Abstract
The recent clinical success of immune checkpoint modulators has stimulated immune-oncology research leading to the identification of new tumor immunology targets. However both, target ...validation and drug development need better preclinical immune oncology models. Translational research further urgently needs such models for identification of clinically relevant biomarkers and defining rational combination strategies. Human patient-derived xenografts (PDX) from different tumor indications transplanted on immunodeficient mice have demonstrated strong predictive power for many drug development programs in cancer research. However, one caveat of PDX models is, that these mice lack a functional immune system, which allows tumor engraftment in the xenogenic host. To overcome these constraints our aim is the development of PDX models on mice with a functional human immune system. This strategy should allow implementing the highly predictable PDX in a functional human immune environment for better drug efficacy and safety studies. We reconstituted a human immune system in mice by engrafting human hematopoietic stem cells in immunodeficient mice. We demonstrate the engraftment of a full set of human immune cells, including T cells, B cells, NK cells, monocytes and dendritic cells in these mice. Tumor expression of PD-L1 is currently evaluated as first biomarker for immunotherapy with the PD1 checkpoint inhibitors. We screened our tumor cell lines and PDX models for PD-L1 expression to identify such a correlation. PD-L1 positive and negative models were transplanted on humanized mice. Most of them showed no difference in tumor growth compared to non-humanized mice (= fully immune resistant). However other PDX showed a delayed growth on the humanized mice (= partly immune resistant), whereas only one model did not grow at all (= immune sensitive), demonstrating a high sensitivity to the innate immune response of this PDX. To evaluate the function of the human immune cells and checkpoints, mice were treated with checkpoint inhibitors ipilimumab and nivolumab. Checkpoint inhibitors alone or in combination led to a minor tumor growth delay and an increased number of activated T-cells in the blood and in the tumor. Our results from experiments with n=19 models revealed a first correlation: stronger tumor growth inhibition on PD-L1 positive PDX and increased sensitivity of “partly immune resistant” PDX. Furthermore we were able to demonstrate, that treatment effects of checkpoint inhibitors could be increased by combination with radiation. Summary: Our humanized PDX models enable appropriate preclinical studies on tumor immune biology, evaluation of new immune therapies and combinations, as well as the identification and validation of biomarkers for tumor immune therapy.
Citation Format: Maria Stecklum, Annika Wulf-Goldenberg, Bernadette Brzezicha, Iduna Fichtner, Jens Hoffmann. Humanized immune-oncology mouse models abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1697. doi:10.1158/1538-7445.AM2017-1697
Abstract
Colorectal carcinoma (CRC) is associated with high incidence and mortality rate, particularly if metastasized to distant sites, such as the peritoneum. At time of first diagnosis 4 to 7 ...percent of the patients suffer from peritoneal metastasis (PM) of CRC. The PM is associated with poor prognosis and limited therapeutic options. Therefore, availability of adequate in vivo models for PM will promote the search for novel prognostic or even predictive biomarkers as well as the evaluation of chemosensitivity of PM towards standard, targeted and novel drugs. In this regard such models could be employed for more individualized concepts to improve the therapeutic outcome of PM patients. During the last decade patient-derived xenograft (PDX) mouse models have gained importance, since they closely resemble the molecular and biological features of the original primary tumors. However, until now no PM PDX models have been established from CRC. We therefore focused on the establishment and characterization of a novel CRC PM panel of PDX as useful platform for preclinical studies.
For the PDX establishment colorectal surgical specimens were subcutaneously (s.c.) transplanted onto immunocompromized NOD scid gamma (NSG) mice. The successfully engrafted tumors were transferred to NMRI nu/nu mice for further passaging. Engrafted tumors were characterized by histopathology, immunohistochemistry and gene expression analyses using real-time RT-PCR. Chemosensitivity of PDX models was evaluated in vivo by application of a panel of conventional chemotherapeutic and of targeted drugs.
For PDX establishment 68 CRC surgical specimens were transplanted onto NSG mice. From those, currently 22 PDX have engrafted and are stably growing on NMRI nu/nu mice. From those, 13 models of 10 patients have been analyzed. Their tumor doubling times ranged between 4 to 28 days. The histopathological evaluation revealed maintenance of the original CRC histology in the PDX. The chemosensitivity testing of conventional and of targeted drugs in the 13 PM PDX models revealed the individual, diverging response of the PDX, such as for 5-FU, irinotecan, oxaliplatin, cetuximab, and erlotinib. For selected PDX, orthotopic transplantation into the peritoneum revealed their potential to form disseminated tumors in kidney, ovaries and abdominal diaphragm.
Our results demonstrate, that this novel panel of PDX maintains the morphology of the patient tumor in early passages, reflect heterogeneous response rates, and can be used as preclinical in vivo platform for translational studies of potential clinical use. In conclusion, this PDX panel of PM of the CRC can be used for further studies to evaluate new biomarker and to test novel therapies or combinations for PM of CRC.
Citation Format: Wolfgang Walther, Eva Pachmayr, Bernadette Brzezicha, Britta Büttner, Beate Rau, Ulrike S. Stein. Patient-derived xenograft models from peritoneal metastasis of colorectal carcinoma as novel platform for biomarker analysis and drug testing abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3853. doi:10.1158/1538-7445.AM2017-3853
Abstract
Background
The preclinical evaluation of novel immune checkpoint modulators is dependent on models with functional human immune cells. In previous experiments, we have demonstrated, that we ...can use either peripheral blood mononuclear cells (PBMC), subset of immune cells like NK cells or hematopoietic stem cells (HSC) to establish a humanized immune system on highly immunodeficient 1st and 2nd generation NOG mice with functional T-, B-, and NK cells, as well as monocytes and dendritic cells. Furthermore, we determined PD-L1 expression as a predictive marker and target for immunotherapy on different patient-derived xenografts (PDX). By co-transplantation of rare PDX, like leukemia or Nuclear-protein-in-testis (NUT) carcinoma, we successfully generated a fully human tumor-immune-cell model in mice. Finally, we evaluated the functionality of the model by the treatment with checkpoint inhibitors like Ipilimumab (Ipi), Nivolumab (Nivo) or Pembrolizumab (Pembro). In parallel, we investigated the functionality of the human immune cells and evaluated concepts for combination therapies i.e. with chemotherapy or radiation.
Methods
HSC-humanized mice were generated by i.v. HSC transplantation and engraftment of immune cells was monitored by FACS analysis. For PBMC-humanized mice, immune cells were implanted i.v.. PDX models from different entities (leukemia or NUT carcinoma) were transplanted on HSC-humanized (HIS) mice and treated with Ipi, Nivo and Pembro alone or in combination with radiation. Blood and tumor samples were analysed by FACS for immune cell infiltration and activation.
Results
The transplanted HSC showed engraftment in mice with proliferation and differentiation. 14 weeks after HSC inoculation up to 20% of the human immune cells in the blood were T-cells, characterized by a high PD-1 expression. We have transplanted PDX from different tumor entities on HSC-humanized mice. Most of the investigated PDX (>70%) successfully engrafted on humanized mice and showed no significant difference in tumor growth compared to growth on non-humanized mice. However, for some PDX we observed a delayed tumor growth or a complete rejection. Engraftment delay seems to correlate with the PD-L1 expression of PDX (the higher PD-L1, then the higher growth delay). Furthermore, our results demonstrate the functionality of the engrafted human immune cells against some PDX. Treatment with Ipi, Nivo or Pembro led to a minor tumor growth delay. Response to checkpoint inhibitors showed a correlation to innate immune response and PD-L1 expression of PDX and could further be increased by combination with radiotherapy.
Conclusions
Our humanized immune-PDX models enable appropriate preclinical translational research on tumor immune biology and the evaluation of new therapies and combinations, as well as the identification and validation of biomarkers for immune therapy, especially in rare PDX models.
Citation Format: Maria Stecklum, Annika Wulf-Goldenberg, Bernadette Brzezicha, Wolfgang Walther, Jens Hoffmann. Preclinical models for translational immuno-oncology research: Rare patient-derived xenografts on humanized mice. 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 5204.
BackgroundThe preclinical evaluation of many novel immune therapies requires the use mouse models with a functional human immune system. In previous studies we have demonstrated that either ...peripheral blood mononuclear cells (PBMCs) or subpopulations of PBMCs such as T- and NK-cells or hematopoietic stem cells (HSC) can be used to establish a humanized immune system in immunodeficient mice system with functional T-, B-, and NK cells as well as monocytes and dendritic cells. By transplanting either cell-line or patient-derived tumor xenografts into humanized mice, we successfully generated a fully human tumor-immune-cell model for several tumor entities. Finally, we validated the functionality of these models using either immune-checkpoint inhibitors, cell therapies, or immune cell engagers.MethodsHSC-humanized mice were generated by i.v. injection of CD34+ stem cells into immunodeficient NOG mice. PBMCs or enriched T- or NK-cell populations from a curated set of blood donors were used to humanize mice by either single or multiple i.v. injections. CDX and PDX models from different entities (i.e. lymphoma, neuroblastoma, and breast cancer) were transplanted into these humanized mice which were used to evaluate novel immune therapy options. The presence of immune cells and their activation status was analyzed by flow cytometry in blood and tumor samplesResultsInjected HSCs successfully engrafted and established a functional human immune system with proliferating and differentiating immune cell populations. Fourteen weeks after injection, up to 20% of the human immune cells in the blood were functional T-cells. Several CDX and PDX models successfully engrafted in these humanized mice without significant differences regarding tumor growth compared to non-humanized mice. Checkpoint inhibitor treatment led to tumor growth delay in selected models and flow cytometry analysis of tumor samples revealed a high number of tumor infiltrating T-cells. A comparison of checkpoint inhibitor activity in a pancreatic cancer PDX model using four different humanized mouse models in parallel (HSC, PBMC, T-or NK cell humanized) revealed most convincing results in terms of tumor growth inhibition for the HSC humanized model.ConclusionsWe have established fully human tumor-immune-cell models for different tumor entities in combination with different donor derived immune cell subsets as effector cells and have demonstrated successful long term engraftment of HSCs. All models have been used for the evaluation of either novel checkpoint inhibitors, cell therapies or immune cell engagers and will allow preclinical and translational studies for the identification of novel therapy options, drug combinations and biomarkers.
Abstract
Background: Acute leukemias and lymphomas encompass a diverse group of hematologic malignancies, posing significant challenges in clinical practice. They often exhibit resistance to ...standard-of-care (SoC) treatments and have a high recurrence rate. Recent advancements in molecular profiling have identified potential drivers for various leukemia and lymphoma subtypes, offering new therapeutic targets. However, the validation of these targets and the development of drugs rely heavily on preclinical models that faithfully represent the different clinical subtypes. These models play a crucial role in refining target selection and facilitating drug development projects.
Methods: AML- and ALL-PDX were derived from bone marrow aspirates or peripheral blood samples, from primary or relapsed acute leukemia patients. Purified cells were transplanted either intravenously (i.v.) and/or subcutaneously (s.c.) into immunodeficient mice. Some mice developed a systemic AML, which was monitored by flow cytometric analysis of blood samples. Non-Hodgkin (NHL) - or Hodgkin lymphoma (HL)-PDX were derived from peripheral blood, lymph node extirpations or core needle biopsies, and were usually transplanted subcutaneously into immunodeficient mice.
Results: More than 45 PDX models from AML, ALL, NHL and HL have been successfully established and characterized. Highly individual response rates to SoC and investigational drugs were observed and correlated with mutation and gene expression data. An individual Human Leucocyte Antigen (HLA) profile of each PDX was determined by RNA-seq and a comprehensive HLA matching analysis of the models and peripheral blood mononuclear cell (PBMC) donors was performed to enable personalized, preclinical immuno-oncology studies.
Conclusions: Our extensively characterized PDX models derived from hematologic malignancies serve as valuable tools for evaluating novel targeted and immunological therapies. These models provide an exceptional platform for identifying and validating new targets, as well as preclinical screening of compounds and combinations for translational research projects in this field.
Citation Format: Antje Siegert, Bernadette Brzezicha, Stephan Fuhrmann, Martin Janz, Clemens Schmitt, Ulrich Keller, Christian Rupp, Wolfgang Walther, Jens Hoffmann. Patient-derived xenograft models from hematological malignancies for preclinical drug development and biomarker research abstract. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A081.
Abstract
Background The preclinical evaluation of novel immune therapies demands humanized mouse models with functional human immune cells. In previous studies we have demonstrated, that either ...peripheral blood mononuclear cells (PBMC), subsets of PBMCs like T- and NK-cells or hematopoietic stem cells (HSC) can be used to establish a humanized immune system with functional T-, B-, and NK cells, as well as monocytes and dendritic cells in immunodeficient mice. By transplantation of cell-line-derived (CDX) or patient-derived (PDX) tumor xenografts on humanized mice, we successfully generated a full human tumor-immune-cell model for different tumor entities. Finally, we validated the functionality of these models using checkpoint inhibitors like Ipilimumab (Ipi), Nivolumab (Nivo), Pembrolizumab (Pembro), cell therapies and immune cell engagers.
Methods HSC-humanized mice were generated by i.v. transplantation of CD34+stem cells to immunodeficient NOG mice. PBMC or isolated T- or NK-cell preparations were used to humanize mice by single or multiple i.v. injections. CDX and PDX from different entities (i.e. lymphoma, neuroblastoma, and breast cancer) were transplanted on those humanized mice. These models were used to evaluate novel immune therapies. Blood and tumor samples were analysed by FACS for immune cell infiltration and activation.
Results The transplanted HSCs engrafted in mice and established a functional human immune system with proliferation and differentiation. 14 weeks after HSC inoculation up to 20% of the human immune cells in the blood were functional T-cells, characterized by a high PD-1 expression. The selected CDX and PDX tumors successfully engrafted on humanized mice without significant differences in tumor growth compared to non-humanized mice. Checkpoint inhibitor treatments induced tumor growth delay in selected models. FACS analysis of tumors revealed an increased percentage of tumor infiltrating T-cells. We identified a set of CDX and PDX models without interference with parallel injection of PBMC, T- or NK-cell preparations for the evaluation of immune cell engagers and other cell therapies.
Conclusions We established human tumor-immune-cell models of different entities using CDX or PDX in combination with different donor derived immune cell subsets as effector cells. We demonstrated successful engraftment of HSC on immunodeficient mouse strains, generating mice with a functional human hematopoiesis. These models have been employed for preclinical evaluation of novel checkpoint inhibitors, cell therapies and immune cell engagers. Our models allow preclinical, translational studies on tumor immune biology as well as evaluation of new therapies, drug combinations and biomarker identification and validation.
Citation Format: Maria Stecklum, Annika Wulf-Goldenberg, Bernadette Brzezicha, Wolfgang Walther, Jens Hoffmann. Humanized mouse models for preclinical evaluation of novel immune cell therapies, checkpoint inhibitors, and immune cell engagers 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 1650.
Abstract
For patients with colorectal cancer (CRC) peritoneal metastases (PM) represent a terminal tumor stage with limited therapeutic options. To increase therapeutic efficacy and overall survival, ...availability of appropriate patient-derived 3D cell culture (PD3D) and patient-derived xenografts (PDX) models of PM could help improving the predictability of drug response for a specific tumor, but also foster the identification of novel biomarkers and therapeutic targets for CRC-PM patients. In this context, we generated the first scaffold-based PD3D and matching PDX models of CRC-PM as platform to test for chemotherapy response and to identify novel biomarkers.
For model establishment, surgical specimens were processed either for PD3D cell culture or transplanted subcutaneously (s.c.) onto immunocompromized NOD scid gamma (NSG) mice. For 3D cell culture models, tissue was dissected, digested and filtered before embedding into a scaffold matrix. For PDX models engrafted tumors were transferred to NMRI nu/nu mice for further passaging. They were characterized by histopathology, immunohistochemistry and gene expression analyses using real-time RT-PCR. Chemosensitivity of both sibling models was evaluated on a panel of conventional chemotherapeutic and of targeted drugs.
The same panel of drugs was used in 15 matched PD3D/PDX models and revealed individual response patterns both in PD3D and PDX. Most interestingly, different drug response pattern was observed in models derived from tumor tissue of the omentum vs. tissue from the peritoneum of the same patient.
Our results demonstrate, that matched models maintain basic characteristics such as the morphology of the patient tumor in early passages, reflect heterogeneous response rates, and can be used as preclinical platform for translational studies of potential clinical use.
Citation Format: Christian RA Regenbrecht, Guido Gambara, Eva Pachmayr, Alessandra Silvestri, Mathias Dahlmann, Bernadette Brzezicha, Britta Buettner, Beate Rau, Ulrich Keilholz, Urike Stein, Wolfgang Walther. Novel patient-derived 3D (PD3D) cell models and matched patient-derived xenografts (PDX) from peritoneal metastasis of colorectal cancer for drug testing and biomarker analysis 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 4099.
Abstract
Background: Glioblastoma (GBM) is the most common malignant brain tumor in adults, with about 90% of tumors developing de novo. Their heterogeneity, aggressiveness and infiltrative growth ...limit success of current standard of care (SoC) therapy and efficacy of new therapeutic approaches. For drug development a large panel of patient-derived tumor cell models is needed to generate most comparable in vitro and in vivo platform reflecting the complex biology of glioblastoma. Glioblastoma cell lines from patient-derived xenografts (PDX) preserve many patient specific characteristics and to perform the essential steps of pre-clinical drug development from in vitro screening to orthotopic in vivo approaches under conditions, which closely resemble the clinical situation. Further, GBM PDX models are useful for immune oncology research.
Methods: A panel of 26 glioblastoma PDX models was established on immunodeficient mice (15 out of these established orthotopically). All models were characterized for drug sensitivity and molecular profile using panel and transcriptome sequencing. From these, three corresponding tumor cell lines were successfully established and characterized. Quality and identity of the models was performed by FACS and PCR analysis. Expression profile, drug sensitivity and growth behavior was determined and compared in vitro and after re-transplantation in vivo with the original PDX glioma model. In addition, we employed GBM PDX models for checkpoint-inhibitor sensitivity in humanized mouse settings.
Results: Drug testing was performed in s.c. and orthotopic models revealed, that best treatment responses in s.c. models (tumor growth inhibition > 50%) were observed for SoC temozolomide (TMZ), irinotecan and bevacizumab. Molecular characterization identified all our models as IDH-wt (R132) with frequent mutations in PARP1, EGFR, TP53, FAT1, and within the PI3K/AKT/mTOR pathway. Their expression profiles resemble proposed mesenchymal, proneural and classical GBM molecular subtypes. Further, treatment of GBM PDX with ipilimumab, nivolumab or pembrozolumab generated minor growth delay.
Conclusions: In drug sensitivity screening, irinotecan or bevacizumab were identified as alternative treatment options in TMZ resistant GBM PDX models. Our data demonstrate, that the established platform of s.c. and orthotopic GBM PDX is valuable for drug development and can well be complemented by a PDX-derived cell lines for in vitro screens. Furthermore, these models can be used for the evaluation of new immune-oncology therapies.
Citation Format: Joshua Alcaniz, Lars Winkler, Maria Stecklum, Hagen Wieland, Antje Siegert, Michael Becker, Bernadette Brzezicha, Wolfgang Walther, Jens Hoffmann. Patient-derived xenograft (PDX) and corresponding cell line models from glioblastoma for drug development, immuno-oncology and translational research. 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 4674.
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Background: Generation of patient-derived models for breast cancer has been difficult. In other histologies, success was higher in refractory disease. With the goal to develop a ...protean resource for testing novel compounds and combinations for treatment refractory breast cancer, we establish patient-derived murine xenograft models (PDX) from tumors clinically unresponsive to at least anthracyclin, platin and taxane. Here we report phenotypic and drug testing data of our established breast cancer models. Methods: On immunodeficient NOG mice, PDX were engrafted from breast cancer tissue samples. The tissues were obtained from patients with disease progression after chemotherapy with three to four drugs. The established PDX were characterized by immunohistochemistry and the PDX and primary tumor are currently undergoing exome and transcriptome sequencing. The PDX were tested for response to all standard chemotherapy agents. The drug testing included platin, taxane, anthracycline, 5-FU, Everolimus, Eribulin and depending on the subtype tamoxifen, olaparib, and a CDK4/6 Inhibitor. Results: Since May 2017 30 breast cancer samples have been processed under rapid and stringent conditions. Currently five models, three TNBC and two hormone receptor positive PDX, have been fully engrafted, phenotypically compared and drug tested. The immunohistochemistry (estrogen/progesterone/androgen/Her2 receptor, Ki-67, CK5/6) of original tumor and PDX were correlative. All patients had shown clinical resistance to platin, anthracyclines, and taxanes in the neoadjuvant or palliative setting. Concordant with the clinical resistance, the PDX models showed only limited or transient sensitivity to single agents. Six more PDX, three TNBC, two HR+ and one Her2+, are currently in various states of engraftment and testing. Conclusions: With our stringent approach, we successfully generated PDX models in 15%, which may rise to 30% with the ongoing models. Phenotype between patient tumors and PDX was consistent. The minor differences in responsiveness to chemotherapy may be due to differences in stromal factors. In summary, the PDX of refractory tumors are a versatile resource for preclinical studies of novel treatment approaches.
Abstract
Acute leukemia, a hematological malignancy, is characterised by a rapid increase of functionally immature blood cells. Acute Myeloid Leukemia (AML) and Acute Lymphoblastic Leukemia (ALL) are ...distinguished by their cells of origin. If untreated, AML and ALL are life-threatening and patients show different responses to current standards of care (SoC) therapy. The majority of acute leukemias are genetically heterogeneous and therefore a challenge for the development of new targeted therapies. There is a need to develop robust animal models to investigate leukemia in vivo. Patient-derived xenograft (PDX) mouse models recapitulate many clinical features of cancer. They have proven to be useful tools for the evaluation of novel therapeutic strategies and biomarkers, and for the study of cancer biology.
The aim of our work was to establish PDX models of AML and ALL and to characterize their sensitivity to different therapeutic agents.
Bone marrow aspirates were obtained from primary and relapsed AML/ALL patients and were intravenously (i.v.) and/or subcutaneously (s.c.) inoculated into immunodeficient mice. Mice were sacrificed when signs of disease were obvious and single cell suspensions of spleens (i.v.) or tumor fragments (s.c.) were transferred to new recipient mice. Chemosensitivity to SoC treatments was tested.
In our study several AML-PDX and ALL-PDX were established. These models show different sensitivity to SoC treatment. The models will be used for further investigations like gene expression or mutation analysis or biomarker identification.
The established PDX models of AML and ALL are suitable tools for preclinical drug development. In order to cover the complex heterogeneity of acute leukemias, the establishment of further PDX models is currently in progress.
Citation Format: Antje Siegert, Bernadette Brzezicha, Annika Wulf-Goldenberg, Iduna Fichtner, Jens Hoffmann. Establishment of patient-derived xenograft models of acute leukemias. abstract. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B35.
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