This protocol describes a strategy for generating 3D prostate organoid cultures from healthy mouse and human prostate cells (either bulk or FAC-sorted single luminal and basal cells), metastatic prostate cancer lesions and circulating tumor cells. This protocol describes a strategy for the generation of 3D prostate organoid cultures from healthy mouse and human prostate cells (either bulk or FACS-sorted single luminal and basal cells), metastatic prostate cancer lesions and circulating tumor cells. Organoids derived from healthy material contain the differentiated luminal and basal cell types, whereas organoids derived from prostate cancer tissue mimic the histology of the tumor. We explain how to establish these cultures in the fully defined serum-free conditioned medium that is required to sustain organoid growth. Starting with the plating of digested tissue material, full-grown organoids can usually be obtained in ∼2 weeks. The culture protocol we describe here is currently the only one that allows the growth of both the luminal and basal prostatic epithelial lineages, as well as the growth of advanced prostate cancers. Organoids established using this protocol can be used to study many different aspects of prostate biology, including homeostasis, tumorigenesis and drug discovery.

Previous studies have described that tumor organoids can capture the diversity of defined human carcinoma types. Here, we describe conditions for long-term culture of human mucosal organoids. Using this protocol, a panel of 31 head and neck squamous cell carcinoma (HNSCC)–derived organoid lines was established. This panel recapitulates genetic and molecular characteristics previously described for HNSCC. Organoids retain their tumorigenic potential upon xenotransplantation. We observe differential responses to a panel of drugs including cisplatin, carboplatin, cetuximab, and radiotherapy in vitro. Additionally, drug screens reveal selective sensitivity to targeted drugs that are not normally used in the treatment of patients with HNSCC. These observations may inspire a personalized approach to the management of HNSCC and expand the repertoire of HNSCC drugs.Significance: This work describes the culture of organoids derived from HNSCC and corresponding normal epithelium. These tumoroids recapitulate the disease genetically, histologically, and functionally. In vitro drug screening of tumoroids reveals responses to therapies both currently used in the treatment of HNSCC and those not (yet) used in clinical practice.See related commentary by Hill and D'Andrea, p. 828.This article is highlighted in the In This Issue feature, p. 813

Organoids are in vitro three-dimensional structures that can be grown from patient tissue. Head and neck cancer (HNC) is a collective term used for multiple tumor types including squamous cell carcinomas and salivary gland adenocarcinomas. Organoids were established from HNC patient tumor tissue and characterized using immunohistochemistry and DNA sequencing. Organoids were exposed to chemo- and radiotherapy and a panel of targeted agents. Organoid response was correlated with patient clinical response. CRISPR-Cas9-based gene editing of organoids was applied for biomarker validation. A HNC biobank consisting of 110 models, including 65 tumor models, was generated. Organoids retained DNA alterations found in HNC. Comparison of organoid and patient response to radiotherapy (primary [n = 6] and adjuvant [n = 15]) indicated potential for guiding treatment options in the adjuvant setting. In organoids, the radio-sensitizing potential of cisplatin and carboplatin could be validated. However, cetuximab conveyed radioprotection in most models. HNC-targeted treatments were tested on 31 models, indicating possible novel treatment options with the potential for treatment stratification in the future. Activating PIK3CA mutations did not predict alpelisib response in organoids. Protein arginine methyltransferase 5 (PRMT5) inhibitors were identified as a potential treatment option for cyclin-dependent kinase inhibitor 2A (CDKN2A) null HNC. Organoids hold potential as a diagnostic tool in personalized medicine for HNC. In vitro organoid response to radiotherapy (RT) showed a trend that mimics clinical response, indicating the predictive potential of patient-derived organoids. Moreover, organoids could be used for biomarker discovery and validation. This work was funded by Oncode PoC 2018-P0003.

Patients diagnosed with head and neck squamous cell carcinoma (HNSCC) are currently treated with surgery and/or radio- and chemotherapy. Despite these therapeutic interventions, 40% of patients relapse, urging the need for more effective therapies. In photodynamic therapy (PDT), a light-activated photosensitizer produces reactive oxygen species that ultimately lead to cell death. Targeted PDT, using a photosensitizer conjugated to tumor-targeting molecules, has been explored as a more selective cancer therapy. Organoids are self-organizing three-dimensional structures that can be grown from both normal and tumor patient-material and have recently shown translational potential. Here, we explore the potential of a recently described HNSCC–organoid model to evaluate Epidermal Growth Factor Receptor (EGFR)-targeted PDT, through either antibody- or nanobody-photosensitizer conjugates. We find that EGFR expression levels differ between organoids derived from different donors, and recapitulate EGFR expression levels of patient material. EGFR expression levels were found to correlate with the response to EGFR-targeted PDT. Importantly, organoids grown from surrounding normal tissues showed lower EGFR expression levels than their tumor counterparts, and were not affected by the treatment. In general, nanobody-targeted PDT was more effective than antibody-targeted PDT. Taken together, patient-derived HNSCC organoids are a useful 3D model for testing in vitro targeted PDT.