Establishment of Pancreatic Organoids from Normal Tissue and Tumors

Establishment of patient-derived adult stem cell-based pancreatic (tumor) organoids was first described in 2015. Since then, multiple laboratories have demonstrated the robustness of this method. We recently described the generation of a pancreatic cancer biobank containing 30 well-characterized tumor organoid models. Here, we describe the applied methods in detail. Use of tumor-selective media prevents contamination with normal cells. Generated organoids can be cryopreserved and can serve as a living biobank of pancreatic cancer for biomarker identification and drug screening. For complete information on the generation and use of this protocol, please refer to Driehuis et al. (2019)

Jaw Bone Invasion of Oral Squamous Cell Carcinoma Is Associated with Osteoclast Count and Expression of Its Regulating Proteins in Patients and Organoids

AIMS: Oral squamous cell carcinoma (OSCC) frequently invades the jaw. The exact mechanism of bone invasion remains unclear. This study investigates (premature) osteoclasts and the expression of its differentiation regulating proteins RANKL, OPG and RANK in patients with OSCC. METHODS: Resection specimens from OSCC patients were divided into NI group (No Invasion), E group (Erosion) or I group (bone Invasion). Tissue sections were stained with Cathepsin K (osteoclast-counting), RANKL, OPG and RANK. The staining intensity was scored on different regions of the tumor: front, center, back and normal mucosa. Immunohistochemistry and qPCR for RANKL/OPG/RANK were performed on five head and neck squamous cell carcinoma (HNSCC) organoids. RESULTS: The mean number of osteoclasts (I group) and premature osteoclasts (E group) was significantly higher compared to the NI group ( p = 0.003, p = 0.036). RANKL expression was significantly higher in the tumor front and tumor center compared to normal mucosa (all groups). In the I group, RANKL and RANK expression was significantly higher in the tumor front compared to the tumor back and there was a trend of higher RANKL expression in the tumor front compared to the E group and NI group. qPCR showed a 20-43 times higher RANKL mRNA expression in three out of five tumor organoids compared to a normal squamous cell organoid line. There was no correlation between protein and mRNA expression in the HNSCC organoids. CONCLUSIONS: These findings suggest that OSCCs induce bone invasion by stimulating osteoclast activation by regulating the production of RANKL and RANK proteins

Patient-derived head and neck cancer organoids allow treatment stratification and serve as a tool for biomarker validation and identification

Background: 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.Methods: 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.Findings: 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.Conclusions: 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.</p

Epidermal growth factor receptor (EGFR) is a target of the tumor-suppressor E3 ligase FBXW7

FBXW7 is an E3 ubiquitin ligase that targets proteins for proteasome-mediated degradation and is mutated in various cancer types. Here, we use CRISPR base editors to introduce different FBXW7 hotspot mutations in human colon organoids. Functionally, FBXW7 mutation reduces EGF dependency of organoid growth by ~10,000-fold. Combined transcriptomic and proteomic analyses revealed increased EGFR protein stability in FBXW7 mutants. Two distinct phosphodegron motifs reside in the cytoplasmic tail of EGFR. Mutations in these phosphodegron motifs occur in human cancer. CRISPR-mediated disruption of the phosphodegron motif at T693 reduced EGFR degradation and EGF growth factor dependency. FBXW7 mutant organoids showed reduced sensitivity to EGFR-MAPK inhibitors. These observations were further strengthened in CRC-derived organoid lines and validated in a cohort of patients treated with panitumumab. Our data imply that FBXW7 mutations reduce EGF dependency by disabling EGFR turnover

Epidermal growth factor receptor (EGFR) is a target of the tumor-suppressor E3 ligase FBXW7

FBXW7 is an E3 ubiquitin ligase that targets proteins for proteasome-mediated degradation and is mutated in various cancer types. Here, we use CRISPR base editors to introduce different FBXW7 hotspot mutations in human colon organoids. Functionally, FBXW7 mutation reduces EGF dependency of organoid growth by ~10,000-fold. Combined transcriptomic and proteomic analyses revealed increased EGFR protein stability in FBXW7 mutants. Two distinct phosphodegron motifs reside in the cytoplasmic tail of EGFR. Mutations in these phosphodegron motifs occur in human cancer. CRISPR-mediated disruption of the phosphodegron motif at T693 reduced EGFR degradation and EGF growth factor dependency. FBXW7 mutant organoids showed reduced sensitivity to EGFR-MAPK inhibitors. These observations were further strengthened in CRC-derived organoid lines and validated in a cohort of patients treated with panitumumab. Our data imply that FBXW7 mutations reduce EGF dependency by disabling EGFR turnover

Dutch National Round Robin Trial on Plasma-Derived Circulating Cell-Free DNA Extraction Methods Routinely Used in Clinical Pathology for Molecular Tumor Profiling

BACKGROUND: Efficient recovery of circulating tumor DNA (ctDNA) depends on the quantity and quality of circulating cell-free DNA (ccfDNA). Here, we evaluated whether various ccfDNA extraction methods routinely applied in Dutch laboratories affect ccfDNA yield, ccfDNA integrity, and mutant ctDNA detection, using identical lung cancer patient-derived plasma samples. METHODS: Aliquots of 4 high-volume diagnostic leukapheresis plasma samples and one artificial reference plasma sample with predetermined tumor-derived mutations were distributed among 14 Dutch laboratories. Extractions of ccfDNA were performed according to local routine standard operating procedures and were analyzed at a central reference laboratory for mutant detection and assessment of ccfDNA quantity and integrity. RESULTS: Mutant molecule levels in extracted ccfDNA samples varied considerably between laboratories, but there was no indication of consistent above or below average performance. Compared to silica membrane-based methods, samples extracted with magnetic beads-based kits revealed an overall lower total ccfDNA yield (-29%; P < 0.0001) and recovered fewer mutant molecules (-41%; P < 0.01). The variant allelic frequency and sample integrity were similar. In samples with a higher-than-average total ccfDNA yield, an augmented recovery of mutant molecules was observed. CONCLUSIONS: In the Netherlands, we encountered diversity in preanalytical workflows with potential consequences on mutant ctDNA detection in clinical practice. Silica membrane-based methodologies resulted in the highest total ccfDNA yield and are therefore preferred to detect low copy numbers of relevant mutations. Harmonization of the extraction workflow for accurate quantification and sensitive detection is required to prevent introduction of technical divergence in the preanalytical phase and reduce interlaboratory discrepancies

Patient-derived head and neck cancer organoids allow treatment stratification and serve as a tool for biomarker validation and identification

Background: 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. Methods: 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. Findings: 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. Conclusions: 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

Pancreatic cancer organoids recapitulate disease and allow personalized drug screening

We report the derivation of 30 patient-derived organoid lines (PDOs) from tumors arising in the pancreas and distal bile duct. PDOs recapitulate tumor histology and contain genetic alterations typical of pancreatic cancer. In vitro testing of a panel of 76 therapeutic agents revealed sensitivities currently not exploited in the clinic, and underscores the importance of personalized approaches for effective cancer treatment. The PRMT5 inhibitor EZP015556, shown to target MTAP (a gene commonly lost in pancreatic cancer)-negative tumors, was validated as such, but also appeared to constitute an effective therapy for a subset of MTAP-positive tumors. Taken together, the work presented here provides a platform to identify novel therapeutics to target pancreatic tumor cells using PDOs

Patient-derived head and neck cancer organoids allow treatment stratification and serve as a tool for biomarker validation and identification

Background: 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. Methods: 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. Findings: 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. Conclusions: 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

Organoids as a tool for fundamental and translational oncology research : Can organoids guide clinical decision making?

Organoids are mini-organs that can be grown in a petri-dish. These structures can be grown from different sources, including patient material. During my PhD, I have established the ‘recipe’ to grow organoids from the lining of the oral cavity, or tumors derived thereof. In the laboratory, these mini-tumors were subsequently exposed to a range of therapies currently used to treat cancer patients (like radiation and chemotherapy). In a small cohort of patients, we could compare the effect of radiation in patients with the response of organoids grown from their tumors. We saw that this organoid response can predict the response of the patient. We are now testing if this predictive potential holds true in a larger group of patients. If it does, this suggests that organoid-tests could help doctors make better informed decisions on which therapy to give to which patients (a concept also known as ‘personalized medicine’). We have also established pancreatic tumor organoids. Here, we saw that for organoids derived from each individual patient, a different drug worked best to kill the tumor cells. This is an interesting finding when you think about the fact that most of these patients will currently receive identical treatment. I have tried to use organoids in more ways to aid or complement the clinic. For example, we used them to study oral mucositis, a common side-effect of chemotherapy given to pediatric leukemia patients. Taken together, I have investigated if and how organoids can be used to develop better treatments for cancer patients