Carcinoma Patient-derived Xenograft Research Articles

Establishment and characterization of ovarian clear cell carcinoma patient-derived xenografts

Establishment and characterization of ovarian clear cell carcinoma patient-derived xenografts

BRCA1 foci test as a predictive biomarker of olaparib response in ovarian cancer patient-derived xenograft models.

Standard therapy for high-grade ovarian carcinoma includes surgery followed by platinum-based chemotherapy and poly-ADP ribose polymerase inhibitors (PARPis). Deficiency in homologous recombination repair (HRD) characterizes almost half of high-grade ovarian carcinomas and is due to genetic and epigenetic alterations in genes involved in HR repair, mainly BRCA1/BRCA2, and predicts response to PARPi. The academic and commercial tests set up to define the HRD status of the tumor rely on DNA sequencing analysis, while functional tests such as the RAD51 foci assay are currently under study, but have not been validated yet and are available for patients. In a well-characterized ovarian carcinoma patient-derived xenograft platform whose response to cisplatin and olaparib, a PARPi, is known, we assessed the association between the BRCA1 foci score, determined in formalin-fixed paraffin-embedded tumor slices with an immunofluorescence technique, and other HRD biomarkers and explored the potential of the BRCA1 foci test to predict tumors' response to cisplatin and olaparib. The BRCA1 foci score was associated with both tumors' HRD status and RAD51 foci score. A low BRCA1 foci score predicted response to olaparib and cisplatin, while a high score was associated with resistance to therapy. As we recently published that a low RAD51 foci score predicted olaparib sensitivity in our xenobank, we combined the two scores and showed that the predictive value was better than with the single tests. This study reports for the first time the capacity of the BRCA1 foci test to identify HRD ovarian carcinomas and possibly predict response to olaparib.

Rational Design and Comparison of Novel 99mTc-Labeled FAPI Dimers for Visualization of Multiple Tumor Types.

Recognizing the significance of SPECT in nuclear medicine and the pivotal role of fibroblast activation protein (FAP) in cancer diagnosis and therapy, this study focuses on the development of 99mTc-labeled dimeric HF2 with high tumor uptake and image contrast. The dimeric HF2 was synthesized and radiolabeled with 99mTc in one pot using various coligands (tricine, TPPTS, EDDA, and TPPMS) to yield [99mTc]Tc-TPPTS-HF2, [99mTc]Tc-EDDA-HF2, and [99mTc]Tc-TPPMS-HF2 dimers. SPECT imaging results indicated that [99mTc]Tc-TPPTS-HF2 exhibited higher tumor uptake and tumor-to-normal tissue (T/NT) ratio than [99mTc]Tc-EDDA-HF2 and [99mTc]Tc-TPPMS-HF2. Notably, [99mTc]Tc-TPPTS-HF2 exhibited remarkable tumor accumulation and retention in HT-1080-FAP and U87-MG tumor-bearing mice, thereby surpassing the monomeric [99mTc]Tc-TPPTS-HF. Moreover, [99mTc]Tc-TPPTS-HF2 achieved acceptable T/NT ratios in the hepatocellular carcinoma patient-derived xenograft (HCC-PDX) model, which provided identifiable contrast and imaging quality. In conclusion, this study presents proof-of-concept research on 99mTc-labeled FAP inhibitor dimers for the visualization of multiple tumor types. Among these candidate compounds, [99mTc]Tc-TPPTS-HF2 showed excellent clinical potential, thereby enriching the SPECT tracer toolbox.

Abstract 945: In vivo anti-tumor activity of onvansertib, a PLK1 inhibitor, combined with gemcitabine or carboplatin in platinum- resistant ovarian carcinoma patient-derived xenograft models

Abstract Background: The standard treatment for high grade ovarian carcinoma (HGOC) is cytoreductive surgery followed by a platinum based therapy. Despite an initial high rate of complete remission, most of the patients relapse with a platinum resistant disease. Patients with resistant tumors have few options, including monotherapy with gemcitabine, and new effective therapeutic alternatives are needed. The Polo-like kinase 1 (PLK1) is a master regulator of mitosis and recent evidences suggest its role in interfering with several DNA repair mechanisms. We investigated the effect of onvansertib (ONVA), a highly selective ATP-competitor PLK1 inhibitor, in combination with carboplatin (CARBO) and gemcitabine (GEM) in platinum-resistant HGOC patient-derived xenografts (PDXs). Methods: The combination of ONVA with either CARBO or GEM was investigated in two platinum-resistant HGOC PDXs, one intrinsically resistant to platinum (MNHOC#315), the other made resistant after repeated in vivo treatments with cisplatin (MNHOC#266R). Tumors were subcutaneously (s.c.; MNHOC#315) or orthotopically (i.p.; MNHOC#266R) transplanted in nude mice, mice were treated for 4 weeks with vehicle, single agents or drug combinations and followed for tumor growth (s.c. model) and survival. Results: CARBO- and GEM-ONVA combinations were well tolerated, with a maximum body weight loss of 6.6%. Both combinations showed strong anti-tumor activities in the two tested models. In MNHOC#315 PDX, CARBO- and GEM-ONVA combinations caused a tumor stabilization that extended beyond the dosing period and resulted in a higher tumor growth inhibition as compared to vehicle and single agents treated mice. In i.p. MNHOC#266R PDX, single agents were inactive, while a statistically significant increase in survival was observed in CARBO/ONVA and in GEM/ONVA treated mice as compared to vehicle treated mice, with a 6.6- and 7-fold increase in mean survival time, respectively. Pharmacodynamic studies are ongoing to decipher the mechanisms underlying the synergic effects of both combinations. Conclusions: The combinations of ONVA with either CARBO or GEM showed potent anti-tumor activities in two platinum-resistant HGOC PDXs. Additional studies in other HGOC platinum-resistant models, both in vivo and in vitro, as well pharmacodynamic studies are ongoing to validate these results and to get insights in the mechanisms underlying their efficacy. Citation Format: Michela Chiappa, Federica Guffanti, Ilaria Mengoli, Maya Ridinger, Giovanna Damia. In vivo anti-tumor activity of onvansertib, a PLK1 inhibitor, combined with gemcitabine or carboplatin in platinum- resistant ovarian carcinoma patient-derived xenograft 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 945.

Abstract 2836: Assessment of therapeutic antibody efficacy without the interference of murine Fc receptors allows for investigation of human antibody-dependent cellular cytotoxicity mediated by NK cells in the FcResolv™ hIL-15 NOG mouse model

Abstract Targeted antibody therapy is applied to treat various cancer types. In addition to the primary mode of action (MOA), which involves direct binding to the tumor antigen, indirect MOA acting through the constant region (Fc) of the antibody can enhance anti-tumor efficacy. Indirect mechanisms engage the innate immune system, mediated by both the complement system (complement-dependent cytotoxicity (CDC)) and immune cells (antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC)). These indirect mechanisms can complicate the evaluation and accurate assessment of antibody-induced ADCC by human NK cells in current mouse models. In immune-deficient mouse strains (e.g. NOG), false positives and/or negatives may occur due to interactions with murine Fc receptors. These can either result in anti-tumor responses via activation of the murine innate immune system or can interfere with the human-targeted therapy's primary MOA. To study the response to anti-cancer antibodies without the interference of these murine Fc receptor interactions and to investigate ADCC mediated by human NK cells, a novel mouse model deficient in Fc receptors and expressing human IL-15 (FcResolv™ hIL-15 NOG) was employed for testing antibody therapies. Methods: Patient-derived xenograft (PDX) tumor models were transplanted into hIL-15 NOG and FcResolv™ hIL-15 NOG mice. A human head and neck squamous cell carcinoma and a lung adenocarcinoma PDX model were both treated with cetuximab. Treatment with pertuzumab and trastuzumab was applied in a breast ductal carcinoma PDX model. Rituximab treatment was tested in two diffuse large B cell lymphoma PDX models. Based on growth kinetics, the lung cancer PDX model was chosen for further testing of ADCC in the NK cell-humanized FcResolv™ hIL-15 NOG mouse. Results and Conclusion: There was no difference in percent tumor growth inhibition between the FcResolv™ hIL-15 NOG and hIL-15 NOG mice with regards to cetuximab treatment in the lung and head and neck cancer or for trastuzumab treatment of breast ductal carcinoma. However, pertuzumab treatment revealed a false positive efficacy, with the false positive effect more pronounced in hIL-15 NOG mice than in FcResolv™ hIL-15 NOG mice. In one of the lymphoma models, a false negative result was observed. Here, rituximab did not show notable tumor inhibition in the hIL-15 NOG mice but did in the FcResolv™ hIL-15 NOG mice. These results demonstrate that FcResolv™ hIL-15 NOG mice serve as a suitable mouse model for a more accurate assessment of the therapeutic efficacy of anti-tumor antibodies. Additionally, evaluation of human-mediated ADCC of therapeutic antibodies in NK cell-humanized FcResolv™ hIL-15 NOG allows detection of effects specifically mediated by human NK cells. Citation Format: Simone Rhein, Maria Stecklum, Monika Buczek, Janell Richardson, Jens Hoffmann. Assessment of therapeutic antibody efficacy without the interference of murine Fc receptors allows for investigation of human antibody-dependent cellular cytotoxicity mediated by NK cells in the FcResolv™ hIL-15 NOG mouse model [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 2836.

Establishment and Thorough Characterization of Xenograft (PDX) Models Derived from Patients with Pancreatic Cancer for Molecular Analyses and Chemosensitivity Testing.

Patient-derived xenograft (PDX) tumor models are essential for identifying new biomarkers, signaling pathways and novel targets, to better define key factors of therapy response and resistance mechanisms. Therefore, this study aimed at establishing pancreas carcinoma (PC) PDX models with thorough molecular characterization, and the identification of signatures defining responsiveness toward drug treatment. In total, 45 PC-PDXs were generated from 120 patient tumor specimens and the identity of PDX and corresponding patient tumors was validated. The majority of engrafted PDX models represent ductal adenocarcinomas (PDAC). The PDX growth characteristics were assessed, with great variations in doubling times (4 to 32 days). The mutational analyses revealed an individual mutational profile of the PDXs, predominantly showing alterations in the genes encoding KRAS, TP53, FAT1, KMT2D, MUC4, RNF213, ATR, MUC16, GNAS, RANBP2 and CDKN2A. Sensitivity of PDX toward standard of care (SoC) drugs gemcitabine, 5-fluorouracil, oxaliplatin and abraxane, and combinations thereof, revealed PDX models with sensitivity and resistance toward these treatments. We performed correlation analyses of drug sensitivity of these PDX models and their molecular profile to identify signatures for response and resistance. This study strongly supports the importance and value of PDX models for improvement in therapies of PC.

GLUT1 Overexpression Enhances 19-28z CAR-T Cell Expansion and Metabolic Fitness

The tumor microenvironment presents many obstacles to effective CAR T cell therapy. One of these obstacles is glucose competition from tumor and myeloid cells to the detriment of CAR T cells. We hypothesize here that CAR T cells with elevated levels of glucose transporter are better fit under such conditions, which will result in improved therapeutic efficacy. We overexpressed either GLUT1 or GLUT3 in CD19-targeted CAR T cells engineered with a clinically validated CAR (19-28z). GLUT1 is ubiquitously expressed in tissues and GLUT3 has a higher affinity for glucose. Upon in vitro antigen stimulation, 1928z-GLUT1 and 1928z-GLUT3 showed greater enrichment of CAR+ T cells and underwent more cell divisions than control 1928z-LNGFR under glucose-limiting conditions. In repetitive antigen stimulation assays, 1928z-GLUT1 expanded significantly more than 1928z-LNGFR cells in 10 mM or 5 mM glucose medium. Surprisingly, 1928z-GLUT3 CAR T cells faired markedly less well than 1928z-LNGFR controls, associated with reduced viability of 1928z-GLUT3 CAR T cells after 2 or 3 exposures to antigen. In the NALM6 acute lymphoblastic leukemia model, low-dose CAR T cell treatment with 1928z-LNGFR and 1928z-GLUT3 did not curtail rapid tumor progression, while 1928z-GLUT1 CAR T cells significantly increased survival. More abundant CAR+ T cells were found in the bone marrow and spleen of 1928z-GLUT1-treated mice. Interestingly, we observed consistent increases in CD62L+CD45RA+ Tscm population in 1928z-GLUT1 cells, including both CD8 and CD4 T cells. We flow-sorted CAR T cells from treated mice and performed SMARTer RNA-seq. GLUT1, but not GLUT3, significantly upregulated TCF7 and genes encoding glycolytic enzymes and electron transport chain, indicating that GLUT1 overexpression enhances both glycolysis and oxidative phosphorylation. To further test GLUT1-CAR-T function in tumor lines with high levels of glucose transporter, we generated a renal cell carcinoma (RCC) patient-derived xenograft (PDX) with ectopic expression of human CD19. CAR-T cells were repetitively stimulated with the RCC PDX under titrated glucose concentrations. 1928z-GLUT1 significantly improved CAR T cell viability compared to 1928z-LNGFR in low glucose conditions. The expansion of GLUT1 CAR-T cells in 10 mM and 5 mM glucose also surpassed that of LNGFR CAR-T cells. In vivo, 1928z-GLUT1-treated mice showed significantly less tumor burden than 1928z-LNGFR-treated mice. Overall, we developed a novel strategy for enhancing the metabolic fitness of 1928z CAR-T cells by constitutively overexpressing GLUT1. GLUT1 but not GLUT3 enhances glycolysis and oxidative phosphorylation in CAR-T cells, which sustains T-cell expansion and increases therapeutic efficacy. This strategy is under investigation with other CAR formats.

Gastroenteropancreatic neuroendocrine carcinoma tumor spheroid drug screen reveals vulnerability to tyrosyl-DNA phosphodiesterase 1 inhibitors

BackgroundGastroenteropancreatic neuroendocrine carcinomas are rare neoplasms with no effective treatments and poor prognosis. Few reliable preclinical models exist for the study of gastroenteropancreatic neuroendocrine carcinomas, limiting investigation of novel treatments. We used tumor spheroids from our recently established gastroenteropancreatic neuroendocrine carcinoma patient-derived xenograft models to systematically screen for compounds with diverse structures to identify potential new categories of therapeutic agents that can target gastroenteropancreatic neuroendocrine carcinomas. MethodsTumor spheroids were derived from our NEC913 and NEC1452 gastroenteropancreatic neuroendocrine carcinoma patient-derived xenograft models. Gastroenteropancreatic neuroendocrine carcinoma spheroids were screened against a library of 885 compounds from the National Cancer Institute Diversity Set VII collection. Cell viability was measured via AlamarBlue assay. After identification of potential therapeutic compounds, synergy screening of a selected group with temozolomide and doxorubicin was performed, and these combinations were further analyzed for γH2AX and phosphorylated-ERK proteins. ResultsWe identified 16 compounds that inhibit over 75% of gastroenteropancreatic neuroendocrine carcinoma spheroid survival. Seven are inhibitors of tyrosyl-DNA phosphodiesterase 1, a DNA repair enzyme working closely with the topoisomerase I complex. When combined with temozolomide or doxorubicin, the tyrosyl-DNA phosphodiesterase 1 inhibitor cytarabine increased the cytotoxic effects of these drugs on NEC1452 cells which was further evidenced by increasing γH2AX and decreasing phosphorylated-ERK in combination treatment compared to temozolomide alone. ConclusionBoth NEC913 and NEC1452 gastroenteropancreatic neuroendocrine carcinoma spheroid lines are useful preclinical models for drug testing. Our library screen revealed these gastroenteropancreatic neuroendocrine carcinoma spheroids are highly sensitive to a novel class of anti-cancer drugs that target nuclear genome stability.

Urokinase-Type Plasminogen Activator Receptor (uPAR) Expression and [64Cu]Cu-DOTA-AE105 uPAR-PET/CT in Patient-Derived Xenograft Models of Oral Squamous Cell Carcinoma

Purpose[64Cu]Cu-DOTA-AE105 urokinase-type plasminogen activator receptor (uPAR)-PET/CT is a novel and promising imaging modality for cancer visualization, although it has not been tested in head and neck cancer patients nor in preclinical models that closely resemble these heterogenous tumors, i.e., patient-derived xenograft (PDX) models. The aim of the present study was to establish and validate oral squamous cell carcinoma (OSCC) PDX models and to evaluate [64Cu]Cu-uPAR-PET/CT for tumor imaging in these models.ProceduresPDX flank tumor models were established by engrafting tumor tissue from three patients with locally advanced OSCC into immunodeficient mice. [64Cu]Cu-DOTA-AE105 was injected in passage 2 (P2) mice, and [64Cu]Cu-uPAR-PET/CT was performed 1 h and 24 h after injection. After the last PET scan, all animals were euthanized, and tumors dissected for autoradiography and immunohistochemical (IHC) staining.ResultsThree PDX models were established, and all of them showed histological stability and unchanged heterogenicity, uPAR expression, and Ki67 expression through passages. A significant correlation between uPAR expression and tumor growth was found. All tumors of all models (n=29) showed tumor uptake of [64Cu]Cu-DOTA-AE105. There was a clear visual concordance between the distribution of uPAR expression (IHC) and [64Cu]Cu-DOTA-AE105 uptake pattern in tumor tissue (autoradiography). No significant correlation was found between IHC (H-score) and PET-signal (SUVmax) (r=0.34; p=0.07).ConclusionsOSCC PDX models in early passages histologically mimic donor tumors and could serve as a valuable platform for the development of uPAR-targeted imaging and therapeutic modalities. Furthermore, [64Cu]Cu-uPAR-PET/CT showed target- and tumor-specific uptake in OSCC PDX models demonstrating the diagnostic potential of this modality for OSCC patients.

Nasopharyngeal carcinoma patient-derived xenograft mouse models reveal potential drugs targeting cell cycle, mTOR, and autophagy pathways

BackgroundNasopharyngeal carcinoma (NPC) is associated with Epstein–Barr virus (EBV) infection. To test preclinical NPC drugs, we established two patient-derived xenograft (PDX) mouse models, EBV-positive PDX-B13 and EBV-negative PDX-Li41, for drug screening. MethodsBased on next generation sequencing (NGS) studies, PDX-B13 had CCND1 copy number (CN) gain but CDKN2A CN loss, whereas PDX-Li41 had CDKN2A and RB1 CN loss, TSC1 (negative regulator of mTOR) frameshift deletion mutation, and increased activation of mTOR, a serine/threonine kinase that governs metabolism, autophagy, and apoptosis. Increased mTOR was also associated with poor NPC prognosis. ResultsEverolimus, an mTOR inhibitor, suppressed tumor growth in the two PDX NPC models and had an additive antitumor effect with palbociclib, a CDK4/6 inhibitor. PDX tumors treated with various drugs or untreated were subjected to RNA sequencing, transcriptome profile analysis, and selective Western blotting to understand the interactions between these drugs and gene expression profiles. Palbociclib also suppressed EB viral nuclear antigen (EBNA1) expression in PDX-B13. Everolimus together with autophagy inhibitor, hydroxychloroquine, had additive anti-tumor effect on PDX-B13 tumor. Immunohistochemistry revealed that high mTOR levels were correlated with poor overall survival in patients with metastatic NPC (N = 90). ConclusionsHigh mTOR levels are a poor prognostic factor in NPC, and cell cycle, mTOR and autophagy pathways may serve as therapeutic targets in NPC. In addition, PDX models can be used for efficiently testing potential NPC drugs.

β-catenin inhibitor ICG-001 suppress cell cycle progression and induce autophagy in endometrial cancer cells.

The incidence of endometrial cancer has been rising in recent years. Gene mutation and high protein expression of β-catenin are commonly detected in endometrioid endometrial cancer. ICG-001 is a β-catenin inhibitor via blocking the complex formation of β-catenin and cAMP response element-binding protein (CREB)-binding protein (CBP). This study aims to investigate the effect of ICG-001 on endometrial cancer inhibition. First, endometrial carcinoma patient-derived xenograft (PDX)-derived organoids and primary cells were used to verify the inhibiting ability of ICG-001 on endometrial cancer. Furthermore, endometrial cancer cell lines were used to investigate the anticancer mechanism of ICG-001. Using MTT assay and tumor spheroid formation assay, ICG-001 significantly reduced the cell viability of HEC-59 and HEC-1A cells. ICG-001 enhanced cisplatin-mediated cytotoxicity. ICG-001 decreased cancer stem cell sphere formation. ICG-001 decreased the protein expressions of CD44, hexokinase 2 (HK2), and cyclin A. ICG-001 lowered the cell cycle progression by flow cytometer analysis. Autophagy, but no apoptosis, was activated by ICG-001 in endometrial cancer cells. Autophagy inhibition by ATG5 silencing enhanced ICG-001-mediated suppression of cell viability, tumor spheroid formation, and protein expression of cyclin A and CD44. This study clarified the mechanism and revealed the clinical potential of ICG-001 against endometrial cancer.

Exportin 1 inhibition prevents neuroendocrine transformation through SOX2 down-regulation in lung and prostate cancers.

In lung and prostate adenocarcinomas, neuroendocrine (NE) transformation to an aggressive derivative resembling small cell lung cancer (SCLC) is associated with poor prognosis. We previously described dependency of SCLC on the nuclear transporter exportin 1. Here, we explored the role of exportin 1 in NE transformation. We observed up-regulated exportin 1 in lung and prostate pretransformation adenocarcinomas. Exportin 1 was up-regulated after genetic inactivation of TP53 and RB1 in lung and prostate adenocarcinoma cell lines, accompanied by increased sensitivity to the exportin 1 inhibitor selinexor in vitro. Exportin 1 inhibition prevented NE transformation in different TP53/RB1-inactivated prostate adenocarcinoma xenograft models that acquire NE features upon treatment with the aromatase inhibitor enzalutamide and extended response to the EGFR inhibitor osimertinib in a lung cancer transformation patient-derived xenograft (PDX) model exhibiting combined adenocarcinoma/SCLC histology. Ectopic SOX2 expression restored the enzalutamide-promoted NE phenotype on adenocarcinoma-to-NE transformation xenograft models despite selinexor treatment. Selinexor sensitized NE-transformed lung and prostate small cell carcinoma PDXs to standard cytotoxics. Together, these data nominate exportin 1 inhibition as a potential therapeutic target to constrain lineage plasticity and prevent or treat NE transformation in lung and prostate adenocarcinoma.

Alteration in molecular properties during establishment and passaging of endometrial carcinoma patient-derived xenografts

Patient-derived xenograft (PDX) tumor models are known to maintain the genomic and phenotypic profiles, including the histopathological structures, of the parental tumors. On the other hand, unique enrichment of single-nucleotide variants or copy number aberrations has been reported in several types of tumors. However, an understanding of endometrial carcinoma PDXs is limited. The purpose of the present study was to clarify the presence or absence of the molecular properties of endometrial carcinomas in PDXs passaged up to eight times. Established PDXs of endometrioid carcinomas maintained their histopathological characteristics, but those of carcinosarcomas predominantly consisted of sarcomatous components when compared to the parental tumors. Alterations in the proportion of cells with positive/negative immunohistochemical staining for estrogen receptor, PTEN, PAX8, and PAX2 were observed, whereas the proportions of cells with AE1/AE3, TP53, ARID1A, PMS2, and MSH6 staining were unchanged. Variants of cancer-associated genes were compared between PDXs and parental tumors. Mutations in POLE and a frameshift deletion in BRCA1 were observed in the parental tumor tissue in each of the six cases, and additional genomic alterations, which were not apparently related to histopathological and immunohistochemical alterations, were found in the PDXs of these cases. The genomic and phenotypic alterations observed between endometrial carcinoma PDXs and parental tumors were partly associated with endometrial cancer-specific characteristics related to cellular differentiation and gene mutations.

Abstract 44: An in vivo platform of pre-characterized renal cell carcinoma (RCC) patient-derived xenograft models allows the preclinical evaluation of patient-tailored intervention strategies

Abstract Renal cell carcinoma (RCC), is the most common kidney cancer of adults, originating in the lining of the proximal convoluted tubule. Prognosis is poor in patients with advanced or metastasized RCC. Drug resistance towards Standard of Care (SoC, incl. everolimus, sorafenib, or sunitinib) drugs develops frequently within months. Therefore, development of novel options to target acquired TKI resistance mechanisms in advanced and metastatic RCC is still an urgent medical need. Preclinical models with high translational relevance can promote the implementation of novel personalized therapies. To evaluate novel targeted therapies and their combinations in preclinical settings, patient-derived xenograft (PDX) models represent valuable tools. Responsible local ethics committees approved usage of patient tissue and all animal procedures. In this study, RCC tissue from 167 patients was collected and xenotransplanted in mice. Partially, a multi-region approach, xenografting tissue from different regions of one tumor, was used. PDX models were characterized by immunohistochemistry (Ki-67, CD31, Pax2 and Pax8 antibodies), gene expression, copy number variations and mutational analyses. To evaluate in vivo drug response of RCC PDX models, mice transplanted with PDX tumors were treated with bevacizumab (i.p.), with everolimus, sorafenib, or sunitinib (p.o.). Adopted clinical response criteria for solid tumors (RECIST) were applied to classify the anti-tumor activity of the tested compounds in RCC PDX models. Next generation sequencing (NGS, panel) and transcriptome data were used to compare primary tumors and metastases. A comprehensive panel of subcutaneous RCC PDX models with well-conserved molecular and pathological features over multiple passages was established. The overall take for the RCC PDX in this study was 21%. Tumor growth characteristics were heterogeneous throughout the different models but were stable during in vivo passaging. Drug screening towards four SoC drugs, targeting the VEGF and PI3K/mTOR pathway, revealed individual and heterogeneous response profiles in the PDX, resembling the clinical situation. Intra-tumor heterogeneity can be assessed via PDX models from multi-tumor regions from one patient in our platform. Development of corresponding in vitro cell culture models from the PDX enables advanced high throughput drug screening in a personalized context. Analyzing novel targeted molecules is possible due to the pre-established molecular characterization of the PDX at the genomic and expression level. In conclusion, we established a new and molecularly characterized panel of RCC PDX models with high relevance for translational preclinical research. Citation Format: Dennis Kobelt, Dennis Gürgen, Michael Becker, Mathias Dahlmann, Susanne Flechsig, Elke Schaeffeler, Florian A. Büttner, Christian Schmees, Regina Bohnert, Jens Bedke, Matthias Schwab, Johann J. Wendler, Martin Schostak, Burkhard Jandrig, Wolfgang Walther, Jens Hoffmann. An in vivo platform of pre-characterized renal cell carcinoma (RCC) patient-derived xenograft models allows the preclinical evaluation of patient-tailored intervention strategies [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 44.

Abstract 6113: Predictive value of HRD testing on the response to PARP inhibitors in ovarian cancer preclinical models

Abstract Since the introduction of Poly(ADP-ribose)polymerase inhibitors (PARPi) in the clinical practice, the search of biomarkers to distinguish homologous recombination defective tumors, eligible for PARPi therapy, has become a priority. Along with testing for BRCA mutations, commercially available tests, such as MyChoice® assay (Myriad Genetics), exploit scores calculated from genomic scarring patterns. Platinum sensitivity is also considered a surrogate marker of PARPi efficacy. However, the prognostic accuracy remains a challenge because these tests can only infer homologous recombination functionality but not directly measure it. Moreover, the association between platinum and PARPi response is imperfect since not all platinum-sensitive patients benefit from PARPi and PARPi response has been observed in platinum-resistant cancers. A panel of 20 high-grade serous ovarian carcinoma patient-derived xenografts (OC-PDXs) were molecularly and pharmacologically characterized to assess predictive markers of response to PARPi therapy. Genomic DNA and total RNA were sequenced searching for BRCA1/2 alterations or sent to Myriad Genetics to obtain HRD scores. Absolute BRCA1 mRNA expression was measured by RT-qPCR. OC-PDXs growing subcutaneously or orthotopically in nude mice were treated with the PARPi olaparib or platinum to test drug sensitivity. The molecular analyses revealed that nearly 90% (17/20) of OC-PDXs are TP53 mutated, thus reflecting the clinical scenario. 50% (10/20) of OC-PDXs harbor mutations in BRCA1/2 genes, while 10% (2/20) lack BRCA1 mRNA expression. MyChoice® assay showed that 65% (13/20) of OC-PDXs score higher than 42, which is the currently used threshold to classify HRD-positive tumors. Correlation analysis confirmed that the HRD score is highly associated with the presence of mutations in BRCA1/2 genes. Preclinical testing showed that 50% of OC-PDXs are sensitive to olaparib. Notably, in 20% of cases the therapeutic response was incorrectly predicted considering either BRCA1/2 mutations or HRD score. 30% of platinum-sensitive OC-PDXs were not responsive to olaparib, confirming that platinum sensitivity not always correlate with response to PARPi. Our results show that tumor genotype and HRD score are strongly associated. Further analyses are required to improve the predictive value of currently used tests to select patients who will benefit the most from PARPi treatment while sparing toxicities in patients that are unlikely to respond. Citation Format: Giulia Dellavedova, Alessandra Decio, Laura Formenti, Luca Porcu, Carmen Ghilardi, Raffaella Giavazzi, Maria Rosa Bani. Predictive value of HRD testing on the response to PARP inhibitors in ovarian cancer preclinical models [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 6113.

A novel xenograft model of human hepatocellular carcinoma in immunocompetent mice based on the microcarrier-6

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignant tumors that threaten human health; thus, the establishment of an animal model with clinical features similar to human hepatocellular carcinoma is of important practical significance. MethodsTaking advantage of the novel microcarrier-6, human HCC cells were injected into immunocompetent mice to establish a novel human HCC patient-derived xenograft (PDX) model. Primary HCC cells were isolated from fresh hepatocellular carcinoma tissues, which were subsequently co-cultured with microcarrier-6 to construct a three-dimensional tumor cell culture model in vitro. The HCC-microcarrier complex was implanted into mice by subcutaneous inoculation, and the tumor formation time, tumor formation rate, and pathological manifestation were recorded. Changes of immune parameters in mice were detected by flow cytometry. ResultsThe success rate was 60% (6/10) in the establishment of hepatocellular carcinoma PDX mouse model, and the total tumor formation rate of the tumor-forming model is 90–100%. H&E staining and immunohistochemical experiments indicate that the model well retained the characteristics of the primary tumor. Interestingly, M2 macrophages in tumor-bearing mice increased significantly, and the levels of CD4+ T cells were significantly reduced. ConclusionsThrough the application of the microcarrier-6 in immunocompetent mice, we successfully established a novel human HCC PDX model, which can be used to better study and further elucidate the occurrence and pathogenic mechanism of HCC, improve the predictability of toxicity and drug sensitivity in HCC.

33 (PB023) - Establishment and characterization of a preclinical platform of subcutaneous renal cell carcinoma (RCC) patient-derived xenograft models to evaluate novel treatment strategies

Background: Renal cell carcinoma (RCC) is a cancer type with an onset mainly during the sixth or seventh decade of the patient’s life. Patients with advanced or metastasized RCC have a poor prognosis. The majority of patients develop drug resistance towards Standard of Care (SoC) drugs within months. Tyrosine kinase inhibitors (TKI) are used in first-line therapy and have been combined with immune checkpoint inhibitors recently. The development of novel therapies targeting acquired TKI resistance mechanisms in advanced and metastatic RCC remains an urgent medical need.

IMPACT OF PHOTOBIOMODULATION THERAPY IN A PATIENT-DERIVED XENOGRAFT MODEL OF ORAL SQUAMOUS CELL CARCINOMA

Objectives This study aimed to evaluate the effect of photobiomodulation therapy (PBMT) in an oral squamous cell carcinoma (OSCC) patient-derived xenograft (PDX) model. Study Design BALB/c nude mice with OSCC-PDX models were divided into nonirradiated and irradiated groups. OSCC-PDX were irradiated daily (660 nm; 100 mW; 6 J/cm2; 0.2 J/point) for 12 weeks. The tumors were collected and submitted to volumetric, histologic, immunohistochemical, and cell cycle analysis. Results No significant differences in the volumetric measurements (P = .89) and in the histopathologic grade (P > .05) were detected between the groups. The immunohistochemical analysis of Ki-67 (P = .9661), H3K9ac (P = .3794), and BMI1 (P = .5182) and the evaluation of the cell cycle phases (P > .05) by flow cytometry also did not demonstrate significant differences between the irradiated and nonirradiated groups. Conclusions In this study, PBMT did not affect the behavior of OSCC-PDX models. This is an important preclinical outcome regarding safety concerns of the use of PBMT in patients with cancer. This study aimed to evaluate the effect of photobiomodulation therapy (PBMT) in an oral squamous cell carcinoma (OSCC) patient-derived xenograft (PDX) model. BALB/c nude mice with OSCC-PDX models were divided into nonirradiated and irradiated groups. OSCC-PDX were irradiated daily (660 nm; 100 mW; 6 J/cm2; 0.2 J/point) for 12 weeks. The tumors were collected and submitted to volumetric, histologic, immunohistochemical, and cell cycle analysis. No significant differences in the volumetric measurements (P = .89) and in the histopathologic grade (P > .05) were detected between the groups. The immunohistochemical analysis of Ki-67 (P = .9661), H3K9ac (P = .3794), and BMI1 (P = .5182) and the evaluation of the cell cycle phases (P > .05) by flow cytometry also did not demonstrate significant differences between the irradiated and nonirradiated groups. In this study, PBMT did not affect the behavior of OSCC-PDX models. This is an important preclinical outcome regarding safety concerns of the use of PBMT in patients with cancer.

Development of Fibroblast Activation Protein Inhibitor-Based Dimeric Radiotracers with Improved Tumor Retention and Antitumor Efficacy.

Fibroblast activation protein (FAP), a fundamental component of the tumor stroma, is overexpressed in cancer-associated fibroblasts (CAFs). As a promising theranostic probe, we evaluated whether the FAP inhibitor (FAPI) dimer (DOTA-2P[FAPI]2) is more effective than its monomeric analogs for FAP-targeted radionuclide therapy. [68Ga]Ga/[177Lu]Lu-DOTA-2P(FAPI)2 were assayed in a stability study, small-animal positron emission tomography (PET) and single-photon emission computed tomography (SPECT), biodistribution, and radionuclide therapy to comprehensively evaluate their preclinical pharmacokinetics. The pharmacokinetics of [68Ga]Ga-DOTA-2P(FAPI)2 and [177Lu]Lu-DOTA-2P(FAPI)2 were determined in FAP-positive hepatocellular carcinoma patient-derived xenografts (PDXs) and HT-1080-FAP cell-derived xenografts (CDXs). [68Ga]Ga-DOTA-2P(FAPI)2 and [177Lu]Lu-DOTA-2P(FAPI)2 were stable in phosphate-buffered saline for 4 h. The tumor retention of [68Ga]Ga-DOTA-2P(FAPI)2 was better than that of [68Ga]Ga-FAPI-46 in HT-1080-FAP CDXs, while healthy organs showed low tracer uptake and fast body clearance. In single-photon emission computed tomography, [177Lu]Lu-DOTA-2P(FAPI)2 showed a higher uptake and longer retention for tumors in both PDXs and CDXs from 1-48 h. [177Lu]Lu-DOTA-2P(FAPI)2 showed the best inhibition of tumor growth in PDXs and CDXs. DOTA-2P(FAPI)2 has increased tumor uptake and retention properties compared to FAPI-46, which significantly improves the use of FAPI-based vectors for PET imaging and radionuclide therapy. [177Lu]Lu-DOTA-2P(FAPI)2 may be safe and effective for the treatment of FAP-positive malignant tumors.

Sorafenib regulates vascular endothelial growth factor by runt-related transcription factor-3 to inhibit angiogenesis in hepatocellular carcinoma

Objective: To investigate the molecular mechanism of sorafenib against hepatocellular carcinoma. Methods: Sorafenib efficacy was screened and verified by the hepatocellular carcinoma patient-derived tumor xenograft (PDX) model. Veterinary B-mode ultrasonography and in vivo confocal laser scanning microscopy were used to observe PDX angiogenesis. Immunohistochemistry was used to observe the expression of proliferation and angiogenesis-related proteins in PDX tissue. Real-time quantitative PCR technology was used to observe the RUNX3 gene in PDX tissues. SPSS 17.0 statistical software was used for statistical analysis. Results: Four cases of PDX were used to screen the efficacy of sorafenib. PDX1 had a significant response to sorafenib, with an inhibition rate of 68.07%. Compared with the control group, sorafenib had significantly inhibited PDX1 relative tumor volume (5.76±2.14 vs. 11.71±2.87, P<0.05). Cell division index (39.50±7.72 vs. 67.10±9.14, P<0.05) and Ki67 expression (288.6±43.40 vs. 531.70±55.60, P<0.05) were significantly decreased. Veterinary B-mode ultrasonography showed evident blood flow signals in PDX1 tumors. In vivo confocal laser scanning microscopy results showed that sorafenib had significantly reduced the total vessel length (1573.00±236.21 vs. 2675.03±162.00, P<0.05) and area (11 145.33±1931.97 vs. 20 105.37±885.93, P<0.05)) of PDX1 tumors. Immunohistochemical results showed that sorafenib had significantly down-regulated the protein expressions of CD34 (27.55±3.76 vs. 45.47±5.57, P<0.05), VEGF (16.33±2.86 vs. 22.77±3.20, P<0.05) and MVD (38.75±6.01 vs. 55.50±8.61, P<0.05). Real-time PCR results showed that sorafenib had significantly up-regulated RUNX3 gene expression (2.14±0.71 vs. 1.00±0.36, P<0.05). However, there was a negative correlation between the expression of RUNX3 gene and the ratio of VEGF-positive cells in sorafenib group (R2=0.509 7). Conclusion: Sorafenib may inhibit the PDX angiogenesis and the growth of hepatocellular carcinoma by regulating the RUNX3-VEGF pathway.