Exon 1-6) -NRG1 (Exon 6-12) fusion transcript model, where the first 6 exons of CD74 fused to exon 6 of NRG1 leading to an in-frame fusion. Validation of this fusion in PDX tissue was accomplished through PCR and Sanger sequencing of the PCR product. We assessed the affinity of the 9-mer neo peptide sequences formed by the translated junction of CD74-NRG1 to Major Histocompatibility Complex (MHC) Class I molecules using the in-silico prediction pipelines MHCNuggets and MixMHCPred-2 and found HLA-A*68:23 to be the MHC allele with highest binding affinity. HLA-Arena was used to investigate the binding affinity between the peptides and the MHC molecules via molecular docking, which revealed 4 peptides to be strong binders to HLA-A*68:23.The immunogenicity of these neoantigenic peptides to HLA-A*68:23 matched Peripheral Blood Mononuclear Cells (PBMCs) will be assessed by the IFN-γ Enzyme Linked Immunosorbent Spot (ELISpot) assay. To elucidate the specific T-cell clonotypes responding to the neo peptides, the 10X Genomics single-cell 5’ Gene Expression Assay, coupled with T Cell Receptor mapping, will be employed. The objective of the study is to establish a robust combinatorial therapeutic strategy leveraging peptide and mRNA vaccine technology to support an effective framework for personalized cancer treatment. Citation Format: Sakuni Rankothgedera, Micah Castillo, Shiyanth Thevasagayampillai, Cole Woody, Aaranyah Kandasamy, Armando Diaz, Michael Wick, Preethi Gunaratne. Discovery of immunogenic neo-peptides from actionable RNA fusions for developing cancer vaccines [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 5006.

Abstract 5136: Development of a highly specific NAPRT monoclonal antibody for the detection of tumors sensitive to pharmacological inhibition of NAMPT

Abstract Introduction: A key component of targeting synthetic lethality in cancer is identifying specific biomarkers, such as sources of NAD+ synthesis, that differentiate between healthy and diseased tissue. In mammals, NAD+ can be synthesized from nicotinamide via nicotinamide phosphoribosyltransferase (NAMPT) or from nicotinic acid (niacin, NA) via nicotinic acid phosphoribosyltransferase (NAPRT). NAPRT loss has been identified across tumor indications as a synthetic lethal sensitizer to small molecule inhibitors of NAMPT. Given the reliance of cancer cells on NAD+ synthesis, blocking NAD+ production is a compelling target for anti-cancer therapeutics. However, identifying tumors with NAPRT loss has been a challenge as NAPRT can be expressed by as many as 14 transcripts, only some of which code for the active enzyme. This complicates transcript-based analyses and necessitates proteomic assessment. Here, we have developed a mouse monoclonal antibody (4A5D7) to specifically detect active NAPRT and to identify indications enriched for NAPRT loss in patients through an IHC-based assay. We further confirm that loss of NAPRT makes cells susceptible to NAMPT inhibition, and ultimately show that our antibody can identify patient-derived xenograft (PDX) models that respond to NAMPT inhibition. Methods We utilized a hybridoma system to produce antibodies against epitopes in the NAPRT active site; clones were subsequently screened for their ability to selectively recognize active NAPRT. After identifying clone 4A5D7, we validated our proprietary antibody against FFPE specimens across multiple cancer indications (TNBC, NSCLC, SCLC, CRC, gastric, ovarian, and prostate). Stained samples were scored by a pathologist and assessed for percent of positive tumor cells, nuclear and/or cytoplasmic staining, and staining intensity. CCLE and TCGA datasets were mined for indications enriched for suspected NAPRT-loss and sensitivity to NAMPT inhibitors. We then selected cell lines and pan-tumor PDX tissue microarrays (TMAs) from indications of interest and characterized samples by western blot and IHC to determine NAPRT status. Cell lines and PDX tumor tissue were exposed to increasing ranges of NAMPTi in the presence or absence of NAPRT-substrate NA to validate our approach. Results: We validated that our proprietary NAPRT monoclonal antibody clone 4A5D7 only detects functional NAPRT and demonstrated improved performance over previous antibodies. We used our antibody to characterize cell lines and PDX tumor samples, and subsequently show that NAPRT-null cell lines and tumor samples are susceptible to NAMPT inhibition, while samples with functional NAPRT can be rescued by NA. Conclusion: Our proprietary antibody can identify NAPRT null tumors and identify samples that will respond to NAMPT inhibition, a critical advancement for patient selection in clinical trials. Citation Format: Josh Spurrier, Emmanuel S. Burgos, Mark R. Lundquist, Keiichiro Tanaka, Samuel Brook, Matt Gozzi, Jae-Sung Yi, Joseph Edmonds, Mitch Raponi. Development of a highly specific NAPRT monoclonal antibody for the detection of tumors sensitive to pharmacological inhibition of NAMPT [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 5136.

Transcriptomic, Proteomic, and Genomic Mutational Fraction Differences Based on HPV Status Observed in Patient-Derived Xenograft Models of Penile Squamous Cell Carcinoma.

Metastatic penile squamous cell carcinoma (PSCC) has only a 50% response rate to first-line combination chemotherapies and there are currently no targeted-therapy approaches. Therefore, we have an urgent need in advanced-PSCC treatment to find novel therapies. Approximately half of all PSCC cases are positive for high-risk human papillomavirus (HR-HPV). Our objective was to generate HPV-positive (HPV+) and HPV-negative (HPV-) patient-derived xenograft (PDX) models and to determine the biological differences between HPV+ and HPV- disease. We generated four HPV+ and three HPV- PSCC PDX animal models by directly implanting resected patient tumor tissue into immunocompromised mice. PDX tumor tissue was found to be similar to patient tumor tissue (donor tissue) by histology and short tandem repeat fingerprinting. DNA mutations were mostly preserved in PDX tissues and similar APOBEC (apolipoprotein B mRNA editing catalytic polypeptide) mutational fractions in donor tissue and PDX tissues were noted. A higher APOBEC mutational fraction was found in HPV+ versus HPV- PDX tissues (p = 0.044), and significant transcriptomic and proteomic expression differences based on HPV status included p16 (CDKN2A), RRM2, and CDC25C. These models will allow for the direct testing of targeted therapies in PSCC and determine their response in correlation to HPV status.

Lipid-Based Self-Microemulsion of Niclosamide Achieved Enhanced Oral Delivery and Anti-Tumor Efficacy in Orthotopic Patient-Derived Xenograft of Hepatocellular Carcinoma in Mice.

We previously identified niclosamide as a promising repurposed drug candidate for hepatocellular carcinoma (HCC) treatment. However, it is poorly water soluble, limiting its tissue bioavailability and clinical application. To overcome these challenges, we developed an orally bioavailable self-microemulsifying drug delivery system encapsulating niclosamide (Nic-SMEDDS). Nic-SMEDDS was synthesized and characterized for its physicochemical properties, in vivo pharmacokinetics and absorption mechanisms, and in vivo therapeutic efficacy in an orthotopic patient-derived xenograft (PDX)-HCC mouse model. Niclosamide ethanolamine salt (NEN), with superior water solubility, was used as a positive control. Nic-SMEDDS (5.6% drug load) displayed favorable physicochemical properties and drug release profiles in vitro. In vivo, Nic-SMEDDS displayed prolonged retention time and plasma release profile compared to niclosamide or NEN. Oral administration of Nic-SMEDDS to non-tumor bearing mice improved niclosamide bioavailability and Cmax by 4.1- and 1.8-fold, respectively, compared to oral niclosamide. Cycloheximide pre-treatment blocked niclosamide absorption from orally administered Nic-SMEDDS, suggesting that its absorption was facilitated through the chylomicron pathway. Nic-SMEDDS (100 mg/kg, bid) showed greater anti-tumor efficacy compared to NEN (200 mg/kg, qd); this correlated with higher levels (p < 0.01) of niclosamide, increased caspase-3, and decreased Ki-67 in the harvested PDX tissues when Nic-SMEDDS was given. Biochemical analysis at the treatment end-point indicated that Nic-SMEDDS elevated lipid levels in treated mice. We successfully developed an orally bioavailable formulation of niclosamide, which significantly enhanced oral bioavailability and anti-tumor efficacy in an HCC PDX mouse model. Our data support its clinical translation for the treatment of solid tumors.

DSBSO-Based XL-MS Analysis of Breast Cancer PDX Tissues to Delineate Protein Interaction Network in Clinical Samples.

Protein-protein interactions (PPIs) are fundamental to understanding biological systems as protein complexes are the active molecular modules critical for carrying out cellular functions. Dysfunctional PPIs have been associated with various diseases including cancer. Systems-wide PPI analysis not only sheds light on pathological mechanisms, but also represents a paradigm in identifying potential therapeutic targets. In recent years, cross-linking mass spectrometry (XL-MS) has emerged as a powerful tool for defining endogenous PPIs of cellular networks. While proteome-wide studies have been performed in cell lysates, intact cells and tissues, applications of XL-MS in clinical samples have not been reported. In this study, we adopted a DSBSO-based in vivo XL-MS platform to map interaction landscapes from two breast cancer patient-derived xenograft (PDX) models. As a result, we have generated a PDX interaction network comprising 2,557 human proteins and identified interactions unique to breast cancer subtypes. Interestingly, most of the observed differences in PPIs correlated well with protein abundance changes determined by TMT-based proteome quantitation. Collectively, this work has demonstrated the feasibility of XL-MS analysis in clinical samples, and established an analytical workflow for tissue cross-linking that can be generalized for mapping PPIs from patient samples in the future to dissect disease-relevant cellular networks.

Molecular characterization of metastatic progression in patient derived xenograft models of malignant uveal melanoma

Aims/Purpose: It has been observed that large UM lesions with significant infiltration of macrophages and CD8+ T cells exhibit specific genetic profiles that increase the risk of liver metastasis. Additionally, recent discoveries of DNA methylation changes in Class 2 tumours undergoing metastasis suggest a potential role of epigenetic alterations in UM progression.Methods: To identify factors involved in UM metastasis, we implanted 25 viable UM tumours (consisting of 11 Class 1 tumours with a good prognosis and 14 Class 2 tumours with a poor prognosis) subcutaneously into immunodeficient NSG mice. From these, we successfully established ten patient‐derived xenografts (PDXs) using Class 2 tumour tissues. Two metastatic PDXs were derived from liver (UM50) and peritoneal (UM58) metastases spontaneously developed on mice.Results: Comprehensive characterization of both primary tumours and PDX tissues was performed using bulk transcriptomics and methylomics techniques. Among the enriched metabolic pathways identified in the liver metastasis‐derived PDX tissue of sample UM50 compared to the PDX tissue from the primary tumour, four were related to epigenetic regulation, including the activation of rRNA expression by ERCC6 and EHMT2, methylation of histones and DNA by Polycomb Repressive Complex 2 (PRC2), regulation of rRNA expression by NAD‐dependent Deacetylase Sirtuin‐1, and the DNA methylation pathway.Conclusions: These findings highlight the potential importance of epigenetic deregulation in UM progression and provide a foundation for future investigations. Further analysis using the Chromium Single Cell ATAC platform to examine chromatin accessibility at the single‐cell level in primary and metastatic PDX tissues of UM50 and UM58 that are ongoing can contribute to unravelling the precise role of epigenetic alterations in UM metastasis. Continued research in this area may lead to the development of novel therapeutic strategies targeting epigenetic mechanisms and improving outcomes for patients with metastatic UM.

Assessing Protein Expression in Patient-Derived Xenografts Using Western Blotting.

The use of patient-derived xenografts (PDXs) in cancer research is increasing due to their ability to closely mimic the features of patient tumors. The ability to quickly and robustly measure protein expression levels in these tissues is a key methodology required in a broad range of experimental designs. Western blotting (WB) is a cost effective and simple tool that is highly specific and sensitive for detecting and quantifying individual proteins, posttranslational modifications and aberrant signaling pathways. Here, we described a method to assess protein expression in PDX tissues using WB to detect proteins involved in cell growth signaling pathways.

Anti-LGALS3BP antibody-drug conjugate treatment induces durable and potent antitumor response in a preclinical model of adenoid cystic carcinoma

ObjectivesAdenoid cystic carcinoma (ACC) is a rare type of cancer that typically arises from glandular tissues, most commonly in the salivary glands. Although relatively rare, it represents a serious clinical issue as the management of the disease is highly complex being the only therapeutic options represented by invasive surgery and/or radiotherapy. In the present study, we have explored the potential of galectin-3 binding protein (LGALS3BP) as a novel target for antibody-drug conjugate (ADC) therapy in ACC. Materials and methodsRNAseq was conducted on a panel of 10 ACC patient-derived xenografts (PDX)s tissues and 6 normal salivary glands to analyze LGALS3BP gene expression. Protein expression was assessed in ACC PDX and primary tumor tissues using immunohistochemistry. Anti-LGALS3BP ADC named 1959-sss/DM4, was tested in high LGALS3BP expressing ACC PDX model ST1502B. ResultsRNAseq analysis revealed that LGALS3BP expression was highly expressed in ACC PDX tissues compared to normal salivary gland tissues. As evaluated by immunohistochemical analysis, LGALS3BP protein was found to be heterogeneously expressed in 10 ACC PDX and in tumor tissues derived from a cohort of 37 ACC patients. Further, treatment with 1959-sss/DM4 ADC led to durable tumor growth inhibition (TGI) in 100% of animals without observed toxicity. ConclusionsOur study provides strong evidence that LGALS3BP is a promising therapeutic target for ACC, warranting further expedited preclinical and clinical investigation.

Establishment of a high-fidelity patient-derived xenograft model for cervical cancer enables the evaluation of patient’s response to conventional and novel therapies

BackgroundRecurrent or metastatic cervical cancer (r/m CC) often has poor prognosis owing to its limited treatment options. The development of novel therapeutic strategies has been hindered by the lack of preclinical models that accurately reflect the biological and genomic heterogeneity of cervical cancer (CC). Herein, we aimed to establish a large patient-derived xenograft (PDX) biobank for CC, evaluate the consistency of the biologic indicators between PDX and primary tumor tissues of patients, and explore its utility for assessing patient’s response to conventional and novel therapies.MethodsSixty-nine fresh CC tumor tissues were implanted directly into immunodeficient mice to establish PDX models. The concordance of the PDX models with their corresponding primary tumors (PTs) was compared based on the clinical pathological features, protein biomarker levels, and genomic features through hematoxylin & eosin staining, immunohistochemistry, and whole exome sequencing, respectively. Moreover, the clinical information of CC patients, RNA transcriptome and immune phenotyping of primary tumors were integrated to identify the potential parameters that could affect the success of xenograft engraftment. Subsequently, PDX model was evaluated for its capacity to mirror patient’s response to chemotherapy. Finally, PDX model and PDX-derived organoid (PDXO) were utilized to evaluate the therapeutic efficacy of neratinib and adoptive cell therapy (ACT) combination strategy for CC patients with human epidermal growth factor receptor 2 (HER2) mutation.ResultsWe established a PDX biobank for CC with a success rate of 63.8% (44/69). The primary features of established PDX tumors, including clinicopathological features, the expression levels of protein biomarkers including Ki67, α-smooth muscle actin, and p16, and genomics, were highly consistent with their PTs. Furthermore, xenograft engraftment was likely influenced by the primary tumor size, the presence of follicular helper T cells and the expression of cell adhesion-related genes in primary tumor tissue. The CC derived PDX models were capable of recapitulating the patient’s response to chemotherapy. In a PDX model, a novel therapeutic strategy, the combination of ACT and neratinib, was shown to effectively inhibit the growth of PDX tumors derived from CC patients with HER2-mutation.ConclusionsWe established by far the largest PDX biobank with a high engraftment rate for CC that preserves the histopathological and genetic characteristics of patient’s biopsy samples, recapitulates patient’s response to conventional therapy, and is capable of evaluating the efficacy of novel therapeutic modalities for CC.

Abstract 4567: Transcriptomic analysis of a 3D engineered cancer model recapitulating stage-dependent heterogeneity in colorectal PDX tumors

Abstract Colorectal cancer (CRC) is the third-most leading cause of cancer-related deaths in the United States. To advance the understanding of CRC tumor progression, models which mimic the tumor microenvironment (TME) and have translatable study outcomes are urgently needed. CRC patient-derived xenografts (PDXs) are promising tools for their ability to recapitulate tumor heterogeneity and key patient tumor characteristics, such as molecular characteristics. However, as in vivo models, CRC PDXs are costly and low-throughput, which leads to a need for equivalent in vitro models. To address this need, we previously established an in vitro model using a tissue engineering toolset with CRC PDX cells. However, it is unclear whether tissue engineering has the capacity to maintain patient- and/or cancer stage-specific tumor heterogeneity. To address this gap, we employed three PDX tumor lines, originated from stage II, III-B, and IV CRC tumors, in the formation of 3D engineered CRC PDX (3D-eCRC-PDX) tissues and performed an in-depth comparison between the 3D-eCRC-PDX tissues and the original CRC-PDX tumors. To form the tissues, CRC-PDX tumors were expanded in vivo and dissociated. The isolated cells were encapsulated within poly(ethylene glycol)-fibrinogen hydrogels and remained viable and proliferative post encapsulation over the course of 29 days in culture. To gain molecular insight into the maintenance of PDX line stage heterogeneity, we performed a transcriptomic analysis using RNA seq to determine the extent to which there were similarities and differences between the CRC-PDX tumors and the 3D-eCRC-PDX tissues. We observed the greatest correspondence in overlapping differentially expressed human genes, gene ontology, and Hallmark gene set enrichment between the 3D-eCRC-PDX tissues and CRC-PDX tumors in the stage II PDX line, while the least correspondence was observed in the stage IV PDX line. The Hallmark gene set enrichment from murine mapped RNA seq transcripts was PDX line-specific which suggested that the stromal component of the 3D-eCRC-PDX tissues was maintained in a PDX line-dependent manner. Consistent with our transcriptomic analysis, we observed that tumor cell subpopulations, including human proliferative (B2M+Ki67+) and CK20+ cells, remained constant for up to 15 days in culture even though the number of cells in the 3D-eCRC-PDX tissues from all three CRC stages increased over time. Yet, tumor cell subpopulation differences in the stage IV 3D-eCRC-PDX tissues were observed starting at 22 days in culture. Overall, our results demonstrate a strong correlation between our in vitro 3D-eCRC-PDX models and the originating in vivo CRC-PDX tumors, providing evidence that these engineered tissues may be capable of mimicking patient- and/or cancer stage-specific heterogeneity. Citation Format: Yuan Tian, Iman Hassani, Benjamin Anbiah, Bulbul Ahmed, William Van Der Pol, Elliot J. Lefkowitz, Peyton C. Kuhlers, Nicole L. Habbit, Martin J. Heslin, Elizabeth A. Lipke, Michael W. Greene. Transcriptomic analysis of a 3D engineered cancer model recapitulating stage-dependent heterogeneity in colorectal PDX tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4567.

Synergistic Effect of Lenvatinib and Chemotherapy in Hepatocellular Carcinoma Using Preclinical Models.

The current study aimed to evaluate the synergistic efficacy of lenvatinib and FOLFOX (infusional fluorouracil (FU), folinic acid, and oxaliplatin) in hepatocellular carcinoma (HCC) using patient-derived xenograft (PDX) and PDX-derived organotypic spheroid (XDOTS) models in vivo and in vitro. PDX and matched XDOTS models originating from three patients with HCC were established. All models were divided into four groups and treated with drugs alone or in combination. Tumor growth in the PDX models was measured and recorded, and angiogenesis and phosphorylation of the vascular endothelial growth factor receptor (VEGFR2), rearranged during transfection (RET), and extracellular signal-regulated kinase (ERK) were detected using immunohistochemistry and Western blot assays. The proliferative ability of XDOTS was evaluated through active staining and immunofluorescence staining, and the effect of the combined medication was evaluated using the Celltiter-Glo luminescent cell viability assay. Three PDX models with genetic characteristics similar to those of the original tumors were successfully established. Combining lenvatinib with FOLFOX led to a higher tumor growth inhibition rate than individual therapies (P < 0.01). Immunohistochemical analysis demonstrated that the combined treatment significantly inhibited the proliferation and angiogenesis of PDX tissues (P < 0.05), and Western blot analysis showed that the combined treatment significantly inhibited the phosphorylation of VEGFR2, RET, and ERK compared with single-agent treatment. Additionally, all three matched XDOTS models were successfully cultured with satisfactory activity and proliferation, and the combined therapies led to better suppression of XDOTS growth compared with individual therapy (P < 0.05). Lenvatinib combined with FOLFOX had a synergistic antitumor effect in HCC PDX and XDOTS models by inhibiting the phosphorylation of VEGFR, RET, and ERK.

A PDX model combined with CD-DST assay to evaluate the antitumor properties of KRpep-2d and oxaliplatin in KRAS (G12D) mutant colorectal cancer

Patient-derived xenograft (PDX) models are more faithful in maintaining the characteristics of human tumors than cell lines and are widely used in drug development, although they have some disadvantages, including their relative low success rate, long turn-around time, and high costs. The collagen gel droplet embedded culture drug sensitivity test (CD-DST) has been used as an in-vitro drug sensitivity test for patients with cancer because of its high success rate of primary cell culture, high sensitivity, and good clinical relevance, but it is based on an in-vitro cell culture and may not simulate the tumor microenvironment accurately. This study aims to combine a PDX model with CD-DST to evaluate the efficiency of antitumor agents. KRpep-2d, a small peptide targeting KRAS (G12D), and oxaliplatin were used to verify the feasibility of this approach. Whole-exome sequencing and Sanger sequencing were first applied to test and validate the KRAS mutation status of a panel of colorectal cancer PDX tissues. One PDX model was verified to carry KRAS (G12D) mutation and was used for in-vivo and the CD-DST drug tests. We then established the PDX mouse model from the patient with the KRAS (G12D) mutation and obtained viable cancer cells derived from the same PDX model. Next, the antitumor abilities of KRpep-2d and oxaliplatin were estimated in the PDX model and the CD-DST. We found that KRpep-2d showed no significant antitumor effect on the xenograft model or on cancer cells derived from the same PDX model. In contrast, oxaliplatin showed significant inhibitory effects in both tests. In conclusion, the PDX model in combination with the CD-DST assay is a comprehensive and feasible method of evaluating the antitumor properties of compounds and could be applied for new drug discovery.

RF27 | LBSUN342 Ar-targeted Linked-read Sequencing Reveals Complex Ar Structural Variants In Castration Resistant Prostate Cancer Models

Abstract Androgen deprivation therapies are employed for the treatment of advanced prostate cancer to suppress the transcriptional activity of the androgen receptor (AR). However, over the course of therapy, AR transcriptional activity re-emerges, and the disease progresses to castration resistant prostate cancer (CRPC). During CRPC progression, AR becomes heavily altered by genomic aberrations that include amplification, missense mutations, and structural variants (SVs). AR SVs are heterogeneous across CRPC specimens, with variability noted in SV class, breakpoint locations, and co-occurrence with amplification. Further variability has been observed for sub-clonal enrichment of AR SVs within tumors. A barrier to understanding the clinical significance of AR SVs is the challenge of deciphering this high degree of variability from short-read DNA-sequencing data. To address this challenge, we performed AR-targeted linked-read DNA-sequencing of CRPC cell line and patient-derived xenograft (PDX) models with the goal of resolving sub-clonal and complex AR SVs. We benchmarked this approach using prostate cancer cell lines and LuCaP86.2 PDX tissue harboring known AR SVs. Using this benchmarking approach, we identified linked AR SVs that co-occurred on single DNA molecules in LuCaP35CR and LuCaP105CR PDX models. We also identified a novel AR SV in the LuCaP77 PDX model and a castration-resistant derivative, LuCaP77CR. These results have revealed heterogeneous trajectories of AR SVs in CRPC and define a new class of complex AR SVs where multiple rearrangements co-occur on single DNA molecules to yield highly rearranged AR gene structures. Presentation: Monday, June 13, 2022 1:05 p.m. - 1:10 p.m.

Abstract B006: Development of a NF1-MPNST-PDX liquid biopsy model using whole-genome sequencing and quantitative PCR of mouse-derived cell-free DNA

Abstract Background: Malignant Peripheral Nerve Sheath Tumors (MPNST) are aggressive, NF1-associated soft tissue sarcomas with a dismal 40% 5-year survival rate, high rates of disease relapse and metastasis, and limited treatment options. Our lab has generated a collection of patient-derived xenograft (PDX) models, which recapitulate the variety of genetic changes that occur in human NF1-MPNST. Longitudinal analysis of PDX tumor burden in preclinical studies is currently limited to imprecise tumor measurement or expensive and time-consuming imaging modalities such as MRI. These methods of analysis fail to allow for the early detection of tumor formation, the assessment of treatment-response dynamics, the formation of resistant subclones, or the detection of molecular residual disease. Liquid biopsies using plasma-derived cell-free DNA (cfDNA) have been successful in addressing these challenges in the context of multiple solid tumor types including MPNST by our group previously (Szymanski et al. 2021). However, liquid biopsy of cfDNA in the context murine model systems is quite limited, primarily due the challenge of collecting sufficient plasma for analysis. We hypothesize that we can detect and longitudinally track tumor burden in response to treatment using human-tumor-specific quantitative PCR (qPCR) and whole-genome sequencing of sequentially collected PDX-bearing murine plasma to develop a NF1-MPNST-PDX liquid biopsy model. Methods: To address this challenge, we sequentially collected ≈50 µL of murine plasma weekly from two independent groups of NF1-MPNST-PDX-bearing mice and corresponding PDX-free controls for up to six weeks. The first group consisted of six PDX-bearing mice who underwent whole-genome sequence of murine-derived plasma cfDNA and paired PDX tissue. The second group consisted of fifteen NF1-MPNST-PDX-bearing mice and five PDX-free controls; plasma cfDNA samples collected from this group underwent human-specific LINE-1 qPCR, which should only detect human DNA derived from implanted PDX and allow for longitudinal tracking of tumor burden. Results: We serially collected ≈50 µL of plasma weekly from NF1-MPNST-PDX mice with no obvious impact on mouse survival or weight. Mean total cfDNA yield from the terminally collected samples was 1.12 ± 1.02 ng per uL plasma (n=21) for PDX-bearing animals with cfDNA fragments canonically appearing between 70 and 450 bps as determined by electropherogram. Whole-genome sequencing of murine plasma cfDNA revealed broad genomic aneuploidy detectable in NF1-MPNST-PDX plasma which corresponded to alterations present in PDX tissue, including MPNST-specific chromosome 8q gain, corresponding with our earlier findings (Dehner et al. 2021). Human-specific LINE-1 qPCR showed specific enrichment of tumor-derived LINE-1 in NF1-MPNST-PDX-bearing mice compared to PDX-free mice which showed no human LINE-1 enrichment. Conclusion: Our data suggest that PDX plasma cfDNA may be an informative biomarker that can be non-invasively collected and used to track tumor burden in NF1-MPNST-PDX models. Citation Format: Paul A. Jones, Wenjia Feng, Xiaochun Zhang, Peter K. Harris, Taylor Sundby, Jeffrey J. Szymanski, Divya Srihari, Faridi Qaium, Jack F. Shern, Aadel A. Chaudhuri, Angela C. Hirbe. Development of a NF1-MPNST-PDX liquid biopsy model using whole-genome sequencing and quantitative PCR of mouse-derived cell-free DNA [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B006.

Effect of Antigen Retrieval on Genomic DNA From Immunodissected Samples.

Immunohistochemical (IHC) staining is an established technique for visualizing proteins in tissue sections for research studies and clinical applications. IHC is increasingly used as a targeting strategy for procurement of labeled cells via tissue microdissection, including immunodissection, computer-aided laser dissection (CALD), expression microdissection (xMD), and other techniques. The initial antigen retrieval (AR) process increases epitope availability and improves staining characteristics; however, the procedure can damage DNA. To better understand the effects of AR on DNA quality and quantity in immunodissected samples, both clinical specimens (KRAS gene mutation positive cases) and model system samples (lung cancer patient-derived xenograft tissue) were subjected to commonly employed AR methods (heat induced epitope retrieval [HIER], protease digestion) and the effects on DNA were assessed by Qubit, fragment analysis, quantitative PCR, digital droplet PCR (ddPCR), library preparation, and targeted sequencing. The data showed that HIER resulted in optimal IHC staining characteristics, but induced significant damage to DNA, producing extensive fragmentation and decreased overall yields. However, neither of the AR methods combined with IHC prevented ddPCR amplification of small amplicons and gene mutations were successfully identified from immunodissected clinical samples. The results indicate for the first time that DNA recovered from immunostained slides after standard AR and IHC processing can be successfully employed for genomic mutation analysis via ddPCR and next-generation sequencing (NGS) short-read methods.

Regulation of TORC1 by MAPK Signaling Determines Sensitivity and Acquired Resistance to Trametinib in Pediatric BRAFV600E Brain Tumor Models.

We investigated why three patient-derived xenograft (PDX) childhood BRAFV600E-mutant brain tumor models are highly sensitive to trametinib. Mechanisms of acquired resistance selected in situ, and approaches to prevent resistance were also examined, which may translate to both low-grade glioma (LGG) molecular subtypes. Sensitivity to trametinib [MEK inhibitor (MEKi)] alone or in combination with rapamycin (TORC1 inhibitor), was evaluated in pediatric PDX models. The effect of combined treatment of trametinib with rapamycin on development of trametinib resistance in vivo was examined. PDX tissue and tumor cells from trametinib-resistant xenografts were characterized. In pediatric models TORC1 is activated through ERK-mediated inactivation of the tuberous sclerosis complex (TSC): consequently inhibition of MEK also suppressed TORC1 signaling. Trametinib-induced tumor regression correlated with dual inhibition of MAPK/TORC1 signaling, and decoupling TORC1 regulation from BRAF/MAPK control conferred trametinib resistance. In mice, acquired resistance to trametinib developed within three cycles of therapy in all three PDX models. Resistance to trametinib developed in situ is tumor-cell-intrinsic and the mechanism was tumor line specific. Rapamycin retarded or blocked development of resistance. In these three pediatric BRAF-mutant brain tumors, TORC1 signaling is controlled by the MAPK cascade. Trametinib suppressed both MAPK/TORC1 pathways leading to tumor regression. While low-dose intermittent rapamycin to enhance inhibition of TORC1 only modestly enhanced the antitumor activity of trametinib, it prevented or retarded development of trametinib resistance, suggesting future therapeutic approaches using rapamycin analogs in combination with MEKis that may be therapeutically beneficial in both KIAA1549::BRAF- and BRAFV600E-driven gliomas.

PET imaging of hepatocellular carcinoma by targeting tumor-associated endothelium using [68Ga]Ga-PSMA-617.

Hepatocellular carcinoma (HCC) is a malignant tumor associated with high morbidity and mortality rates. In many non-prostate solid tumors such as HCC, prostate-specific membrane antigens (PSMA) are overexpressed in tumor-associated endothelial cells. Therefore, the aim of this study was to evaluate the performance of [68Ga]Ga-PSMA-617 PET imaging on HCC with different animal models, including cell line-derived xenografts (CDX) and patient-derived xenografts (PDX), and to explore its mechanisms of function. [68Ga]Ga-PSMA-617 was prepared. The expression level of PSMA in two human hepatocellular cancer cells (HepG2 and HuH-7) was evaluated, and the cellular uptakes of [68Ga]Ga-PSMA-617 were assayed. HepG2 and HuH-7 subcutaneous xenograft models, HepG2 orthotopic xenograft models, and four different groups of PDX models were prepared. Preclinical pharmacokinetics and performance of [68Ga]Ga-PSMA-617 were evaluated in different types of HCC xenografts models using small animal PET and biodistribution studies. Low PSMA expression level of HepG2 and HuH-7 cells was observed, and the cellular uptake and blocking study confirmed the non-specificity of the PSMA-targeted probe binding to HepG2 and HuH-7 cells. In the subcutaneous xenograft models, the tumor uptakes at 0.5 h were 0.76 ± 0.12%ID/g (HepG2 tumors) and 0.78 ± 0.08%ID/g (HuH-7 tumors), respectively, which were significantly higher than those of the blocking groups (0.23 ± 0.04%ID/g and 0.20 ± 0.04%ID/g, respectively). In the orthotopic xenograft models, PET images clearly displayed the tumor locations based on the preferential accumulation of [68Ga]Ga-PSMA-617 in tumor tissue versus normal liver tissue, suggesting the possibility of using [68Ga]Ga-PSMA-617 PET imaging to detect primary HCC lesions in deep tissue. In the four different groups of HCC PDX models, PET imaging with [68Ga]Ga-PSMA-617 provided clear tumor uptakes with prominent tumor-to-background contrast, further demonstrating its potential for the clinical imaging of PSMA-positive HCC lesions. The staining of tumor tissue sections with CD31- and PSMA-specific antibodies visualized the tumor-associated blood vessels and PSMA expression on endothelial cells in subcutaneous, orthotopic tissues, and PDX tissues, confirming the imaging with [68Ga]Ga-PSMA-617 might be mediated by targeting tumorassociated endothelium. In this study, in vivo PET on different types of HCC xenograft models illustrated high uptake within tumors, which confirmed that [68Ga]Ga-PSMA-617 PET may be a promising imaging modality for HCC by targeting tumorassociated endothelium.

Abstract 3112: Metabolic hallmarks of rare renal cell carcinoma patient-derived xenograft models

Abstract Renal medullary carcinoma (RMC) and fumarate hydratase (FH)-deficient renal cell carcinoma (RCC) are rare but highly aggressive malignancies that are often refractory to the therapies used for the more common RCCs. FH-deficient RCC most often occurs in individuals with hereditary leiomyomatosis and renal cell carcinoma syndrome (HLRCC) and is characterized by loss of FH activity, a key metabolic enzyme. Bevacizumab plus erlotinib (B+E) is a preferred first-line therapy for FH-deficient RCC. RMC is characterized by loss of SMARCB1, which is involved in ATP-dependent chromatin remodeling. Platinum-based chemotherapy consisting of either cisplatin or carboplatin plus paclitaxel is the preferred first-line therapy for RMC. We developed patient-derived xenograft (PDX) models of tumors exposed to the commonly used first-line therapies to allow functional characterization of the metabolic hallmarks of treatment-experienced RMC and FH-deficient RCC. Methods: Resected tumor tissue was immediately implanted into NSG male and female mice. After tumor engraftment, animals were euthanized, and the tumor tissue implanted into another set of NSG mice. PDX tissues were confirmed to be genetically identical to the original patient tissue by short tandem repeat analysis. Histology was confirmed to be identical between original patient tumor and PDX by a trained clinical pathologist. Four tumors from each PDX model and four normal human kidney tissue samples were analyzed using reverse phase protein array (RPPA). Expression of proteins shown to be highly expressed by RPPA were further validated by Western blots. Results: We successfully generated one PDX model for RMC and one for FH-deficient RCC, respectively. The RMC model was generated from a 28-year old male with sickle cell trait, who had previously received cisplatin plus paclitaxel followed carboplatin plus paclitaxel. His lesion was characterized as ypT3apN1pMx and histology was consistent with RMC. The FH-deficient RCC model was generated from a 24-year-old female with HLRCC, who had been previously treated with B+E. Her lesion was characterized as pT3apN1pM1 and histology consistent with FH-deficient RCC. Germline testing revealed a R233H pathogenic mutation in the FH gene. RPPA revealed higher expression of hexokinase II (10x HLRCC, 16x RMC), lactate dehydrogenase A (27x HLRCC, 17x RMC), and glutaminase (4x HLRCC) in PDX tissue compared to normal kidney tissue. Western blots confirmed higher expression of glutaminase 1 in both HLRCC and RMC compared to normal kidney. Furthermore, we observed substantially higher hypoxia inducible factor 2α (HIF2α) protein expression in PDX tissue from FH-deficient RCC compared to RMC PDX tumors and normal kidney. Conclusions: Our study revealed distinct metabolic features in PDX models of FH-deficient RCC following treatment with B+E, and of platinum-experienced RMC, these differences may confer targetable vulnerabilities. Citation Format: Manuel Ozambela, Alberto Pieretti, Graciela M. Nogueras Gonzalez, Tapati Maity, Lei Wang, Carolyn De La Cerda, Breanna Alonzo, Luis Segarra, Angelita Alaniz, Priya Rao, Nizar Tannir, Jose A. Karam, Christopher G. Wood, Pavlos Msaouel, Niki M. Zacharias. Metabolic hallmarks of rare renal cell carcinoma patient-derived xenograft 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 3112.