Application of a 3D hydrogel-based model to replace use of animals for passaging patient-derived xenografts

Abstract

Purpose: This 3D in vitro cancer model for propagation of patient-derived cells, using a synthetic self-assembling peptide gel, allows the formation of a fully characterised, tailorable tumour microenvironment. Unlike many existing 3D cancer models, the peptide gel is inert, apart from molecules and motifs deliberately added or produced by cells within the model.

Methods: Breast cancer patient-derived xenografts (PDXs) were disaggregated and embedded in a peptide hydrogel. Growth was monitored by microscopic examination and at intervals, cells were extracted from the gels and passaged on into fresh gels. Passaged cells were assessed by qPCR and immunostaining techniques for the retention of characteristic markers.

Results: Breast cancer PDXs were shown to be capable of expansion over four or more passages in the peptide gel. Contaminating mouse cells were found to be rapidly removed by successive passages. The resulting human cells were shown to be compatible with a range of common assays useful for assessing survival, growth and maintenance of heterogeneity.

Conclusions: Based on these findings, the hydrogel has the potential to provide an effective and practical breast cancer model for the passage of PDXs which will have the added benefits of being relatively cheap, fully-defined and free from the use of animals or animal products. Encapsulated cells will require further validation to confirm the maintenance of cell heterogeneity, genotypes and phenotypes across passage, but with further development, including the addition of bespoke cell and matrix components of the tumour microenvironment, there is clear potential to model other cancer types.

Supplementary information: The online version contains supplementary material available at 10.1007/s44164-023-00048-x.

Keywords: 3D culture; Breast cancer; Hydrogel; In vitro model; PDX; Tumour microenvironment.

Fig. 1

PDX-derived cells can be grown in peptide gel. a Diagram showing process of cell derivation, peptide gel generation and seeding of cells in peptide gel (Created with BioRender.com). b Diagram showing arrangement of peptide gel in transwell insert format (Created with BioRender.com). c Brightfield image showing PDX-A-derived cells grown in peptide gel for 8 days, scale bar shows 200 µm. d Live-dead staining of PDX-A- or PDX-B-derived cells grown for 8 days in peptide gel, scale bar 100 μm. e Live-dead staining of PDX-A-derived cells grown for 11 days in peptide gel after passage in NSG or Rag mice. Both live/dead stained (live green, dead red) with 100 μm scale bar. All gels used 10 mg/ml peptide

Fig. 2

Optimisation of passage of breast cancer PDX in peptide gel. a Diagram of optimised elements in passage process, showing variants kept for the final process in blue (light) boxes and variants discarded as less effective in orange (dark) boxes. b (i–vi) Brightfield microscope images of several PDX-A-in-peptide-gel wells during optimisation of passage, with orange (dark) and blue (light) swatches below corresponding to elements in (a) representing the process used to derive them, where (vi) shows PDX-A cells generated using the final passage process. Scale bars 200 µm. c (i–vi) Histograms showing cluster sizes for each condition in (b), measured using FIJI in micrometres. d Diagram showing passage number nomenclature, Created with BioRender.com

Fig. 3

PDXs derived from different subtypes of breast cancer can be passaged with high resulting cell viability. a Live/dead staining (live cells stained green, dead cells stained red) of PDX-A- and PDX-B-derived cells after 8 days of growth after first seeding (G1) in 10 mg/ml peptide gel. b The same cultures following passage, in gel 2, gel 3, and gel 4. Scale bar 100 µm. c Box and whisker plot of qPCR data showing reduction of murine component to almost undetectable levels from before G1 (P0) to between G1 and G2 (P1) to between G3 and G4 (P3)

Fig. 4

PDX-derived cells passaged in the peptide gel show F-actin (phalloidin) and cytokeratin 18 staining. Phalloidin and CK18 staining shown in red and green alongside DAPI stain for nuclei (blue) in G1 and G5 for cells derived from PDXs A, B, C and D. Scale bar 50 µm

Fig. 5

Histological sectioning and staining can be performed on PDX-derived cells in peptide gels. a H&E staining of 4 µm sections of cells from four PDXs grown in peptide gel to G5. b Ki67 with IgG control immunohistochemical staining of nearby sections of the same samples, showing unambiguous staining with varied Ki67 expression between PDXs. Scale bars 100 µm

Fig. 6

Laminin staining demonstrates the capacity of PDX-derived cells, passaged in the peptide gel, to define their microenvironment. Top: clusters of cells derived from PDX-A, PDX-B, PDX-C and PDX-D, passaged in peptide gel to gel 5 and stained for laminin (red), F-actin (phalloidin, green) and DNA (DAPI, blue). Bottom: red channel (laminin) isolated from top images, showing a “nest” of laminin for each cell cluster. Scale bar 50 µm

Fig. 7

PDX lines retain characteristic spheroid formation after extended passage in peptide gels. a Schematic of experimental setup. b PDX-C and PDX-D morphology in peptide gels immediately prior to cell harvesting and spheroid formation (scale bar 100 µm). c Box and whisker plot showing quantification of spheroid circularity for each passage method and d representative images demonstrating the successful formation of spheroids with characteristic, line-specific morphology (scale bar 250 µm)