Established Patient-derived Xenograft Models Research Articles

Abstract A034: Single cell RNA sequencing of triple negative breast cancer patient-derived xenograft model identifies CRABP1 of cancer-associated fibroblast as a key regulator of breast cancer metastasis

Abstract Triple-negative breast cancer (TNBC) is associated with a high risk of distant metastasis, particularly to the lung and liver. Both intrinsic characteristics of cancer cells and the tumor microenvironment (TME) influence TNBC growth and metastasis. The TME, comprising stromal, immune, and endothelial cells, exhibits heterogeneity both between and within cell types. Identifying the molecular characteristics of TME cells that affect cancer progression is crucial. We aim to investigate this using single-cell RNA sequencing of patient-derived xenograft (PDX) models to explore TME cells and genes involved in TNBC metastasis. We established PDX models by transplanting tumor tissues from 26 TNBC patients into immunodeficient mice. These models were categorized based on their metastatic potential and patient outcomes: non-metastatic (n=5) and metastatic (n=4). Single-cell RNA sequencing of nine PDX tumors revealed significant differences in gene expression of murine stromal cells between metastatic and non-metastatic models. We identified ten genes (Serpinb2, Spp1, Tnc, Thbs1, Timp1, Il11, Mt2, Crabp1, Cck, Mt1) that were highly expressed in the stromal cells of metastatic PDX models. To test if TNBC cells influence these fibroblast genes, we exposed NIH3T3 fibroblasts to conditioned media from the 4T1 TNBC cell line. In three independent experiments, only Crabp1 expression in NIH3T3 cells was consistently increased by 4T1- conditioned media. CRABP1, a retinoic acid-binding protein, is known for its role in differentiation and proliferation by regulating MAPK signaling. Although Crabp1-positive cancer-associated fibroblasts have been noted, its role in TNBC metastasis was previously unexplored. We investigated CRABP1 function using a CRABP1 knockdown NIH3T3 cell line. CRABP1 knockdown led to reduced invasion and migration of 4T1 cells in trans-well assays and decreased invasiveness of 4T1 spheroids in a collagen matrix. Additionally, CRABP1 knockdown NIH3T3 cells showed reduced proliferation in vitro and fewer alpha-SMA positive cancer-associated fibroblasts in co-injected 4T1 tumors in BALB/C mice. In summary, CRABP1, identified through single-cell RNA sequencing of stromal cells in TNBC PDX models, is a potential regulator of TNBC metastasis, affecting cancer cell migration, invasion, and fibroblast proliferation. Citation Format: Woohang Heo, Yujeong Her, Sieun Yang, Dakyung Lee, Rokhyun Kim, Jong-Il Kim, Hyeong-Gon Moon. Single cell RNA sequencing of triple negative breast cancer patient-derived xenograft model identifies CRABP1 of cancer-associated fibroblast as a key regulator of breast cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A034.

Abstract 5469: ITCC-P4, a preclinical proof-of-concept drug testing platform as a tool for pharmacological screening in pediatric tumor models

Abstract Cancer remains the main cause of disease-related death in childhood. Pediatric tumors are characterized by a low mutational burden and high intertumoral heterogeneity, with multiple subtypes compared to their adult counterparts. The lack of access to many innovative therapies remains one of the main challenges in the pediatric oncology, especially for the 25% of patients who experience relapses. In this context, the need for the development of a well characterized collection of pediatric models, to provide large scale preclinical testing, is capital for the subsequent identification and prioritization of promising novel therapeutic options. The EU funded “Innovative Therapies for Children with Cancer-Pediatric Preclinical Proof-of-Concept Platform” (ITCC-P4) consortium is a unique public-private collaborative project consisting of academic and industrial partners that aimed at establishing a collection of >400 patient-derived xenograft (PDX) models representing the most common high-risk pediatric cancers. The project involved various aspects of model development including the thorough molecular and pharmacological characterization. XenTech’s participation was focused on the development and preclinical in vivo drug testing of Ewing sarcoma (n=17), hepatoblastoma (n=10), rhabdoid tumors (n=6), synovial sarcoma (n=2), rhabdomyosarcoma (n=2) and other tumors (n=6), as part of overall cohort. PDXs were obtained by transplantation of post-surgery tumor specimens, either by grafting tumor fragments into the interscapular region or subcutaneously in the right flank of nude, NOD-Scid or NOD-Scid gamma mice. Tumor xenografts were amplified by serial transplantation, and tissue samples were retained at early passages for molecular characterization. Fragments from established PDX models where frozen to generate a revivable ITCC-P4 PDX collection. Then, proof-of-concept drug testing was conducted, in a single mouse trial format: each tumor type (n=X PDX models) was treated with a dedicated panel of Standard-of-Care (SoC;n=3) and novel targeted therapies (n=6), or combinations of 2 or 3 novel targeted therapies; for each PDX model n=1 mouse being included per treatment. All molecular and drug-testing data obtained by the different partners are being centralized in the R2 repository (https://r2.amc.nl), providing a powerful tool for data integration, visualization and interpretation of the results. A unique collection of well characterized pediatric PDX models derived from the most relevant pediatric tumor types was enabled by a strong public-private collaborative project. This large cohort is now available for preclinical testing of novel therapeutic agents within a non-for-profit spinoff company, ITCC-P4 gGmbH (www.itccp4.com), offering new perspectives to the identification of promising treatment options for children with cancer. Citation Format: Emilie Indersie, Sophie Branchereau, Brice Fresneau, Christophe Chardot, Didier Surdez, Alexandra Saint-Charles, Maria Eugénia Marques da Costa, Ángel M. Carcaboso, Katia Scotlandi, Massimo Moro, Heinrich Kovar, Jan-Henning Klusmann, Klaus-Michael Debatin, Simon Bomken, Louis Chesler, Chris Jones, Beat Schäfer, Marco Wachtel, Johannes Gojo, Walter Berger, Christina Guttke, Maureen Hattersley, Frédéric Colland, Ashley Strougo, Dennis Gürgen, Jens Hoffmann, Julia Schueler, Pablo M. Aviles, María José Guillén, Aniello Federico, Apurva Gopisetty, Justyna Anna Wierzbinska, Andreas Schlicker, Sara Colombetti, Olaf Heidenreich, Fatima Iradier, Nicole Huebener, Natalie Jäger, Jan Koster, Marcel Kool, Gudrun Schleiermacher, Jan J. Molenaar, Birgit Geoerger, David J. Shields, Hubert N. Caron, Louis F. Stancato, Stefan M. Pfister, Gilles Vassal, Eva-Maria Rief, Olivier Déas. ITCC-P4, a preclinical proof-of-concept drug testing platform as a tool for pharmacological screening in pediatric tumor models [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 5469.

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.

Consistency between Primary Uterine Corpus Malignancies and Their Corresponding Patient-Derived Xenograft Models.

Patient-derived xenograft (PDX) models retain the characteristics of tumors and are useful tools for personalized therapy and translational research. In this study, we aimed to establish PDX models for uterine corpus malignancies (UC-PDX) and analyze their similarities. Tissue fragments obtained from 92 patients with uterine corpus malignancies were transplanted subcutaneously into immunodeficient mice. Histological and immunohistochemical analyses were performed to compare tumors of patients with PDX tumors. DNA and RNA sequencing were performed to validate the genetic profile. Furthermore, the RNA in extracellular vesicles (EVs) extracted from primary and PDX tumors was analyzed. Among the 92 cases, 52 UC-PDX models were established, with a success rate of 56.5%. The success rate depended on tumor histology and staging. The pathological and immunohistochemical features of primary and PDX tumors were similar. DNA sequencing revealed similarities in gene mutations between the primary and PDX tumors. RNA sequencing showed similarities in gene expressions between primary and PDX tumors. Furthermore, the RNA profiles of the EVs obtained from primary and PDX tumors were similar. As UC-PDX retained the pathological and immunohistochemical features and gene profiles of primary tumors, they may provide a platform for developing personalized medicine and translational research.

In Vivo Ablation of NFκB Cascade Effectors Alleviates Disease Burden in Myeloproliferative Neoplasms

ABAL and TK equal contributors Multilevel regulation of NFκB activation exists in response to microenvironmental cues. Aberrant hyperactivation of the NFκB cascade propagates oncogenic signaling and inflammation, which together augment disease burden in myeloproliferative neoplasms (MPNs). Here, we systematically ablated NFκB signaling effectors to identify core dependencies using a series of primary samples and syngeneic and patient-derived xenograft (PDX) mouse models. We first examined the impact of Rela abrogation in the Jak2 V617F knock-in mouse model that exemplifies polycythemia vera hallmarks and generated Jak2V617FRela flox/flox Ubc-Cre-ERT2 ( Jak2- Rela fl) mice. Evident suppression of leukocytosis was observed within a week of Rela excision and sustained until endpoint. Elevated hematocrit, monocytes, and CD45.2 myeloid cells were suppressed in both the peripheral blood (PB) and bone marrow (BM) and Jak2- Rela flmice had significant reduction of splenomegaly. We then adopted a second MPN model, where MPL W515L retroviral expression produces a phenotype resembling myelofibrosis. Consistent reduction of pathologic WBC counts, monocytes, and PB and BM CD45.2 myeloid cells were observed in MPL- Rela fl mice. Rela ablation also reduced hematocrit, platelet counts, and CD45.2 GFP+ cells. Importantly, we observed a significant attenuation of disease hallmarks with a decrease of spleen weight and dysmegakaryopoiesis, enhanced survival, and reduction of BM reticulin deposition. We next established PDX models where CD34+ cells from MF patients and healthy BM donors (NBM) were transduced with an sgRNA targeting RELA and engrafted into NSGS mice. Across two MPN PDXs, there was significant suppression of leukemic cells and improvement of disease burden, reduced spleen weights and prolonged survival, and subsequent BM histology revealing improved sclerosis, fibrosis, and dysmegakaryopoiesis. In contrast, NSGS mice engrafted with NBM CD34+ cells did not yield disease symptoms, nor did RELA targeting in healthy cells affect hematopoiesis or measured disease hallmarks. RNA-seq on CD34+ cells from four MPN patients following RELA abrogation revealed augmented and altered expression of genes that are 1) regulators of survival and proliferative pathways: MDM2, TP53INP1, and FAS 2) major ferroptosis mediator GPX4, and 3) cell cycle effectors: CDKN1A (p21), E2F4, and CHEK2. In further investigation of NFκB pathway effectors, we genetically targeted upstream effector Myd88 and transduced Kit+ cells from Myd88-null mice with MPL W515L. Consistent with Rela-knockout, Myd88-deficiency also diminished pathogenic WBC and monocyte count upsurgence, inhibited the accumulation of BM CD45.2 myeloid cells, and reduced spleen weights. The rapid lethality governed by mutant MPL was also delayed upon Myd88 loss, consistent with reduced pathology in spleen and BM sections. While Myd88 and Rela activate NFκB, miR-146a is repressive. We performed further knock-out of MIR146A using PDX models and transplanted MIR146A-KO CD34+ cells into NSGS mice and observed enhanced leukemic engraftment, increased spleen weights, earlier lethality, and evidence of BM sclerosis or fibrosis across both MF PDXs. Importantly, these disease parameters remained unchanged in a NBM PDX following MIR146A-KO. Lastly, in our WashU Cohort of RNA-sequencing performed on 90 CD34+ samples encompassing the spectrum of MPN and secondary acute myeloid leukemia (AML) with NBM controls, we observed significant upregulation of IRAK4 and IL1B in MF and sAML compared to NBM, and their significant expression correlation in MPN. We then pharmacologically inhibited IRAK4 using a small molecule, CA-4948, which is currently under evaluation in phase 1/2a trials of relapsed/refractory AML/myelodysplastic syndrome. In PDX experiments, CA-4948 treatment effectively reduced leukemic engraftment and spleen weights of two MF models without inducing toxicity in a NBM PDX. Through qRT-PCR and mass cytometry, we demonstrate that CA-4948 treatment dampened inflammatory cytokine production induced by IL-1β in primary MPN CD14+ monocytes. Overall, we demonstrate that targeting mediators including Rela, Myd88, and IRAK4 specifically alleviated MPN disease burden without toxicity to healthy tissue and further establish CA-4948 as a promising therapeutic avenue for the treatment of MPN.

Therapeutic target biomarkers of patient-derived xenograft models of gastric-type cervical adenocarcinoma

BackgroundMost cervical adenocarcinomas are associated with human papillomavirus (HPV). Gastric-type cervical adenocarcinoma (GAS), an HPV-independent adenocarcinoma, shows an aggressive clinical feature, resulting in a poor prognosis. Resistance to chemotherapy poses a difficulty in managing patients with metastatic GAS. We aimed to establish patient-derived xenografts (PDXs) of tumors from two patients with GAS and evaluated protein biomarkers for drug development using immunohistochemistry. MethodsTwo PDXs were established 78 and 48 days after transplanting the patient’s tumor tissues into immunodeficient mice, respectively. PDX and patient’s tumor samples were stained for HER2, HER3, PMS2, MSH6, PanTrk, and ARID1A to evaluate biomarkers for therapeutic targets. In addition, whole exome sequencing and RNA sequencing were performed on available samples. ResultsThe pathological findings in morphological features and immunohistochemical profiles from the established PDXs were similar to those from the patients’ surgical tumor specimens. HER3 was overexpressed in the patient’s tumors, and the corresponding PDX tumors and HER2 was weakly stained in both types of tumor samples. In all PDX and patient tumor samples, PMS2, MSH6, and ARID1A were retained, and PanTrk was not expressed. In addition, a total of 10 samples, including tumor tissue samples from 8 other GAS patients, were evaluated for HER3 expression scores, all of which were 2 + or higher. ConclusionsIn summary, we evaluated biomarkers for therapeutic targets using newly established PDX models of GAS. Frequent HER3 overexpression and HER2 expression in GAS tumors suggest the possibility of new treatments for patients with GAS by targeting HER3 and HER2.

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.

Establishment and validation of preclinical models of SMARCA4-inactivated and ARID1A/ARID1B co-inactivated dedifferentiated endometrial carcinoma

ObjectiveDedifferentiated endometrial cancer (DDEC) is an uncommon and clinically highly aggressive subtype of endometrial cancer characterized by genomic inactivation of SWItch/Sucrose Non-Fermentable (SWI/SNF) complex protein. It responds poorly to conventional systemic treatment and its rapidly progressive clinical course limits the therapeutic windows to trial additional lines of therapies. This underscores a pressing need for biologically accurate preclinical tumor models to accelerate therapeutic development. MethodsDDEC tumor from surgical samples were implanted into immunocompromised mice for patient-derived xenograft (PDX) and cell line development. The histologic, immunophenotypic, genetic and epigenetic features of the patient tumors and the established PDX models were characterized. The SMARCA4-deficienct DDEC model was evaluated for its sensitivity toward a KDM6A/B inhibitor (GSK-J4) that was previously reported to be effective therapy for other SMARCA4-deficient cancer types. ResultsAll three DDEC models exhibited rapid growth in vitro and in vivo, with two PDX models showing spontaneous development of metastases in vivo. The PDX tumors maintained the same undifferentiated histology and immunophenotype, and exhibited identical genomic and methylation profiles as seen in the respective parental tumors, including a mismatch repair (MMR)-deficient DDEC with genomic inactivation of SMARCA4, and two MMR-deficient DDECs with genomic inactivation of both ARID1A and ARID1B. Although the SMARCA4-deficient cell line showed low micromolecular sensitivity to GSK-J4, no significant tumor growth inhibition was observed in the corresponding PDX model. ConclusionsThese established patient tumor-derived models accurately depict DDEC and represent valuable preclinical tools to gain therapeutic insights into this aggressive tumor type.

Establishment and Characterization of Patient-Derived Xenograft Models of Anaplastic Thyroid Carcinoma and Head and Neck Squamous Cell Carcinoma.

Patient-derived xenograft (PDX) models faithfully preserve the histological and genetic characteristics of the primary tumor and maintain its heterogeneity. Pharmacodynamic results based on PDX models are highly correlated with clinical practice. Anaplastic thyroid carcinoma (ATC) is the most malignant subtype of thyroid cancer, with strong invasiveness, poor prognosis, and limited treatment. Although the incidence rate of ATC accounts for only 2%-5% of thyroid cancer, its mortality rate is as high as 15%-50%. Head and neck squamous cell carcinoma (HNSCC) is one of the most common head and neck malignancies, with over 600,000 new cases worldwide each year. Herein, detailed protocols are presented to establish PDX models of ATC and HNSCC. In this work, the key factors influencing the success rate of model construction were analyzed, and the histopathological features were compared between the PDX model and the primary tumor. Furthermore, the clinical relevance of the model was validated by evaluating the in vivo therapeutic efficacy of representative clinically used drugs in the successfully constructed PDX models.

Abstract 3831: Utilizing decellularized triple-negative breast cancer patient-derived tumor models as drug testing platforms

Abstract Triple-negative breast cancer (TNBC) is an aggressive, heterogenous disease that lacks approved targeted therapeutics. Due to the complexity of tumor microenvironment (TME) interactions in TNBC, it is crucial to identify therapeutic targets that regulate the extracellular matrix (ECM). Traditional 2D TNBC models do not accurately mimic the cell-matrix interactions and structural matrix proteins that are key in maintaining the unique TME in individual patient tumors. Here, we introduce our decellularized TNBC patient-derived xenograft (PDX) models to address this gap in knowledge. Utilizing our decellularized models, we can investigate the unique ECM composition and cell-matrix interactions in PDX models from understudied patients with diverse clinical presentations. We can also use our decellularized tumor scaffolds as drug testing models. We utilize our tissue decellularization method on PDX tumor scaffolds derived from two of our established TNBC PDX models (TU-BcX-56S and TU-BcX-4QX). Decellularization of tumor scaffolds was confirmed by DNA quantification and histological analysis (H&E, Masson’s Trichrome, and Movat’s staining). MDA-MB-231 cells expressing GFP and luciferase were seeded onto scaffolds and treated with 100nM of Paclitaxel, a microtubule-stabilizing cytotoxic chemotherapeutic, or DMSO for 48 hours. Concordantly, cells in 2D culture were treated with 100nM of Paclitaxel or DMSO for 48 hours. Using the IVIS imaging system, we measured the bioluminescence of cells on our seeded scaffolds before treatment and at 24 and 48 hours after treatment. Cells in 2D culture showed a significant decrease in bioluminescent signal (p-value < 0.05) in the Paclitaxel group compared to the DMSO group at 48 hours. Cells seeded on TU-BcX-56S scaffold also showed a significant decrease in bioluminescent signal (p-value < 0.05) in the Paclitaxel group compared to the DMSO group at 48 hours. Interestingly, cells seeded on TU-BcX-4QX scaffold showed a decrease in bioluminescence in both the treatment and control groups at 24 and 48 hours, albeit there was no significant difference. Using qPCR, we investigated scaffold regulation of ECM gene composition in our treatment and control groups. These results demonstrate that the unique ECM composition and architecture of these scaffolds are important drivers of cell behavior. Here, we demonstrate that our decellularized PDX tumor scaffold models can be used as drug testing platforms. Furthermore, our models recapitulate key structural matrix proteins in the TNBC TME, can be used to investigate ECM onco-architecture and composition, and can be used in therapeutic discovery research. Citation Format: Maryl Wright, Khoa Nguyen, Elizabeth Martin, Melyssa Bratton, Bridgette Collins-Burow, Matthew Burow. Utilizing decellularized triple-negative breast cancer patient-derived tumor models as drug testing platforms [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 3831.

Abstract 3115: Establishment and characterization of patient-derived breast cancer models for cancer studies

Abstract BACKGROUND: Despite the progress and development of targeted agents and Immunotherapeutics, breast cancer is still the leading cause of death among women worldwide. At present, targeted therapy for breast cancers include endocrine blockage therapy for estrogen receptor positive (ER+) tumors and trastuzumab or lapatinib for HER2 positive (HER2+) tumors and CDK inhibitors for HER negative (HER-) tumors as well as PARP inhibitors for BRCA1/2 mutations. There is still a high unmet need for triple negative breast cancer (TNBC) and relapsed breast cancers with specific mutations. Breast cancer is a highly heterogeneous disease and reliable breast cancer models are needed to develop new clinical therapies and better reflect tumor biology. Patient-derived xenograft (PDX) models have been widely used for understanding tumor characteristics and represent human heterogeneity more faithfully and thus are suitable for mouse clinical studies for predicting drug efficacy in the clinic. Here we report the establishment and characterization of a panel of breast cancer (BC) PDX for preclinical research. METHODS: Tumor samples from patients’ breast tumor or metastastic lesion obtained via surgery, biopsy, or pleural effusions/ascites were engrafted subcutaneously in immunodeficient mice to establish PDX models. The models were classified and characterized by histopathology, immunohistochemistry (IHC), and breast cancer specific biomarkers such as estrogen receptor (ER), progesterone receptor (PR) and HER2. Genomic analysis of these models was performed by next generation sequencing (NGS). In these breast cancer models, the effects of estrogen on tumor growth and targeted endocrine therapy such as Fulvestrant were evaluated. The change of tumor volume over time was used to determine the growth rate and response to treatment in mice. RESULTS: A series of BC PDX models derived from primary patient breast tumor or metastatic lesions were successfully established and characterized. Key characteristics of parental tumors, such as histopathology, clinical markers, gene expression and copy number, as well as estrogen dependence was retained. The IHC data indicated various expression levels of ER, PR and HER2 across the panel, with TNBC being the dominant subtype. ESR1 mutation, which may play an important role in BC progression and endocrine therapy resistance, was also identified in an estrogen-independent model. In vivo, these models showed distinct tumor growth rates and different pharmacodynamic responses to standard of care agents. For example, ESR1 mutant model was responsive to Fulvestrant. CONCLUSIONS: The established BC PDX panel represent a range or ER/PR/HER2 expression status from primary and advanced cancer providing a series of preclinical models for breast cancer research and potential for mouse clinical trial enrollment. Citation Format: Leilei Chen, Xueying Yang, Likun Zhang, Xiaobo Chen, Henry Q.X. Li, Jingjing Wang. Establishment and characterization of patient-derived breast cancer models for cancer studies [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 3115.

The efficiency of anlotinib in osteosarcoma with chemoresistance: Exploratory therapy based on PDX models and next-generation sequencing.

11527 Background: It’s urgent to find a new approach for the treatment of progressed osteosarcoma in neoadjuvant chemotherapy. Using patient-derived xenograft (PDX) models and next-generation sequencing (NGS), we have explored the efficiency of anlotinib in these patients. Methods: Frozen osteosarcoma tissues were used to establish PDX models. Ten samples of each PDX model were randomized to treatment or control group. The treatment group were gavaged at a volume of 0.1 ml/10g body weight for 4 weeks, while the control group were administered with vehicle at the same dose orally. Tumor volume and body weight were measured every 3 days, and the tumour were removed and weighed after 28 days. The number of mitotic cells and tumor necrosis rate were detected by Hematoxylin-Eosin (HE) staining. Immunohistochemistry (IHC) was used to evaluate the number of apoptotic cells and the expression of VEGFR-2, PDGFR-β, FGFR-1, c-Kit and their phosphorylated proteins, CD 31. Additionally, patients who had progressed during neoadjuvant chemotherapy (NAC) were treated with combination of anlotinib. After four cycles NAC, we performed salvage surgery and maintained with anlotinib. The change of tumor size was evaluated for tumor response. We used IHC and NGS to analyze the expression of drug targets. Results: 21 PDX models were established successfully from 43 samples. Tumor specimens from patients, who had pulmonary metastasis, local recurrence or chemoresistance, were easier to establish PDX models (P < 0.001, P = 0.046, P = 0.013). Six models were selected randomly for anlotinib. After anlotinib administration, the tumor inhibition rates were 18.82%, 45.98%, 85.86%, 83.38%, 84.57% and 86.42%. Anlotinib could not only inhibit the mitosis, induce tumor cell apoptosis and necrosis, but also decrease the expression of drug targets. The expression of VEGFR-2, PDGFR-β and CD31 were positively associated with tumor response. Five patients had received anlotinib during NAC. Four patients had tumor regression (69.4%, 28%, 19%, 8.7%), including two with high expression of VEGFR-2 mRNA or/and PDGFR-β and one with medium expression. Conclusions: Based on the results of PDX models and NGS, we suggested that osteosarcoma with high expression of VEGFR-2 or/and PDGFR-β was sensitive to anlotinib, which was alternative for patients progressed during NAC.

Camptothecin Delivery via Tumor-Derived Exosome for Radiosensitization by Cell Cycle Regulation on Patient-Derived Xenograft Mice.

Purpose: While radiotherapy remains the leading clinical treatment for many tumors, its efficacy can be significantly hampered by the insensitivity of cells in the S phase of the cell cycle to such irradiation. Methods: Here, we designed a highly targeted drug delivery platform in which exosomes were loaded with the FDA-approved anti-tumor drug camptothecin (CPT) which is capable of regulating cell cycle. The utilized exosomes were isolated from patient tumors, enabling the personalized treatment of individuals to ensure better therapeutic outcomes. Results: This exosome-mediated delivery strategy was exhibited robust targeted to patient-derived tumor cells in vitro and in established patient-derived xenograft models. By delivering CPT to tumor cells, this nanoplatform was able to decrease cell cycle arrest in the S phase, increasing the frequency of cells in the G1 and G2/M phases such that they were more radiosensitive. Conclusion: This therapeutic approach was able to substantially enhance the sensitivity of patient-derived tumors to ionizing radiation, thereby improving the overall efficacy of radiotherapy without the need for a higher radiation dose.

Identification of Prognostic Markers of Gynecologic Cancers Utilizing Patient-Derived Xenograft Mouse Models.

Simple SummaryAs a preclinical model for personalized cancer therapy, the patient-derived xenograft (PDX) model is suitable because it provides the best representation of the original tumor, but it still has many limitations. We analyzed gynecologic cancer PDX models with their clinical information and gene expression profiling, and found that the success rate of PDX correlated with the patient’s tumor grade and prognosis. Moreover, we showed that the faster the tumor progressed in PDXs, the poorer the prognosis in ovarian cancer patients. We confirmed that the differentially expressed genes (DEGs) selected according to PDX engraftment status could be a prognosis marker of ovarian clear cell cancer patients. This study paves the way for a better application of the PDX in gynecologic cancer.Patient-derived xenografts (PDXs) are important in vivo models for the development of precision medicine. However, challenges exist regarding genetic alterations and relapse after primary treatment. Thus, PDX models are required as a new approach for preclinical and clinical studies. We established PDX models of gynecologic cancers and analyzed their clinical information. We subcutaneously transplanted 207 tumor tissues from patients with gynecologic cancer into nude mice from 2014 to 2019. The successful engraftment rate of ovarian, cervical, and uterine cancer was 47%, 64%, and 56%, respectively. The subsequent passages (P2 and P3) showed higher success and faster growth rates than the first passage (P1). Using gynecologic cancer PDX models, the tumor grade is a common clinical factor affecting PDX establishment. We found that the PDX success rate correlated with the patient’s prognosis, and also that ovarian cancer patients with a poor prognosis had a faster PDX growth rate (p < 0.0001). Next, the gene sets associated with inflammation and immune responses were shown in high-ranking successful PDX engraftment through gene set enrichment analysis and RNA sequencing. Up-regulated genes in successful engraftment were found to correlate with ovarian clear cell cancer patient outcomes via Gene Expression Omnibus dataset analysis.

Establishment of childhood hepatoblastoma xenografts and evaluation of the anti-tumour effects of anlotinib, oxaliplatin and sorafenib.

Hepatoblastoma (HB) is a common primary malignant liver tumour in children, mainly treated by means of traditional chemotherapy using platinum and doxorubicin (ADM). There has been limited progress in the research and development of new drugs for treating HB. A tumour biopsy from a child with HB was implanted into immunodeficient mice. The primary tumour and patient-derived xenograft (PDX) tumour were extensively characterised by histology, immunohistochemistry (IHC), and humanisation identification. We used the PDX model to evaluate the anti-tumour effects of anlotinib oxaliplatin (L-OHP) and sorafenib on childhood HB. The established PDX model maintained the histological characteristics of the primary tumour. Anlotinib, L-OHP, and sorafenib can significantly inhibit the tumour growth in the PDX model. There was no obvious damage of the drugs to the heart, liver and kidney of the mice, and the side effects observed were light. We have successfully established a PDX model of childhood HB. The model retains important molecular characteristics of human primary tumours. Using the model, it was found that anlotinib, L-OHP, and sorafenib have a good inhibitory effect on the growth of childhood HB. This provides a preliminary research basis for the clinical application of the drugs.

Abstract 3020: Human leukocyte antigen (HLA) typing of a broad panel of cancer patient-derived xenograft (PDX) models for immune therapies

Abstract Background In immuno-oncology research, an appropriate microenvironment for both human tumor cells and human immune cells is an important but challenging factor for successful development of relevant preclinical models. Humanized patient-derived xenograft (PDX) models can support establishment of such an environment based on matching human leukocyte antigen (HLA) profiles of PDX and compatible human immune cell populations. In this study, we determined the individual HLA types of a broad panel of 291 established PDX models from 18 different tumor entities. Furthermore, we performed comprehensive HLA matching analyses of all PDX models and 13 peripheral blood mononuclear cell (PBMC) donors to enable personalized studies with HLA-matched PDX. Methods The HLA profile of each PDX was determined comprising HLA class I, II and non-class types in 4-digit resolution. Matching analyses were performed according to donor-recipient HLA matching criteria recommended by the Blood and Marrow Transplant Clinical Trials Network (BMT CTN). For comparative HLA profile analyses, allele frequencies of 8862 German stem cell donors provided by The Allele Frequency Net Database were downloaded. Results The individual HLA profiles of all PDX models were generated comprising HLA-A, -B and -C (class I), HLA-DQA1, -DQB1, -DRB1, -DPA1 and -DPB1 (class II) as well as HLA-H (non-class) types. Comparative analyses of HLA profiles revealed that obtained frequencies of PDX HLA alleles are comparable with frequencies of a representative population of 8862 German stem cell donors. HLA profile matching analyses performed for all PDX models and PBMC donors resulted in 15 matches regarding PDX from head and neck, lung, mammary and pancreatic carcinoma as well as from endometrial cancer, glioma, lymphoma, melanoma and sarcoma. Dependence of immune therapy efficacy on matched HLA profiles was shown exemplarily for a head and neck squamous cell cancer (HNSCC) PDX. The HNSCC PDX was humanized with PBMC from 5 donors representing different degrees of HLA matching (33 - 100 %). Treatment with the checkpoint inhibitor Nivolumab caused a more powerful tumor growth inhibition in matched HLA profiles compared to non-matched profiles. This indicated a relation between antitumoral immune therapy effect and HLA matching in preclinical models. Conclusions We generated a comprehensive HLA profile portfolio containing information of a broad panel of PDX models. Matched HLA profiles of PDX models and PBMC donors enable personalized, preclinical immune-oncology studies to support the development of novel therapeutic strategies. Citation Format: Theresia Conrad, Annika Wulf-Goldenberg, Maria Stecklum, Michael Becker, Konrad Klinghammer, Jens Hoffmann. Human leukocyte antigen (HLA) typing of a broad panel of cancer patient-derived xenograft (PDX) models for immune therapies [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 3020.

Implantation of Patient-Derived Acute Leukemia Cells into the Hematopoietic Niche of Immunocompromised Mice Favors Faithful Recapitulation of the Disseminated Growth of Human Leukemia

The aim of this study was to determine the influence of the engraftment site on the tumor biology and drug sensitivity of a panel of hematological patient derived xenografts (PDX). PDX cells (3x10e6 cells/mouse) were injected intratibially (i.t.), intrasplenically (i.s.) or subcutaneously (s.c.) into NOG (NOD/Shi-scid/IL-2Rγnull) mice. Leukemic cell engraftment was determined by flow cytometry (FC) in bone marrow (BM), peripheral blood (PB) and spleen during the course and at the end of the experiment. Overall survival (OS) served as an additional read-out. In nine models, sensitivity towards cytarabine (Cy) was evaluated across the different implantation sites. Up to now our group has established 26 PDX of acute leukemia (23 AML, one ALL, two APL) covering a broad range of the different genetic subtypes: amongst other molecular alterations models carrying PML-RARA or AML1-ETO fusion proteins are included in the collection. 24/26 lines engrafted when injected i.t., 17/22 developed tumors after i.s. implantation and 16/19 established tumors post s.c. injection of T cell depleted PB or BM of patients. Hence, for the majority of the models the engraftment capacity per se was not dependent on the injection site. Yet, some models could exclusively be propagated in a specific setting: one AML model, grew solely i.t. or i.s., whereas another AML and one APL could be propagated only when injected s.c.. The implantation site did influence tumor growth behavior: Mean OS ranged from 161.3 (±22.3) days for i.t. to 93.1 (±13.1) days for s.c. propagation. I.s. transplanted mice had to be sacrificed after 137.6 (±25.2) days. The differences in mean OS between the three settings were not statically significant (one way ANOVA). Nevertheless, the OS times within single models differed significantly depending on the implantation technique (p ranging from < 0.005 - 0.017, Log-rank (Mantel-Cox) test). The dissemination pattern of individual lines was as well affected by the injection site. In general, infiltration of the investigated hematopoietic organs was significantly higher when cells were engrafted i.t. or i.s as compared to the s.c. approach (p< 0.0001, one way ANOVA). The mean BM (i.t.: 58.1 ± 8.1%; i.s.: 51.3 ± 13.4%; s.c.: < 0.01%), spleen (i.t.: 40.4 ± 8.8%; i.s.: 52 ± 11.5%; s.c.: < 0.01%) and PB (i.t.: 36.6 ± 8.1%; i.s.: 36.4 ± 12.0 %; s.c.: 10.4 ± 7.2 %) infiltration rate across all 26 models was similar in the i.t. and the i.s. setting but considerably higher than in the s.c approach. Significant differences could be detected between individual models depicting characteristic engraftment patterns independent of the injection site (p ranging from < 0.003 - 0.02, one way ANOVA). The expression pattern of the six investigated surface markers (CD45, CD3, CD34, CD33, CD38 & HLA-ABC) was not influenced by the application route. Every model depicted its distinct expression pattern similar to the expression pattern of the donor sample. Of note, the expression pattern was stable across different passages in the murine host. Cy was highly active in five AML and one ALL model: OS was significantly prolonged in the s.c. as well as in the disseminated setting (p ranging from < 0.001 - 0.003, Log-rank (Mantel-Cox) test). Three other AML models depicted a less pronounced sensitivity towards Cy (p ranging from < 0.005 - 0.007, Log-rank (Mantel-Cox) test) both growing s.c. or i.t.. Thus, drug sensitivity was not influenced by the injection site of the leukemic cells. To the best of our knowledge, this is the first time that such a thorough side-by-side comparison was performed in acute leukemia PDX models. Taken together, the application route has a major impact on the tumor biology of the PDX model: i.t. and i.s but not s.c. implantation induced a disseminated growth pattern mimicking consistently the human disease. Up to now, implanting leukemic cells i.t. led to the highest engraftment rate (92%) within the panel of established AML PDX models, indicating the importance of the hematopoietic niche as a supportive tumor microenvironment. Therefore, the establishment of favorable genetic AML subtypes should be preferably done by i.t. implantation. Nevertheless, the consistent expression of surface markers as well as sensitivity towards Cy independent of the implantation site, justifies the use of s.c. implanted AML PDX for screening approaches in an early stage of the drug development process. DisclosuresSchueler:Charles River Research Services Germany GmbH: Employment. Lenhard:Charles River Research Services Germany GmbH: Employment. Klingner:Charles River Research Services Germany GmbH: Employment. Oswald:Charles River Research Services Germany GmbH: Employment.

Establishment and characterization of an ovarian yolk sac tumor patient-derived xenograft model.

The lack of appropriate preclinical models of ovarian yolk sac tumor (OYST) is currently hindering the pursuit of new methods of treatment and investigation of the pathogenesis of the disease. We developed and characterized an OYST patient-derived xenograft (PDX) model in this study. Tumor fragments from a patient with an OYST were implanted subcutaneously into BALB/c Nude mice. Engrafted xenografts were compared with the original tumor according to histology, immunohistochemistry, humanized identified, and drug efficacy testing with in vivo treatment programs. There was a high degree of histologic and immunohistochemical (IHC) resemblance between the established PDX model and its corresponding human tumors. Bleomycin, etoposide, and cisplatin (JEB) chemotherapy regimens were effective in clinical patients and were effective in the OYST PDX model; therefore, the effect of PDX intervention was consistent with clinical outcomes of OYSTs. We have successfully established an OYST PDX model. This OYST model preserves the basic molecular features of the primary human tumor, thereby providing a valuable method to preclinically evaluate new treatments and explore disease pathogenesis.

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis.

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately represent the disease. Patient derived xenograft (PDX) models of CNS metastasis have been shown to better represent the phenotypic and molecular characteristics of the human disease, as well as better reflect the heterogeneity and clonal dynamics of human patient tumors compared to historic cell line models. There are multiple sites that can be used to implant patient-derived tissue when setting up preclinical trials, each with their own advantages and disadvantages, and each suited for studying different aspects of the metastatic cascade. Here, the protocol describes how to establish PDX models and present three different approaches for utilizing CNS metastasis PDX models in pre-clinical studies, discussing each of their applications and limitations. These include flank implantation, orthotopic injection in the brain, and intracardiac injection. Subcutaneous flank implantation is the easiest to monitor and, therefore, most convenient for pre-clinical studies. In addition, metastases to brain and other tissues from flank implantation were observed, indicating that the tumor has undergone multiple steps of metastasis, including intravasation, extravasation, and colonization. Orthotopic injection in the brain is the best option for recapitulating the brain tumor microenvironment and is useful for determining the efficacy of biologics to cross the blood-brain barrier (BBB) but bypasses most steps of the metastatic cascade. Intracardiac injection facilitates metastasis to the brain and is also useful for studying organ tropism. While this method forgoes earlier steps of the metastatic cascade, these cells will still have to survive circulation, extravasate, and colonize. The utility of a PDX model, is therefore, impacted by the route of tumor inoculation and the choice of which one to utilize should be dictated by the scientific question and overall goals of the experiment.

Abstract PR009: Characterization of patient-derived xenograft models of endometrial cancer

Abstract The appropriate models to use for studies of cancer development, progression, drug response and resistance to therapy remain hotly contested. Leading models include cell lines, genetically-modified mice, patient-derived organoids and patient-derived xenografts (PDX). Progress in studies of endometrial cancer is hampered in part by the lack of extensively characterized research models. Our group recently performed next-generation sequencing on the five most commonly-studied endometrial cancer cell lines, Ishikawa H, ECC1, Hec50co, KLE and RL95-2. Our data, which includes histology, mutation screening, MLH1 promoter methylation, copy number variation and microsatellite instability (MSI), conclude that none of the five models cleanly fit any extant post-hoc classification scheme, including the four clusters defined by TCGA: POLE ultra-mutated (cluster 1), MSI hypermutated (cluster 2), copy-number low (endometrioid, cluster 3), and copy-number high (serous-like, cluster 4). We hypothesized that cell lines may not fully represent the true biology of endometrial cancer. Therefore, we have now established PDX models of endometrial cancer in hopes of creating a more representative set of research tools. We generated PDX models by implanting fresh endometrial tumor tissue (hysterectomy specimens) subcutaneously into athymic mice. Of 21 implanted tumors, 8 successfully grafted (take rate=38%). After the initial growth in mice (passage 1 or P1), tumors could be excised, cryopreserved and stored for extended periods in liquid nitrogen. Cryopreserved tumors retain the original phenotype of the P1 engrafted PDX tumor, maintain the ability to proliferate and can be utilized for subsequent larger-scale xenograft studies. Histology is preserved for 5-7 passages, including the architectural pattern of abnormal glands, sheets and occasional papillary configurations. Additionally, some PDX models could be cultured in 2D. We next undertook an extensive morphologic and molecular characterization of three PDX models, denoted EC-PDX1, 2 and 3. EC-PDX1 is serous by histology but contains a POLE mutation (P286R) along with mutations in MSH2, ARID1A and TP63, suggestive of a cluster 1 POLE ultramutated phenotype. Expression of estrogen and progesterone receptors (ER and PR) were low or absent. EC-PDX1 was sensitive to cisplatin in vivo. EC-PDX2 is a high-grade endometrioid model devoid of ER, PR and p53 by immunohistochemistry. Mutational analysis identified a frame shift mutation in exon 8 of p53, which results in a premature stop codon and supports the null staining. These data suggest that EC-PDX2 is most similar to copy-number high cluster 4. EC-PDX3 is low-grade endometrioid with robust expression of ER and PR and wild-type levels of p53. This model harbors activating mutations in PI3KCA (E545K) and KRAS (G13D) and a stop gained mutation in ARID1A. Based on these features, we bin EC-PDX3 as a copy-number low cluster 3. We conclude that PDX models may be superior to cancer cell lines for some studies of the biology and therapeutic response of endometrial cancer. Citation Format: Yuping Zhang, Eric J. Devor, Donghai Dai, Jesus Gonzalez-Bosquet, Kristina W. Thiel, Kimberly K. Leslie. Characterization of patient-derived xenograft models of endometrial cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PR009.