Name: patient-derived tumor explants as a "live" preclinical platform for predicting drug resistance in patients
Uploaded: 2021-02-07T14:30:00
Description: Watch this Scientific Journal Video about Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients at JoVE.com

We thank the surgeons and pathologists at University Hospitals of Leicester NHS Trust for providing surgical resected tumor tissue. We also thank the Histology facility within Core Biotechnology Services for help with tissue processing and sectioning of FFPE tissue blocks and Kees Straatman for support with use of the Vectra Polaris. This research was supported and funded by the Explant Consortium comprising four partners: The University of Leicester, The MRC Toxicology Unit, Cancer Research UK Therapeutic Discovery Laboratories, and LifeArc. Additional support was provided by the CRUK-NIHR Leicester Experimental Cancer Medicine Centre (C10604/A25151). Funding for GM, CD, and NA was provided by Breast Cancer Now&#39;s Catalyst Programme (2017NOVPCC1066), which is supported by funding from Pfizer.

1. Tissue collection
<ol>
	<li>After surgery, transfer freshly resected human tumor specimens into a tube containing 25 mL of fresh culture medium (Dulbecco&#8217;s modified Eagle medium supplemented with 4.5 g/L glucose and L-glutamine&#160;+ 1% (v/v) fetal calf serum + 1% penicillin&#8211;streptomycin) and stored on ice. Process the explant within 2 h of surgery in a sterile class II hood.</li>
</ol>
2. Explant preparation
<ol>
	<li>Clean al...

This paper describes the methods for generation, drug treatment, and analysis of PDEs and highlights the advantages of the platform as a preclinical model system. Ex vivo&#160;culturing of a freshly resected tumor, which does not involve its deconstruction, allows for the retention of the tumor architecture13,24 and thus, the spatial interactions of cellular components in the TME as well as intratumoral heterogeneity. This method demonstrates how, by using a tumo...

The development of effective and safe anticancer agents requires appropriate preclinical models that can also provide insight into mechanisms of action that can facilitate the identification of predictive and pharmacodynamic biomarkers. Inter- and intratumor heterogeneity1,2,3,4,5 and the TME6,7,8,9,10,11,12 are known to influence anticancer drug responses, and many existing preclinical cancer models such as cell lines, organoids, and mouse models are not able to fully accommodate these crucial features. An &#34;ideal&#34; model is one that can recapitulate the complex spatial interactions of malignant with non-malignant cells within tumors as well as reflect the regional differences within tumors. This article focuses on PDEs as an emerging platform that can fulfil many of these requirements13.
The first example of the use of human PDEs, also known as histocultures, dates back to the late 1980s when Hoffman et al. generated slices of freshly resected human tumors and cultured them in a collagen matrix14,15. This involved establishing a 3D culture system that preserved tissue architecture, ensuring the maintenance of stromal components and cell interactions within the TME. Without deconstructing the original tumor, Hoffman et al.16 heralded a new approach of translational research, and since this time, many groups have optimized different explant methods with the aim of preserving the tissue integrity and generating accurate drug response data17,18,19,20,21,22,23,24, although some differences between protocols are evident. Butler et al. cultured explants in gelatin sponges to help the diffusion of nutrients and drugs through the specimen20,21,25, whereas Majumder et al. created a tumor ecosystem by culturing explants on top of a matrix composed of tumor and stromal proteins in the presence of autologous serum derived from the same patient22,23.
More recently, our group set up a protocol whereby explants are generated by fragmentation of tumors into 2 - 3 mm3-sized pieces that are then placed without additional components on permeable membranes at the air-liquid interface of a culture system24. Taken together,&#160;these numerous studies have demonstrated that PDEs allow the culture of intact, live fragments of human tumors that retain the spatial architecture and regional heterogeneity of the original tumors. In original experiments, explants or histocultures were usually subjected to homogenization following drug treatment, after which various viability assays were applied to the homogenized samples such as the histoculture drug response assay20,21, the MTT (3-(6)-2,5-diphenyltetrazolium bromide) assay, the lactate dehydrogenase assay, or the resazurin-based assay26,27,28. Recent progress in endpoint analysis techniques, particularly digital pathology, have now expanded the repertoire of endpoint tests and assays that can be performed on explants29,30. To apply these new technologies, instead of homogenization, explants are fixed in formalin, embedded in paraffin (FFPE) and then analyzed using immunostaining techniques, allowing spatial profiling. Examples of this approach have been documented for non-small cell lung cancer (NSCLC), breast cancer, colorectal cancer, and mesothelioma explants whereby immunohistochemical staining for the proliferation marker, Ki67, and the apoptotic marker, cleaved poly-ADP ribose polymerase (cPARP), was used to monitor changes in cell proliferation and cell death24,31,32,33,34.
Multiplexed immunofluorescence is particularly amenable for spatial profiling of drug responses in explants at endpoint35. For example, it is possible to measure the relocalization and spatial distribution of specific classes of immune cells, such as macrophages or T cells, within the TME upon drug treatment13,36,37,38, and investigate whether a therapeutic agent can favor the transition from &#34;cold tumor&#34; to &#34;hot tumor&#34;39. In recent years, this group has focused on the derivation of PDEs from different tumor types (NSCLC, renal cancer, breast cancer, colorectal cancer, melanoma) and the testing of a range of anticancer agents including chemotherapies, small-molecule inhibitors, and immune checkpoint inhibitors (ICIs). Endpoint analysis methods have been optimized to include multiplexed immunofluorescence to allow spatial profiling of biomarkers for viability as well as biomarkers for different constituents of the TME.

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			<td>Barnstead NANOpure Diamond</td>
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			<td>Costar Multiple Well Cell Culture Plates</td>
			<td>Corning Incorporated</td>
			<td>3516</td>
			<td>6 multiwell plate</td>
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			<td>DAPI Dilactate</td>
			<td>Life Technologies</td>
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			<td>100 x 17 mm Dish, Nunclon Delta</td>
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			<td>DMEM (1x) Dubelcco&#39;s Modified Eagle Medium + 4.5 g/L D-Glucose + 110 mg/mL Sodium Pyruvate</td>
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			<td>Merck</td>
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			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>3609</td>
			<td>Reagent for TE buffer</td>
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			<td>Eosin</td>
			<td>CellPath</td>
			<td>RBC-0100-00A</td>
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			<td>Foetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10500-064</td>
			<td>For use in tissue culture medium</td>
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			<td>37% Formaldehyde</td>
			<td>Fisher (Acros)</td>
			<td>119690010</td>
			<td>10% Formalin</td>
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			<td>iGenix, microwave oven IG2095</td>
			<td>iGenix</td>
			<td>IG2095</td>
			<td>Microwave used for antigen retreival</td>
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			<td>Industrial methylated spirit (IMS)</td>
			<td>Genta Medical</td>
			<td>199050</td>
			<td>99% Industrial Denatured Alcohol (IDA)</td>
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			<td>InForm Advanced Image Analysis Software</td>
			<td>Akoya Biosciences</td>
			<td>InForm</td>
			<td></td>
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			<td>Leica ASP3000 Tissue Processor</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td>Automated Vacuum Tissue Processor</td>
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			<td>Leica Arcadia H and C</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td>Embedding wax bath</td>
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			<td>Leica RM2125RT</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td>Rotary microtome</td>
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			<td>Leica ST4040 Linear Stainer</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td>H&#38;E stainer</td>
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			<td>Mayer&#39;s Haematoxylin</td>
			<td>Sigma</td>
			<td>GHS132-1L</td>
			<td>Staining reagent</td>
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			<td>Millicell Cell Culture Inserts, 30 mm, hydrophilic PTFE, 0.4 &#181;m</td>
			<td>Merck Milipore</td>
			<td>PICMORG50</td>
			<td>Organotypic culture insert disc</td>
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			<td>Novolink Polymer Detection System</td>
			<td>Leica Biosystems</td>
			<td>RE7150-K</td>
			<td>DAB staining kit</td>
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			<td>OPAL 480</td>
			<td>Akoya Biosciences</td>
			<td>FP1500001KT</td>
			<td>Fluorophore with Dimethyl Sulfoxide (DMSO) diluent</td>
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			<td>OPAL 520</td>
			<td>Akoya Biosciences</td>
			<td>FP1487001KT</td>
			<td>Fluorophore with Dimethyl Sulfoxide (DMSO) diluent</td>
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			<td>OPAL 570</td>
			<td>Akoya Biosciences</td>
			<td>FP1488001KT</td>
			<td>Fluorophore with Dimethyl Sulfoxide (DMSO) diluent</td>
		</tr>
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			<td>OPAL 620</td>
			<td>Akoya Biosciences</td>
			<td>FP1495001KT</td>
			<td>Fluorophore with Dimethyl Sulfoxide (DMSO) diluent</td>
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			<td>OPAL 650</td>
			<td>Akoya Biosciences</td>
			<td>FP1496001KT</td>
			<td>Fluorophore with Dimethyl Sulfoxide (DMSO) diluent</td>
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			<td>OPAL 690</td>
			<td>Akoya Biosciences</td>
			<td>FP1497001KT</td>
			<td>Fluorophore with Dimethyl Sulfoxide (DMSO) diluent</td>
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			<td>OPAL 780 / OPAL TSA-DIG Reagent</td>
			<td>Akoya Biosciences</td>
			<td>FP1501001KT</td>
			<td>Fluorophore with Dimethyl Sulfoxide (DMSO) diluent and TSA-DIG reagent</td>
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			<td>Opal Polymer HRP Ms Plus Rb, 1x</td>
			<td>Perkin Elmer</td>
			<td>ARH1001EA</td>
			<td>HRP polymer</td>
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			<td>Penicillin/streptomycin solution</td>
			<td>Fisher Scientific</td>
			<td>11548876</td>
			<td>For use in tissue culture medium</td>
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			<td>PhenoChart Whole Slide Contextual Viewer</td>
			<td>Akoya Biosciences</td>
			<td>PhenoChart</td>
			<td>Viewer software for scanned images</td>
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			<td>Phosphate Buffered Saline Tablets</td>
			<td>Thermo Scientific Oxoid</td>
			<td>BR0014G</td>
			<td>PBS</td>
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			<td>1x Plus Amplification Diluent</td>
			<td>Perkin Elmer</td>
			<td>FP1498</td>
			<td>Fluorophore diluent</td>
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			<td>Prolong Diamond Antifade Mountant</td>
			<td>Invitrogen</td>
			<td>P36961</td>
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			<td>Slide Carrier</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>To load slides into Slide Carrier Hotel for scanning with Vectra Polaris</td>
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			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>S/3160/63</td>
			<td>10% Formalin</td>
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			<td>Sodium Hydroxide pellets</td>
			<td>Fisher Scientific</td>
			<td>S/4920/53</td>
			<td>Reagent for citrate buffer</td>
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			<td>Tenatex Toughened Wax - Pink (500 g)</td>
			<td>KEMDENT</td>
			<td>1-601</td>
			<td>Dental wax surface</td>
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			<td>Thermo Scientific&#160;Shandon Sequenza Slide Rack for Immunostaining Center</td>
			<td>Fisher Scientific</td>
			<td>10098889</td>
			<td>Holder for slides and slide clips</td>
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			<td>Thermo Scientific Shandon Plastic Coverplates</td>
			<td>Fisher Scientific</td>
			<td>11927774</td>
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			<td>Tris(hydroxymethyl)aminomethane (Tris)</td>
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			<td>VectaShield</td>
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patient-derived tumor explants as a "live" preclinical platform for predicting drug resistance in patients

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable preclinical models that can accurately predict patient responses. One of the disadvantages of existing preclinical models is the inability to contextually preserve the human tumor microenvironment (TME) and accurately represent intratumoral heterogeneity, thus limiting the clinical translation of data. By contrast, by representing the culture of live fragments of human tumors, the patient-derived explant (PDE) platform allows drug responses to be examined in a three-dimensional (3D) context that mirrors the pathological and architectural features of the original tumors as closely as possible. Previous reports with PDEs have documented the ability of the platform to distinguish chemosensitive from chemoresistant tumors, and it has been shown that this segregation is predictive of patient responses to the same chemotherapies. Simultaneously, PDEs allow the opportunity to interrogate molecular, genetic, and histological features of tumors that predict drug responses, thereby identifying biomarkers for patient stratification as well as novel interventional approaches to sensitize resistant tumors. This paper reports PDE methodology in detail, from collection of patient samples through to endpoint analysis. It provides a detailed description of explant derivation and culture methods, highlighting bespoke conditions for particular tumors, where appropriate. For endpoint analysis, there is a focus on multiplexed immunofluorescence and multispectral imaging for the spatial profiling of key biomarkers within both tumoral and stromal regions. By combining these methods, it is possible to generate quantitative and qualitative drug response data that can be related to various clinicopathological parameters and thus potentially be used for biomarker identification.

Multispectral imaging of mIF-stained histological sections permits identification and phenotyping of individual cell populations and identification of tumor and stromal components in the explant TME (Figure 2). Multispectral imaging is particularly useful for the analysis of tissues with high intrinsic autofluorescence, such as tissue with a high collagen content, as it allows the autofluorescence signal to be deconvoluted from other signals and excluded from subsequent analysis. Subsequent ...

Watch this Scientific Journal Video about Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients at JoVE.com

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

This paper describes methods for the generation, drug treatment, and analysis of patient-derived explants for assessing tumor drug responses in a live, patient-relevant, preclinical model system.

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable ...

The patient-derived explants are a short term method providing immediate drug response data that can accurately predict patient outcomes. The PD platforms allows drug responses to be examined in a 3D context that mirrors the pathological and architectural features of the regional tumors as closely as possible. Explants have the potential to offer novel insights into the metabolism and mechanism of action of a drug and to facilitate the identification of new pharmacodynamic biomarkers.Before starting the experiment, clean all surgical equipment with 70%industrial methylated spirits solution. Fill a 10 centimeter culture dish with 25 milliliters of fresh medium on ice. Using tweezers, transfer the specimen onto a dental wax surface And use two skin graft blades to slice the tissue into fragments of approximately two to three cubic millimeters.Transfer the explants into a 10 centimeter dish and place six to nine pieces of the tissue into a one milliliter tube containing 10%non buffered formalin solution for a 24 hour incubation at room temperature. Fill the desired number of wells of a six well plate with 1.5 milliliters of fresh medium per well. And place an organotypic culture insert dish into each well so that it floats on top of the medium.Place six to nine explants onto each insert disk and store for a 16 hour incubation in a 5%carbon dioxide incubator to allow recovery. The next day, add fresh medium and drug to each well of a new plate. Include a well for the vehicle control.When all drug treatments have been prepared, use tweezers to transfer one insert to each well of the new six well plate for a 24 to 48 hour incubation in the carbon dioxide incubator. After the drug treatment, transfer the discs into new six well plates containing 10%formalin. And add a few drops of 10%formalin to the top of each explant to ensure complete coverage with the fixative for 24 hours of incubation at room temperature.The next day, soak an appropriate number of small histology sponges in 70%industrial methylated solution. And place the sponges inside histology cassettes. When all sponges have been placed, transfer all the explants from the same drug treatment condition onto a single sponge with the drug treated side of each explant contacting the inserted disc.Place another presoaked sponge on top of each explant and close the cassette to secure the explants in the cassette. Then submerge the cassettes in 70%industrial methylated solution before proceeding with the histological processing. After scanning, perform a training analysis and load the scans containing the stamps for training into the appropriate analysis software.In single mode view, navigate through the images and select the scans to analyze and the scan with intrinsic fluorescence. with the intrinsic fluorescence scan on a single view, click the autofluorescence picker to draw a segment on an area of unstained tissue. Click edit markers and colors and enter the marker names in the box associated with each fluorophore.Click prepare images to allow the software to subtract the intensity of intrinsic fluorescence from all the uploaded images. Click the segment tissue step on top of the screen to open the tissue segmentation training window. In the tissue categories panel, enter the name of each tissue category to be segmented.Click draw, to draw regions around groups of cells in the tissue category of interest. Then switch to the next tissue category and draw new regions. When all the training regions have been defined, use train tissue segmenter, and wait for the training accuracy to settle.Possibly to a value close to 100%Click segment all to segment the tissues. After verifying the quality of the tissue segmentation, click cell segmentation and select the cellular compartments to segment. To configure the nuclear component, use the typical intensity slider to adjust the threshold used to detect the nuclear pixels.Live feedback is provided by the preview window. To split the clusters of nuclear pixels, in the nuclear components splitting panel, select the button that best describes the staining quality of the nuclei and use the slider bar to adjust the splitting sensitivity. Then click segment all to segment all of the images.When all of the images have been segmented, in the phenotypes panel, add the list of phenotypes to be detected and enter the name of the phenotypes into the corresponding text box. Click edit phenotypes to select a particular cell and select the corresponding phenotype in the dropdown menu. After completing the selection for training, click the train classifier to evaluate the result of each phenotype.Proceed with phenotype all, save the project and click export. Select the batch analysis tab and load the project in the batch algorithm or project box. To export the images and tables, select all of the boxes in the export options, click add slides and load all the images containing the stamps for the previously prepared batches.Then click run to the start batch analysis of one stamp and export the corresponding data when it has been acquired. Multi-spectral imaging of NSCLC explant tissue stained for markers of cell viability, permits the identification and phenotyping of individual cell populations, as well as the identification of tumor and stromal components within the explant tumor micro environment. Tissue and cell segmentation of cell viability marker stains multi-spectral images as demonstrated, can be used to quantify cell death and proliferation in patient derived explants treated with drugs of interest.For example, in this analysis, in tissue samples from tumors treated with Nivolumab, a higher number of dying cells were identified compared to the control treated tumor samples. In addition, as illustrated, the ability to extract spatial information from stained dissections, allows the calculation of intercell distances. Explants can also be used for extensive spatial profiling or mass cytometry, which allows hundreds of biomarkers to be profiled simultaneously at subsidiary resolution while preserving the 3D structure of the sample.PDs could predict the efficacy of immunotherapy. For example, it could be possible to distinguish sensitive or resistant cases to immune checkpoint inhibitors and investigate the mechanisms underpinning this distinction.

patient-derived-tumor-explants-as-live-preclinical-platform-for

Research

JoVE Journal

Cancer Research

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Predicting In Vivo Payloads Delivery using a Blood-brain Tumor-barrier in a Dish

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of brain tumors, the molecular selectivity of BBB also shields the neoplastic cells by blocking the delivery of peripherally administered chemotherapies. The development of novel drugs (including nanoparticles) targeting malignant brain tumors ideally requires the use of preclinical animal models to study the drug&#8217;s transcytosis and antitumor efficacy. In order to comply with the 3R principle (refine, reduce, and replace) to reduce the number of laboratory animals in experimental setup and perform the high-throughput screening of a large library of antitumor agents, we developed a reproducible in vitro human and murine mimic of the blood-brain tumor-barrier (BBTB) using three-layered cultures of endothelial cells, astrocytes, and patient-derived glioblastoma spheres. For higher scalability and reproducibility, commercial cell lines or immortalized cells have been used in tailored conditions to allow the formation of a barrier resembling the actual BBB. Here we describe a protocol to obtain a BBTB mimic by culturing endothelial cells in contact with astrocytes at specific cell densities on inserts. This BBTB mimic can be used, for instance, for the quantification and confocal imaging of the nanoparticle passage through the endothelial and astrocytic barriers, in addition to the evaluation of the tumor cell targeting within the same assay. Moreover, we show that the obtained data can be used to predict the behavior of nanoparticles in preclinical animal models. In a broader perspective, this in vitro model could be adapted to other neurodegenerative diseases for the determination of the passage of new therapeutic molecules through the BBB and/or be supplemented with brain organoids to directly evaluate the efficacy of drugs.

Drug targeting to central nervous system tumors is a major challenge. Here we describe a protocol to produce an in vitro mimic of the blood-brain tumor-barrier using murine and/or human cells and discuss their relevance for the predictability of central nervous system tumor targeting in vivo.

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of ...

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and basic cancer researches. Generations of PDX models in traditional host mice are time-consuming and only working for a small proportion of samples. Recently, zebrafish PDX (zPDX) has emerged as a unique host system, with the characteristics of small-scale and high efficiency. Here, we describe an optimized methodology for generating a dual fluorescence-labeled tumor xenograft model for comparative chemotherapy assessment in zPDX models. Tumor cells and fibroblasts were enriched from freshly-harvested or frozen pancreatic cancer tissue at different culture conditions. Both cell groups were labeled by lentivirus expressing green or red fluorescent proteins, as well as an anti-apoptosis gene BCL2L1. The transfected cells were pre-mixed and co-injected into the 2 dpf larval zebrafish that were then bred in modified E3 medium at 32 &#176;C. The xenograft models were treated by chemotherapy drugs and/or BCL2L1 inhibitor, and the viabilities of both tumor cells and fibroblasts were investigated simultaneously. In summary, this protocol allows researchers to quickly generate a large amount of zPDX models with a heterogeneous tumor microenvironment and provides a longer observation window and a more precise quantitation in assessing the efficiency of drug candidates.

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform.

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture models. PDOs have been successfully generated from a variety of human tumors to recapitulate the architecture and function of tumor tissue accurately and efficiently. However, PDOs are unsuitable for an in vitro high-throughput assay system (HTS) or cell analysis using 96-well or 384-well plates when evaluating anticancer drugs because they are heterogeneous in size and form large clusters in culture. These cultures and assays use extracellular matrices, such as Matrigel, to create tumor tissue scaffolds. Therefore, PDOs have a low throughput and high cost, and it has been difficult to develop a suitable assay system. To address this issue, a simpler and more accurate HTS was established using PDOs to evaluate the potency of anticancer drugs and immunotherapy. An in vitro HTS was created that uses PDOs established from solid tumors cultured in 384-well plates. An HTS was also developed for assessment of antibody-dependent cellular cytotoxicity activity to represent the immune response using PDOs cultured in 96-well plates.

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

A highly accurate in vitro high-throughput assay system was developed to evaluate anticancer drugs using patient-derived tumor organoids (PDOs), similar to cancer tissues but are unsuitable for in vitro high-throughput assay systems with 96-well and 384-well plates.

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D tumoroids. Since patient derived tumor organoids can retain the characteristics of the original tumor, these reliable preclinical models enable cancer drug screening and the study of drug resistance mechanisms. However, CRC related death in patients is mostly associated with the presence of metastatic disease. It is therefore essential to evaluate the efficacy of anti-cancer therapies in relevant in vivo models that truly recapitulate the key molecular features of human cancer metastasis. We have established an orthotopic model based on the injection of CRC patient-derived cancer cells directly into the cecum wall of mice. These tumor cells develop primary tumors in the cecum that metastasize to the liver and lungs, which is frequently observed in patients with advanced CRC. This CRC mouse model can be used to evaluate drug responses monitored by microcomputed tomography (&#181;CT), a clinically relevant small-scale imaging method that can easily identify primary tumors or metastases in patients. Here, we describe the surgical procedure and the required methodology to implant patient-derived cancer cells in the cecum wall of immunodeficient mice.

This protocol describes the orthotopic implantation of patient-derived cancer cells in the cecum wall of immunodeficient mice. The model recapitulates advanced colorectal cancer metastatic disease and allows for the evaluation of new therapeutic drugs in a clinically relevant scenario of lung and liver metastases.

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...