Development of human pancreatic cancer avatars as a model for dynamic immune landscape profiling and personalised therapy

Description

Notes

Methods

Acquisition of tissue and blood samples

The study (REC number 19/A056) was approved for the collection of tumour and healthy pancreatic tissue by the Oxford Radcliffe BioBank. The collection of the specimens was supported by the Oxford Centre for Histopathology Research. All patients recruited to the study provided written consent confirming voluntary participation and permission for tissue donation for research. Biopsy punch samples (5mm diameter) were obtained by the pathologist at the John Radcliffe Hospital following surgery provided that clear surgical margins could be determined.

Sectioning and culture of live tumour slices

Samples were transported on ice in unsupplemented low-glucose DMEM media before suspension in agarose scaffolds. Following a manual wash in LG DMEM media, biopsy punches were suspended in 4% low-gelling-temperature agarose and cooled. The agarose scaffold structure was generated by melting the solution and suspending the slice in a small embedding mould. 250 µm sections were generated using the Leica VT1200 vibratome (blade angle +21°C, speed 1.5 mms^-1, amplitude 2 mm) in a bath of ice-cold PBS and transported in LG DMEM media on ice.

Alvetex perfusion plates (REPROCELL) were used to conduct dynamic perfusion experiments, with syringe pumps maintaining a constant flow of 10 µLmin^-1. The apparatus was assembled within a sterile tissue culture hood. To construct the circuit, 60 mL syringes were connected to silicone tubing and flushed with 70% ethanol before washing in unsupplemented LG DMEM media. Alvetex 12-well tissue culture inserts were activated in 70% ethanol for 2 minutes before washing in LG DMEM media. Long-term culture of tissue slices was conducted using LG DMEM complete pancreatic medium (see reagents table) at +37°C and 5% CO₂.

Treatment of slices in perfusion culture

All ex vivo treatment of avatars was commenced on day 0 (day of tumour acquisition). Systemic chemotherapy was prepared in DMSO. Drug delivery commenced after the avatars had been created and subsequently placed in the perfusion plate, via a inflow circuit comprising a syringe and tubing, attached to the perfusion plate and connected to the perfusion pump. After the intended time period for drug delivery, the infusion was stopped, and the perfusion plate removed from the incubator and placed within a tissue culture hood. The inflow circuit was removed and the inflow channel on the perfusion plate temporarily occluded using a 2ml syringe. An inflow circuit was then created, and the syringe filled with culturing media. The inflow circuit was primed with media (from the attached syringe) to remove any air bubbles present in the tubing prior to attachment to the perfusion plate to ensure that there was a constant column of media from the syringe and the tubing to the perfusion plate. For treatment with metformin and ascorbic acid, 20 mM metformin and 100 mM ascorbic acid was perfused for 5 days, with freshly made-up solutions applied each day. Gemcitabine concentration was perfused for one hour at a concentration of 250 mM.

GeoMX spatial transcriptomic analysis

Spatial transcriptomic analysis was provided by NanoString Technologies, Inc. through their GeoMX® DSP Technology Access Program Grant. Formalin-fixed paraffin-embedded (FFPE) samples were provided cut at 5 µm thickness and mounted on negatively-charged slides. Samples were sent to the NanoString Technology Access Program labs for analysis using the GeoMX Digital Spatial Profiler. Slides were incubated with oligonucleotide-antibody conjugates with photocleavable linkers. UV light was then used to selectively release oligonucleotide barcodes which were read and quantified through sequencing. Four immunofluorescent markers were used for morphology definition, facilitating region of interest (ROI) selection. A total of 68 ROIs were selected across treated and control samples. The Whole Transcriptome Atlas was used for sample profiling, with genes mapped to barcodes using an in-house algorithm produced by NanoString Technologies, Inc., generating spatially resolved transcriptional data. Quality control involved assessing raw read threshold, percent-aligned reads, and sequencing saturation. A quantification limit was set based on a negative probe signal (mean + two standard deviations). Reads were filtered based on their expression in >5% AOIs and counts were normalised to account for differences in AOI size and cellularity.

Multiplexed immunofluorescence and HALO analysis

The multiplex IF staining of avatars was performed in collaboration with the Oxford Translational Histopathology Lab. Slides were stained using the Leica BOND RXm autostainer machine (Leica, Microsystems) whilst following the OPAL™ protocol 28 (AKOYA Biosciences). A total of 6 staining cycles were subsequently performed.  The following primary antibody-opal fluorophore pairing was used: CD4 – Opal 520, CD8 – Opal 570, CD20 – Opal 480, Foxp3 – Opal 620, CD68 – Opal 690, pan Cytokerratin – Opal 780. In adherence to the manufacturer's instructions, the primary antibodies were incubated for one hour and subsequently detected with the BOND™ 4 Polymer Refine Detection System (DS9800, Leica Biosystems). The DAB step was replaced by the opal fluorophores which consisted of a 10 minute incubation with no haematoxylin step. The antigen retrieval step was performed with Epitope Retrieval (ER) Solution 2 (AR9640, Leica 8 Biosystems) for 20 minutes at 100 °C. This was performed prior to the application of each primary antibody. VECTASHIELD® Vibrance™ Antifade Mounting Medium with DAPI 10 (H-1800-10, Vector Laboratories.) was used to mount each slide. The AKOYA Biosciences Vectra® Polaris™ was used in order to obtain whole slide scans and multispectral images (MSI). A batch analysis of every MSIs was performed using the inForm 2.4.8 software. The final step consisted of fusing the multiple batched analysed MSIs on the HALO (Indica Labs) software. This resulted in the creation of a spectrally unmixed reconstructed whole image of the avatar. Analysis of the multiplex IF was performed on the Indica Labs HALO® (version 3.0.311.407). This software allows for deconvolution of the image by selecting individual fluorophores for analysis. The initial step was teaching the software a Random Forest Classifier module in which the images were segmented into tumour and stromal regions. Manual annotation of the slides was performed to exclude areas with staining artefacts. Cell detection and subsequent phenotyping was performed using the Indica Labs - HighPlex FL v3.1.0 (fluorescent images). Individual cells were defined by their expression of the specific markers: Tumour (DAPI+ panCytokeratin+), CD4 helper (DAPI+CD4+), CD8 cytotoxic (DAPI+CD8+), regulatory T-cell (DAPI+CD4+Foxp3+), B cells (DAPI+CD20+) and Macrophages (DAPI+CD68+).