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Model Selection for the Preclinical Development of New Drug–Radiotherapy Combinations

  • J. Singh
    Affiliations
    Global Translational Science, Varian, a Siemens Healthineers company, Palo Alto, California, USA
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  • S. Hatcher
    Affiliations
    Global Translational Science, Varian, a Siemens Healthineers company, Palo Alto, California, USA
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  • A.A. Ku
    Affiliations
    Global Translational Science, Varian, a Siemens Healthineers company, Palo Alto, California, USA
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  • Z. Ding
    Affiliations
    Global Translational Science, Varian, a Siemens Healthineers company, Palo Alto, California, USA
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  • F.Y. Feng
    Affiliations
    Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California, USA

    Division of Hematology and Oncology, Department of Medicine, University of California, San Francisco, California, USA

    Department of Radiation Oncology, University of California, San Francisco, California, USA

    Department of Urology, University of California, San Francisco, California, USA
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  • R.A. Sharma
    Affiliations
    Global Translational Science, Varian, a Siemens Healthineers company, Palo Alto, California, USA

    UCL Cancer Institute, University College London, London, UK
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  • S.X. Pfister
    Correspondence
    Author for correspondence: S.X. Pfister, Global Translational Science, Varian Medical Systems, Palo Alto, CA, 94304, USA.
    Affiliations
    Global Translational Science, Varian, a Siemens Healthineers company, Palo Alto, California, USA
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Open AccessPublished:August 31, 2021DOI:https://doi.org/10.1016/j.clon.2021.08.008

      Abstract

      Radiotherapy plays an essential role in the treatment of more than half of all patients with cancer. In recent decades, advances in devices that deliver radiation and the development of treatment planning software have helped radiotherapy attain precise tumour targeting with minimal toxicity to surrounding tissues. Simultaneously, as more targeted drug therapies are being brought into the market, there has been significant interest in improving cure rates for cancer by adding drugs to radiotherapy to widen the therapeutic window, the difference between normal tissue toxicity and treatment efficacy. The development of new combination therapies will require judicious adaptation of preclinical models that are routinely used for traditional drug discovery. Here we highlight the strengths and weaknesses of each of these preclinical models and discuss how they can be used optimally to identify new and clinically beneficial drug–radiotherapy combinations.

      Key words

      Statement of Search Strategies Used and Sources of Information

      This paper reflects the authors' views based on the existing literature searched using Pubmed and other widely available search engines.

      Introduction

      Radiotherapy is a cornerstone of both curative and palliative cancer care. It is regularly used as an adjuvant therapy to other treatment modalities, such as surgery and chemotherapy. With the escalating cost of discovering novel therapeutics with each progressive year and low clinical approval rate, there is a significant need to repurpose already approved drugs for combination therapy [
      • DiMasi J.A.
      • Grabowski H.G.
      • Hansen R.W.
      Innovation in the pharmaceutical industry: new estimates of R&D costs.
      ,
      • Dominguez L.W.
      • Willis J.S.
      Research and development costs of new drugs.
      ]. Combination drug therapies promise to bring novel and effective therapies to patients at a much faster rate and a fraction of the cost [
      • Sun W.
      • Sanderson P.E.
      • Zheng W.
      Drug combination therapy increases successful drug repositioning.
      ]. One such treatment modality that significantly benefits patients is identifying novel drug–radiotherapy combinations, including the class of drugs termed ‘radiosensitisers’. These drugs make tumours more susceptible to radiation therapy, thus increasing the therapeutic index [
      • Wardman P.
      Chemical radiosensitizers for use in radiotherapy.
      ]. Since the Food and Drug Administration's approval of cetuximab with radiotherapy in 2006 for locally advanced head and neck cancer, there has been increased interest in discovering novel drug–radiotherapy combinations [
      • Bonner J.A.
      • Harari P.M.
      • Giralt J.
      • Azarnia N.
      • Shin D.M.
      • Cohen R.B.
      • et al.
      Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck.
      ].
      Radiosensitisers improve the therapeutic window by increasing tumour cell killing upon exposure to radiation or allowing lower doses of radiation to be used to achieve the same efficacy [
      • van Bijsterveldt L.
      • Durley S.C.
      • Maughan T.S.
      • Humphrey T.C.
      The challenge of combining chemo- and radiotherapy with checkpoint kinase inhibitors.
      ,
      • Venkatesulu B.P.
      • Krishnan S.
      Radiosensitization by inhibiting DNA repair: turning the spotlight on homologous recombination.
      ]. The clinical significance of radiosensitisers is supported by several randomised clinical trials that have shown that delivering systemic chemotherapy concurrently with radiotherapy leads to better overall survival in many different cancers [
      • Lawrence Y.R.
      • Vikram B.
      • Dignam J.J.
      • Chakravarti A.
      • Machtay M.
      • Freidlin B.
      • et al.
      NCI-RTOG translational program strategic guidelines for the early-stage development of radiosensitizers.
      ]. Except for cetuximab, most of these radiosensitisers have been cytotoxic drugs, such as cisplatin, temozolomide, 5-fluorouracil (5-FU) and Mitomycin. For example, in non-metastatic inoperable non-small cell lung cancer, cisplatin combined with radiotherapy has been shown to significantly improve patient survival [
      • Schaake-Koning C.
      • van den Bogaert W.
      • Dalesio O.
      • Festen J.
      • Hoogenhout J.
      • van Houtte P.
      • et al.
      Effects of concomitant cisplatin and radiotherapy on inoperable non-small-cell lung cancer.
      ]. Concurrent addition of cisplatin and 5-FU to a radiotherapy regimen has been shown to be beneficial in locally advanced cervical cancer patients [
      • Keys H.M.
      • Bundy B.N.
      • Stehman F.B.
      • Muderspach L.I.
      • Chafe W.E.
      • Suggs 3rd, C.L.
      • et al.
      Cisplatin, radiation, and adjuvant hysterectomy compared with radiation and adjuvant hysterectomy for bulky stage IB cervical carcinoma.
      ,
      • Rose P.G.
      • Bundy B.N.
      • Watkins E.B.
      • Thigpen J.T.
      • Deppe G.
      • Maiman M.A.
      • et al.
      Concurrent cisplatin-based radiotherapy and chemotherapy for locally advanced cervical cancer.
      ,
      • Morris M.
      • Eifel P.J.
      • Lu J.
      • Grigsby P.W.
      • Levenback C.
      • Stevens R.E.
      • et al.
      Pelvic radiation with concurrent chemotherapy compared with pelvic and para-aortic radiation for high-risk cervical cancer.
      ]. In muscle-invasive bladder cancer, chemotherapy drugs 5-FU and mitomycin C, when combined with radiotherapy, significantly improved locoregional control of bladder cancer with no significant increase in toxicity [
      • James N.D.
      • Hussain S.A.
      • Hall E.
      • Jenkins P.
      • Tremlett J.
      • Rawlings C.
      • et al.
      Radiotherapy with or without chemotherapy in muscle-invasive bladder cancer.
      ]. Similarly, in epidermoid anal cancer, a combination of radiotherapy and infused 5-FU and mitomycin C was superior to radiotherapy alone [
      • Northover J.
      • Glynne-Jones R.
      • Sebag-Montefiore D.
      • James R.
      • Meadows H.
      • Wan S.
      • et al.
      Chemoradiation for the treatment of epidermoid anal cancer: 13-year follow-up of the first randomised UKCCCR Anal Cancer Trial (ACT I).
      ,
      • UKCCCR Anal Cancer Trial Working Party
      Epidermoid anal cancer: results from the UKCCCR randomised trial of radiotherapy alone versus radiotherapy, 5-fluorouracil, and mitomycin. UKCCCR Anal Cancer Trial Working Party. UK Co-ordinating Committee on Cancer Research.
      ,
      • Flam M.
      • John M.
      • Pajak T.F.
      • Petrelli N.
      • Myerson R.
      • Doggett S.
      • et al.
      Role of mitomycin in combination with fluorouracil and radiotherapy, and of salvage chemoradiation in the definitive nonsurgical treatment of epidermoid carcinoma of the anal canal: results of a phase III randomized intergroup study.
      ,
      • Bartelink H.
      • Roelofsen F.
      • Eschwege F.
      • Rougier P.
      • Bosset J.F.
      • Gonzalez D.G.
      • et al.
      Concomitant radiotherapy and chemotherapy is superior to radiotherapy alone in the treatment of locally advanced anal cancer: results of a phase III randomized trial of the European Organization for Research and Treatment of Cancer Radiotherapy and Gastrointestinal Cooperative Groups.
      ]. Furthermore, the clinical benefits of concomitant administration of temozolomide with radiotherapy in newly diagnosed glioblastoma have also been reported [
      • Stupp R.
      • Mason W.P.
      • van den Bent M.J.
      • Weller M.
      • Fisher B.
      • Taphoorn M.J.
      • et al.
      Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.
      ].
      A comprehensive literature search on Pubmed for radiosensitisers identified over 7000 publications on drug–radiotherapy combinations. As shown in Figure 1, chemotherapies such as cisplatin, gemcitabine, 5-FU and paclitaxel are among the most studied drugs combined with radiation, consistent with the current clinical practice of using these combinations. In addition to the cytotoxic drugs [
      • van Bijsterveldt L.
      • Durley S.C.
      • Maughan T.S.
      • Humphrey T.C.
      The challenge of combining chemo- and radiotherapy with checkpoint kinase inhibitors.
      ], recent studies have suggested that newly developed targeted therapies, such as pyrotinib, regorafenib, pertuzumab, ibrutinib and tepotinib, can also act as radiosensitisers (Figure 1).
      Fig 1
      Fig 1Heatmap showing the landscape of radiosensitisers published between 1990 and 2020. Each column represents a year while each row represents one radiosensitiser, which is ranked by the total number of publications in PubMed from 1990 to 2020 (drugs at the bottom has the largest total number of publications). The colour intensity on the heatmap indicates the percentage of the publication for a given drug published in a given year compared with all the publications for that drug. For example, nine of 244 cisplatin papers were published in 2020. The percentage is calculated as 9/244 × 100% = 3.7%. One of one pyrotinib paper was published in 2020, so the percentage of publication for pyrotinib in 2020 is 100%. It indicates that pyrotinib is a newly discovered radiosensitiser. The darker the colour, the more radiosensitiser papers were published in that year. The top right region of the heatmap shows the newly published radiosensitisers, because they only have a few publications in recent years. The bottom rows of the heatmap shows the well-studied radiosensitisers, because they have more publications and the papers are published evenly across the years.
      Despite a large number of preclinical publications, there are very few Food and Drug Administration-approved drug–radiotherapy combinations. In addition to the lack of a clear regulatory path, one limitation in identifying clinically relevant new drug–radiotherapy combinations is the lack of accurate and predictive preclinical models. Therefore, this overview will explore the preclinical models available to date and discuss their strengths and weakness for drug–radiotherapy preclinical research.

      Preclinical Models for the Identification of Novel Drug–Radiotherapy Combinations

      Preclinical models in oncology are essential tools for the discovery of novel cancer therapeutics. Various models, such as in vitro two-dimensional assays and in vivo xenografts, have been routinely used in the laboratory to test the efficacy of radiotherapy. More recently, higher complexity tumour models, such as three-dimensional tumour spheroids, have shown the potential to test translational hypotheses to discover novel radiosensitisers. We will explore these models in detail and discuss the significance of these models in deciphering the immune system's role in the patient's response to radiotherapy [
      • Demaria S.
      • Ng B.
      • Devitt M.L.
      • Babb J.S.
      • Kawashima N.
      • Liebes L.
      • et al.
      Ionizing radiation inhibition of distant untreated tumors (abscopal effect) is immune mediated.
      ].

       In Vitro Two-dimensional Models of Cancer

      Two-dimensional or monolayer cultures have played an instrumental role in discovering novel cancer therapeutics. Over the years, several two-dimensional assays were optimised using immortalised tumour cell lines to assess the antiproliferative activity of ionising radiation. Based on Puck and Marcus' [
      • Puck T.T.
      • Marcus P.I.
      Action of X-rays on mammalian cells.
      ] seminal work published in 1956, the Clonogenic Survival Assay (CSA) was developed to measure a single cancer cell's ability to establish colonies upon exposure to the therapeutic agent. Traditionally, clonogenic assays suffer from low throughput and are labour intensive (Figure 2), although the effort to miniaturise this assay for screening purposes has been reported [
      • Lin S.H.
      • Zhang J.
      • Giri U.
      • Stephan C.
      • Sobieski M.
      • Zhong L.
      • et al.
      A high content clonogenic survival drug screen identifies mek inhibitors as potent radiation sensitizers for KRAS mutant non-small-cell lung cancer.
      ].
      Fig 2
      Fig 2Summary of the commonly used preclinical tumour models for novel therapeutic discovery. Preclinical tumour models are either developed from primary tissue or immortalised tumour cells. Primary human tumour tissue can be dissociated as an organoid (patient-derived organoids; PDOs), sliced (patient-derived explants; PDEs) or implanted in immunocompromised mice as patient-derived xenografts (PDXs). These models (shown on the left) are complex, often low throughput, but have significant resemblance of human tumour. By contrast, models developed from immortalised cell lines (shown on the right) are grown as two- or three-dimensional cultures. Both model systems offer high throughput screening capability and provide an excellent tool for molecular mechanism studies. However, these models lack translational potential as they do not fully recapitulate the complexities of the actual tumour. Immortalised human and mouse tumour cells can also be injected into immunocompromised mice (xenograft) or immunocompetent mice (syngeneic and genetically engineered mouse model), thus providing an opportunity to study therapeutic efficacy in vivo.
      A notable advancement from the clonogenic assay is the soft agar assay developed by West et al. [
      • West C.M.
      • Davidson S.E.
      • Roberts S.A.
      • Hunter R.D.
      Intrinsic radiosensitivity and prediction of patient response to radiotherapy for carcinoma of the cervix.
      ]. The assay was initially designed to characterise the intrinsic response of tumour-derived cells at 2 Gy [
      • Deacon J.
      • Peckham M.J.
      • Steel G.G.
      The radioresponsiveness of human tumours and the initial slope of the cell survival curve.
      ]. Since the initial publication, several subsequent papers have shown that the assay can correlate with local tumour control in some tumour types, such as cervical cancer, but has had varied success in head and neck cancer [
      • West C.M.
      • Davidson S.E.
      • Roberts S.A.
      • Hunter R.D.
      Intrinsic radiosensitivity and prediction of patient response to radiotherapy for carcinoma of the cervix.
      ,
      • Stausbol-Gron B.
      • Overgaard J.
      Relationship between tumour cell in vitro radiosensitivity and clinical outcome after curative radiotherapy for squamous cell carcinoma of the head and neck.
      ,
      • Bjork-Eriksson T.
      • West C.
      • Karlsson E.
      • Mercke C.
      Tumor radiosensitivity (SF2) is a prognostic factor for local control in head and neck cancers.
      ]. Like the CSA, a significant drawback to this assay is its throughput and the requirement of primary tumour human tissues.
      By contrast, high throughput screening (HTS) assays have enabled researchers to identify novel therapeutic candidates from drug libraries that consist of hundreds or thousands of agents. These are usually short-term assays and measure the therapeutic agent's antiproliferative activity. For an assay to be compatible with the HTS workflow, it has to be scalable, reproducible and has a good signal-to-noise ratio. One such commercially available reagent amenable in the HTS workflow is a Cell-Titer Glo (CTG) assay. This assay measures cellular ATP, roughly proportional to cell viability, and has a robust signal-to-noise ratio [
      • Crouch S.P.
      • Kozlowski R.
      • Slater K.J.
      • Fletcher J.
      The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity.
      ]. This assay was used to study the radiosensitising effect of epidermal growth factor receptor inhibitor, which was similar to shorter-term cyto60 (fluorescent nucleic acid stain), thus paving the path for these assays to discover novel radiosensitisers [
      • Wang M.
      • Morsbach F.
      • Sander D.
      • Gheorghiu L.
      • Nanda A.
      • Benes C.
      • et al.
      EGF receptor inhibition radiosensitizes NSCLC cells by inducing senescence in cells sustaining DNA double-strand breaks.
      ].
      Several studies have shown a good correlation between the CTG assay and clonogenic assay results [
      • Yard B.D.
      • Adams D.J.
      • Chie E.K.
      • Tamayo P.
      • Battaglia J.S.
      • Gopal P.
      • et al.
      A genetic basis for the variation in the vulnerability of cancer to DNA damage.
      ]. Since then, multiple groups have used the CTG assay for drug–radiotherapy screening. For example, Abazeed et al. [
      • Abazeed ME
      • Adams DJ
      • Hurov KE
      • Tamayo P
      • Creighton CJ
      • Sonkin D
      • et al.
      Integrative radiogenomic profiling of squamous cell lung cancer.
      ] screened 18 lung squamous cell carcinoma cell lines and identified nuclear factor erythroid 2-related factor 2 as a regulator of radiation sensitivity. In another study, Qi et al. [
      • Liu Q.
      • Wang M.
      • Kern A.M.
      • Khaled S.
      • Han J.
      • Yeap B.Y.
      • et al.
      Adapting a drug screening platform to discover associations of molecular targeted radiosensitizers with genomic biomarkers.
      ] used the CTG assay across 32 cancer cell lines using 18 radiosensitising agents. They concluded that the CTG assay could accurately predict the relative radiosensitising potential of targeted therapeutic agents. Apart from CTG, other biochemical assays, such as MTT and the resazurin assay to determine cell viability, have also been reported to explore drug–radiotherapy combinations [
      • Liu Q.
      • Wang M.
      • Kern A.M.
      • Khaled S.
      • Han J.
      • Yeap B.Y.
      • et al.
      Adapting a drug screening platform to discover associations of molecular targeted radiosensitizers with genomic biomarkers.
      ,
      • Carter R.
      • Cheraghchi-Bashi A.
      • Westhorpe A.
      • Yu S.
      • Shanneik Y.
      • Seraia E.
      • et al.
      Identification of anticancer drugs to radiosensitise BRAF-wild-type and mutant colorectal cancer.
      ]. These assays measure a biochemical by-product of cellular metabolism as a surrogate for cellular viability. Thus, results obtained from such studies may be confounded by metabolic alteration of tumour cells and may not correlate with cellular proliferation. Validation of results to CSAs is therefore essential.
      An alternative to biochemical detection of cell viability is to use automated image-based assays to quantify cellular proliferation. High content screening involves imaging platforms that allow a user to capture and quantify cells using brightfield or fluorescent images, thus indirectly measuring cellular proliferation [
      • Lema C.
      • Varela-Ramirez A.
      • Aguilera R.J.
      Differential nuclear staining assay for high-throughput screening to identify cytotoxic compounds.
      ,
      • Zock J.M.
      Applications of high content screening in life science research.
      ]. The main advantage of these imaging-based methods is that they directly quantify cell number rather than a surrogate biochemical product. In addition to cell count, a few high content imaging screens have also measured γH2AX foci in cell lines to identify drug–radiotherapy combinations [
      • Fu S.
      • Yang Y.
      • Das T.K.
      • Yen Y.
      • Zhou B.S.
      • Zhou M.M.
      • et al.
      gamma-H2AX kinetics as a novel approach to high content screening for small molecule radiosensitizers.
      ,
      • Goglia A.G.
      • Delsite R.
      • Luz A.N.
      • Shahbazian D.
      • Salem A.F.
      • Sundaram R.K.
      • et al.
      Identification of novel radiosensitizers in a high-throughput, cell-based screen for DSB repair inhibitors.
      ]. However, these screens require expensive instruments, thus limiting their widespread use.

       Tumour Spheroids

      The cancer cell lines grown in two-dimensional cultures assume that tumour cells are homogeneous and grow as a monolayer with abundant nutrients, oxygen and a physiological pH [
      • Duval K.
      • Grover H.
      • Han L.H.
      • Mou Y.
      • Pegoraro A.F.
      • Fredberg J.
      • et al.
      Modeling physiological events in 2D vs. 3D cell culture.
      ]. By contrast, the patient's tumour is a combination of neoplastic cells and tumour microenvironment (TME), consisting of transformed epithelial cells, tumour-infiltrating lymphocytes, cancer-associated fibroblasts, extracellular matrix (ECM), growth factors, chemokines and resident host cells that help to promote tumour progression and metastasis [
      • Spill F.
      • Reynolds D.S.
      • Kamm R.D.
      • Zaman M.H.
      Impact of the physical microenvironment on tumor progression and metastasis.
      ]. Therefore tumor spheroids were developed to resemble the morphology of primary tumours, which provides a significant improvement from two-dimensional models [
      • Lee J.M.
      • Mhawech-Fauceglia P.
      • Lee N.
      • Parsanian L.C.
      • Lin Y.G.
      • Gayther S.A.
      • et al.
      A three-dimensional microenvironment alters protein expression and chemosensitivity of epithelial ovarian cancer cells in vitro.
      ] (Table 1).
      Table 1A summary of the advantages and limitations of all the major preclinical models.
      Preclinical modelAdvantagesDisadvantageReference
      In vitro models
      Two-dimensional cell culture
      • Easy to set up
      • Cost-effective
      • Consistent and reproducible data
      • Amenable to HTS
      • Great for biochemical and MOA studies
      • Easier downstream processing and data analysis
      • Least physiological
      • Does not accurate mimic tumour microenvironment
      • Cells grow in surplus oxygen and nutrients
      [
      • Kapalczynska M.
      • Kolenda T.
      • Przybyla W.
      • Zajaczkowska M.
      • Teresiak A.
      • Filas V.
      • et al.
      2D and 3D cell cultures – a comparison of different types of cancer cell cultures.
      ]
      Spheroid cultures
      • Relatively easy to set up
      • Presence of ECM
      • Amenable to HTS
      • Mimic tumour microenvironment better than two-dimensional cultures
      • No vasculature
      • Uses immortalised cell lines
      • Non-native tumour microenvironment
      • Expensive to set up
      • Less physiological than other complex in vitro models such as PDOs, explants and in vivo models
      [
      • Vinci M.
      • Gowan S.
      • Boxall F.
      • Patterson L.
      • Zimmermann M.
      • Court W.
      • et al.
      Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation.
      ,
      • Nath S.
      • Devi G.R.
      Three-dimensional culture systems in cancer research: focus on tumor spheroid model.
      ,
      • Zanoni M.
      • Piccinini F.
      • Arienti C.
      • Zamagni A.
      • Santi S.
      • Polico R.
      • et al.
      3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained.
      ]
      Ex vivo models
      Patient-derived organoids (PDOs)
      • Derived from primary human tumors
      • Somewhat amenable to HTS
      • Presence of immune cells
      • No vasculature
      • Non-native tumour microenvironment
      • Limited throughput
      • Slow tumour organoid growth
      [
      • Bar-Ephraim Y.E.
      • Kretzschmar K.
      • Clevers H.
      Organoids in immunological research.
      ,
      • Drost J.
      • Clevers H.
      Organoids in cancer research.
      ,
      • Kretzschmar K.
      Cancer research using organoid technology.
      ]
      Patient-derived explant slices (PDEs)
      • Actual human tumour slices
      • Mimic in vivo tumour environment
      • Presence of intratumour immune cells
      • Host-immune cell interaction preserved as that of the actual tumour
      • Closed system with no cell trafficking
      • Difficult to set up with low throughput
      • Expensive to set up
      • Can only be cultured for few days
      • Less reproducible than other models due to tumour variability from patient to patient
      [
      • Powley I.R.
      • Patel M.
      • Miles G.
      • Pringle H.
      • Howells L.
      • Thomas A.
      • et al.
      Patient-derived explants (PDEs) as a powerful preclinical platform for anti-cancer drug and biomarker discovery.
      ]
      In vivo models
      Xenografts
      • Easy to set up,
      • Well-characterised and predictable
      • Lack of immune system
      • Uses immortalised human cell lines
      [
      • Morton C.L.
      • Houghton P.J.
      Establishment of human tumor xenografts in immunodeficient mice.
      ]
      Patient-derived xenografts (PDX)
      • Preservation of human tumour morphology
      • Lack of immune system
      • Expensive
      [
      • Tentler J.J.
      • Tan A.C.
      • Weekes C.D.
      • Jimeno A.
      • Leong S.
      • Pitts T.M.
      • et al.
      Patient-derived tumour xenografts as models for oncology drug development.
      ,
      • Olson B.
      • Li Y.
      • Lin Y.
      • Liu E.T.
      • Patnaik A.
      Mouse models for cancer immunotherapy research.
      ]
      Syngeneic rodent models
      • Well-characterised
      • Presence of mouse immune system
      • Limited immortalised mouse tumor cell lines available
      • Limited number of mouse strains
      • Mouse immune system is different from that of human
      [
      • Olson B.
      • Li Y.
      • Lin Y.
      • Liu E.T.
      • Patnaik A.
      Mouse models for cancer immunotherapy research.
      ]
      Genetically engineered mouse models (GEMMs)
      • de novo murine tumors in a natural immune-proficient microenvironment
      • Presence of mouse immune system
      • Mouse immune system is different from that of human
      • Time-consuming and expensive
      [
      • Kersten K.
      • de Visser K.E.
      • van Miltenburg M.H.
      • Jonkers J.
      Genetically engineered mouse models in oncology research and cancer medicine.
      ]
      ECM, extracellular matrix; HTS, high throughput screening; MOA, mechanism of action.
      Spheroids can either be grown from tumour cells alone or cocultured with various cell types, such as fibroblasts, endothelial and immune cells, to mimic the crosstalk among different cellular compartments of patients' tumours (Figure 3) [
      • Courau T.
      • Bonnereau J.
      • Chicoteau J.
      • Bottois H.
      • Remark R.
      • Assante Miranda L.
      • et al.
      Cocultures of human colorectal tumor spheroids with immune cells reveal the therapeutic potential of MICA/B and NKG2A targeting for cancer treatment.
      ,
      • Giannattasio A.
      • Weil S.
      • Kloess S.
      • Ansari N.
      • Stelzer E.H.
      • Cerwenka A.
      • et al.
      Cytotoxicity and infiltration of human NK cells in in vivo-like tumor spheroids.
      ,
      • Lanuza P.M.
      • Vigueras A.
      • Olivan S.
      • Prats A.C.
      • Costas S.
      • Llamazares G.
      • et al.
      Activated human primary NK cells efficiently kill colorectal cancer cells in 3D spheroid cultures irrespectively of the level of PD-L1 expression.
      ,
      • Lazzari G.
      • Nicolas V.
      • Matsusaki M.
      • Akashi M.
      • Couvreur P.
      • Mura S.
      Multicellular spheroid based on a triple co-culture: a novel 3D model to mimic pancreatic tumor complexity.
      ]. Although spheroids lack the vasculature and cellular heterogeneity of a primary tumour, their gene expression profiles closely mimic patient tumours [
      • Luca A.C.
      • Mersch S.
      • Deenen R.
      • Schmidt S.
      • Messner I.
      • Schafer K.L.
      • et al.
      Impact of the 3D microenvironment on phenotype, gene expression, and EGFR inhibition of colorectal cancer cell lines.
      ]. Due to the three-dimensional nature of the tumour spheroid, a gradient of nutrients, pH and oxygen develops over time. Outer cells of the spheroid undergo rapid proliferation, whereas interior cells display extensive hypoxia, necrosis and apoptosis [
      • Costa E.C.
      • Moreira A.F.
      • de Melo-Diogo D.
      • Gaspar V.M.
      • Carvalho M.P.
      • Correia I.J.
      3D tumor spheroids: an overview on the tools and techniques used for their analysis.
      ] and become quiescent over time [
      • Wallace D.I.
      • Guo X.
      Properties of tumor spheroid growth exhibited by simple mathematical models.
      ]. While providing a more physiological TME over two-dimensional culture, spheroid models are still amenable to many routine downstream two-dimensional assays [
      • Han K.
      • Pierce S.E.
      • Li A.
      • Spees K.
      • Anderson G.R.
      • Seoane J.A.
      • et al.
      CRISPR screens in cancer spheroids identify 3D growth-specific vulnerabilities.
      ]. Metabolism-based HTS assays for two-dimensional cultures, such as the resazurin assay, acid phosphatase, lactate dehydrogenase, CTG and MTT assay, have been used to monitor spheroid growth [
      • Yeon S.E.
      • No da Y.
      • Lee S.H.
      • Nam S.W.
      • Oh I.H.
      • Lee J.
      • et al.
      Application of concave microwells to pancreatic tumor spheroids enabling anticancer drug evaluation in a clinically relevant drug resistance model.
      ,
      • Walzl A.
      • Unger C.
      • Kramer N.
      • Unterleuthner D.
      • Scherzer M.
      • Hengstschlager M.
      • et al.
      The resazurin reduction assay can distinguish cytotoxic from cytostatic compounds in spheroid screening assays.
      ,
      • Ho W.Y.
      • Yeap S.K.
      • Ho C.L.
      • Rahim R.A.
      • Alitheen N.B.
      Development of multicellular tumor spheroid (MCTS) culture from breast cancer cell and a high throughput screening method using the MTT assay.
      ,
      • Friedrich J.
      • Ebner R.
      • Kunz-Schughart L.A.
      Experimental anti-tumor therapy in 3-D: spheroids – old hat or new challenge?.
      ]. The recent introduction of ultra-low attachment spheroid plates coupled with the introduction of high content screening platforms has made it possible to culture and analyse spheroids on a large scale [
      • Costa E.C.
      • Moreira A.F.
      • de Melo-Diogo D.
      • Gaspar V.M.
      • Carvalho M.P.
      • Correia I.J.
      3D tumor spheroids: an overview on the tools and techniques used for their analysis.
      ]. Quantitative brightfield and fluorescence microscopy have been shown to be particularly useful for characterising the spheroid size, morphology and distribution of dead and live cells [
      • Al-Ramadan A.
      • Mortensen A.C.
      • Carlsson J.
      • Nestor M.V.
      Analysis of radiation effects in two irradiated tumor spheroid models.
      ,
      • Leek R.
      • Grimes D.R.
      • Harris A.L.
      • McIntyre A.
      Methods: using three-dimensional culture (spheroids) as an in vitro model of tumour hypoxia.
      ].
      Fig 3
      Fig 3Cellular organisation of commonly used preclinical models. Tumour cells can either be cultured as a monolayer (A) or assembled as a three-dimensional spheroid (B) using techniques that promote cell–cell interactions over cell–surface interactions. These cultures can be grown as a homogenous mixture of tumour cells or cocultured with immune cells, fibroblasts or endothelial cells (C), recapitulating tumour heterogeneity. Human explants (D) preserve the cellular composition, organisation and complexity of the parent tumour, providing an excellent opportunity to test novel therapeutics in the context of a native tumour environment. The orthotopic tumour model (E) offers an opportunity to study human or mouse tumour cells in the context of an organ in which they are implanted.
      It is worth noting that the unique three-dimensional characteristics of tumour spheroids have proven useful to understand the clinical significance of novel drugs. For example, prior KRAS two-dimensional screens identified two different KRAS mutant cell lines that either required or did not require KRAS oncogene for survival [
      • Singh A.
      • Greninger P.
      • Rhodes D.
      • Koopman L.
      • Violette S.
      • Bardeesy N.
      • et al.
      A gene expression signature associated with "K-Ras addiction" reveals regulators of EMT and tumor cell survival.
      ]. When developing KRAS inhibitors, this was a limitation, as mutation status might not be sufficient to select a specific patient population. However, Janes et al. [
      • Janes M.R.
      • Zhang J.
      • Li L.S.
      • Hansen R.
      • Peters U.
      • Guo X.
      • et al.
      Targeting KRAS mutant cancers with a covalent G12C-specific inhibitor.
      ] used three-dimensional tumour spheroids to show that all KRAS G12C cells were dependent on KRAS, suggesting that KRAS dependency may be an artefact of two-dimensional cell culture.
      Similarly, three-dimensional spheroid work has been used to test the efficacy of radiation on the proliferative capacity of tumour cells [
      • Al-Ramadan A.
      • Mortensen A.C.
      • Carlsson J.
      • Nestor M.V.
      Analysis of radiation effects in two irradiated tumor spheroid models.
      ,
      • Olive P.L.
      • Durand R.E.
      Drug and radiation resistance in spheroids: cell contact and kinetics.
      ,
      • Bruningk S.C.
      • Rivens I.
      • Box C.
      • Oelfke U.
      • Ter Haar G.
      3D tumour spheroids for the prediction of the effects of radiation and hyperthermia treatments.
      ,
      • Khan S.
      • Bassenne M.
      • Wang J.
      • Manjappa R.
      • Melemenidis S.
      • Breitkreutz D.Y.
      • et al.
      Multicellular spheroids as in vitro models of oxygen depletion during FLASH irradiation.
      ]. Narayan et al. [
      • Narayan R.S.
      • Fedrigo C.A.
      • Brands E.
      • Dik R.
      • Stalpers L.J.
      • Baumert B.G.
      • et al.
      The allosteric AKT inhibitor MK2206 shows a synergistic interaction with chemotherapy and radiotherapy in glioblastoma spheroid cultures.
      ] tested the efficacy of AKT inhibition with irradiation and temozolomide and concluded that synergy was only seen in the spheroid cultures and not adherent two-dimensional cultures. Recent work characterised FLASH irradiation's mechanism in tumour spheroids and confirmed that spheroids receiving FLASH radiation experienced more oxygen depletion than those receiving conventional radiation [
      • Khan S.
      • Bassenne M.
      • Wang J.
      • Manjappa R.
      • Melemenidis S.
      • Breitkreutz D.Y.
      • et al.
      Multicellular spheroids as in vitro models of oxygen depletion during FLASH irradiation.
      ]. Interestingly, no significant difference was detected for the FLASH effect in well-oxygenated two-dimensional cultured cells, further highlighting the strength of three-dimensional models over two-dimensional models for radiotherapy.
      Although the spheroid model is widely recognised, technical challenges remain for their broader adoption. Three-dimensional culture models are often more complicated, cumbersome and less characterised than two-dimensional cultures (Table 1) [
      • Friedrich J.
      • Seidel C.
      • Ebner R.
      • Kunz-Schughart L.A.
      Spheroid-based drug screen: considerations and practical approach.
      ]. In addition, not all cancer cell lines form spheroids of uniform shape and size [
      • Vinci M.
      • Gowan S.
      • Boxall F.
      • Patterson L.
      • Zimmermann M.
      • Court W.
      • et al.
      Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation.
      ]. Furthermore, spheroids lack vasculature, and drugs may accumulate on the periphery of large spheroids, yielding inconsistent and sometimes confusing results (Table 1) [
      • Mehta G.
      • Hsiao A.Y.
      • Ingram M.
      • Luker G.D.
      • Takayama S.
      Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy.
      ]. Thus, more work is needed to improve the applicability of tumour spheroids for preclinical research to discover new drug–radiotherapy combinations.

       Patient-derived Tumour Organoids

      Like spheroids, patient-derived organoids (PDOs) are another preclinical model offering three-dimensional complexity [
      • Gilazieva Z.
      • Ponomarev A.
      • Rutland C.
      • Rizvanov A.
      • Solovyeva V.
      Promising applications of tumor spheroids and organoids for personalized medicine.
      ]. Whereas spheroids are formed from immortalised tumour cell lines, organoids are developed from resected patient tumour samples. PDOs have been established from several epithelial tumour indications, including colon, liver, pancreas, prostate and lung [
      • Bar-Ephraim Y.E.
      • Kretzschmar K.
      • Clevers H.
      Organoids in immunological research.
      ,
      • Drost J.
      • Clevers H.
      Organoids in cancer research.
      ,
      • Kretzschmar K.
      Cancer research using organoid technology.
      ]. PDOs typically use freshly resected tumour tissue, which is mechanically and/or enzymatically dissociated. The dissociated cells are seeded into matrigel or another natural or synthetic extracellular matrix substrate [
      • Drost J.
      • Clevers H.
      Organoids in cancer research.
      ,
      • Pasch C.A.
      • Favreau P.F.
      • Yueh A.E.
      • Babiarz C.P.
      • Gillette A.A.
      • Sharick J.T.
      • et al.
      Patient-derived cancer organoid cultures to predict sensitivity to chemotherapy and radiation.
      ,
      • Yao Y.
      • Xu X.
      • Yang L.
      • Zhu J.
      • Wan J.
      • Shen L.
      • et al.
      Patient-derived organoids predict chemoradiation responses of locally advanced rectal cancer.
      ] in multiwell plates [
      • Pasch C.A.
      • Favreau P.F.
      • Yueh A.E.
      • Babiarz C.P.
      • Gillette A.A.
      • Sharick J.T.
      • et al.
      Patient-derived cancer organoid cultures to predict sensitivity to chemotherapy and radiation.
      ,
      • Yao Y.
      • Xu X.
      • Yang L.
      • Zhu J.
      • Wan J.
      • Shen L.
      • et al.
      Patient-derived organoids predict chemoradiation responses of locally advanced rectal cancer.
      ]. Organoid cultures can be propagated for multiple weeks and may be cryopreserved for banking [
      • Drost J.
      • Clevers H.
      Organoids in cancer research.
      ,
      • Kretzschmar K.
      Cancer research using organoid technology.
      ,
      • Yao Y.
      • Xu X.
      • Yang L.
      • Zhu J.
      • Wan J.
      • Shen L.
      • et al.
      Patient-derived organoids predict chemoradiation responses of locally advanced rectal cancer.
      ].
      Organoid models have been developed to recapitulate the tissue heterogeneity of the TME, and two distinct methodologies have been reviewed by Bar-Ephraim et al. [
      • Bar-Ephraim Y.E.
      • Kretzschmar K.
      • Clevers H.
      Organoids in immunological research.
      ]. The authors designated one approach as ‘holistic’ [
      • Bar-Ephraim Y.E.
      • Kretzschmar K.
      • Clevers H.
      Organoids in immunological research.
      ] and described Neal et al.'s [
      • Neal J.T.
      • Li X.
      • Zhu J.
      • Giangarra V.
      • Grzeskowiak C.L.
      • Ju J.
      • et al.
      Organoid modeling of the tumor immune microenvironment.
      ] air–liquid interface method of culturing freshly minced tumours, without enzymatic dissociation, suspended in collagen gel. This method was reported to preserve diverse cellular composition, including the tumour epithelium, stromal cells and endogenous immune cells [
      • Neal J.T.
      • Li X.
      • Zhu J.
      • Giangarra V.
      • Grzeskowiak C.L.
      • Ju J.
      • et al.
      Organoid modeling of the tumor immune microenvironment.
      ]. The second approach, referred to as ‘reductionist’, incorporates autologous immune cells isolated from peripheral blood and cocultures them with primary tumour organoids [
      • Bar-Ephraim Y.E.
      • Kretzschmar K.
      • Clevers H.
      Organoids in immunological research.
      ]. Such methods allow for the interrogation of specific cellular relationships and functionality, restricted to only the selected immune subsets included in the coculture.
      PDOs have been used as a predictive model for the response to chemotherapy–radiation combinations. Yao et al. [
      • Yao Y.
      • Xu X.
      • Yang L.
      • Zhu J.
      • Wan J.
      • Shen L.
      • et al.
      Patient-derived organoids predict chemoradiation responses of locally advanced rectal cancer.
      ] described the generation, characterisation and in vitro chemoradiation treatment of a PDO biobank from colorectal cancer primary tumours. PDOs were tested for response to X-ray irradiation, 5-FU and irinotecan, and these responses were compared back to patient responses to neoadjuvant chemoradiation treatment [
      • Yao Y.
      • Xu X.
      • Yang L.
      • Zhu J.
      • Wan J.
      • Shen L.
      • et al.
      Patient-derived organoids predict chemoradiation responses of locally advanced rectal cancer.
      ]. Similarly, Pasch et al. [
      • Pasch C.A.
      • Favreau P.F.
      • Yueh A.E.
      • Babiarz C.P.
      • Gillette A.A.
      • Sharick J.T.
      • et al.
      Patient-derived cancer organoid cultures to predict sensitivity to chemotherapy and radiation.
      ] reported establishing PDOs from multiple tumour types that maintained crucial phenotypic and molecular characteristics of the original tissue. These organoids provided cellular-level quantification of drug responses to predict treatment outcomes accurately, thus paving a path for precision medicine.
      Although the PDO model has in vivo-like physiology, there are a number of limitations (Table 1). Currently, PDO protocols have primarily been restricted to epithelial cancers [
      • Drost J.
      • Clevers H.
      Organoids in cancer research.
      ,
      • Kretzschmar K.
      Cancer research using organoid technology.
      ]. Additionally, given that PDOs are frequently generated from biopsy, the model may only partially represent the tumour [
      • Bar-Ephraim Y.E.
      • Kretzschmar K.
      • Clevers H.
      Organoids in immunological research.
      ]. Another caveat inherent to this model is the loss of original tissue architecture with the tissue's dissociation. Lastly, PDO models, in comparison with tumour cell line models, are significantly more labour-intensive and time-consuming and consequently less amenable to HTS (Table 1) [
      • Drost J.
      • Clevers H.
      Organoids in cancer research.
      ].

       Patient-derived Explant Culture

      Like the PDOs described in the previous section, patient-derived explant culture uses fresh tumour tissue resected from patients, preserving endogenous cellular heterogeneity (Figure 3). Such organotypic culture models keep the native tissue architecture of the TME intact, offering an opportunity to investigate the role of spatial proximity between various cell types [
      • Powley I.R.
      • Patel M.
      • Miles G.
      • Pringle H.
      • Howells L.
      • Thomas A.
      • et al.
      Patient-derived explants (PDEs) as a powerful preclinical platform for anti-cancer drug and biomarker discovery.
      ]. Tumours can be stratified into either immune-infiltrated ‘hot’ or ‘cold’ tumours with little to no immune infiltrate or ‘immune excluded’ where the immune cells are present but have not infiltrated the tumour parenchyma [
      • Powley I.R.
      • Patel M.
      • Miles G.
      • Pringle H.
      • Howells L.
      • Thomas A.
      • et al.
      Patient-derived explants (PDEs) as a powerful preclinical platform for anti-cancer drug and biomarker discovery.
      ]. This allows the investigation of drug and/or radiotherapy mechanisms of action in the context of known immune infiltration state.
      An early application of patient-derived ex vivo culture in the mid-1980s was the Histoculture Drug Response Assay, which was reported to show high accuracy for predicting a patient's response and resistance to chemotherapy [
      • Freeman A.E.
      • Hoffman R.M.
      In vivo-like growth of human tumors in vitro.
      ,
      • Vescio R.A.
      • Redfern C.H.
      • Nelson T.J.
      • Ugoretz S.
      • Stern P.H.
      • Hoffman R.M.
      In vivo-like drug responses of human tumors growing in three-dimensional gel-supported primary culture.
      ,
      • Vescio R.A.
      • Connors K.M.
      • Kubota T.
      • Hoffman R.M.
      Correlation of histology and drug response of human tumors grown in native-state three-dimensional histoculture and in nude mice.
      ]. Interestingly, broad use of histoculture modelling was not adopted at that time and this may be in part due to the gaining popularity of other preclinical models, such as two- and three-dimensional cell line models and patient-derived xenografts [
      • Powley I.R.
      • Patel M.
      • Miles G.
      • Pringle H.
      • Howells L.
      • Thomas A.
      • et al.
      Patient-derived explants (PDEs) as a powerful preclinical platform for anti-cancer drug and biomarker discovery.
      ]. In more recent years, with recognition of the impact of the TME, there may be reinvigorated interest in complex histoculture models. This is evident with the use of histoculture models in preclinical immunotherapy drug discovery efforts, including recent reports studying pharmacodynamic response for a PARP14 inhibitor [
      • Schenkel L.B.
      • Molina J.R.
      • Swinger K.K.
      • Abo R.
      • Blackwell D.J.
      • Lu A.Z.
      • et al.
      A potent and selective PARP14 inhibitor decreases protumor macrophage gene expression and elicits inflammatory responses in tumor explants.
      ] and regulatory T cell depleting anti-CCR8 antibody [
      • Campbell J.R.
      • McDonald B.R.
      • Mesko P.B.
      • Siemers N.O.
      • Singh P.B.
      • Selby M.
      • et al.
      Fc-optimized anti-CCR8 antibody depletes regulatory t cells in human tumor models.
      ].
      Several protocols culturing fresh human tumour tissue have been described [
      • Freeman A.E.
      • Hoffman R.M.
      In vivo-like growth of human tumors in vitro.
      ]. Freshly resected tumour tissue is typically either diced into small fragments [
      • Powley I.R.
      • Patel M.
      • Miles G.
      • Pringle H.
      • Howells L.
      • Thomas A.
      • et al.
      Patient-derived explants (PDEs) as a powerful preclinical platform for anti-cancer drug and biomarker discovery.
      ,
      • Freeman A.E.
      • Hoffman R.M.
      In vivo-like growth of human tumors in vitro.
      ,
      • Bayin N.S.
      • Ma L.
      • Thomas C.
      • Baitalmal R.
      • Sure A.
      • Fansiwala K.
      • et al.
      Patient-specific screening using high-grade glioma explants to determine potential radiosensitization by a TGF-beta small molecule inhibitor.
      ,
      • Centenera M.M.
      • Raj G.V.
      • Knudsen K.E.
      • Tilley W.D.
      • Butler L.M.
      Ex vivo culture of human prostate tissue and drug development.
      ,
      • Dohmen A.J.C.
      • Sanders J.
      • Canisius S.
      • Jordanova E.S.
      • Aalbersberg E.A.
      • van den Brekel M.W.M.
      • et al.
      Sponge-supported cultures of primary head and neck tumors for an optimized preclinical model.
      ] or sliced using a vibratome [
      • Powley I.R.
      • Patel M.
      • Miles G.
      • Pringle H.
      • Howells L.
      • Thomas A.
      • et al.
      Patient-derived explants (PDEs) as a powerful preclinical platform for anti-cancer drug and biomarker discovery.
      ,
      • Centenera M.M.
      • Raj G.V.
      • Knudsen K.E.
      • Tilley W.D.
      • Butler L.M.
      Ex vivo culture of human prostate tissue and drug development.
      ,
      • Gerlach M.M.
      • Merz F.
      • Wichmann G.
      • Kubick C.
      • Wittekind C.
      • Lordick F.
      • et al.
      Slice cultures from head and neck squamous cell carcinoma: a novel test system for drug susceptibility and mechanisms of resistance.
      ,
      • Koerfer J.
      • Kallendrusch S.
      • Merz F.
      • Wittekind C.
      • Kubick C.
      • Kassahun W.T.
      • et al.
      Organotypic slice cultures of human gastric and esophagogastric junction cancer.
      ,
      • Merz F.
      • Gaunitz F.
      • Dehghani F.
      • Renner C.
      • Meixensberger J.
      • Gutenberg A.
      • et al.
      Organotypic slice cultures of human glioblastoma reveal different susceptibilities to treatments.
      ,
      • Misra S.
      • Moro C.F.
      • Del Chiaro M.
      • Pouso S.
      • Sebestyen A.
      • Lohr M.
      • et al.
      Ex vivo organotypic culture system of precision-cut slices of human pancreatic ductal adenocarcinoma.
      ]. The tissue is either supported by a membrane insert [
      • Powley I.R.
      • Patel M.
      • Miles G.
      • Pringle H.
      • Howells L.
      • Thomas A.
      • et al.
      Patient-derived explants (PDEs) as a powerful preclinical platform for anti-cancer drug and biomarker discovery.
      ,
      • Bayin N.S.
      • Ma L.
      • Thomas C.
      • Baitalmal R.
      • Sure A.
      • Fansiwala K.
      • et al.
      Patient-specific screening using high-grade glioma explants to determine potential radiosensitization by a TGF-beta small molecule inhibitor.
      ,
      • Centenera M.M.
      • Raj G.V.
      • Knudsen K.E.
      • Tilley W.D.
      • Butler L.M.
      Ex vivo culture of human prostate tissue and drug development.
      ,
      • Gerlach M.M.
      • Merz F.
      • Wichmann G.
      • Kubick C.
      • Wittekind C.
      • Lordick F.
      • et al.
      Slice cultures from head and neck squamous cell carcinoma: a novel test system for drug susceptibility and mechanisms of resistance.
      ,
      • Koerfer J.
      • Kallendrusch S.
      • Merz F.
      • Wittekind C.
      • Kubick C.
      • Kassahun W.T.
      • et al.
      Organotypic slice cultures of human gastric and esophagogastric junction cancer.
      ,
      • Merz F.
      • Gaunitz F.
      • Dehghani F.
      • Renner C.
      • Meixensberger J.
      • Gutenberg A.
      • et al.
      Organotypic slice cultures of human glioblastoma reveal different susceptibilities to treatments.
      ,
      • Misra S.
      • Moro C.F.
      • Del Chiaro M.
      • Pouso S.
      • Sebestyen A.
      • Lohr M.
      • et al.
      Ex vivo organotypic culture system of precision-cut slices of human pancreatic ductal adenocarcinoma.
      ] or a collagen sponge [
      • Powley I.R.
      • Patel M.
      • Miles G.
      • Pringle H.
      • Howells L.
      • Thomas A.
      • et al.
      Patient-derived explants (PDEs) as a powerful preclinical platform for anti-cancer drug and biomarker discovery.
      ,
      • Freeman A.E.
      • Hoffman R.M.
      In vivo-like growth of human tumors in vitro.
      ,
      • Centenera M.M.
      • Raj G.V.
      • Knudsen K.E.
      • Tilley W.D.
      • Butler L.M.
      Ex vivo culture of human prostate tissue and drug development.
      ,
      • Dohmen A.J.C.
      • Sanders J.
      • Canisius S.
      • Jordanova E.S.
      • Aalbersberg E.A.
      • van den Brekel M.W.M.
      • et al.
      Sponge-supported cultures of primary head and neck tumors for an optimized preclinical model.
      ], which helps to preserve tissue structure and prolong the survival of stromal cells [
      • Centenera M.M.
      • Raj G.V.
      • Knudsen K.E.
      • Tilley W.D.
      • Butler L.M.
      Ex vivo culture of human prostate tissue and drug development.
      ]. Majumder et al. [
      • Majumder B.
      • Baraneedharan U.
      • Thiyagarajan S.
      • Radhakrishnan P.
      • Narasimhan H.
      • Dhandapani M.
      • et al.
      Predicting clinical response to anticancer drugs using an ex vivo platform that captures tumour heterogeneity.
      ] investigated even further complex growth substrates, reporting that grade-matched tumour-stromal matrix proteins conserved baseline tumour tissue characteristics more effectively than mismatched tumour-stromal matrix proteins.
      Both human tumour slice culture and fragment explants have been used to study drug–radiation combinations ex vivo. Merz et al. [
      • Merz F.
      • Gaunitz F.
      • Dehghani F.
      • Renner C.
      • Meixensberger J.
      • Gutenberg A.
      • et al.
      Organotypic slice cultures of human glioblastoma reveal different susceptibilities to treatments.
      ] reported using human glioblastoma slice culture to investigate ex vivo exposure to irradiation, temozolomide or the two in combination. Single treatments of irradiation and temozolomide resulted in cell death, whereas no synergy was observed in the combination treatment. Bayin et al. [
      • Bayin N.S.
      • Ma L.
      • Thomas C.
      • Baitalmal R.
      • Sure A.
      • Fansiwala K.
      • et al.
      Patient-specific screening using high-grade glioma explants to determine potential radiosensitization by a TGF-beta small molecule inhibitor.
      ] described using high-grade glioma explants from seven patients to test ex vivo treatment of transforming growth factor-β inhibitor, LY364947, as a radiosensitiser. These applications of human tumour slice and explant cultures provide evidence of using fresh tissue for ex vivo modelling to complement tumour cell line and murine models.
      Although patient-derived explant models recapitulate the complexities of the TME, there are several limitations (Table 1). This model relies on collaboration with clinicians, surgeons and pathologists to have a source of freshly resected tumour tissue accessible [
      • Powley I.R.
      • Patel M.
      • Miles G.
      • Pringle H.
      • Howells L.
      • Thomas A.
      • et al.
      Patient-derived explants (PDEs) as a powerful preclinical platform for anti-cancer drug and biomarker discovery.
      ,
      • Centenera M.M.
      • Raj G.V.
      • Knudsen K.E.
      • Tilley W.D.
      • Butler L.M.
      Ex vivo culture of human prostate tissue and drug development.
      ]. Additionally, not all tissue samples can be successfully cultured due to necrosis [
      • Powley I.R.
      • Patel M.
      • Miles G.
      • Pringle H.
      • Howells L.
      • Thomas A.
      • et al.
      Patient-derived explants (PDEs) as a powerful preclinical platform for anti-cancer drug and biomarker discovery.
      ]. Moreover, the tumour tissue can only be maintained for a short duration of time, and the number of available tissue slices restricts assay throughput (Table 1) [
      • Powley I.R.
      • Patel M.
      • Miles G.
      • Pringle H.
      • Howells L.
      • Thomas A.
      • et al.
      Patient-derived explants (PDEs) as a powerful preclinical platform for anti-cancer drug and biomarker discovery.
      ,
      • Centenera M.M.
      • Raj G.V.
      • Knudsen K.E.
      • Tilley W.D.
      • Butler L.M.
      Ex vivo culture of human prostate tissue and drug development.
      ,
      • Gerlach M.M.
      • Merz F.
      • Wichmann G.
      • Kubick C.
      • Wittekind C.
      • Lordick F.
      • et al.
      Slice cultures from head and neck squamous cell carcinoma: a novel test system for drug susceptibility and mechanisms of resistance.
      ,
      • Misra S.
      • Moro C.F.
      • Del Chiaro M.
      • Pouso S.
      • Sebestyen A.
      • Lohr M.
      • et al.
      Ex vivo organotypic culture system of precision-cut slices of human pancreatic ductal adenocarcinoma.
      ]. Finally, studies using the explant model are confined to mechanisms within the TME, as this closed system model lacks the systemic signalling and immune trafficking observed in vivo [
      • Powley I.R.
      • Patel M.
      • Miles G.
      • Pringle H.
      • Howells L.
      • Thomas A.
      • et al.
      Patient-derived explants (PDEs) as a powerful preclinical platform for anti-cancer drug and biomarker discovery.
      ,
      • Centenera M.M.
      • Raj G.V.
      • Knudsen K.E.
      • Tilley W.D.
      • Butler L.M.
      Ex vivo culture of human prostate tissue and drug development.
      ].

       In Vivo Tumour Models

      Animal models provide one of the most advanced preclinical models to develop novel drug therapeutics. These models bridge the gap between in vitro experiments and first in human studies, providing useful information about efficacy, mechanism of action, pharmacokinetics and pharmacodynamics and determining dose-associated toxicities. Murine models are one of the most commonly used preclinical in vivo models due to their relatively low cost, ease of handling and known genetic information.
      Based on the tumour cell line and tumour tissue used for the study, commonly used rodent tumour models are classified into the following categories: (i) syngeneic tumour models, which involve implanting immortalised murine tumour cells in the immune-proficient mice [
      • Dovedi S.J.
      • Adlard A.L.
      • Lipowska-Bhalla G.
      • McKenna C.
      • Jones S.
      • Cheadle E.J.
      • et al.
      Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade.
      ], (ii) cell line-derived xenografts, where immortalised human tumour cell lines are implanted in an immunodeficient mouse [
      • Morton C.L.
      • Houghton P.J.
      Establishment of human tumor xenografts in immunodeficient mice.
      ], (iii) patient-derived xenografts, where patient tumour tissue is implanted directly into immunodeficient mice [
      • Tentler J.J.
      • Tan A.C.
      • Weekes C.D.
      • Jimeno A.
      • Leong S.
      • Pitts T.M.
      • et al.
      Patient-derived tumour xenografts as models for oncology drug development.
      ,
      • Jung J.
      Human tumor xenograft models for preclinical assessment of anticancer drug development.
      ], and (iv) genetically engineered mouse models, where genetic alteration of one or several genes thought to be involved in carcinogenesis are either deleted, mutated or overexpressed [
      • Kersten K.
      • de Visser K.E.
      • van Miltenburg M.H.
      • Jonkers J.
      Genetically engineered mouse models in oncology research and cancer medicine.
      ] (Table 1). Tumour cells are either implanted ectopically or orthotopically injected into the organ directly (Figure 3E). Although the orthotopic model allows investigation of the potential influence of the murine microenvironment on the organ of interest, it is sometimes difficult to irradiate the tumour at the site of tumour implantation. Moreover, it is hard to monitor tumour sizes over time accurately. On the other hand, subcutaneous tumour models are widely used due to their simplicity and ease with which subcutaneous tumours can be implanted and irradiated without affecting other mouse organs. However, these models have a caveat that they are less physiological and are rarely metastatic.
      These in vivo models have successfully been used to test the efficacy of radiotherapy in combination with some of the known cytotoxic drugs such as mitomycin, cisplatin, 5-FU, as well for some of the newer drugs such as olaparib, a PARP inhibitor, which are still part of the first line of cancer care [
      • Budach W.
      • Paulsen F.
      • Welz S.
      • Classen J.
      • Scheithauer H.
      • Marini P.
      • et al.
      Mitomycin C in combination with radiotherapy as a potent inhibitor of tumour cell repopulation in a human squamous cell carcinoma.
      ,
      • Chaudary N.
      • Pintilie M.
      • Hedley D.
      • Hill R.P.
      • Milosevic M.
      • Mackay H.
      Hedgehog inhibition enhances efficacy of radiation and cisplatin in orthotopic cervical cancer xenografts.
      ,
      • Urick M.E.
      • Chung E.J.
      • Shield 3rd, W.P.
      • Gerber N.
      • White A.
      • Sowers A.
      • et al.
      Enhancement of 5-fluorouracil-induced in vitro and in vivo radiosensitization with MEK inhibition.
      ,
      • Senra J.M.
      • Telfer B.A.
      • Cherry K.E.
      • McCrudden C.M.
      • Hirst D.G.
      • O'Connor M.J.
      • et al.
      Inhibition of PARP-1 by olaparib (AZD2281) increases the radiosensitivity of a lung tumor xenograft.
      ]. Although both syngeneic and xenograft models have been used to study tumour radioresponse in vivo [
      • Chandler B.C.
      • Moubadder L.
      • Ritter C.L.
      • Liu M.
      • Cameron M.
      • Wilder-Romans K.
      • et al.
      TTK inhibition radiosensitizes basal-like breast cancer through impaired homologous recombination.
      ,
      • Yoon S.S.
      • Stangenberg L.
      • Lee Y.J.
      • Rothrock C.
      • Dreyfuss J.M.
      • Baek K.H.
      • et al.
      Efficacy of sunitinib and radiotherapy in genetically engineered mouse model of soft-tissue sarcoma.
      ,
      • Znati S.
      • Carter R.
      • Vasquez M.
      • Westhorpe A.
      • Shahbakhti H.
      • Prince J.
      • et al.
      Radiosensitisation of hepatocellular carcinoma cells by vandetanib.
      ], these models have limitations (Table 1). For syngeneic models, there are fewer murine tumour cell lines available than human tumour cell lines. Furthermore, the results can be challenging to interpret due to inherent differences in mouse and human biology. In contrast, human tumour xenografts may better represent human cell radioresponse and activity of DNA repair molecules [
      • Banuelos C.A.
      • Banath J.P.
      • MacPhail S.H.
      • Zhao J.
      • Eaves C.A.
      • O'Connor M.D.
      • et al.
      Mouse but not human embryonic stem cells are deficient in rejoining of ionizing radiation-induced DNA double-strand breaks.
      ,
      • Kahn J.
      • Tofilon P.J.
      • Camphausen K.
      Preclinical models in radiation oncology.
      ] than syngeneic models, but may not be an excellent model to study the immune response after radiation as they lack the host immune system (Table 1). Besides widespread use of rodent tumour models, canine and feline tumour models have been used for radiation research [
      • Nolan M.W.
      • Kent M.S.
      • Boss M.K.
      Emerging translational opportunities in comparative oncology with companion canine cancers: radiation oncology.
      ]. These animals inherently develop cancers that resemble human malignancies, thus providing an excellent opportunity to study the aetiology of cancer progression [
      • Dow S.
      A role for dogs in advancing cancer immunotherapy research.
      ]. Additionally, their immune system closely resembles humans, thus making them a potentially more favourable model system for studying the impact of radiation on the host immune system.

      Conclusion

      In summary, no single preclinical model can fully recapitulate the intricacies of patient tumours. Although two-dimensional models are amenable to HTS, they do not offer the complexity of the TME. In contrast, human organoids and histoculture have the advantage of mimicking the TME of an actual tumour. However, they are challenging to establish, being more variable and not amenable to HTS. The optimal approach to discover novel drug–radiotherapy combinations is to choose the most appropriate model according to the scientific question and drug modalities and carefully orchestrate at least two preclinical models to validate the findings.

      Conflicts of Interest

      J. Singh, S. Hatcher, A.A. Ku, Z. Ding, R.A. Sharma and S.X. Pfister are employees of Varian, a Siemens Healthineers company. F.Y. Feng serves as a consultant for Varian, a Siemens Healthineers company.
      The open access APC for this article has been paid by Varian, a Siemens Healthineers company. All articles were independently peer-reviewed and accepted as per journal policy prior to any open access funding arrangements.

      Acknowledgments

      We thank all members of the Global Translational Science team at Varian Medical Systems for their input and feedback. Apologies to all the colleagues whose work we were not able to cite due to space limitations.

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