Patient-derived Organoid Models Research Articles

Abstract 3070: Non-small cell lung cancer patient-derived organoids as high-throughput chemotherapy testing platforms

Abstract Background: Lung cancer is associated with phenotypic, genotypic, and transcriptomic heterogeneity among patients that poses a threat to the development of precision medicine. Despite being considered as potential drug screening platforms, patient-derived xenograft (PDX) models are limited by low rates and prolonged durations of tumor engraftment. To overcome these major limitations, patient-derived organoid (PDO) models have been developed for standard of care drug testing and the outcome of which is driven by differential transcriptome trajectories. Experimental methods: Single cell suspension from ten surgically resected patient primary non-small cell lung cancer (NSCLC) tissues (adenocarcinoma and squamous cell carcinoma) was mixed with matrigel (growth factor reduced) and cultured in organoid growth medium. Organoids were characterized for structural (Hematoxylin & Eosin staining) and pathological marker (immunohistochemistry for cytokeratin (CK) 5/6, CK7, Napsin A, Thyroid transcription factor-1 (TTF-1) and p40) similarities with primary tumor. Upon passaging, organoids were seeded in triplicates, allowed to acclimate for 24h and subjected to standard of care chemotherapy testing versus vehicle control. PDOs and corresponding primary tumors were subjected to bulk RNA sequencing to understand the transcriptome driven outcome in chemotherapy sensitive versus resistant organoids. Results: Organoid growth was monitored by light microscopy. PDOs exhibited different growth kinetics indicating the inter-tumor heterogeneity. Time for PDOs to reach 100-200 μm ranged from 6 days to 14 days and exhibited structural features and pathological marker expression similar to their corresponding primary tumors reflecting respective histological subtypes - adenocarcinoma or squamous cell carcinoma. Chemotherapy testing (1 μg/ml Carboplatin plus 0.5 μg/ml Paclitaxel) showed differential responses (day 6 of treatment). Based on the scoring, PDOs were categorized as partially sensitive, very sensitive or resistant (based on organoid growth or regression in presence of chemotherapeutic drugs). Results of RNA sequencing may reveal distinct transcriptomic signatures and molecular pathways reflecting in differential chemoresponses. Conclusions and future perspectives: PDOs capitulate structural and pathological marker expression of patient primary tumors and serve as versatile, high-throughput drug testing platforms to treat NSCLC patients. Further, identification of key transcriptomic signatures and molecular pathways resulting in chemoresistance will be critical in formulating personal precision medicine to treat NSCLC patients. Citation Format: Yariswamy Manjunath, Kanve Nagaraj Suvilesh, Iuliia Innokenteva, Wesley Warren, Eric T. Kimchi, Kevin F. Staveley-O’Carroll, Guangfu Li, Jonathan B. Mitchem, Jussuf T. Kaifi. Non-small cell lung cancer patient-derived organoids as high-throughput chemotherapy testing platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3070.

Abstract 5460: Preclinical characterization of QLH11906, a novel pan-RAF inhibitor

Abstract Background: The type 1/2 BRAF inhibitors (e.g., vemurafenib) have been used for patients with BRAFV600E-mutant tumors. But the application of the inhibitors was restricted to a few types of tumors (e.g., melanoma). Common adverse effects such as pruritus and skin papilloma have limited the usage. It could be attributed to the paradoxical activation of the MAPK pathway. The paradoxical activation also leads to tumor promotion in BRAF wild-type melanoma and skin toxicity in 30%-70% patients with KRAS mutations. Therefore, development of type II pan-RAF inhibitors which avoid MAPK paradoxical activation is necessary. Methods: The selective pan-RAF inhibitor QLH11906 was tested on A/B/C RAF kinases and a representative kinase panel. Both 2D and 3D proliferation studies were conducted for cell lines with BRAF and various KRAS mutations, followed by both immunoprecipitation and ERK phosphorylation assays to elucidate the mode of action. Combination studies with MEK inhibitors were implemented in both KRAS-mutant cell lines and BRAF/KRAS-mutant patient derived organoids (PDO). Single-agent and combination studies were also carried out in vivo on both BRAF/KRAS-mutant cell line-derived xenograft (CDX) and KRAS-mutant patient-derived xenograft (PDX) models. Results: QLH11906 inhibited wild-type A/B/C RAF and BRAFV600E with the IC50 of 4.9, 1.9, 0.5, and 1.1 nM, respectively, and showed no inhibition at 1 μM in a selectivity panel of 28 kinases except for DDR1 and DDR2. Under 2D culture condition, QLH11906 inhibited proliferation of various BRAF/KRAS-mutant cell lines (IC50 300-1700 nM) and suppressed ERK phosphorylation (IC50 20-600 nM). In 3D cultures, QLH11906 showed higher activity in proliferation suppression in both HCT116 (KRASG13D, IC50 24 nM 3D vs 1186 nM 2D) and Calu-6 (KRASQ61K, IC50 85 nM 3D vs 1411 nM 2D). In immunoprecipitation studies with HCT116 and Calu-6 cells, both BRAF homodimers and BRAF/CRAF heterodimers could be detected under the treatment of QLH11906. QLH11906 showed synergistic effect when combined with MEK inhibitors trametinib or cobimetinib in KRAS-mutant cell lines, including A549 (KRASG12S), Calu-6, HCT116, NCIH2122 (KRASG12C) and Panc1005 (KRASG12D), and 77%-99% tumor suppression was reached at the expected clinical Ctrough concentrations. Synergistic effects with trametinib were observed in PDO models including pancreatic cancers (KRASG12D and KRASG12V), colon cancers (KRASG12D and KRASG13D) and lung cancers (KRASG12V). QLH11906 also promoted tumor shrinkage in vivo as a single agent in Calu-6, HCT116, and Colo-205 (BRAFV600E) CDX models or combined with trametinib in PDX models (KRASG12C lung cancers and KRASG12C colon cancers) Conclusion: QLH11906 is a highly selective pan-RAF inhibitor with promising antitumor activity both in vitro and in vivo. It is hopeful to be a therapeutic agent for BRAF and KRAS-mutant tumor patients. Citation Format: Zhe Cheng, Fei Chen, Xile Liu, Cong Gao, Linchao Jia, Yuanzhi Lao. Preclinical characterization of QLH11906, a novel pan-RAF inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5460.

Abstract 5457: QLH11811, a selective 4th-generation EGFR inhibitor for osimertinib-resistant EGFR-mutant NSCLC

Abstract Background: The 3rd-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), like osimertinib, provide marked clinical benefit for EGFR-mutant non-small cell lung cancer (NSCLC) patients with extended overall survival vs former EGFR TKIs (e.g., gefitinib). Approximate 10%-24% of NSCLC patients acquired C797S mutation when treated with osimertinib. Unfortunately, limited treatments are available for patients after osimertinib resistance. QLH11811 is a new generation EGFR TKI designed to target the EGFR with ex19del/L858R/T790M/C797S mutations. Here, we disclosed its preclinical data to support its clinical development in EGFR-mutant NSCLC. Methods: The inhibitory activity of QLH11811 on mutated and wild-type EGFR was tested in engineered cell lines and patient-derived organoid (PDO). The in vivo antitumor activity of QLH11811 was evaluated in the patient-derived xenograft (PDX) model with cis EGFR ex19del/T790M/C797S triple mutations, and the H1975 (cis EGFR L858R/T790M/C797S and cis EGFR ex19del/T790M/C797S), PC-9 (EGFR ex19del), and Ba/F3 (EGFR ex19del/C797S) cell line-derived xenograft (CDX) models. The pharmacokinetic (PK) profile was investigated in animals, and the human PK profile was projected using allometric scaling method. Results: QLH11811 displayed potent anti-proliferation activity against Ba/F3 (EGFR ex19del/T790M/C797S, L858R/T790M/C797S, ex19del/C797S, or L858R/C797S), PC-9 (EGFR ex19del/T790M/C797S), H1975 (EGFR L858R/T790M/C797S, L858R/T790M), H3255 (L858R) and HCC827 (ex19del) with IC50 of 2.6, 3.1, 2.4, 4.1, 51, 50, 27, 21, and 11 nM, respectively. QLH11811 also showed excellent selectivity when compared the above values with its IC50 against Ba/F3 (EGFR wild-type, 61 nM) and A431 (EGFR wild-type, 440nM). QLH11811 demonstrated excellent inhibitory activities against seven osimertinib-resistant PDO models. Daily oral QLH11811 significantly inhibited tumor growth at all doses tested (P <0.001) in the PDX model, the H1975, PC-9, and Ba/F3 CDX models. QLH11811 had good PK profile in mice, rats, dogs, and monkeys, with the absolute bioavailability at 71%, 29%, 80% and 42%. The human PK parameters were obtained by allometric scaling method, and the efficacious dose in human was projected to 103 mg, daily. Conclusion: The in vitro and in vivo preclinical data demonstrated QLH11811 is a highly potent and selective 4th-generation EGFR TKI with activity against the osimertinib-resistant NSCLC with EGFR C797S mutation. The preclinical PK data supported the efficacious dose of QLH11811 in human would be 103 mg. Citation Format: Shansong Zheng, Wei Deng, Qingmei Zheng, Yingying Yang, Na Li, Tan Pang, Xueying Feng, Simon Taylor, Lina Ma, Yaqiong Wu, Ziwei Zhao. QLH11811, a selective 4th-generation EGFR inhibitor for osimertinib-resistant EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5457.

Abstract 2639: KB-0742 is active in preclinical MYC high models of TNBC, ovarian, and DLBCL cancers

Abstract MYC is an early-response gene downstream of many receptor complexes, and a component that promotes several pro-growth signal transduction pathways. As a result, MYC expression is highly regulated, but once dysregulated in cancer, MYC promotes tumorigenesis via activation of cell growth, proliferation, and angiogenesis. CDK9 is both an upstream and downstream cofactor of MYC. CDK9 promotes MYC expression through its recruitment to super enhancers at the MYC amplicon and acts as a cofactor to drive gene expression programs. Previously, we profiled pan-cancer sensitivity to KB-0742, a potent, selective, and orally bioavailable small-molecule inhibitor of CDK9, and identified MYC-amplified cancers as being especially sensitive to KB-0742. To better understand the connection between MYC and KB-0742 sensitivity, 11 different tumor types were evaluated preclinically using immortalized cell lines, patient-derived cell lines (PDCs), patient-derived organoids (PDOs), and patient-derived xenografts (PDXs). The 11 indications included head and neck, esophageal, gastric, liver, non-small cell lung cancer (NSCLC), pancreatic, ovarian, triple-negative breast cancer (TNBC), prostate, colon, and lymphoma. For 5 of the indications (head and neck, esophageal, gastric, liver, and colon), little activity was observed in cell lines and in vivo with weak correlations to MYC amplification. TNBC, ovarian cancer, and lymphoma showed good responses to KB-0742 treatment. For TNBC and ovarian, immortalized and patient-derived cell lines indicated lower IC50 values with increased MYC amplification or expression, but the results were not statistically significant. However, in vivo assessments using 15 PDX models showed good correlation of tumor growth inhibition (TGI) and MYC amplification/expression, with some tumor regressions observed in several ovarian models. Two PDO models of TNBC with different treatment histories were also treated with KB-0742 and compared to 4 standard-of-care (SOC) compounds. KB-0742 showed much greater activity in the models, with maximal inhibition rates of 100% and 89%. Cell-line screens of blood cancer-derived cell lines showed that lymphocyte-derived cell lines were the most sensitive to KB-0742 as compared to all other cancer types. We tested several PDX models for response to our compound. Treatment with KB-0742 resulted in TGIs of over 50% in several lymphoma models, including a TGI of 56% in 1 model of double-hit diffuse large B-cell lymphoma. These data support the continual development of KB-0742, which is currently being tested in a phase 1/2 clinical trial (NCT04718675). The dose-escalation phase is opened to relapsed or refractory solid tumors or non-Hodgkin lymphoma. Once a recommend phase 2 dose is determined, we plan to open dedicated expansion arms enrolling patients with MYC over-expression/amplification within the tumor types supported by these studies. Citation Format: Melinda A. Day, Douglas C. Saffran, Tressa Hood, Nikolaus Obholzer, Akanksha Pandey, Charles Y. Lin, Pavan Kumar, Jorge DiMartino. KB-0742 is active in preclinical MYC high models of TNBC, ovarian, and DLBCL cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2639.

Patient-Derived Organoid Model in the Prediction of Chemotherapeutic Drug Response in Colorectal Cancer.

As an emerging technology in precision medicine, the patient-derived organoid (PDO) technology has been indicated to provide novel modalities to judge the sensitivity of individual tumors to cancer drugs. In this work, an in vitro model of colorectal cancer (CRC) was established using the PDO culture, and it is demonstrated that the PDO samples preserved, to a great extent, the histologic features and marker expression of the original tumor tissues. Subsequently, cancer drugs 5-FU, oxaliplatin, and irinotecan were selected and screened on five CRC PDO samples, while the patient-derived organoid xenograft (PDOX) model was applied for comparison. The receiver operating characteristic (ROC) curve was drawn according to the IC50 data from the PDO model and the relative tumor proliferation rate (T/C%) from PDOX. Interestingly, the area under the ROC curve was 0.84 (95% CI, 0.64-1.04, P value = 0.028), which suggested that the IC50 of cancer drugs from the PDO model was strongly correlated with PDOX responses. In addition, the optimal sensitivity cutoff value for drug screening in CRC PDOs was identified at 10.35 μM, which could act as a reference value for efficacy evaluation of 5-FU, oxaliplatin, and irinotecan in the colorectal cancer drug screening. Since there are no unified criteria to judge the sensitivity of drugs in vitro, our work provides a method for establishing in vitro evaluation criteria via PDO and PDOX model using the patient tissues received from local hospitals, exhibiting potential in clinical cancer therapy and precision medicine.

MEDB-52. Organoids as preclinical models to improve and personalize disease outcome for sonic hedgehog medulloblastoma

Four main medulloblastoma (MB) molecular subgroups are known, including the sonic hedgehog (SHH) subgroup, which represents ~25% of MB cases. The 5-year overall survival of SHH-MB is ~80%. However, survival between patients is highly diverse and dependent on the driver mutation(s) of the tumor. Patients with TP53 mutated tumors (often accompanied with MYCN and/or GLI2 amplifications) don’t respond well to current therapies and have a 10-year overall survival below 20%. Therefore, there is a need for new and more tailored therapies for these patients. In this study we aim to screen patient-derived organoid models of TP53-mutated SHH MB with a library of ~200 different compounds. We have optimized the cultures of two PDX-derived and one patient-derived organoid line in vitro. The lines will be screened in a high-throughput manner and the best hits and combinations will be validated in corresponding in vivo PDX models. To further assess the role of specific mutations in therapy outcome of TP53-mutated SHH MB, cerebellar organoids generated from human iPSCs were genetically modified with overexpression of dominant-negative P53 (DNP53) alone or in combination with MYCN and/or GLI2. Introduction of DNP53 and MYCN overexpression in cerebellar organoids at day 28/35 leads to the outgrowth of a Ki67-positive proliferating mass after three weeks, indicating tumor growth. Further analyses are ongoing to see how they match SHH-MB patient tumors. These genetically engineered organoid models may elucidate the role of specific mutations in therapy response and/or resistance. In addition, as tumors in these genetically engineered cerebellar organoids arise in a microenvironment of normal cerebellar cell types, initial safety of drugs on cerebellar cells can be assessed. In conclusion, different organoid models of TP53-mutated SHH MB will enable us to find more effective treatments and to better understand how to treat patients with different mutation combinations.

Micro-organospheres retain patient tumor microenvironment for precision immuno-oncology.

2573 Background: Current patient-derived organoid (PDO) models are largely devoid of immune components. We developed a precision microfluidic and membrane platform to generate patient-derived micro-organospheres (MOS) that retain tumor-resident immune and stromal components for personalized immuno-oncology (IO) assays. Methods: MOS were generated from lung, kidney, and colorectal cancer patients. The composition and function of patient tumor-resident immune cells in MOS were characterized by flow cytometry, single-cell RNA-seq, antibody staining, and TCR-seq. High-content and longitudinal imaging with AI analyses were used to quantify tumor cell death and immune cell dynamics inside MOS in response to IO therapies including checkpoint inhibitors, T cell bispecific antibodies, and adoptive tumor infiltrating lymphocyte (TIL) therapies, followed by single-cell analyses. Results: Tumor and stromal cells quickly form tissue niches inside MOS to sustain the viability and function of encapsulated immune cells. MOS derived from lung and kidney cancer patients respond to Nivolumab, indicated by the Annexin V apoptosis marker. ESK1* (TCB antibody targeting HLA-A2/WT1) induces killing in eight lung tumor patients derived MOS. We further developed a MOS T-cell potency assay as autologous TILs or PBMC efficiently infiltrate MOS from lung, kidney and CRC patients and induce tumor cell apoptosis. Adjunctive therapies combining TCB with TILs enhanced the potency of adoptive cell therapy against lung tumor. Based on the data, three ongoing and upcoming personalize IO clinical trials will further validate the ability of the MOS assay to predict patient response to TCB, checkpoint combinations, and adoptive cell therapy. Conclusions: MOS provide a rapid and scalable personalized platform for developing and testing IO therapies such as checkpoint inhibitors, bispecific antibodies, and T cell therapies on patient tumor models that still retain the original tumor microenvironments.

A 107-gene Nanostring assay effectively characterizes complex multiomic gastric cancer molecular classification in a translational patient-derived organoid model.

4049 Background: Multi-omics profiling of gastric cancer (GC) has produced numerous molecular classification systems. However, widespread clinical implementation and testing of molecular subtypes are currently limited. Here, we develop, validate and implement a custom Nanostring assay capable of allocating GC molecular subtypes to clinical specimens in a translational patient-derived organoid model. Methods: Using publicly available whole-transcriptome data, machine learning models were developed to predict GC molecular subtypes from 376 Cancer Genome Atlas (TCGA) and 1797 Tumour Microenvironment Score (TME) patients. Models were generated using feature selection with 10-fold nested cross-validation. GC biopsies from 10 local patients were preserved in paraffin (tumour) and established as an organoid culture (organoid). Gene expression was measured using Nanostring. The allocation of molecular subtypes was internally and externally validated using gold-standard reference features in public databases comprising 2202 GC patients and 10 tumour-organoid pairs, respectively. We evaluated the concordance of tumour-organoid molecular subtypes and explored the correlation between subtype scores and in-vitro chemotherapy response. Results: Classification models for TCGA (57 genes) and TME (50 genes) predicted subtypes with an accuracy ± standard deviation of 89.46% ± 0.04 and 89.33% ± 0.02, respectively. Subtype assignment of microsatellite instability (MSI) in reference to capillary electrophoresis was found to have 99.3% [95% CI 97.4-99.9, n = 277] internal and 100% [95% CI 83.2-100, n = 20] external accuracy. In reference to Epstein-Barr Virus (EBV) in-situ hybridization, EBV type internal and external accuracy was 98.7% [95% CI 97.4-99.5, n = 552] and 100% [95% CI 83.2-100, n = 20], respectively. TCGA Genomically Stable (GS) scores followed a previously characterized enrichment of diffuse-type histology compared to intestinal-type in internal and external cohorts (Dunn’s Test, p < 0.0001 and p < 0.05, n = 1471 and n = 15, respectively). Statistically similar subtype scores (Paired Wilcoxon, p > 0.05) were found for tumour-organoid pairs. Discordance occurred in three tumour-organoid pairs. In-vitro Drug Sensitivity Score was not statistically efficacious in any molecular subtype, but Pearson correlation identified increasing efficacy with increasing EBV and MSI scores. Conclusions: Patient-derived organoids generally recapitulate the molecular subtype of parent tumours; however, in specific cases, subtype discordance occurs. Although additional external validation is required, our 107 gene assay effectively captures multi-omics classification systems in GC and allows future inquiry into the prognostic and therapeutic implications of these molecular subtypes.

A novel patient-derived organoid model for triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy for screening personalized drugs.

e12625 Background: Triple-negative breast cancer (TNBC) patients with residual disease after neoadjuvant chemotherapy (NAC) have high risk of recurrence. In this study, we developed a novel organoid culture method for growing TNBC patient samples after NAC, with the goal of generating models that more faithfully recapitulates the key features of the primary tumor for screening personalized drugs. Methods: We established organoid lines derived from TNBC patients who had residual tumors after anthracycline and taxane±platinum based NAC as well as normal human mammary tissues. The histopathology signatures of organoid lines were characterized by H&E staining and immunohistochemistry. The genetic and transcriptional features of organoids were analyzed by targeted sequencing and RNA sequencing. A set of clinical drugs were screened for their ability to suppress cancer organoids in vitro and the consistency was also analyzed between clinical response and drug efficacy of organoid models. Results: A total of 10 cancer organoid lines were successfully established, and cultured stably for more than 4 months. For comparison, 2 organoids derived from normal mammary tissues were generated as well. Our success rate for establishing cancer organoids from TNBC patient samples after NAC was 83% (10 out of 12 samples), and for organoids from normal mammary tissues, it was 100% (2 out of 2 samples). These organoid lines closely recapitulated the histopathology, genetic and transcriptional signature, and intratumor heterogeneity of the primary tissues. We found that active PI3K/AKT/mTOR pathways signaling is always required for cancer organoids from TNBC patient samples after NAC, while Wnt pathway activation is dispensable for generation of them in comparison to the organoid cultures from normal breast tissues. More importantly, we found that the anti-Trop-2 antibody-drug conjugate sacituzumab govitecan (IMMU-132) showed higher sensitivity to most cancer organoids from TNBC patient samples after NAC (8 out of 10 samples) with IC50 ranging from 0.3 nM to 86 nM in contrast to 5-FU (5 out of 10 samples) with IC50 ranging from 0.9 μM to 10.2 μM in vitro, whose prodrug capetabine is considered as a standard option in the postneoadjuvant setting. These results were also confirmed in patient-derived tumor xenograft models. The consistency of drug efficacy in the established cancer organoids and the patient responses in the clinic reached to 70% (7 out of 10 samples). Conclusions: We established a novel organoid culture system for TNBC patient samples after NAC, which was potentially a useful platform to explore molecular characteristics of TNBC patients after NAC and discover personalized drugs for intensive treatment.

Identification of a neural development gene expression signature in colon cancer stem cells reveals a role for EGR2 in tumorigenesis

SummaryRecent evidence demonstrates that colon cancer stem cells (CSCs) can generate neurons that synapse with tumor innervating fibers required for tumorigenesis and disease progression. Greater understanding of the mechanisms that regulate CSC driven tumor neurogenesis may therefore lead to more effective treatments. RNA-sequencing analyses of ALDHPositive CSCs from colon cancer patient-derived organoids (PDOs) and xenografts (PDXs) showed CSCs to be enriched for neural development genes. Functional analyses of genes differentially expressed in CSCs from PDO and PDX models demonstrated the neural crest stem cell (NCSC) regulator EGR2 to be required for tumor growth and to control expression of homebox superfamily embryonic master transcriptional regulator HOX genes and the neural stem cell and master cell fate regulator SOX2. These data support CSCs as the source of tumor neurogenesis and suggest that targeting EGR2 may provide a therapeutic differentiation strategy to eliminate CSCs and block nervous system driven disease progression.

Integration of Tumor Microenvironment in Patient-Derived Organoid Models Help Define Precision Medicine of Renal Cell Carcinoma.

Renal cell carcinoma (RCC) is a common urological tumor, with a poor prognosis, as the result of insensitivity to chemotherapy and radiotherapy. About 20%–30% of patients with RCC have metastasis at the first diagnosis, so only systemic treatment is possible. Due to the heterogeneity of renal tumors, responses to drugs differ from person to person. Consequently, patient-derived organoid, highly recapitulating tumor heterogeneity, becomes a promising model for high-throughput ex vivo drug screening and thus guides the drug choice of patients with RCC. Systemic treatment of RCC mainly targets the tumor microenvironment, including neovasculature and immune cells. We reviewed several methods with which patient-derived organoid models mimic the heterogeneity of not only tumor epithelium but also the tumor microenvironment. We further discuss some new aspects of the development of patient-derived organoids, preserving in vivo conditions in patients with RCC.

Establishment of a patient-derived organoid model and living biobank for nasopharyngeal carcinoma

Background Recurrent nasopharyngeal carcinoma (NPC) remains a major challenge for clinicians and scientists. Tumor organoid is a revelational disease model that highly resembled the heterogeneity and histopathological characteristics of original tumors. This study aimed to optimize the modeling process of patient-derived NPC organoids (NPCOs), and establish a living-biobank of NPCs to study the mechanism and explore the more effective treatment of the disease.MethodsSixty-two fresh NPC tissue samples and 15 normal mucosa samples were collected for 3-dimensional (3D) organoid culture. The organoids were confirmed using hematoxylin and eosin assays. The expression levels of CD133, CD44, BMI-1, and Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) were detected by immunohistochemistry (IHC) and in situ hybridization (ISH). Recurrent NPCOs were frozen in liquid nitrogen for 6 months and then resuscitated and passaged.ResultsWe identified a novel two-step enzymatic strategy for the treatment of NPC and nasal mucosa specimens and an optimal medium for culturing NPCOs and nasal mucosa organoids (NMOs). Organoid cultures were generated from 34 primary NPC samples, 28 recurrent NPC samples, and 15 normal mucosa samples. The success rates for primary NPCO, recurrent NPCO, and NMO formation were 47.06%, 81.25%, and 86.5%, respectively. All the NPCOs were EBER positive and CK7 negative. Recurrent NPCOs had higher expressions of stem cell markers, including BMI-1, CD44, and CD133. Additionally, recurrent NPCOs could be cultured to passage 4 and frozen and revived repeatedly, while primary NPCOs were challenging to culture.Conclusions In summary, we successfully established a living biobank using the NPCO model, which has enormous potential in basic and clinical research on NPC.

Patient-Derived Bladder Cancer Organoid Models in Tumor Biology and Drug Testing: A Systematic Review.

Simple SummaryPrimary culture of cancer cells from patient tumors in a physiologically relevant system can provide information about tumor biology, disentangle the role of different cell types within the tumors, and give information about drug sensitivity for the development of cancer-targeted therapies and precision medicine. This requires the use of well-characterized and easily expandable tumor models. This review focuses on 3D models developed from primary human tissue including normal urothelium or bladder cancer samples, the characteristics of the models, and to what extent the organoids represent the diversity observed among human tumors.Bladder cancer is a common and highly heterogeneous malignancy with a relatively poor outcome. Patient-derived tumor organoid cultures have emerged as a preclinical model with improved biomimicity. However, the impact of the different methods being used in the composition and dynamics of the models remains unknown. This study aims to systematically review the literature regarding patient-derived organoid models for normal and cancer tissue of the bladder, and their current and potential future applications for tumor biology studies and drug testing. A PRISMA-compliant systematic review of the PubMED, Embase, Web of Sciences, and Scopus databases was performed. The results were analyzed based on the methodologies, comparison with primary tumors, functional analysis, and chemotherapy and immunotherapy testing. The literature search identified 536 articles, 24 of which met the inclusion criteria. Bladder cancer organoid models have been increasingly used for tumor biology studies and drug screening. Despite the heterogeneity between methods, organoids and primary tissues showed high genetic and phenotypic concordance. Organoid sensitivity to chemotherapy matched the response in patient-derived xenograft (PDX) models and predicted response based on clinical and mutation data. Advances in bioengineering technology, such as microfluidic devices, bioprinters, and imaging, are likely to further standardize and expand the use of organoids.

Mitochondrial Calcium Uniporter Drives Metastasis and Confers a Targetable Cystine Dependency in Pancreatic Cancer.

Elevated mitochondrial calcium uptake in PDAC promotes metastasis but exposes cystine addiction and ferroptosis sensitivity that could be targeted to improve pancreatic cancer treatment.

Patient-Derived Tumor Organoids: New Progress and Opportunities to Facilitate Precision Cancer Immunotherapy

Cancer immunotherapy has revolutionized the field of cancer treatment in recent years. However, not all patients receiving cancer immunotherapy exhibit durable responses, and reliable, high-throughput testing platforms are urgently needed to guide personalized cancer immunotherapy. The ability of patient-derived tumor organoids to recapitulate pivotal features of original cancer tissues makes them useful as a preclinical model for cancer research and precision medicine. Nevertheless, many challenges exist in the translation of tumor organoid research to clinical decision making. Herein we discuss the applications of patient-derived tumor organoid models and the advances and potential of using complex immune-organoid systems as testing platforms to facilitate precision cancer immunotherapy. In addition, we highlight intriguing applications of tumor organoids with novel multi-omics in preclinical cancer research, highlighting genetic editing, proteomics, and liquid biopsy.

Aurora kinase blockade drives de novo addiction of cervical squamous cell carcinoma to druggable EGFR signalling.

Oncogenic signalling confers tumour-progression advantages; thus, its pharmacological blockade is the best strategy for cancer chemotherapy. However, drug resistance and heterogeneous dependency of tumour hamper their therapeutic potential, suggesting the necessity for a new ubiquitous modality based on evading drug resistance. Here, we proposed a de novo addiction to oncogenic signalling (Dead-On) concept, wherein specific blockade of target molecules forces cancer cells to develop dependency on an oncogenic signalling. In cervical squamous cell carcinoma cells, Aurora A/B dual blockade elicited rapid addiction to EGFR-Erk signalling, and its pharmacological/genetic inhibition synergistically enhanced anti-cancer activities in vitro, in vivo, and in a patient-derived organoid model. The signal activation was independent of EGFR genetic status, it was triggered by receptor accumulation on the plasma membrane via Rab11-mediated endocytic recycling machinery. These findings support our novel Dead-On concept which may lead to drug discovery as well as expand the adaptation of approved targeted drugs.

Use of Patient-Derived Organoids as a Treatment Selection Model for Colorectal Cancer: A Narrative Review.

Simple SummaryColorectal cancer (CRC) is the third most common type of cancer globally. Despite successful treatment, it has a 40% chance of recurrence within five years after surgery. While neoadjuvant chemotherapy is offered for stage IV cancers, it comes with a risk of resistance and disease progression. CRC tumors vary biologically, recur frequently, and pose a significant risk for cancer-related mortality, making it increasingly relevant to develop methods to study personalized treatment. A tumor organoid is a miniature, multicellular, and 3D replica of a tumor in vitro that retains its characteristics. Here, we discuss the current methods of culturing organoids and the correlation of drug response in organoids with clinical responses in patients. This helps us to determine whether organoids can be used for treatment selection in a clinical setting. Based on the studies included, there was a strong correlation between treatment responses of organoids and clinical treatment responses.Surgical resection is the mainstay in intended curative treatment of colorectal cancer (CRC) and may be accompanied by adjuvant chemotherapy. However, 40% of the patients experience recurrence within five years of treatment, highlighting the importance of improved, personalized treatment options. Monolayer cell cultures and murine models, which are generally used to study the biology of CRC, are associated with certain drawbacks; hence, the use of organoids has been emerging. Organoids obtained from tumors display similar genotypic and phenotypic characteristics, making them ideal for investigating individualized treatment strategies and for integration as a core platform to be used in prediction models. Here, we review studies correlating the clinical response in patients with CRC with the therapeutic response in patient-derived organoids (PDO), as well as the limitations and potentials of this model. The studies outlined in this review reported strong associations between treatment responses in the PDO model and clinical treatment responses. However, as PDOs lack the tumor microenvironment, they do not genuinely account for certain crucial characteristics that influence therapeutic response. To this end, we reviewed studies investigating PDOs co-cultured with tumor-infiltrating lymphocytes. This model is a promising method allowing evaluation of patient-specific tumors and selection of personalized therapies. Standardized methodologies must be implemented to reach a “gold standard” for validating the use of this model in larger cohorts of patients. The introduction of this approach to a clinical scenario directing neoadjuvant treatment and in other curative and palliative treatment strategies holds incredible potential for improving personalized treatment and its outcomes.

Establishment of a mouse model of pancreatic cancer using human pancreatic cancer cell line S2-013-derived organoid

A well-established preclinical model of pancreatic cancer needs to be established to facilitate research on new therapeutic targets. Recently established animal models of pancreatic cancer, including patient-derived tumor models and organoid models, are used for pre-clinical drug testing and biomarker discovery. These models have useful characteristics over conventional xenograft mouse models based on cell lines in preclinical studies, but still cannot accurately predict the clinical outcomes of new treatments and have not yet been broadly implemented in research. We employed pancreatic cancer organoid culture methods using the pancreatic cancer cell line S2-013, and performed pathological and immunohistochemical analyses to characterize tumor xenografts obtained from a mouse model implanted with S2-013 cell line-derived organoids. Serum levels of the pancreatic cancer tumor marker CA19-9 were measured by ELISA. We generated human pancreatic cancer organoids using a co-culture of S2-013 cells, human endothelial cells derived from human umbilical vein endothelial cells, and human mesenchymal stem cells, and established a mouse model with subcutaneously transplanted human pancreatic cancer organoids (S2-013-organoid model). Although blood clotting crater-like formation developed in the middle of subcutaneous xenografts in the S2-013-conventional model, created by subcutaneously injecting S2-013 cells into the right flank of nude mice, the size of xenografts in the S2-013-organoid model gradually increased without crater-like formation. Importantly, tumor xenografts obtained from the S2-013-organoid model exhibited a clinical human pancreatic cancer tissue-like cellular morphology, tissue architecture, and polarity, and actively formed cancer stroma containing mature blood vessels with the high expression of the vascular tight junction marker CD31. In subcutaneous xenografts of S2-013-conventional mice, no blood vessel density or widely expanding areas of necrotic regions were present. Consequently, serum levels of CA19-9 in the S2-013-organoid model correlated with tumor volumes. In addition, epithelial–mesenchymal transition, the conversion of epithelial cells to the mesenchymal phenotype, was observed in tumor xenografts of the S2-013-organoid model. The S2-013-organoid model provides tumor xenografts consisting of clinical human pancreatic cancer-like tissue formation with the effective development of vascularized stroma, and may be valuable for facilitating studies on pre-clinical drug testing and biomarker discovery.

Repurposing econazole as a pharmacological autophagy inhibitor to treat pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the highest mortality among carcinomas. The pathogenesis of PDAC requires elevated autophagy, inhibition of which using hydroxychloroquine has shown promise. However, current realization is impeded by its suboptimal use and unpredictable toxicity. Attempts to identify novel autophagy-modulating agents from already approved drugs offer a rapid and accessible approach. Here, using a patient-derived organoid model, we performed a comparative analysis of therapeutic responses among various antimalarial/fungal/parasitic/viral agents, through which econazole (ECON), an antifungal compound, emerged as the top candidate. Further testing in cell-line and xenograft models of PDAC validated this activity, which occurred as a direct consequence of dysfunctional autophagy. More specifically, ECON boosted autophagy initiation but blocked lysosome biogenesis. RNA sequencing analysis revealed that this autophagic induction was largely attributed to the altered expression of activation transcription factor 3 (ATF3). Increased nuclear import of ATF3 and its transcriptional repression of inhibitor of differentiation-1 (ID-1) led to inactivation of the AKT/mammalian target of rapamycin (mTOR) pathway, thus giving rise to autophagosome accumulation in PDAC cells. The magnitude of the increase in autophagosomes was sufficient to elicit ER stress-mediated apoptosis. Furthermore, ECON, as an autophagy inhibitor, exhibited synergistic effects with trametinib on PDAC. This study provides direct preclinical and experimental evidence for the therapeutic efficacy of ECON in PDAC treatment and reveals a mechanism whereby ECON inhibits PDAC growth.

Diversifying preclinical research tools: expanding patient-derived models to address cancer health disparities

Cancer health disparities define a critical healthcare issue for racial/ethnic minorities in the USA. Key findings have led to cancer treatment improvements tailored to minority patients, but such successes have been rare. Here, we highlight how the use of patient-derived xenograft (PDX) and organoid models could resolve current blocks toward precision cancer health equity.