A technology that visualizes tumor stem cells with clinically relevant tracers could have a broad impact on cancer diagnosis and treatment. The AC133 epitope of CD133 currently is one of the ...best-characterized tumor stem cell markers for many intra- and extracranial tumor entities. Here we demonstrate the successful noninvasive detection of AC133 ⁺ tumor stem cells by PET and near-infrared fluorescence molecular tomography in subcutaneous and orthotopic glioma xenografts using antibody-based tracers. Particularly, microPET with ⁶⁴Cu-NOTA-AC133 mAb yielded high-quality images with outstanding tumor-to-background contrast, clearly delineating subcutaneous tumor stem cell-derived xenografts from surrounding tissues. Intracerebral tumors as small as 2–3 mm also were clearly discernible, and the microPET images reflected the invasive growth pattern of orthotopic cancer stem cell-derived tumors with low density of AC133 ⁺ cells. These data provide a basis for further preclinical and clinical use of the developed tracers for high-sensitivity and high-resolution monitoring of AC133 ⁺ tumor stem cells.
BackgroundThe field of cancer immunology is rapidly moving towards innovative therapeutic strategies, resulting in the need for robust and predictive preclinical platforms reflecting the ...immunological response to cancer. Well characterized preclinical models are essential for the development of predictive biomarkers in the oncology as well as the immune-oncology space. In the current study, gold standard preclinical models are being refined and combined with novel image analysis tools to meet those requirements.MethodsA panel of 14 non-small cell lung cancer patient-derived xenograft models (NSCLC PDX) was propagated in humanized NOD/Shi-scid/IL-2Rnull mice. The models were comprehensively characterized for relevant phenotypic and molecular features, including flow cytometry, immunohistochemistry, histology, whole exome sequencing and cytokine secretion.ResultsModels reflecting hot (>5% tumor-infiltrating lymphocytes/TILs) as opposed to cold tumors (<5% TILs) significantly differed regarding their cytokine profiles, molecular genetic aberrations, stroma content, and programmed cell death ligand-1 status. Treatment experiments including anti cytotoxic T-lymphocyte-associated protein 4, anti-programmed cell death 1 or the combination thereof across all 14 models in the single mouse trial format showed distinctive tumor growth response and spatial immune cell patterns as monitored by computerized analysis of digitized whole-slide images. Image analysis provided for the first time qualitative evaluation of the extent to which PDX models retain the histological features from their original human donors.ConclusionsDeep phenotyping of PDX models in a humanized setting by combinations of computational pathology, immunohistochemistry, flow cytometry and proteomics enables the exhaustive analysis of innovative preclinical models and paves the way towards the development of translational biomarkers for immuno-oncology drugs.
We systematically analyzed multiple myeloma (MM) cell lines and patient bone marrow cells for their engraftment capacity in immunodeficient mice and validated the response of the resulting xenografts ...to antimyeloma agents.
Using flow cytometry and near infrared fluorescence in-vivo-imaging, growth kinetics of MM cell lines L363 and RPMI8226 and patient bone marrow cells were investigated with use of a murine subcutaneous bone implant, intratibial and intravenous approach in NOD/SCID, NOD/SCID treated with CD122 antibody and NOD/SCID IL-2Rγ(null) mice (NSG).
Myeloma growth was significantly increased in the absence of natural killer cell activity (NSG or αCD122-treated NOD/SCID). Comparison of NSG and αCD122-treated NOD/SCID revealed enhanced growth kinetics in the former, especially with respect to metastatic tumor sites which were exclusively observed therein. In NSG, MM cells were more tumorigenic when injected intratibially than intravenously. In NOD/SCID in contrast, the use of juvenile long bone implants was superior to intratibial or intravenous cancer cell injection. Using the intratibial NSG model, mice developed typical disease symptoms exclusively when implanted with human MM cell lines or patient-derived bone marrow cells, but not with healthy bone marrow cells nor in mock-injected animals. Bortezomib and dexamethasone delayed myeloma progression in L363- as well as patient-derived MM cell bearing NSG. Antitumor activity could be quantified via flow cytometry and in vivo imaging analyses.
Our results suggest that the intratibial NSG MM model mimics the clinical situation of the disseminated disease and serves as a valuable tool in the development of novel anticancer strategies.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Patient-derived xenografts (PDX) have emerged as an important translational research tool for understanding tumor biology and enabling drug efficacy testing. They are established by transfer of ...patient tumor into immune compromised mice with the intent of using them as Avatars; operating under the assumption that they closely resemble patient tumors. In this study, we established 27 PDX from 100 resected gastric cancers and studied their fidelity in histological and molecular subtypes. We show that the established PDX preserved histology and molecular subtypes of parental tumors. However, in depth investigation of the entire cohort revealed that not all histological and molecular subtypes are established. Also, for the established PDX models, genetic changes are selected at early passages and rare subclones can emerge in PDX. This study highlights the importance of considering the molecular and evolutionary characteristics of PDX for a proper use of such models, particularly for Avatar trials.
In up to 30% of non-small cell lung cancer (NSCLC) patients, the oncogenic driver of tumor growth is a constitutively activated epidermal growth factor receptor (EGFR). Although these patients gain ...great benefit from treatment with EGFR tyrosine kinase inhibitors, the development of resistance is inevitable. To model the emergence of drug resistance, an EGFR-driven, patient-derived xenograft (PDX) NSCLC model was treated continuously with Gefitinib in vivo. Over a period of more than three months, three separate clones developed and were subsequently analyzed: Whole exome sequencing and reverse phase protein arrays (RPPAs) were performed to identify the mechanism of resistance. In total, 13 genes were identified, which were mutated in all three resistant lines. Amongst them the mutations in NOMO2, ARHGEF5 and SMTNL2 were predicted as deleterious. The 53 mutated genes specific for at least two of the resistant lines were mainly involved in cell cycle activities or the Fanconi anemia pathway. On a protein level, total EGFR, total Axl, phospho-NFκB, and phospho-Stat1 were upregulated. Stat1, Stat3, MEK1/2, and NFκB displayed enhanced activation in the resistant clones determined by the phosphorylated vs. total protein ratio. In summary, we developed an NSCLC PDX line modelling possible escape mechanism under EGFR treatment. We identified three genes that have not been described before to be involved in an acquired EGFR resistance. Further functional studies are needed to decipher the underlying pathway regulation.
Abstract
While the use of bioluminescent proteins for molecular imaging is a powerful technology to support our knowledge of complex processes, with the advent of near infrared-labeling capabilities ...optical imaging can be pursuit with deeper tissue penetration, a better signal to background ratio and a quicker image acquisition without the need to inject substrates in preclinical oncology mouse models. This is specifically important in orthoptic brain tumors, when the substrate concentration in the tumor tissue might be influenced by the blood-brain barrier. In our recent study, we validated the feasibility of stable or transient transfection of human brain tumor cell lines and patient derived xenografts to determine tumor load in an intracranial setting. As a proof-of principle, we stably transfected the human glioblastoma cell line U87MG with an iRFP713 construct and followed tumor growth of intracranially implanted NSG mice. As a follow-up, five PDX of pediatric brain tumors (two Ependymoma, two Medulloblastoma, one high grade Glioblastoma) were transiently transfected with the same construct 24h before intracranial implantation. Tumor load was determined twice a week with the Pearl trilogy system (LiCor, Germany) and animals were examined for neurological symptoms every day. When the overall condition of the mice got deteriorated, animals were taken down and brains fixed in formalin. The tumor load and localization was confirmed by H&E and human-specific IHC for LamininB. The comparison between non-transfected and transfected lines of one specific tumor model, revealed that the tumor growth was very similar in both settings. The tumor take rate was as well not affected. In the subsequent analyses we could show that the tumor load linearly correlated with the imaging signal. Moreover, the iRFP713 signal was stable post-resection of the tumor. Thus, it was possible to correlate the tumor load quantification via iRFP713-Protein expression and LamininB-IHC within one slide. The transient transfection was successful for the five tested models. When termination criteria were reached, over 90% of the tumor tissue were expressing the iRFP713 protein. The more general applicability for larger cohorts of mice (> 20) and drug testing experiments will be evaluated soon. We believe the superior optical properties of iRFP713 will be a valuable asset to overcome some of the complications inherent to imaging live animals and a powerful tool for preclinical drug development in clinical relevant mouse models of brain cancer.
Citation Format: Kanstantsin Lashuk, Jacqueline Bersano, Dorothee Lenhard, Eva Oswald, Kerstin Klingner, Julia Schüler. Development of an iRFP713-based orthotopic patient derived brain cancer model to study tumor development in vivo 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 2806.
Abstract
Patient-derived tumor xenografts (PDX) play a major role in the development of new cancer therapies and their strengths and weaknesses have gradually been elucidated. In the current study we ...established a melanoma PDX from donor patient tissue. In addition, we were able to create two sublines from spontaneous metastases occurring in the murine host during the establishment phase of the original model. All three lines were characterized by tumor growth kinetics, antitumoral activity against standard of care Temozolomide and patho-histological examination. Furthermore, whole exome sequencing and RNAseq data of primary PDX and its metastases are available. Two out of the three sublines have corresponding cell lines for 2D and 3D testing. The PDX model was developed from a biopsy of a 68 year old woman undergoing surgery due to a non-pretreated melanoma. After seven subcutaneous passages in immune-compromised mice, individual animals showed tumor growth in the liver as well as the spleen. We were able to passage and characterize those metastases in parallel to the original model. The human origin of the lines as well as cell lines established in 2D was confirmed by str-analysis. All three in vivo lines depicted distinct growth kinetics: The doubling times varied significantly (Kruskal-Wallis, p< 0.0018) between 12.34 days (primary tumor, MEXF 2090P) and 30.78 day (spleen metastasis, MEXF 2090S) and 19.01 days doubling time for the liver metastasis model (MEXF 2090L). MEXF 2090S thereby depicted the slowest growth rate in our melanoma PDX panel (24 models, 10.09 days mean doubling time). The patho-histological examination revealed a well differentiated melanoma with low stroma content (3-5%) in all three lines. The molecular analysis (whole exome sequencing) identified distinct differences. Nevertheless, the potential driver mutations, Tp53 (R213Q) and Nras (Q61K), were recognized in all investigated samples. Temozolomide was applied to all three lines in vivo. 6 mice per group and line were treated either with Temozolomide (40 mg/kg/d, iv, twice a week for three weeks) or the control vehicle (10% DMSO, 90% NaCl). The primary tumor depicted statistically significant antitumoral activity with a T/C (test vs control) value of 36% (p< 0.05, t test, two-tailed) two weeks after the last treatment (experiment day 32). The two lines derived of metastases were resistant against the alkylating agent depicting T/C values of 76% (2090L) and 79% (2090S), respectively. Spontaneous metastases are a rare event in PDX models growing subcutaneously in NMRI nude mice. Thus, shedding some light into the biology of the metastatic event in the current model will help to understand and influence the metastatic process in general. In any case, those lines can serve as indispensable tools in the oncology drug development pipeline.
Citation Format: Kerstin Klingner, Dorothee Lenhard, Elke Simon, Anne-Lise Peille, Julia Schüler. Metastases of a temozolimide-sensitive patient-derived melanoma xenograft model show distinct biologic features and developed resistance against temozolomide abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2169.
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.
Schueler: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.
Abstract
Patient-derived tumor xenografts (PDX) play a major role in the development of new cancer therapies and their strengths and weaknesses have gradually been elucidated. Large panels of solid ...cancer PDX are available to screen innovative compounds, identify new targets, and study tumor biology. In the current study we established a melanoma PDX from donor patient tissue. In addition, we were able to create two sublines from spontaneous metastases occurring in the murine host during the establishment phase of the original model. All three lines were characterized by tumor growth kinetics, antitumoral activity against standard-of-care temozolomide, and pathohistologic examination. Furthermore, molecular examination of primary PDX and its metastases is under way as well as cell line establishment. The PDX model was developed from a biopsy of a 68-year-old woman undergoing surgery due to a non-pretreated melanoma. After seven subcutaneous passages in immune-compromised mice, individual animals showed tumor growth in the liver as well as the spleen. We were able to passage and characterize those metastases in parallel to the original model. The human origin of the lines as well as one cell line established in 2D from the primary PDX was confirmed by str-analysis. All three in vivo lines depicted distinct growth kinetics: The doubling times varied significantly (Kruskal-Wallis, p< 0.0018) between 12.34 days (primary tumor, MEXF 2090P) and 30.78 days (spleen metastasis, MEXF 2090S) and 19.01 days doubling time for the liver metastasis model (MEXF 2090L). The pathohistologic examination revealed a well-differentiated melanoma with low stroma content (3-5%) in all three lines. The molecular analysis (whole exome sequencing) is ongoing. So far the primary tumor has been characterized and two potential driver mutations, Tp53 (R213Q) and Nras (Q61K), were recognized. To characterize the models in more detail, temozolomide was applied to all three lines. 6 mice per group and line were treated either with temozolomide (40 mg/kg/d, iv, twice a week for three weeks) or the control vehicle (10% DMSO, 90% NaCl). The primary tumor depicted statistically significant antitumoral activity with a T/C (test vs control) value of 36% (p< 0.05, t test, two-tailed) two weeks after the last treatment (experiment day 32). The two lines derived of metastases were resistant against the alkylating agent, depicting T/C values of 76% (2090L) and 79% (2090S), respectively. Further characterization of the PDX model and its different sublines will help to elucidate the resistance mechanism behind these data. Spontaneous metastases are a rare event in PDX models growing subcutaneously in NMRI nude mice. Thus, shedding some light into the biology of the metastatic event in the current model will help to understand and influence the metastatic process in general. In any case, those lines can serve as indispensable tools in the oncology drug development pipeline.
Citation Format: Kerstin Klingner, Dorothee Lenhard, Elke Simon, Anne-Lise Peille, Julia Schüler. Establishment and characterization of a melanoma patient-derived xenograft model comprising three different sublines with distinct biologic features abstract. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A019.
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