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

Canine lymphoma (cL) is a common type of neoplasia in dogs with an estimated incidence rate of 20–100 cases per 100,000 dogs and is in many respects comparable to non-Hodgkin lymphoma in humans. Although the exact cause is unknown, environmental factors and genetic susceptibility are thought to play an important role. cL is not a single disease, and a wide variation in clinical presentations and histological subtypes is recognized. Despite this potential variation, most dogs present with generalized lymphadenopathy (multicentric form) and intermediate to high-grade lymphoma, more commonly of B-cell origin. The most common paraneoplastic sign is hypercalcemia that is associated with the T-cell immunophenotype. Chemotherapy is the treatment of choice and a doxorubicin-based multidrug protocol is currently the standard of care. A complete remission is obtained for most dogs and lasts for a median period of 7–10 months, resulting in a median survival of 10–14 months. Many prognostic factors have been reported, but stage, immunophenotype, tumor grade, and response to chemotherapy appear of particular importance. Failure to respond to chemotherapy suggests drug resistance, which can be partly attributed to the expression of drug transporters of the ABC-transporter superfamily, including P-gp and BCRP. Ultimately, most lymphomas will become drug resistant and the development of treatments aimed at reversing drug resistance or alternative treatment modalities (e.g. immunotherapy and targeted therapy) are of major importance. This review aims to summarize the relevant data on cL, as well as to provide an update of the recent literature.

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.

In mice, intestinal tuft cells have been described as a long-lived, postmitotic cell type. Two distinct subsets have been identified: tuft-1 and tuft-2 (ref. ¹). By combining analysis of primary human intestinal resection material and intestinal organoids, we identify four distinct human tuft cell states, two of which overlap with their murine counterparts. We show that tuft cell development depends on the presence of Wnt ligands, and that tuft cell numbers rapidly increase on interleukin-4 (IL-4) and IL-13 exposure, as reported previously in mice^2,3,4. This occurs through proliferation of pre-existing tuft cells, rather than through increased de novo generation from stem cells. Indeed, proliferative tuft cells occur in vivo both in fetal and in adult human intestine. Single mature proliferating tuft cells can form organoids that contain all intestinal epithelial cell types. Unlike stem and progenitor cells, human tuft cells survive irradiation damage and retain the ability to generate all other epithelial cell types. Accordingly, organoids engineered to lack tuft cells fail to recover from radiation-induced damage. Thus, tuft cells represent a damage-induced reserve intestinal stem cell pool in humans.

Canine lymphoma is typically treated with a doxorubicin-based multidrug chemotherapy protocol. Although this is often initially successful, tumour recurrence is common and frequently refractory to treatment. Failure to respond to chemotherapy is thought to represent drug resistance and has been associated with active efflux of cytostatic drugs by transporter proteins of the ATP-binding cassette (ABC) family, including P-glycoprotein (ABCB1), MRP1 (ABCC1) and BCRP (ABCG2). In this study, ABC transporter mRNA expression was assessed in 63 dogs diagnosed with multicentric lymphoma that were treated with a doxorubicin-based chemotherapy protocol. Expression of ABCB1, ABCB5, ABCB8, ABCC1, ABCC3, ABCC5 and ABCG2 mRNA was quantified in tumour samples (n = 107) obtained at the time of diagnosis, at first tumour relapse and when the tumour was no longer responsive to cytostatic drugs while receiving chemotherapy. Expression data were related to patient demographics, staging, treatment response and drug resistance (absent, intrinsic, acquired). ABC transporter expression was independent of sex, weight, age, stage or substage, but T cell lymphoma and hypercalcaemia were associated with increased ABCB5 and ABCC5 expression, and decreased ABCC1 mRNA expression. Drug resistance occurred in 35/63 (55.6%) dogs and was associated with increased ABCB1 mRNA expression in a subset of dogs with B cell lymphoma, and with increased ABCG2 and decreased ABCB8, ABCC1 and ABCC3 mRNA expression in T cell lymphomas. ABC transporter expression in the pre-treatment sample was not predictive of the length of the first disease-free period or overall survival. Glucocorticoids had no effect on ABC transporter mRNA expression. In conclusion, drug resistance in canine multicentric lymphoma is an important cause of treatment failure and is associated with upregulation of ABCB1 and ABCG2 mRNA.