Development of personalised human immunocompetent ex vivo models of primary and secondary liver cancers using precision cut tissue slice technology

Background: In the treatment of clear cell renal cell carcinoma (ccRCC), nivolumab is an established component of the first-line therapy with a favorable impact on progression free survival and overall survival. However, treatment-related adverse effects occur and, to date, there is no approved predictive biomarker for patient stratification. Thus, the aim of this study was to establish an ex vivo tissue slice culture model of ccRCC and to elucidate the impact of nivolumab on tumor infiltrating immune cells. Methods: Fresh tumor tissue of ccRCC was treated with the immune checkpoint inhibitor nivolumab using ex vivo tissue slice culture (TSC). After cultivation, tissue slices were formalin-fixed, immunohistochemically stained and analyzed via digital image analysis. Results: The TSC model was shown to be suitable for ex vivo pharmacological experiments on intratumoral immune cells in ccRCC. PD1 expression on tumor infiltrating immune cells was dose-dependently reduced after nivolumab treatment (p < 0.01), whereas density and proliferation of tumor infiltrating T-cells and cytotoxic T-cells were inter-individually altered with a remarkable variability. Tumor cell proliferation was not affected by nivolumab. Conclusions: This study could demonstrate nivolumab-dependent effects on PD1 expression and tumor infiltrating T-cells in TSC of ccRCC. This is in line with results from other scientific studies about changes in immune cell proliferation in peripheral blood in response to nivolumab. Thus, TSC of ccRCC could be a further step to personalized medicine in terms of testing the response of individual patients to nivolumab.

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Ex vivo tissue slice culture system to measure drug-response rates of hepatic metastatic colorectal cancer

BMC Cancer ◽

10.1186/s12885-019-6270-4 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 1

Author(s):

Steve Z. Martin ◽

Daniel C. Wagner ◽

Nina Hörner ◽

David Horst ◽

Hauke Lang ◽

...

Keyword(s):

Colorectal Cancer ◽

Colorectal Carcinoma ◽

Metastatic Colorectal Cancer ◽

Drug Response ◽

Tumor Tissue ◽

Ex Vivo ◽

Tissue Slice ◽

Patient Specific ◽

Slice Culture ◽

Tissue Slices

Abstract Background The lack of predictive biomarkers or test systems contributes to high failure rates of systemic therapy in metastasized colorectal carcinoma, accounting for a still unfavorable prognosis. Here, we present an ex vivo functional assay to measure drug-response based on a tissue slice culture approach. Methods Tumor tissue slices of hepatic metastases of nine patients suffering from colorectal carcinoma were cultivated for 72 h and treated with different concentrations of the clinically relevant drugs Oxaliplatin, Cetuximab and Pembrolizumab. Easy to use, objective and automated analysis routines based on the Halo platform were developed to measure changes in proliferative activity and the morphometric make-up of the tumor. Apoptotic indices were assessed semiquantitatively. Results Untreated tumor tissue slices showed high morphological comparability with the original “in vivo”-tumor, preserving proliferation and stromal-tumor interactions. All but one patients showed a dosage dependent susceptibility to treatment with Oxaliplatin, whereas only two patients showed responses to Cetuximab and Pembrolizumab, respectively. Furthermore, we identified possible non-responders to Cetuximab therapy in absence of RAS-mutations. Conclusions This is the first time to demonstrate feasibility of the tissue slice culture approach for metastatic tissue of colorectal carcinoma. An automated readout of proliferation and tumor-morphometry allows for quantification of drug susceptibility. This strongly indicates a potential value of this technique as a patient-specific test-system of targeted therapy in metastatic colorectal cancer. Co-clinical trials are needed to customize for clinical application and to define adequate read-out cut-off values.

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Glioblastoma Tissue Slice Tandem-Cultures for Quantitative Evaluation of Inhibitory Effects on Invasion and Growth

Cancers ◽

10.3390/cancers12092707 ◽

2020 ◽

Vol 12 (9) ◽

pp. 2707

Author(s):

Vasile Sidorcenco ◽

Luisa Krahnen ◽

Marion Schulz ◽

Janina Remy ◽

Donat Kögel ◽

...

Keyword(s):

Brain Tissue ◽

Ex Vivo ◽

Single Cells ◽

Tissue Slice ◽

In Vivo Studies ◽

Tumor Xenograft ◽

Normal Brain ◽

Inhibitory Effects ◽

Tumor Mass

Glioblastomas (GBMs) are the most malignant brain tumors and are essentially incurable even after extensive surgery, radiotherapy, and chemotherapy, mainly because of extensive infiltration of tumor cells into the adjacent normal tissue. Thus, the evaluation of novel drugs in malignant glioma treatment requires sophisticated ex vivo models that approach the authentic interplay between tumor and host environment while avoiding extensive in vivo studies in animals. This paper describes the standardized setup of an organotypic brain tissue slice tandem-culture system, comprising of normal brain tissue from adult mice and tumor tissue from human glioblastoma xenografts, and explore its utility for assessing inhibitory effects of test drugs. The microscopic analysis of vertical sections of the slice tandem-cultures allows for the simultaneous assessment of (i) the invasive potential of single cells or cell aggregates and (ii) the space occupying growth of the bulk tumor mass, both contributing to malignant tumor progression. The comparison of tissue slice co-cultures with spheroids vs. tissue slice tandem-cultures using tumor xenograft slices demonstrates advantages of the xenograft tandem approach. The direct and facile application of test drugs is shown to exert inhibitory effects on bulk tumor growth and/or tumor cell invasion, and allows their precise quantitation. In conclusion, we describe a straightforward ex vivo system mimicking the in vivo situation of the tumor mass and the normal brain in GBM patients. It reduces animal studies and allows for the direct and reproducible application of test drugs and the precise quantitation of their effects on the bulk tumor mass and on the tumor’s invasive properties.

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292 Ex vivo profiling of PD-1 blockade using an organotypic tissue slice model in solid tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.292 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A316-A316

Author(s):

Lina Ding ◽

Kristin Sullivan ◽

Chensheng Zhou ◽

Jimena Trillo-Tinoco ◽

Anne Lewin ◽

...

Keyword(s):

T Cell ◽

Tumor Microenvironment ◽

Cancer Immunotherapy ◽

Ex Vivo ◽

Tissue Slice ◽

Mechanisms Of Resistance ◽

Original Tumor ◽

Organotypic Tissue ◽

The Impact ◽

Secretome Profiling

BackgroundTumor explant models provide a powerful ex vivo tool to evaluate complex biological mechanisms in a controlled environment. Ex vivo models retain much of the original tumor biology, heterogeneity, and tumor microenvironment, and therefore provide a useful preclinical platform and functional approach to assess drug responses rapidly and directly.MethodsTo explore mechanisms of resistance to cancer immunotherapy, we established an organotypic tissue slice Air-Liquid Interface (ALI) ex vivo system utilizing surgical tumor specimens from patients to assess the impact of the clinically utilized anti-PD-1 antibody nivolumab (OPDIVO). In the present study, we built a real-world patient cohort comprised of six tumor types: non-small cell lung cancer, melanoma, pancreatic ductal adenocarcinoma, breast cancer, prostate cancer, and colorectal cancer. We assessed tissue morphology, histology, PD-L1 IHC (CPS and TPS), CD8 T cell topology, proliferation in the tumor and stromal compartments, and secretome profiling.ResultsOur tumor slice model highly recapitulated features of the original tumor, including tumor architecture, immune phenotypes, and the prognostic markers. To identify responses to aPD-1 treatment, we compared baseline values for the cultured tumor slices with values at different timepoints post treatment. Secretome profiling of tissue explant supernatants using a panel of 94 analytes, revealed alterations to cytokines produced in the tumor microenvironment in response to aPD-1 treatment. We found that soluble expression patterns were associated with T-cell patterns (inflamed, excluded and desert) and PD-L1 score (CPS and TPS) in tumor tissues. These cytokines mediate critical functions across the immune cell cycle. Ongoing efforts to characterize T cell activation, exhaustion, tumor intrinsic responses and microenvironment composition using Imaging Mass Cytometry will be presented.ConclusionsIn this study, we demonstrated the feasibility of using fresh, surgically resected human tumors to test aPD-1 responses in an ex vivo system. Further, this model system has the potential to drive discovery and translational efforts by evaluating mechanisms of resistance to cancer immunotherapy and evaluate new single agent or combination therapies in the ex vivo setting.

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The investigation of virus-driven intrahepatic cholangiocarcinoma through dysregulated hepatic differentiation

Access Microbiology ◽

10.1099/acmi.ac2020.po0821 ◽

2020 ◽

Vol 2 (7A) ◽

Author(s):

Alexandria Kilvington ◽

Laura Matthews ◽

Stephen Griffin

Keyword(s):

Gene Expression ◽

Intrahepatic Cholangiocarcinoma ◽

Ex Vivo ◽

Expression Patterns ◽

Gene Expression Pattern ◽

Induced Pluripotent Stem Cell ◽

Hcv Infection ◽

Hepatic Differentiation ◽

Specific Differentiation ◽

Liver Cancers

Intrahepatic Cholangiocarcinoma (iCCA), bile duct cancer, is increasing in incidence worldwide. Infection with hepatitis C virus (HCV) is a known risk factor for developing iCCA. Recent studies of HCV associated hepatocellular carcinoma (HCC) have demonstrated that HCV infection induces oncogenic gene expression patterns resulting from altered epigenetic profiles. Importantly, whilst ∼90% of patients treated with new direct acting antivirals (DAAs) attain a sustained virological response, the risk of HCC and other malignancies is not reduced by the same extent as previous interferon therapies. We, and others, have shown that this may be related to an imprinted gene expression pattern persisting post-treatment. The cellular origin of primary liver cancers can vary due to liver cell plasticity. However, the incidence of mixed iCCA-HCC tumour phenotypes and the dual iCCA/HCC risk associated with HCV infection led us to hypothesise that virus-infected hepatic progenitor cells (HPC) might be the source of virus-driven malignancies. Accordingly, we demonstrated that adult HPCs were susceptible to HCV infection ex vivo. Moreover, models of hepatic differentiation revealed that HCV disrupts this process via hijacking the HIPPO signalling pathway with oncogenic hallmarks persisting following viral cure. To explore HCV-induced alterations during cholangiocyte-specific differentiation, we have chosen to exploit the immortalised non-transformed HPC line, HepaRG and induced pluripotent stem cell derived HPCs. We will describe endeavours to develop a robust model of HCV-mediated perturbation of cholangiocyte-specific differentiation in order to identify new treatments that complement DAA therapy in order to eliminate the risk of malignancy.

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Correlation of γ-radioactivity and Scanning Electron Microscopy in measurement of retention of 111Indium-labeled canine endothelial cells on synthetic vascular grafts

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156419 ◽

1989 ◽

Vol 47 ◽

pp. 886-887

Author(s):

E.J. Prendiville ◽

S. Laliberté Verdon ◽

K. E. Gould ◽

K. Ramberg ◽

R. J. Connolly ◽

...

Keyword(s):

Blood Flow ◽

Ex Vivo ◽

Vascular Grafts ◽

Retention Data ◽

Vascular Prostheses ◽

Group 4 ◽

Human Fibronectin ◽

Insufficient Time ◽

Group 3 ◽

Group 2

Endothelial cell (EC) seeding is postulated as a mechanism of improving patency in small caliber vascular grafts. However the majority of seeded EC are lost within 24 hours of restoration of blood flow in previous canine studies . We postulate that the cells have insufficient time to fully develop their attachment to the graft surface prior to exposure to hemodynamic stress. We allowed EC to incubate on fibronectin-coated ePTFE grafts for four different time periods after seeding and measured EC retention after perfusion in a canine ex vivo shunt circuit.Autologous canine EC, were enzymatically harvested, grown to confluence, and labeled with 30 μCi 111 Indium-oxine/80 cm 2 flask. Four groups of 5 cm x 4 mm ID ePTFE vascular prostheses were coated with 1.5 μg/cm.2 human fibronectin, and seeded with 1.5 x 105 EC/ cm.2. After seeding grafts in Group 1 were incubated in complete growth medium for 90 minutes, Group 2 were incubated for 24 hours, Group 3 for 72 hours and Group 4 for 6 days. Grafts were then placed in the canine ex vivo circuit, constructed between femoral artery and vein, and subjected to blood flow of 75 ml per minute for 6 hours. Continuous counting of γ-activity was made possible by placing the seeded graft inside the γ-counter detection crystal for the duration of perfusion. EC retention data after 30 minutes, 2 hours and 6 hours of flow are shown in the table.

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