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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Cancer Research

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

Published: February 7th, 2021

DOI:

10.3791/62130

1Leicester Cancer Research Center, University of Leicester, 2MRC Toxicology Unit

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 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.

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,<....

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1. Tissue collection

  1. After surgery, transfer freshly resected human tumor specimens into a tube containing 25 mL of fresh culture medium (Dulbecco’s modified Eagle medium supplemented with 4.5 g/L glucose and L-glutamine + 1% (v/v) fetal calf serum + 1% penicillin–streptomycin) and stored on ice. Process the explant within 2 h of surgery in a sterile class II hood.

2. Explant preparation

  1. Clean al.......

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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 .......

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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 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.......

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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,....

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Name Company Catalog Number Comments
Acetic acid Sigma 320099 Staining reagent
Antibody Diluent / Block, 1x Perkin Elmer ARD1001EA Antibody diluent/blocking buffer
Barnstead NANOpure Diamond Barnstead Ultra Pure (UP) H2O machine
Citric Acid Monohydrate Sigma-Aldrich C7129 Reagent for citrate buffer
Costar Multiple Well Cell Culture Plates Corning Incorporated 3516 6 multiwell plate
DAPI Dilactate Life Technologies D3571
100 x 17 mm Dish, Nunclon Delta ThermoFisher Scientific 150350 100 mm diameter dish for tissue culture
DMEM (1x) Dubelcco's Modified Eagle Medium + 4.5 g/L D-Glucose + 110 mg/mL Sodium Pyruvate Gibco (Life Technologies) 10569-010 Tissue culture medium (500 mL)
DPX mountant VWR 360294H Mounting medium
DPX mountant Merck 6522 Mounting medium
Ethylenediaminetetraacetic acid (EDTA) Sigma-Aldrich 3609 Reagent for TE buffer
Eosin CellPath RBC-0100-00A Staining reagent
Foetal Bovine Serum Gibco 10500-064 For use in tissue culture medium
37% Formaldehyde Fisher (Acros) 119690010 10% Formalin
iGenix, microwave oven IG2095 iGenix IG2095 Microwave used for antigen retreival
Industrial methylated spirit (IMS) Genta Medical 199050 99% Industrial Denatured Alcohol (IDA)
InForm Advanced Image Analysis Software Akoya Biosciences InForm
Leica ASP3000 Tissue Processor Leica Biosystems Automated Vacuum Tissue Processor
Leica Arcadia H and C Leica Biosystems Embedding wax bath
Leica RM2125RT Leica Biosystems Rotary microtome
Leica ST4040 Linear Stainer Leica Biosystems H&E stainer
Mayer's Haematoxylin Sigma GHS132-1L Staining reagent
Millicell Cell Culture Inserts, 30 mm, hydrophilic PTFE, 0.4 µm Merck Milipore PICMORG50 Organotypic culture insert disc
Novolink Polymer Detection System Leica Biosystems RE7150-K DAB staining kit
OPAL 480 Akoya Biosciences FP1500001KT Fluorophore with Dimethyl Sulfoxide (DMSO) diluent
OPAL 520 Akoya Biosciences FP1487001KT Fluorophore with Dimethyl Sulfoxide (DMSO) diluent
OPAL 570 Akoya Biosciences FP1488001KT Fluorophore with Dimethyl Sulfoxide (DMSO) diluent
OPAL 620 Akoya Biosciences FP1495001KT Fluorophore with Dimethyl Sulfoxide (DMSO) diluent
OPAL 650 Akoya Biosciences FP1496001KT Fluorophore with Dimethyl Sulfoxide (DMSO) diluent
OPAL 690 Akoya Biosciences FP1497001KT Fluorophore with Dimethyl Sulfoxide (DMSO) diluent
OPAL 780 / OPAL TSA-DIG Reagent Akoya Biosciences FP1501001KT Fluorophore with Dimethyl Sulfoxide (DMSO) diluent and TSA-DIG reagent
Opal Polymer HRP Ms Plus Rb, 1x Perkin Elmer ARH1001EA HRP polymer
Penicillin/streptomycin solution Fisher Scientific 11548876 For use in tissue culture medium
PhenoChart Whole Slide Contextual Viewer Akoya Biosciences PhenoChart Viewer software for scanned images
Phosphate Buffered Saline Tablets Thermo Scientific Oxoid BR0014G PBS
1x Plus Amplification Diluent Perkin Elmer FP1498 Fluorophore diluent
Prolong Diamond Antifade Mountant Invitrogen P36961 Mounting medium
Slide Carrier Perkin Elmer To load slides into Slide Carrier Hotel for scanning with Vectra Polaris
Sodium Chloride Fisher Scientific S/3160/63 10% Formalin
Sodium Hydroxide pellets Fisher Scientific S/4920/53 Reagent for citrate buffer
Tenatex Toughened Wax - Pink (500 g) KEMDENT 1-601 Dental wax surface
Thermo Scientific Shandon Sequenza Slide Rack for Immunostaining Center Fisher Scientific 10098889 Holder for slides and slide clips
Thermo Scientific Shandon Plastic Coverplates Fisher Scientific 11927774 Slide clips
Tris(hydroxymethyl)aminomethane (Tris) Sigma-Aldrich 252859 Reagent for TE buffer
VectaShield Vecta Laboratories H-1000-10 Mounting medium
Vectra Polaris Slide Scanner Perkin Elmer Vectra Polaris Slide scanner
Xylene Genta Medical XYL050 De-waxing agent

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