WO2021159162A1

WO2021159162A1 - Alten

Info

Publication number: WO2021159162A1
Application number: PCT/AU2020/050129
Authority: WO
Inventors: David Gallego Ortega; Andrew Man-kit LAW; Thomas Cox; Joanna SKHINAS
Original assignee: Garvan Institute Of Medical Research
Priority date: 2020-02-14
Filing date: 2020-02-14
Publication date: 2021-08-19

Abstract

The present disclosure is directed to an in vitro method for encapsulating animal tissue in an alginate-based hydrogel, termed ALTEN (Alginate-based Tissue Encapsulation). Encapsulated organoids produced using the ALTEN method retain cell viability e.g., during tissue culture and storage, and maintain tissue architecture and cell heterogeneity in the original tissue sample. The disclosure also relates to the use of ALTEN encapsulated tissues e.g., encapsulated tumour organoids, for drug-screening, in particular, in the context of personalised cancer therapies.

Description

"ALTEN"

TECHNICAL FIELD

The present disclosure is directed to an in vitro method for encapsulating animal tissue in an alginate-based hydrogel, termed ALTEN (Alginate-based Tissue Encapsulation). The disclosure also relates to the use of ALTEN encapsulated tissues for drug-screening, in particular, in the context of personalised cancer therapies, as well as the use of ALTEN to preserve tissue samples for transport and/or storage.

BACKGROUND

The shift from a traditional “one-size-fits-all” approach for cancer therapy to a precision medicine-based approach has highlighted the need for new systems to enable drug screening and testing of treatment efficacy in biologically relevant models. Tumour organoids and tumouroids are emerging as an attractive organism model to assess drug responses in cancer therapy. Bridging conventional in vitro drug testing and patient derived xenografts (PDXs), tumouroids amalgamate the throughput capacity of 2D screening platforms with the biological relevance of in vivo systems. However, tumour-derived organoids are limited in their ability to mirror individualised patient responses to treatment. These organoids typically grow in highly specialised media containing growth factors and inhibitors of cell-differentiation pathways that support the self-renewal and propagation capacity of the organoid. Although tumour-derived organoids generally retain genomic characteristics of their primary tumour [1], they are limited in their ability to capture cell heterogeneity. This is because they are exposed to clonal drift and lack the typical complexity of tumours, which are formed by ensembles of cells from multiple lineages. Cancer-associated cell species play an important role in tumour behaviour in response to stimuli [2], and there is compelling evidence showing that the specific cell niche physiognomy strongly influences drug sensitivity and acquisition of drug resistance. Additionally, tumour organoid biobanks from extemporal system organisms do not necessarily resemble the specific context of the source patient. As such, they too are unable to capture the cellular states of a tumour immediately after the patient is subjected to neoadjuvant treatment. Thus, the pharmacological heterogeneity is likely to be defined by a combination of: i) the diversity of genetic aberrations at the subclonal level, ii) epigenetic factors, and iii) the specific transcriptional state, which is greatly determined by the signals emanating from the tumour ecosystem. For at least these reasons, there is a need for improved drug screening systems to enable rapid analysis of the therapeutic sensitivity of candidate drugs directly in whole tissue specimens. SUMMARY

The present disclosure is based on the development of a novel in vitro method for encapsulating animal tissue in an alginate-based hydrogel, termed ALTEN (Alginate-based Tissue Encapsulation). The present inventors have shown that the ALTEN method is capable of sustaining viability of tumour samples ex vivo from both mouse and patient samples. By encapsulating tissues in permeable alginate spheres that mimic the stiffness of the extra cellular matrix (ECM) and microenvironment of tumours, the inventors have shown that transcriptional networks of ex vivo tumours can be preserved in culture whilst maintaining the intratumoural heterogeneity that 2D monolayer cultures are unable to. The inventors have also demonstrated the utility of the ALTEN platform in ex vivo drug testing of clinically relevant biospecimens, which retain the appropriate heterotypic cross-talk that occurs between cancer cells, immune cells, stromal cells, and the ECM. Thus, ALTEN can be used as a tool for the multidimensional analysis of drug responses to tumour tissues in situ , including immunomodulation of tumour infiltrated immune cells. ALTEN also provides a platform for the systematic and standardised study of high-resolution molecular responses to cancer therapy in patient samples from multiple hospitals.

Accordingly, in one example, the present disclosure provides a method for preserving viability of cells and/or tissue architecture in a tissue sample obtained from an animal, said method comprising:

(a) obtaining tissue fragments from the tissue sample; and

(b) encapsulating one or more of the tissue fragments in an alginate-based hydrogel to form one or more encapsulated organoids, wherein the one or more encapsulated organoids comprise viable cells and retain the tissue architecture of the tissue sample.

In one example, the alginate-based hydrogel gels at room temperature. In one example, the alginate-based hydrogel has a physiologically acceptable pH (e.g., about neutral). In one example, the alginate-based hydrogel gels at room temperature and has a physiologically acceptable pH (e.g., about neutral).

In one example, the one or more encapsulated organoids are formed by encapsulating the one or more tissue fragments in alginate-based droplets on a hydrophobic surface, and contacting the droplets with a cross-linking agent to form beads or spheroids in which the organoids are encapsulated.

In one example, the method further comprises culturing the one or more encapsulated organoids in tissue culture media. For example, the tissue culture media may comprise one or more nutrients and/or factors necessary to sustain viability of the tissue.

In one example, the tissue culture is a static culture system.

In one example, the one or more tissue fragments are about 0.5mm to about 3mm in diameter. For example, the one or more tissue fragments may be about 1mm to about 2mm in diameter. For example, the one or more tissue fragments may be about lmm in diameter. For example, the one or more tissue fragments may be about 1.5 mm in diameter. For example, the one or more tissue fragments may be about 2mm in diameter.

In one example, the method further comprises preparing the tissue fragments from the tissue sample. The tissue sample may be a tissue biopsy. The tissue sample may be a surgical resection.

In one example, the one or more encapsulated organoids retain cell heterogeneity of the tissue sample and/or retain cell-to-cell communication function of the tissue sample. In one example, the one or more encapsulated organoids retain cell heterogeneity of the tissue sample and retain cell-to-cell communication function of the tissue sample.

In one example, the tissue sample is a tumour sample. For example, the tumour may be a solid tumour. For example, the tumour may be cancerous. In one example, the cancer is selected from a breast cancer, prostate cancer, cervical cancer, colorectal cancer, colonic cancer, rectal cancer, pancreatic cancer, lung cancer, liver cancer, kidney cancer, oesophageal cancer, head and neck cancer, ovarian cancer, bladder cancer, uterine cancer, testicular cancer, glioblastoma, gastric cancer and melanoma. In one example, the cancer is breast cancer.

In each of the foregoing examples, the cancer may be a primary cancer. Alternatively, the cancer may be a metastasis.

According to an example in which the tissue sample is a tumour sample, the one or more encapsulated organoids are encapsulated tumour organoids. In one example, the encapsulated tumour organoids retain the tumour microenvironment.

In one example, the method further comprises obtaining the tissue sample from the animal. For example, the method may comprise taking a biopsy from the animal. For example, the method may comprise surgically resecting the tissue e.g., a tumour, from the animal.

The animal may be a mammalian animal. In one example, the animal is a human. In another example, the animal is a non-human animal. The present disclosure also provides a method of determining responsiveness of a cancer to one or more anti-cancer drug candidates, said method comprising:

(a) preparing one or more encapsulated tumour organoids from a cancer patient derived tumour sample, wherein the encapsulated tumour organoids are prepared by performing the method as described herein;

(b) culturing the one or more encapsulated tumour organoids in the presence of one or more anti-cancer drug candidates;

(c) contacting the encapsulated tumour organoids with an agent which liberates the tissue fragments from the alginate-based hydrogel; and

(d) performing one or more assays or analytical techniques on the liberated tissue fragments or cells obtained therefrom to determine responsiveness of the cancer to the one or more anti-cancer drug candidates.

In one example, the method of determining responsiveness of a cancer to one or more anti-cancer drug candidates comprises dissolving the alginate hydrogel to liberate the tissue fragments. For example, the alginate-based hydrogel may be dissolved by contacting the encapsulated tumour organoids with a chelating agent. For example, the chelating agent may be a solution comprising sodium citrate and EDTA.

The method of determining responsiveness of a cancer to one or more anti cancer drug candidates may further comprise the step of classifying the cancer as being responsive or non-responsive to the candidate drug based on the outcome of the one or more assays or analytical techniques. In accordance with an example in which the cancer is classified as being responsive to the candidate drug, the method may further comprise treating the cancer patient with an anti-cancer drug candidate to which the cancer responds. Alternatively, the method may further comprise prescribing the cancer patient treatment with the anti-cancer drug candidate to which the cancer responds.

In one example, the method comprises isolating individual cells from the liberated tissue fragments prior to performing the one or more assays or analytical techniques. For example, cells may be isolated from the tissue fragments by enzymatic digestion and/or a mechanism separation method.

In one example, the one or more assays or analytical techniques measure one or more of cell death, cell viability, cell proliferation, apoptosis, change in cell motility, change in cell adhesion, change in gene expression and/or a biomarker of anyone thereof, in the tumour organoid following exposure to the anti-cancer drug candidate(s). For example, the one or more assays or analytical techniques may be selected from single-cell RNA sequencing (scRNA-seq), single-cell genomic sequencing, assay for transposase-accessible chromatin using sequencing (ATAC-seq), transcriptome sequencing, genomic sequencing, polymerase chain reaction (PCR), fluorescent in situ hybridisation (FISH), microscopy and/or an immune-detection assay selected from the group consisting of immunofluorescence (IF), Western blot, enzyme-linked immunosorbent assay (ELISA), fluorescence activated cell sorting (FACS) and immunohistochemistry (IHC).

In one example, the patient derived tissue sample is a tumour sample. For example, the tumour may be a solid tumour. For example, the tumour may be cancerous. In one example, the cancer is selected from a breast cancer, prostate cancer, cervical cancer, colorectal cancer, colonic cancer, rectal cancer, pancreatic cancer, lung cancer, liver cancer, kidney cancer, oesophageal cancer, head and neck cancer, ovarian cancer, bladder cancer, uterine cancer, testicular cancer, glioblastoma, gastric cancer and melanoma. In one example, the cancer is breast cancer.

The present disclosure also provides a method of culturing one or more encapsulated organoids prepared according to the method described herein in the presence of one or more growth factors, immunomodulatory agents and/or other agents that modify cell status.

The present disclosure also provides an encapsulated organoid prepared by the method described herein. For example, an encapsulated tumour organoid.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1. ALTEN-engineered hydrospheres precisely preserve tumour specimens and their microenvironment including cell diversity, extracellular matrix and 3D tissue architecture. A) Photograph of an ALTEN-engineered mammary tumour hydrosphere with magnified brightfield and fluorescent image. Green fluorescence indicating cancer cells. B) Tumour 3D architecture of an ALTEN-engineered mammary tumoursphere visualised by multiphoton microscopy, collagen as measured by second- harmonic generation (SHG) (magenta) and cancer cells (green) in both 2D (left) and 3D projection (right). C) Cell number of 4T1.2-mCherry mammary tumour recovered after ALTEN ex vivo culture for 3 and 7 days, quantified after tissue recovery and digestion using FACS. Total cell number (left) and cell viability (DAPI) (right) are shown in comparison with the fresh tissue (day 0); and D) the survival of cancer, immune, and stromal cell populations in the same cell preparations defined by (CD45 and mCherry). E) Representative photomicrographs of histology of H&E, picrosirius red and immunohistochemistry of Ki67 and Cleaved-Caspase 3 of ALTEN-engineered 4T1.2- mCherry mammary tumour hydrospheres after 3 and 7 days of culture compared with fresh tissue (baseline/ day 0), and F) their respective quantification. G) Representative photomicrographs of histology of H&E, picrosirius red and immunohistochemistry of Ki67 and Cleaved-Caspase 3 of ALTEN-engineered MMTV-PyMT mammary tumour hydrospheres after 1 and 3 days of culture compared with fresh tissue (baseline/ day 0) treated with Vehicle control (Veh) or 2uM Doxorubicin (Dox); and their quantification: Ki67 (H), Cleaved-Caspase 3 (I) and picrosirius red, and (J) cell their numbers assayed by FACS. L) Cellular composition of composition of the baseline (red) compared with the vehicle treated ALTEN-engineered MMTV-PyMT mammary tumour hydrospheres at day 1 (green) and 3 (blue) assayed using scRNAseq and visualised using UMAP dimensional reduction, and their cell diversity (M) as defined by K-means clustering and differential gene expression analysis; bottom panel shows the percentage of each cell cluster in each timepoint (-5,000 cells in each timepoint).

Figure 2. Molecular analysis of Doxorubicin (Dox) response in ALTEN-engineered MMTV-PyMT mammary tumour hydrospheres using scRNAseq; and immunemodulation of gastric patient tumour excised from a patient biopsy. A)

UMAP visualisation of the cellular clusters upon Dox treatment compared to vehicle (Veh) treated hydrospheres (-5000 cells per codition). B) K-means clustering and differential gene expression analysis; bottom panel shows the percentage of each cell cluster in each timepoint showing the effects of Dox in the proportion of cell clusters.

C) Dox response signature analysis (metascore) showing cells that express genes consistent with a response to Dox (warm colours). Upper right corresponds to Veh treated cells and bottom right panel to Dox treated; Left panel is a composite of all cells analysed. D) Individual expression of representative genes of the Dox response signature by cluster (as defined in panel B) (Red Veh, Green Dox). E) Differential gene expression analysis of the effects of Dox (units in log2 scale). F) Trajectory analysis (Monocle) along the gene expression of the doxorubicin signature identifying 3 major cell fates (axis). Right panel shows the differential distribution of the Veh (Red) and the Dox (green) cells. G) Visualisation of the monocle defined cell fates in UMAP dimensions. H) Unsupervised definition of cell fates within the major axis showing the differential effects of Dox treatment. I) single-cell RNAseq of IL2 (green) treatment of ALTEN-engineered gastric tumour pieces from a clinical biopsy compared to Veh treated tumour hydrosperes. J) K-means clustering analysis revealing tumour cell diversity in the Veh and IL2 treated. Including the identification of a Tcell cluster (9). K) Gene expression of genes involved in Tcell activation showing increased response in IL2 treated tumour hydrospheres.

Figure 3. A) Overview of the ALTEN-engineering process of tumour encapsulation. B) FACS analysis of cancer cells using a cell tracer dye on ALTEN-engineered mammary 4T1.2-mCherry tumour hydrospheres cultured over period of 3 and 7 days. C) Maximum projections of multiphoton microscopy images of ALTEN-engineered mammary 4T1.2-mCherry tumour hydrospheres cultured over period of 3 days. Red cancer cells; green collagen fibres (SHG).

Figure 4. A) Cell viability after recovery of ex vivo cultured 4T1.2 tumours over 7 days comparing conventional scaffold dependent ex vivo cultures (Air-liquid-interface on collagen sponge, ALI) vs ALTEN. B) Dox perfusion on ALTEN hydrospheres. C) FACS analysis of cell number recovery of different cell species after ALTEN culture of MMTV-PyMT mammary tumours with Dox treatment of Veh control (immune,

CD45+, cancer mCherry positive, stroma double negative), and D) their cell viability using DAPI exclusion.

Figure 5. A) Cellular viability of clinical breast tumours after culturing in ALTEN for 24 hours assessed by FACS using EpCAM and CD45. B) H&E images from resected tumour pieces from patients with various cancer types after ALTEN ex vivo culture over 1, 2 or 3 days compared with fresh tissue at day 0 prior encapsulation (baseline).

Figure 6. Extended analysis of cell diversity and composition of ALTEN-engineered tumours using scRNAseq. A) Quality control metrics on scRNAseq comparing fresh tissue prior encapsulation (baseline (red)) vs ALTEN cultured MMTV-PyMT tumours for 1 (green) and 3 days (blue). nCount, molecules identified; nFeature, gene identities identified; percent.mito, ratio mitochondrial/nuclear genes. B) identification of the major cell lineages in the tumours comparing (baseline (upper right)) vs ALTEN cultured MMTV-PyMT tumours for 1 (middle-right) and 3 days (bottrom-right), and the percentage bottom panel. C) signatures of the main lineages identified using the Xcell signature algorithm, by cluster as defined in 1. D) UMAP plots featuring the expression of canonical markers of different cell types. E) Top gene expression by cell cluster as defined in figure 1. F) cell cycle signatures comparing fresh tissue prior encapsulation (baseline) vs ALTEN cultured MMTV-PyMT tumours for 1 and 3 days, and their percentages. Figure 7. A) Quality control measures of the Veh and Dox treated ALTEN tumour hydrospheres after 3 days of culturing. B) Identification of the major cell lineages in the tumours comparing ALTEN cultured MMTV-PyMT tumours for 3 days with a Veh or Dox treatment and their percentage. Bottom panel signatures of the main lineages identified using the Xcell signature algorithm, by cluster as defined in 2. C)

Percentages of cells in each cell cycle phase identified using gene signatures, comparing ALTEN cultured MMTV-PyMT tumours Veh- or Dox-treated for 3 days.

D) Maintenance of the proportion of different species of myeloid derived suppressor cells (MDSC) associated to cancer tissue using GM-CSF alone or in combination with FBS or tumour cell conditioned media (TES). M-MDSC, monocytic MDSC; PMN- MDSC, polymorphonucleated-MDSCs.

Figure 8. A) Quality control measures of the locally cultured (Sydney, Syd) and transported (Melbourne, Mel) ALTEN MMTV-PyMT mammary tumour hydrospheres after 3 days of culturing, and their UMAP representation (-5,000 cells per condition). Cell diversity (C), main cell lineage composition (D) and cell cycle (E) preservation between Syd and the Mel sample.

Figure 9. A) Single-cell RNAseq of IL2 (green) treatment of ALTEN-engineered gastric tumour pieces from a clinical biopsy compared to Veh treated tumour hydrosperes (patient 2). B) K-means clustering analysis revealing tumour cell diversity in the Veh and IL2 treated. Including the identification Tcells clusters. C) Expression of the top differential genes by cluster. D) UMAP visualisation of the gene expression of canonical markers. F) Gene expression of genes involved in Tcell activation showing increased response in IL2 treated tumour hydrospheres.

DETAILED DESCRIPTION

General

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, feature, composition of matter, group of steps or group of features or compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, features, compositions of matter, groups of steps or groups of features or compositions of matter.

Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.

Any example or embodiment of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise.

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

Unless otherwise indicated, the recombinant DNA, recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley- Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", is understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers. The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

Methods of encapsulating tissue

An in vitro method for encapsulating animal tissue in an alginate-based hydrogel, termed ALTEN (Alginate-based Tissue Encapsulation), is provided. The method of the disclosure is capable of preserving or maintaining the viability of cells in a tissue sample obtained from a subject, whilst maintaining the original tissue architecture and cell diversity within the tissue sample. This has the advantage of retaining cell-to-cell communication within the tissue same e.g., such as the tumour microenvironment (TME) in the case of tumour-derived tissue samples.

In one example, a method for preserving viability of cells and/or tissue architecture in a tissue sample obtained from an animal is provided, said method comprising:

(a) obtaining tissue fragments from the tissue sample; and

As used herein, the term "hydrogel" refers to a physically or chemically cross- linked polymer network that is able to absorb large amounts of water and is a common material for forming tissue engineering scaffolds. They can be classified into different categories depending on various parameters including the preparation method, the charge, and the mechanical and structural characteristics. Reference can be made to S. Van Vlierberghe et ah, "Biopolymer -Based Hydrogels As Scaffolds for Tissue Engineering Applications: A Review," Biomacromolecules, 2011, 12(5), pp. 1387- 1408, which is incorporated herein by reference. Hydrogels are an appealing scaffold material because they are structurally similar to the extracellular matrix of many tissues, can often be processed under relatively mild conditions.

An “alginate-based hydrogel” shall thereby be understood to refer to a hydrogel formed by cross-linking alginate molecules. The term " alginate " as used herein refers to any number of derivatives of alginic acid known in the art, examples of which include, but are not limited to, calcium, sodium or potassium salts, or propylene glycol alginate. Alginate-based hydrogels are known in the art. The mechanical properties of these gels can be modulated depending on the divalent cation used to achieve cross- linking. Bivalent and trivalent cations such as Ca2+, Ba2+, Mg2+, Fe2+ and A13+ covalently bind alginate G blocks to form a three-dimensional structure called “egg box”. F or example, the use of barium or strontium instead of calcium leads to more rigid gels. M and G blocks can be combined in different sequences or alternately ensuring that bivalent cation cross-linked polymer chains form a 3D structure capable of binding large amounts of water, drugs and bioactive substances for supporting tissues.

Alginate-based hydrogels are particularly suited to the method of the disclosure since the cross-linked alginate is easily dissolved using a chelating agent, making it possible to retrieve embedded tissue without the need for harsh chemicals or mechanical forces that could damage the encapsulated tissue. This can be contrasted with other hydrogels and/or biomimetic 3D scaffolds known in the art e.g., collagen and matrigel, which contain cell adhesion proteins or other factors which make liberation of tissue from the hydrogel difficult. Alginate-based hydrogels are also well suited to the method of the disclosure because they do not inherently contain any growth factors, cytokines and/or binding site for mammalian cells which might alter a cell’s state and augment the architecture of the encapsulated tissue. As a result, organoids encapsulated in a alginate-based hydrogels recapitulate the original tissue architecture over time. An additional advantage of using alginate-based hydrogels is that alginate is a clear material that allows high-end image analysis using multiphoton or high-resolution confocal microscopy of the embedded tissue.

Alginate-based hydrogels that are particularly suited to the method of the disclosure are those that gel at room temperature and have a physiologically acceptable pH (i.e., substantially neutral).

In one example, the alginate-based hydrogel will not comprise collagen or matrigel.

The term “tissue” refers to a group or layer of similarly specialized cells which together perform certain special functions.

The terms “organoid” or “organotypic structure” are used interchangeably to refer to a heterogeneous 3D agglomeration of cells that recapitulates aspects of cellular self-organization, architecture and signalling interactions present in the native tissue or organ from which the tissue fragment was obtained or isolated. Accordingly, an organoid or tissue fragment of the disclosure which is “encapsulated” in a alginate- based hydrogel i.e., “an encapsulated organoid”, shall be understood to mean an organoid or tissue fragment as described herein which is enclosed, covered or otherwise enveloped in an alginate-based hydrogel. An encapsulated organoid or encapsulated tissue fragment of the disclosure will be wholly contained within the encapsulating alginate-based hydrogel.

As described herein, the method of the disclosure preserves the viability of cells within a tissue fragment obtained from a tissue sample. A “viable cell” in the context of the present disclosure shall be understood to mean a cell that is capable of surviving and substantially maintaining its extant biological function under suitable biological conditions (e.g., in vitro or ex vivo). In one example, the method of the present disclosure preserves viability of at least about 50%, preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, and most preferably at least about 90% or more of the cells within the encapsulated organoid.

By preserving “tissue architecture” in a tissue sample, it shall be understood that the encapsulated organoids of the disclosure retain the same, or substantially the same, architecture or microarchitecture of the tissue sample from which the encapsulated tissue fragment was obtained or isolated. That is, the tissue architecture of the encapsulated organoid is the same or substantially the same as the tissue architecture of the tissue same from which the organoid was derived or obtained. By preserving the “architecture” or “microarchitecture” of the tissue sample, it shall be understood that at least about 50%, preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, and most preferably at least about 90% or more of the cells of the population within the encapsulated organoid maintain, in vitro, their physical and/or functional contact with at least one cell or non-cellular substance with which they are in physical and/or functional contact in vivo in the tissue sample from which the fragment is obtained or isolated. An encapsulated organoid in which the tissue architecture is preserved will preferably retain cell heterogeneity of the tissue original sample and cell-to-cell communication function of the tissue sample. For example, in accordance with an example in which the tissue fragment is obtained from a tumour tissue sample, the encapsulated organoid produced by the method of the disclosure preserves the tumour microenvironment.

Methodologies for encapsulation using hydrogels are known in the art and contemplated herein. However, in one particular example, the method of the disclosure comprises forming droplets of an alginate-based hydrogel on a hydrophobic surface (e.g., parafilm), embedding tissue fragments from an animal into the alginate-based droplets so that the tissue fragments are wholly encapsulated within the alginate droplets, and then contacting the droplets with a cross-linking agent to form beads or spheroids in which the organoids are encapsulated. The alginate droplets may be formed by any suitable method and/or instrumentation e.g., a dropper, pipette or robot, and may be varied in size to suit the tissue fragments to be encapsulated.

Any suitable cross-linking agent may be used in the method of the disclosure.

In one example, the cross-linking agent is a solution containing calcium chloride e.g., as described in the Examples herein. However, a skilled person will appreciated that other cross-linking agents may be used.

A tissue fragment for encapsulation may be prepared by any suitable means.

For example, tissue fragments for encapsulation in accordance with the method of the disclosure may be prepared from a larger tissue sample (e.g., a tissue biopsy or surgical resection material) by cutting fragment of suitable size therefrom. The tissue fragments may be from about 0.1mm to about 3mm in diameter. For example, a tissue fragment for encapsulation in accordance with the method of the disclosure may be from about 0.5mm to about 2.5mm in diameter, or about 1mm to about 2mm in diameter. In one example, the tissue fragment is about 1mm to about 2mm in diameter.

In one example, the tissue fragments are obtained from a healthy tissue sample. That is, a tissue sample obtained from an individual not suffering from a disease or condition.

In another example, the tissue sample are obtained from an individual suffering from a disease or condition e.g., the tissue sample may be obtained for pathology. In one example, the tissue sample may be obtained from an individual suffering from cancer. For example, the tissue sample from which the tissue fragments are obtained may be a tumour sample or comprise tumour tissue. In accordance with an example in which the tissue sample is from an individual suffering from cancer, the cancer may be a solid tumour cancer. For example, the cancer may be selected from a breast cancer, prostate cancer, cervical cancer, colorectal cancer, colonic cancer, rectal cancer, pancreatic cancer, lung cancer, liver cancer, kidney cancer, oesophageal cancer, head and neck cancer, ovarian cancer, bladder cancer, uterine cancer, testicular cancer, glioblastoma, gastric cancer and melanoma. The cancer may be a primary cancer or a secondary cancer. In one example, the tissue sample is a tumour from a primary cancer. In another example, the tissue sample is from a metastasis.

In accordance with examples in which the tissue fragment which is encapsulated is obtained from a tumour sample, the resulting encapsulated organoid will be an encapsulated tumour organoid. In this regard, the encapsulation of the tumour organoid will preserve the tumour microenvironment, consisting with both benign and malign cells. As used herein, the term “animal” shall be understood to encompass both human or non-human animals. The “non-human animal” may be a primate, livestock (e.g. sheep, horses, cattle, pigs, donkeys), companion animal (e.g. pets such as dogs and cats), laboratory test animal (e.g. mice, rabbits, rats, guinea pigs), performance animal (e.g. racehorses, camels, greyhounds) or captive wild animal. In one example, the animal from which the tissue sample is derived is a mammal. In one example, the animal from which the tissue sample is derived is a human.

Following encapsulation, the one or more encapsulated organoids may be subjected to one or more tissue culture steps. Methods for tissue culture and medium therefor are known in the art and a skilled person would be able to select an appropriate culture medium and conditions based on the needs of the specific tissue. Appropriate culture medium to be employed in the methods of the disclosure will preferably support viability, and proliferation of cells within the organoid. Exemplary tissue culture methods and conditions are described in the Examples herein. In one example, the tissue culture system used is a static tissue culture system. In one example, a perfusion culture system is not used.

In some examples, the method of the disclosure may comprise culturing the one or more encapsulated organoids in the presence of one or more growth factors, cytokines, immunomodulatory agents and/or other agents that modify cell status.

As used herein, an “immunomodulatory agent” shall be understood to mean any agent that modulates an immune response. “Modulate”, as used herein, refers to inducing, enhancing, stimulating, or directing an immune response.

Immunomodulatory agents are known in the art.

As used herein, the term "growth factors" and “cytokines” both refer to proteins and polypeptides which are capable of inducing biological effects on a cell or tissue, such as the stimulation of cellular growth, proliferation and cellular differentiation.

Both terms are used interchangeable. Examples are interleukins, interferons, fibroblast growth factors, insulin and insulin-like growth factors, chemokines, colony-stimulating factors and tumour necrosis factors. Typically, cytokines and growth factors bind to cells via specific receptors.

Storage and transport

Encapsulated organoids produced by the method of the disclosure retain cell viability and tissue architecture under storage and transport conditions. Thus the ALTEN method described herein may have applications for storage and transport of biopsies (e.g., for clinical pathology or research purposes), as well as bio-banking of clinically-relevant samples.

Accordingly, in one example, the present disclosure provides a method for storing an animal tissue sample comprising at least the steps of:

(a) encapsulating the tissue sample in an alginate-based hydrogel by performing the method described herein to thereby produce an encapsulated organoid; and

(b) maintaining the encapsulated organoid under storage conditions.

In another example, the present disclosure provides a method for transporting an animal tissue sample comprising at least the steps of:

(b) transporting the encapsulated organoid from one location to another.

In one example, the encapsulated organoid is stored/transported at a temperature of from about -10° C to about 25° C. For example, the encapsulated organoid may be stored/transported at a temperature of from between about 0° C. and about 25° C. For example, the encapsulated organoid may be stored/transported at a temperature of from between about 4° C. and about 10° C (e.g., such as may be the case for conventional laboratory refrigeration equipment). In one example, the encapsulated organoid is stored/transported at ambient temperature (e.g., as may be the case during transport).

The methods of storing/transporting an animal tissue sample as disclosed herein will be useful in maintaining the cell viability and/or tissue architecture in the tissue sample after storage and/or transport for several hours to several days, and even to several weeks or months without losing enough cellular viability and/or tissue integrity that would render the tissue sample unsuitable for clinical or research applications, such as those described herein. For example, the encapsulated organoid prepared and stored/transported according to the methods of the disclosure will retain at least about 60%-80% or more of its original viability after storage/transport of the encapsulated organoid. In one example, the encapsulated organoid will retain at least about 70% of its original viability after storage/transport. In one example, the encapsulated organoid will retain at least about 75% of its original viability after storage/transport. In one example, the encapsulated organoid will retain at least about 80% of its original viability after storage/transport. In one example, the encapsulated organoid will retain at least about 85% of its original viability after storage/transport. In one example, the encapsulated organoid will retain at least about 90% of its original viability after storage/transport. Drug screening

Encapsulated organoids produced by the method of the disclosure are particularly useful in drug screening applications, such as in the case of personalised medicine which aims to deliver personalised interventions based on genetic, molecular and environmental data gathered for an individual patient. Accordingly, in one example, the present disclosure provides a method of determining responsiveness of a patient to one or more drug candidates, comprising:

(a) preparing one or more encapsulated organoids from a patient derived tissue sample, wherein the encapsulated organoids are prepared by performing the method described herein;

(b) culturing the one or more encapsulated organoids in the presence of one or more drug candidates;

(c) contacting the encapsulated organoids with an agent which liberates the tissue fragments from the alginate-based hydrogel; and

(d) performing one or more assays or analytical techniques on the liberated tissue fragments or cells obtained therefrom to determine responsiveness of the subject to the one or more drug candidates.

In another example, the present disclosure provides a method of assessing toxicity of one or more drug candidates, comprising:

(d) performing one or more assays or analytical techniques on the liberated tissue fragments or cells obtained therefrom to determine the level of toxicity of the one or more drug candidates to the tissue and/or cells.

In one particular example, the patient derived tissue sample may be a patient derived tumour sample from a patient suffering from cancer. Accordingly, the present disclosure provides a method of determining responsiveness a cancer to one or more anti-cancer drug candidates, said method comprising:

(a) preparing one or more encapsulated tumour organoids from a patient derived tumour sample, wherein the encapsulated tumour organoids are prepared by performing the method described herein using the patient derived tumour sample; (b) culturing the one or more encapsulated tumour organoids in the presence of one or more anti-cancer drug candidates;

As described herein, the patient derived tumour sample may be a biopsy or a tissue resection of a solid tumour. The solid tumour may be any solid tumour. In some examples, the solid tumour is associate with a cancer selected from the group consisting of breast cancer, prostate cancer, cervical cancer, colorectal cancer, colonic cancer, rectal cancer, pancreatic cancer, lung cancer, liver cancer, kidney cancer, oesophageal cancer, head and neck cancer, ovarian cancer, bladder cancer, uterine cancer, testicular cancer, glioblastoma, gastric cancer and melanoma. In one example, the cancer is a primary cancer. In another example, the cancer is metastatic.

In each of the foregoing examples, determining responsiveness of a cancer to an anti-cancer drug may comprise testing difference dosages of an anti-cancer drug to determine a dose of an anti-cancer drug to which a patient is likely to respond. Similarly, the method may comprise testing difference dosages of an anti-cancer drug to determine a dose of an anti-cancer drug to which a patient is likely to respond and which is not toxic to tissue and/or cells therefrom.

In one example, the method comprises culturing the or each encapsulated tumour organoid in the presence of a single anti-cancer drug candidate to determine responsiveness of the cancer(s) to the single anti-cancer drug candidate.

In another example, the method may comprise culturing the or each encapsulated tumour organoid in the presence of a combination of anti-cancer drug candidates to determine responsiveness of the cancer(s) to the one or more different combinations of anti-cancer drug candidates.

As described herein, one of the advantages of using an alginate-based hydrogel as the biomimetic scaffold for encapsulation of tissue is the ability to retrieve or liberate the tissue from the hydrogel without the need for harsh chemicals or mechanical forces that could damage the encapsulated tissue. In this regard, cross- linked alginate may be dissolved using a solution containing a chelating agent. Accordingly, in one example, the method comprises dissolving the alginate-based hydrogel to liberate the tissue fragments therefrom. Suitable chelating agents and solutions comprising same will be known to a person of skill in the art. However, in one example, the method comprises the use of a solution comprising sodium citrate and EDTA to dissolve the alginate hydrogel and thereby liberate the tissue. An exemplary solution based on sodium citrate and EDTA is described in the Examples herein.

The method of the disclosure may further comprise the step of isolating one or more individual cells from the tissue to enable the performance of downstream assays or analytical techniques which look at the effect of the drug candidate on individual cells. Methods for isolating cells from tissue are known in the art, but may include, for example, enzymatic digestion and/or a mechanism separation methods.

In order to assess the efficacy and/or toxicity of a drug candidate, the method of the disclosure may comprises performing one or more assays or analytical techniques to measure one or more of cell death, cell viability, cell proliferation, apoptosis, change in cell motility, change in cell adhesion, change in gene expression and/or a biomarker of anyone thereof, in the organoid following exposure to the drug candidate(s). For example, the one or more assays or analytical techniques may be selected from single cell RNA sequencing (scRNA-seq), single-cell genomic sequencing, assay for transposase-accessible chromatin using sequencing (ATAC-seq), transcriptome sequencing, genomic sequencing, polymerase chain reaction (PCR), fluorescent in situ hybridisation (FISH), microscopy and/or an immune-detection assay selected from the group consisting of immunofluorescence (IF), Western blot, enzyme-linked immunosorbent assay (ELISA), fluorescence activated cell sorting (FACS) and immunohistochemistry (IHC). Other analytical methods are known in the art and are also contemplated herein.

Based on the outcome of those one or more assays or analytical techniques, it may be possible to determine whether or not a disease, disease subtype, or patient, will respond to treatment with a particular drug candidate or combination of drug candidates. For example, in the case of an encapsulated tumour organoid which has been cultured in the presence of an anti-cancer drug candidate, one or more of: an increase in cell death, a reduction in cancer cell viability, a reduction in cancer cell proliferation, an increase in apoptosis, a reduction in cell motility, a change in cell adhesion, a change in expression of a cancer-associated gene and/or a biomarker of anyone thereof, may be indicative that the patient from which the tumour sample was derived will be responsive to treatment with the drug candidate.

The method may further comprise the step of treating the cancer patient with an anti-cancer drug candidate to which the cancer is responsive. As used herein, the terms "treating", "treat" or "treatment" and variations thereof, refer to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. It therefore follows that treatment of cancer, includes reduce or eliminate at least one symptom of the cancer. For example, treatment of cancer may include, but is not limited to, increasing apoptosis of cancer cells and/or inhibiting uncontrolled cell division in the tumor or tumor microenvironment and/or reducing tumor size and/or preventing or suppressing or delaying metastasis or progression of the cancer. An individual is successfully "treated", for example, if one or more of the above treatment outcomes are achieved.

EXAMPLES

Example 1. Development and evaluation of ALTEN

This Example describes ALTEN (ALginate-based Tissue ENgineering), a versatile and cost-effective in situ drug screening system that enables rapid analysis of therapeutic sensitivity directly in whole tissue specimens.

1.1 General materials and methods

1.1.1 Cell culture

The mCherry-expressing mammary adenocarcinoma 4T1.2 and 67NR (provided by R. Anderson) were cultured in Minimum essential medium (MEM) Alpha with 5% v/v fetal bovine serum (FBS). Cell lines were tested for mycoplasma and were cultured in 37°C at 5% CO2. Cells were passaged when confluence reached 80%.

1.1.2 Development of tumour bearing mice

Mice were maintained following the Australian code of practice for the care and use of animals for scientific purposes observed by the Garvan Institute of Medical Research/St. Vincent's Hospital Animal Ethics Committee (AEC). Mice were provided with food and water ad libitum. For the implantable mammary tumour models, 8- week-old female Balb/c mice (ABR, Mossvale, New South Wales, Australia) were injected intraductally with 67NR (2xl0⁵) or 4T1.2 (5xl0⁵) tumour cells in the fourth inguinal mammary glands. Animals were anaesthetically induced at 1 L/minute oxygen with 4% Isoflurane and maintained at lL/minute oxygen with 2% Isoflurane. The nipple of one side of the fourth inguinal mammary gland was removed using spring scissors and cells injected via a Hamilton syringe with a 30 gauge blunt-ended fixed 1/2-inch needle. Animals were then placed in a half on/half off recovery box over a heat pad to prevent hypothermia after surgery. Animals were consistently monitored weekly post-surgery until tumours were collected. The MMTV-PyMT animals were used as a primary mammary tumour model. At 12-14 weeks old, tumours were collected from all mammary glands.

1.1.3 Tissue encapsulation in alginate

Tissues were cut into 1 mm³ size explants using surgical scissors and were encapsulated into 20 pL of alginate droplets on top of a hydrophobic surface. The droplets were transferred to a cross-linking agent, 0.1M calcium chloride solution, for 10 min to allow the formation of the alginate beads. Excess CaCb was then washed off with DMEM media. Beads were cultured individually in a 48 well plate at 37°C in 5% CO2 with DMEM media supplemented with 10% FBS, 72U insulin, 2mM glutamine, lOmM HEPES, lOng/mL EGF, lOng/mL cholera toxin. Tissues treated with doxorubicin (Selleckchem S1208) were added to a final concentration of 2mM. The media was changed every second day.

Sodium alginate (NovaMatrix, Sandvika, Norway) was prepared in dFhO at a concentration of 1% (w/v) and dissolved overnight at 37°C in a rotary suspension mixer. The alginate solution was then filter sterilised with a 0.22 mM filter.

Alginate beads were collected and subjected to 55mM sodium citrate in 0.1 M EDTA for 10 min to dissolve the alginate. The recovered tissue was then washed in PBS with 2% FBS. The wash solution was discarded and the tissue was used for downstream processes.

1.1.4 Tissue digestion and flow cytometry

Tissues were digested enzymatically with 15,000 U collagenase (Sigma C9891) and 5000 U hyaluronidase (Sigma H3506) in MEM media at 37°C for 1 hour at 200rpm within a shaking incubator. The samples were then further digested in 2.5% trypsin with 1 mM EGTA in PBS for 1 min at 37°C. Red blood cells were lysed with 155 mM ammonium chloride for 3 min at 37°C and the sample was filtered through a 40 mM filter. Flow cytometry was performed using the BD FACS Symphony for analysis and FACS Aria III for sorting. The following antibodies were used for flow cytometry EpCAM (Clone G8.8), CD45 (Clone 30-F11), CDllb (Clone Ml/70), F4/80 (Clone BM8), Ly6C (Clone HK1.4), Ly6G (Clone 1A8), CD3 (Clone 17A2), CD4 (Clone GK1.5), CD8 (Clone 53-6.7), B220 (Clone RA3-6B2). Cell proliferation was monitored with CellTrace™ Cell Proliferation Kit following the manufacturer’s instructions. Flow cytometry data was analysed using the software package FlowJo (version 10.4.2). PyMT tumours used for single cell RNA-sequencing were processed following the 10 X Genomics Chromium Single Cell Protocol following the manufacturer specifications as stated in 10X Genomics user guide Chromium Single Cell 3' Reagent Kits v3 (Document: CG000183 Rev A).

1.1.5 Immunohistochemistry

Tissues were fixed in 10% neutral buffered formalin for 24 hours at room temperature. After fixation, tissues were cut into 4 pm sections and were stained for H&E, IHC (Ki67 and CC3) and picrosirius red. Fibrillar collagen was stained with 0.1% picrosirius red according to manufacturer’s instructions. Polarized light imaging was performed on a Leica DM6000 fitted with a polarizer in combination with a transmitted light analyser [3] Quantification of Ki67, CC3, and picrosirius red staining was performed using the automated image analysis [4]

1.1.6 Multi-photon Microscopy

Second Harmonic Generation (SHG) collagen signal was acquired using a 25x 0.95 NA water objective on an inverted Leica DMI 6000 SP8 confocal microscope. Excitation source was a Ti: Sapphire femtosecond laser cavity (Coherent Chameleon Ultra II), tuned to a wavelength of 880 nm and operating at 80 MHz. Signal intensity was recorded with RLD HyD detectors (420/40 nm)). For tumour samples, 5 representative regions of interest (512 pm x 512 pm) per tumour were imaged, for CDMs 3 representative areas of 3 technical replicates over a 3D z-stack (20 pm depth; 20 pm depth for CDM, with a z-step size of 1.26 pm). SHG signal coverage in tumour samples was measured with ImageJ (National Institutes of Health, Bethesda, MD, USA). For CDMs mean SHG intensity was measured using Matlab (Mathworks)

1.1.7 Bioinformatic analysis of single cell data

The sequencing output was analysed using the McCarrolTsw lab protocol (Drop-Seq Laboratory Protocol version 3.1 (12/28/15) Evan Macosko, Melissa Goldman Harvard Medical School (http://mccarrolllab.org/dropseq/) [5] and the Cell Ranger package (10X Genomics), a custom genome (hg38, mm 10 plus Trinity assemblies of transgene sequences [6], and gene annotation (GRCh38, encode vM14 plus). Seurat (v Seurat_2.3.4, [7] was the main platform for downstream analysis.

A total of 25,000 cells were sequenced (5,000 of each condition). Firstly, we removed low-quality cells by modelling mitochondrial to nuclear gene content to <5% [5]and considering differences between homeostatic tissues and tumours. We subsequently removed outlier cells that contained more than 4,000 genes as they could potentially constitute cell doublets. Thus, DGE matrices were trimmed for quality metrics (>200 genes, <5% mitochondrial genes, and identified genes expressed in at least 3 cells). Informative genes were identified based on expression and variance and organised into principal components. Two thirds of the total variation of the system was defined by the first 16 PCs. Downstream analysis was performed according to Butler et al with UMI number regression and 16 principal components of variable genes being used for dimensional reduction (tSNE or UMAP) and cluster calling [7]

Monocle (v2 ole, [8] was used to assemble cells assigned to epithelial clusters along a pseudotime vector generated from cell response gene signatures. States were assigned using DDRTree according to the manual.

1.2 Preservation of the tumour ecosystem by ALTEN

The ALTEN process is conveniently illustrated in Fig. 3. As shown, tumour pieces of 0.1 - 0.2 mm were encapsulated in an alginate-based hydrogel forming whole-tumour organoid or “tumoroid” using the methodology described in Example 1. The tuneable stiffness of the alginate matrix allows to accurately mimic the microenvironment for subsequent ex vivo culture of the tumoroids (Fig. 1 A). Alginate is a non-refringent material that allows high-end image analysis using multiphoton or high-resolution confocal microscopy of the embedded tissue (Fig. IB). The biomimetic properties of the alginate-hydrogel allow the recovery of tumour pieces without damaging the tissue, thus enabling multidimensional downstream analysis of cellular responses by conventional techniques, but also sensitive techniques such as droplet- based single-cell RNAseq.

Unlike in vitro generated tumour organoids, ALTEN whole tumour organoids preserve the original complexity of the tumour ecosystem, maintaining original three- dimensional features, tumour architecture and cell heterogeneity, thus retaining cell-to- cell/ECM communication (Fig. IB). The widely used mouse models of breast cancer: the luminal MMTV-PyMT and two Triple-Negative Breast Cancer (TNBC) models, 67NR and 4T1, were used to illustrate the capacity of ALTEN to preserve tumour properties of cells and demonstrate its utility as a drug screening method.

ALTEN tumouroid viability cultured for 3 and 7 days was assayed by FACS.

The overall cell recovery after tissue dissociation of 4T1 tumours was not significantly different compared to the original fresh sample and cell viability of the recovered cells was not affected by the culture conditions (Fig. 1C). When looking at specific cell lineages, only a reduction of immune cells was identified (Fig. ID), presumably due to the short-lived nature of some of the immune cell species. Importantly, a live cell tracer analysis showed that cancer cells kept on proliferating during the culturing period of time (Fig. 3B). Cell proliferation and death was confirmed by Ki67 and cleaved caspase 3 (CC3) immunohistochemistry staining on the cultured ALTEN tumouroids (Fig. IE and F). Although the overall viability of the cells was not significantly different upon ALTEN tumouroid culture, regions of cells undergoing apoptosis were identified (Fig. 1G), suggesting that the tumours are in a proliferation-to-cell death equilibrium, consistent with the typical evolution of a tumour. Extracellular matrix (ECM) components and the overall 3D architecture of the tumour was also maintained in ALTEN as shown by multi-photon microscopy (Fig. IB, Fig. IE, Fig. 3C). Similar results were obtained using the 67NR syngeneic breast cancer model (Fig. 4D).

Clinical biopsies of an array of different cancer types, including breast, pancreatic, colorectal and gastric cancer, were also preserved using ALTEN (Fig. 5A and B). Finally, ALTEN outperformed conventional techniques based on Air-to-Liquid Interface (ALI) for ex vivo culture of tumour specimens (Fig. 4A). Taken together, these data demonstrate the versatility of ALTEN for long-term culture of ex-vivo tumours preserving high tumour cell viability and tissue architecture.

1.3 Drug screening using ALTEN

To demonstrate its utility as a drug screening method, the ALTEN platform was used to assay drug sensitivity in whole tumour samples harvested from spontaneous MMTV-PyMT mammary carcinomas. Similar to patient tumour specimens, MMTV- PyMT tumours are inherently heterogeneous, thus the cellular diversity of MMTV- PyMT tumours and their metastasis was modelled using FACS analysis which defined five major areas within the tumours. In order to characterise the early transcriptomic events of drug sensitivity, randomised MMTV-PyMT ALTEN-engineered tumouroids were cultured in ALTEN in the presence of a suboptimal concentration of Doxorubicin- e (Dox), or a vehicle, for 24h and 72h. Similar to the 4T1 and 67NR models, cell viability and tissue architecture was preserved in spontaneous MMTV-PyMT tumours (Fig. 1G- K and Fig. 4C and D). As expected, Dox-treated samples presented decreased cell proliferation and increased apoptosis, as assayed by Ki67 and CC3 immunostaining respectively. These results indicate that Dox permeated the alginate matrix and accessed the tissue, as confirmed by the staining of the matrices in a dose- dependent manner (Fig. 4B).

The inventors then used unbiased droplet-based scRNAseq to investigate molecular Dox responses in 25,000 tumour cells. Transcriptomically, ALTEN cultured cells produced a similar output and comparable quality than the fresh sample (Fig. 6A). The impact of ALTEN ex vivo culturing on the cell diversity and molecular repertoire of the tumouroids was also investigated with a freshly prepared scRNAseq capture (baseline - Oh) of the same tumours. A dimensional reduction UMAP visualisation showed that cells analysed from the baseline sample clustered together with the vehicle treated cells at both 24h and 72h, and that all cell clusters identified were represented in the 3 samples (Fig. 1L). Unsupervised clustering analysis identified 12 distinct cell clusters (Fig. 1M). Importantly, tumour cell diversity was conserved during the ALTEN culturing conditions in all main cellular lineages [9] (Fig. 1M right panel and Fig. 6B) and the identified clusters annotated using specific canonical markers for the PyMT tumours [10] Fig. 6D and E). The inventors were able to transcriptomically define both cancer cells and their associated species in a similar proportion to those identified using FACS analysis. Finally, ALTEN conserved a similar proportion of cells undergoing cell cycle (Fig. 6F).

The early transcriptomic effects of Dox in the ALTEN cultured tumoroids was also investigated. Dox-treated samples produced transcriptional profiles similar to untreated cells (Fig. 7A). Cell diversity was comparable between the Dox-treated and the control sample (Fig2A-B and Fig. 7B). However, a Dox response signature [11, 12]identified a differential progressive effect in cancer cells over time (Fig. 2C) - some of the individual gene markers are shown in Fig. 2D. The cell cycle was increasingly impaired in a subpopulation of cells, consistent with the action of Dox (Fig. 7C). Cells in which cell cycle was arrested robustly expressed signatures of Dox response, indicating that these cells were sensitive to Dox and presumably dying. Interestingly, genes that are down-regulated in Dox-treated cells are related to p53 activation, identifying p53-driven apoptosis as one of the mechanism of action of Dox using scRNAseq (Fig. 2E). The sequential relationship of cancer cells through the doxorubicin sensitivity axis was analysed using unbiased pseudotime alignment, classifying 3 main cell states (Fig. 2F). The treated and control cells were found to be distributed across three distinct states: “non-responsive state 1” (SI) exhibited by those cells that were not sensitive to Dox; “Dox-sensitive state” (S2) exhibited by those cells that were sensitive to Dox; and “Dox-responsive state” (S3) exhibited by those cells which were responsive upon treatment with Dox. An overlap of states SI, S2 and D3 on the UMAP coordinates (Fig. 2G), and the k-means definition of cell diversity (Fig. 2H), shows that clusters 2,3 and 4 exhibit features of Dox resistance, characterised by a unique differential expression of Phlda3, a p53-regulated repressor of Akt (Fig. 2D). Taken together, these results suggest that cancer cells that are able to elicit a p53 driven inhibition of cell proliferation response are better adapted to the cytotoxic effects of Dox, presumably though a mechanism driven by Phlda. Taken together, this high- resolution molecular analysis demonstrates that ALTEN ex vivo culturing is a bona fide system to preserve the molecular repertoire, to preserve cell diversity of tumour specimens and to produce high-resolution portraits of chemotherapy response in situ.

1.4 Immunomodulation of ALTEN encapsulated tissues

One of the advantages of ALTEN is that it is able to preserve the assembly of the tumour ecosystem, thereby preserving immune and stromal cell viability. Due to the short-lived nature of immune cells, the inventors explored the utility of ALTEN as a testing platform for immunomodulation.

Briefly, the highly metastatic 4T1 breast cancer model was used to ALTEN- encapsulate lung tissue containing metastatic nodules together with a rich and immunosuppressive myeloid cell infiltration. Metastatic tissue was cultured for 3 and 7 days in the presence of GM-CSF, a potent stimulator of cancer entrained Myeloid- Derived Suppressor Cells (MDSCs) (CD45+/CDllb+/F4-80-/Ly6Clow/Ly6Ghigh) tumours and their metastasis.

Metastanoids treated with GM-CSF showed increased lifespan of MDSCs (Fig. 7D). These results indicate that ALTEN culture conditions are amenable to modification in order to study specific cell populations, such as chemotherapy -resistant cells or cancer-associated immune cells.

To further confirm these findings and evaluate ALTEN translational utility, biospecimens from tumour resections of two gastric cancer patients were encapsulated, generating a n-of-1 clinical trial. Human biospecimens were randomised and exposed to IL2 or a vehicle control for 24hrs. Cultured tissue was processed and analysed using single cell RNAseq as performed with the mouse tissue (described above). Figure 21 and J show an UMAP representation of the n-of-1 trial of the first patient with gastric metaplasia. Similar to the observations in the mouse tissue, ALTEN was able to retain cell diversity and the sampling of the n-of-1 trial resulted in an even distribution of the cell species into the two testing conditions. The IL2 treated tissue showed a noticeable increase in the number of T lymphocytes, CD4 and CD8 cells, and enhanced expression of genes involved in CTL function (Fig. 2K). Thus, a positive immunomodulation of tumour-rejecting cell species. By contrast, a less prominent effect was observed on T lymphocytes in the tissue treated with vehicle control (Fig. 9). These results indicate that ALTEN is a suitable platform for rapid analysis of the effects of immunomodulation directly in immune cells infiltrated in tumours. Coupled with scRNAseq technology, ALTEN presents an unprecedented means to measure immunotherapy sensitivity in a clinically-relevant setting.

1 5 ALTEN for transportation of tumour biopsies

The capacity of ALTEN to preserve cell viability and the three-dimensional tumour architecture during transport of tumour biopsies was also tested.

Briefly, MMTV PyMT tumour pieces were embedded in ALTEN and sent interstate at room temperature using a regular courier. The ALTEN hydrogels were received the following day and processed normally. Following digestion, 80% cell viability was achieved from the transported ALTEN hydrogels. Single cell RNAseq performed on the tissues transported in ALTEN hydrogels produced a similar readout (Fig. 8A) and recapitulated the cell diversity observed in the baseline sample (Fig. 8B- D). Based on these observations, it may be concluded that ALTEN has the capacity to preserve cell viability and three-dimensional tumour architecture during transport.

T6 Conclusions

In conclusion, ALTEN platform provides an innovative and consistent 3D biosystem to sustain the viability of tumours ex vivo from both mouse and patient samples. By creating a permeable alginate sphere that mimics the stiffness of the ECM and microenvironment of tumours, the transcriptional networks of ex vivo tumours can be preserved in culture whilst maintaining the intratumoural heterogeneity that 2D monolayer cultures are unable to. The ALTEN platform permits accurate ex vivo drug testing of clinically relevant biospecimens, while preserving the appropriate heterotypic cross-talk that occurs between cancer cells, immune cells, stromal cells, and the ECM.

ALTEN can be used as a tool for the multidimensional analysis of drug responses to tumour tissues in situ , including immunomodulation of tumour infiltrated immune cells. In addition, ALTEN provides an alternative to drug testing in PDX models in a quick and reliable way, and enables a systematic and standardised capacity to study high-resolution molecular responses to cancer therapy in patient samples from multiple hospitals.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. REFERENCES

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Claims

1 A method for preserving viability of cells and/or tissue architecture in a tissue sample obtained from an animal, said method comprising:

(a) obtaining tissue fragments from the tissue sample; and

2. The method of claim 1, wherein the alginate-based hydrogel gels at room temperature and has a physiologically acceptable pH.

3. The method of claim 1 or 2, wherein the one or more encapsulated organoids are formed by encapsulating the one or more tissue fragments in alginate-based droplets on a hydrophobic surface, and contacting the droplets with a cross-linking agent to form beads or spheroids in which the organoids are encapsulated.

4. The method of any one of claims 1 to 3, further comprising culturing the one or more encapsulated organoids in tissue culture media.

5. The method of claim 4, wherein the tissue culture is a static culture.

6. The method of any one of claims 1 to 5, wherein the one or more tissue fragments are about 0.5mm to about 3mm in diameter.

7. The method of claim 6, wherein the one or more tissue fragments are about lmm to about 2mm in diameter.

8. The method of any one of claims 1 to 7, comprising preparing the tissue fragments from the tissue sample.

9. The method of any one of claims 1 to 8, wherein the tissue sample is a biopsy or tissue resection.

10. The method of any one of claims 1 to 9, wherein the one or more encapsulated organoids retain cell heterogeneity of the tissue sample and/or retain cell-to-cell communication function of the tissue sample.

11. The method of claim 10, wherein the tissue sample is a tumour sample.

12. The method of claim 11, wherein the tissue sample is from a breast cancer, prostate cancer, cervical cancer, colorectal cancer, colonic cancer, rectal cancer, pancreatic cancer, lung cancer, liver cancer, kidney cancer, oesophageal cancer, head and neck cancer, ovarian cancer, bladder cancer, uterine cancer, testicular cancer, glioblastoma, gastric cancer and melanoma.

13. The method of claim 12, wherein the cancer is a primary cancer or metastasis.

14. The method of any one of claims 1 to 13, wherein the one or more encapsulated organoids are encapsulated tumour organoids.

15. The method of claim 14, wherein the encapsulated tumour organoids retain the tumour microenvironment.

16 The method of any one of claims 1 to 15, further comprising obtaining the tissue sample from the animal.

17. The method of any one of claims 1 to 16, wherein the animal is a human.

18. The method of any one of claims 1 to 16, wherein the animal is a non-human animal.

19. A method of determining responsiveness of a cancer to one or more anti-cancer drug candidates, said method comprising:

(a) preparing one or more encapsulated tumour organoids from a cancer patient derived tumour sample, wherein the encapsulated tumour organoids are prepared by performing the method of any one of claims 1 to 18;

(b) culturing the one or more encapsulated tumour organoids in the presence of one or more anti-cancer drug candidates; (c) contacting the encapsulated tumour organoids with an agent which liberates the tissue fragments from the alginate-based hydrogel; and

20. The method of claim 19, wherein the method comprises dissolving the alginate hydrogel to liberate the tissue fragments.

21. The method of claim 19 or 20, wherein the alginate-based hydrogel is dissolved by contacting the encapsulated tumour organoids with a solution comprising sodium citrate and EDTA.

22. The method of any one of claims 19 to 21, comprising classifying the cancer as being responsive or non-responsive to the candidate drug based on the outcome of the one or more assays or analytical techniques.

23. The method of any one of claims 19 to 22, comprising isolating individual cells from the liberated tissue fragments prior to performing the one or more assays or analytical techniques.

24. The method of claim 23, wherein cells are isolated from the tissue fragments by enzymatic digestion and/or a mechanism separation method.

25. The method of any one of claims 19 to 24, wherein the one or more assays or analytical techniques measure one or more of cell death, cell viability, cell proliferation, apoptosis, change in cell motility, change in cell adhesion, change in gene expression and/or a biomarker of anyone thereof, in the tumour organoid following exposure to the anti-cancer drug candidate(s).

26. The method of any one of claims 19 to 25, wherein the one or more assays or analytical techniques are selected from single-cell RNA sequencing (scRNA-seq), single-cell genomic sequencing, assay for transposase-accessible chromatin using sequencing (ATAC-seq), transcriptome sequencing, genomic sequencing, polymerase chain reaction (PCR), fluorescent in situ hybridisation (FISH), microscopy and/or an immune-detection assay selected from the group consisting of immunofluorescence (IF), Western blot, enzyme-linked immunosorbent assay (ELISA), fluorescence activated cell sorting (FACS) and immunohistochemistry (IHC).

27. The method of any one of claims 19 to 26, wherein the patient derived tumour sample is a biopsy or tissue resection of a solid tumour.

28. The method of any one of claims 19 to 27, wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, cervical cancer, colorectal cancer, colonic cancer, rectal cancer, pancreatic cancer, lung cancer, liver cancer, kidney cancer, oesophageal cancer, head and neck cancer, ovarian cancer, bladder cancer, uterine cancer, testicular cancer, glioblastoma, gastric cancer and melanoma.

29. The method of any one of claims 1 to 28, wherein the one or more encapsulated organoids are cultured in the presence of one or more growth factors, immunomodulatory agents and/or other agents that modify cell status.

30. The method of any one of claims 19 to 28, further comprising treating the cancer patient with an anti-cancer drug candidate to which the cancer responds.

31. An encapsulated organoid prepared by the method of any one of claim 1 to 29.

32. The encapsulated organoid of claim 31, which is an encapsulated tumour organoid.