Patient-derived 3D Research Articles

Pheno-seq \u2013 linking visual features and gene expression in 3D cell culture systems

Patient-derived 3D cell culture systems are currently advancing cancer research since they potentiate the molecular analysis of tissue-like properties and drug response under well-defined conditions. However, our understanding of the relationship between the heterogeneity of morphological phenotypes and the underlying transcriptome is still limited. To address this issue, we here introduce “pheno-seq” to directly link visual features of 3D cell culture systems with profiling their transcriptome. As prototypic applications breast and colorectal cancer (CRC) spheroids were analyzed by pheno-seq. We identified characteristic gene expression signatures of epithelial-to-mesenchymal transition that are associated with invasive growth behavior of clonal breast cancer spheroids. Furthermore, we linked long-term proliferative capacity in a patient-derived model of CRC to a lowly abundant PROX1-positive cancer stem cell subtype. We anticipate that the ability to integrate transcriptome analysis and morphological patho-phenotypes of cancer cells will provide novel insight on the molecular origins of intratumor heterogeneity.

Prospective Validation of an Ex Vivo, Patient-Derived 3D Spheroid Model for Response Predictions in Newly Diagnosed Ovarian Cancer

Although 70–80% of newly diagnosed ovarian cancer patients respond to first-line therapy, almost all relapse and five-year survival remains below 50%. One strategy to increase five-year survival is prolonging time to relapse by improving first-line therapy response. However, no biomarker today can accurately predict individual response to therapy. In this study, we present analytical and prospective clinical validation of a new test that utilizes primary patient tissue in 3D cell culture to make patient-specific response predictions prior to initiation of treatment in the clinic. Test results were generated within seven days of tissue receipt from newly diagnosed ovarian cancer patients obtained at standard surgical debulking or laparoscopic biopsy. Patients were followed for clinical response to chemotherapy. In a study population of 44, the 32 test-predicted Responders had a clinical response rate of 100% across both adjuvant and neoadjuvant treated populations with an overall prediction accuracy of 89% (39 of 44, p < 0.0001). The test also functioned as a prognostic readout with test-predicted Responders having a significantly increased progression-free survival compared to test-predicted Non-Responders, p = 0.01. This correlative accuracy establishes the test’s potential to benefit ovarian cancer patients through accurate prediction of patient-specific response before treatment.

Advanced 3D printed model of middle cerebral artery aneurysms for neurosurgery simulation

BackgroundNeurosurgical residents are finding it more difficult to obtain experience as the primary operator in aneurysm surgery. The present study aimed to replicate patient-derived cranial anatomy, pathology and human tissue properties relevant to cerebral aneurysm intervention through 3D printing and 3D print-driven casting techniques. The final simulator was designed to provide accurate simulation of a human head with a middle cerebral artery (MCA) aneurysm.MethodsThis study utilized living human and cadaver-derived medical imaging data including CT angiography and MRI scans. Computer-aided design (CAD) models and pre-existing computational 3D models were also incorporated in the development of the simulator. The design was based on including anatomical components vital to the surgery of MCA aneurysms while focusing on reproducibility, adaptability and functionality of the simulator. Various methods of 3D printing were utilized for the direct development of anatomical replicas and moulds for casting components that optimized the bio-mimicry and mechanical properties of human tissues. Synthetic materials including various types of silicone and ballistics gelatin were cast in these moulds. A novel technique utilizing water-soluble wax and silicone was used to establish hollow patient-derived cerebrovascular models.ResultsA patient-derived 3D aneurysm model was constructed for a MCA aneurysm. Multiple cerebral aneurysm models, patient-derived and CAD, were replicated as hollow high-fidelity models. The final assembled simulator integrated six anatomical components relevant to the treatment of cerebral aneurysms of the Circle of Willis in the left cerebral hemisphere. These included models of the cerebral vasculature, cranial nerves, brain, meninges, skull and skin. The cerebral circulation was modeled through the patient-derived vasculature within the brain model. Linear and volumetric measurements of specific physical modular components were repeated, averaged and compared to the original 3D meshes generated from the medical imaging data. Calculation of the concordance correlation coefficient (ρc: 90.2%–99.0%) and percentage difference (≤0.4%) confirmed the accuracy of the models.ConclusionsA multi-disciplinary approach involving 3D printing and casting techniques was used to successfully construct a multi-component cerebral aneurysm surgery simulator. Further study is planned to demonstrate the educational value of the proposed simulator for neurosurgery residents.

Abstract 469: Patient-derived sarcoma models: First results from the SARQMA study

Abstract Objective: The development of new targeted therapeutics has allowed an essential improvement in carcinoma treatment. For sarcomas, however, the main approach is still the combination of surgery, chemotherapy and radiation, which is due to their high heterogeneity, with more than 70 histopathological subtypes, and the limited knowledge of the molecular drivers of tumor development and progression. Here we show that patient-derived 3D (PD3D) cell culture models allow for an in vitro system to systematically test compounds and combos in a semi-automated way, generating a pre-clinical dataset that in combination with clinical data, genomic and proteomics profiles may help to better understand the biology and ultimately identify more potent treatment regimens. Methods: Fresh surgical specimen underwent several steps of dissociation. Cell aggregates were then seeded into 24w plates in matrix-like scaffolds and grown until &amp;gt;100μm. Organoids were then harvested, transferred to 384w plates and treated with a set of compounds that resemble standard-of-care treatments and novel compounds. Finally, viability was calculated. In parallel, protein extracts were used for DigiWest, a multiplexed protein profiling assay allowing to interrogate up to 800 (phospho)-proteins. Results: Of 49 cases with a biopsy, 23 had too little material available. Of these 23 samples, 5 were reported as sarcoma tissue afterwards, while 5 of the taken 26 samples were not sarcomas according to the final histopathology. Of all sarcomas, 90% (19/21) were taken from tumors localized at the extremities, the rest was located at the trunk. 52% (11/21) were growing in short term cell culture at least to passage 1 (p1), 28% (6/21) were growing for long term analyses. Two of those six were myxoid liposarcomas, two were undifferentiated pleomorphic sarcomas or classified as not otherwise specified (UPS/NOS), one was a myxoid liposarcoma and one a biphasic synovial sarcoma. Here we show that it is feasible to generate organoids from sarcoma tissues for extensive characterization in order to better understand their biology and mechanisms of treatment. High-throughput drug screening allows for an profiling of pharmacokinetic properties of individual sarcomas. Using the same material for additional (phospho)-proteomics provides multiple layers of understanding. Conclusion: At present, the structure of clinical trials is not amenable to N of 1 studies, so applying the information garnered from this platform, particularly combination therapy drug screens, remains a significant hurdle. The major limitation to the establishment of organoid cultures was insufficient amounts of fresh tissue with viable tumor cells. Increasing the tumor tissue available for organoid production would lead to a greater success rate. Nevertheless, by their recapitulation of the donor tissue architecture, they provide an interesting and important tool to study the huge variety of soft-tissue tumors. Citation Format: Manuela Gaebler, Alessandra Silvestri, Peter Reichardt, Eva Wardelmann, Guido Gambara, Johannes Haybaeck, Philipp Stroebel, Maya Niethard, Gerrit Erdmann, Christian R. Regenbrecht. Patient-derived sarcoma models: First results from the SARQMA study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 469.

45 - A preclinical model to assess the interaction between patient-derived 3D microtumors and tumor infiltrating immune cells in prostate cancer

45 - A preclinical model to assess the interaction between patient-derived 3D microtumors and tumor infiltrating immune cells in prostate cancer

Three-Dimensional Organoids Reveal Therapy Resistance of Esophageal and Oropharyngeal Squamous Cell Carcinoma Cells.

Background & AimsOropharyngeal and esophageal squamous cell carcinomas, especially the latter, are a lethal disease, featuring intratumoral cancer cell heterogeneity and therapy resistance. To facilitate cancer therapy in personalized medicine, three-dimensional (3D) organoids may be useful for functional characterization of cancer cells ex vivo. We investigated the feasibility and the utility of patient-derived 3D organoids of esophageal and oropharyngeal squamous cell carcinomas.MethodsWe generated 3D organoids from paired biopsies representing tumors and adjacent normal mucosa from therapy-naïve patients and cell lines. We evaluated growth and structures of 3D organoids treated with 5-fluorouracil ex vivo.ResultsTumor-derived 3D organoids were grown successfully from 15 out of 21 patients (71.4%) and passaged with recapitulation of the histopathology of the original tumors. Successful formation of tumor-derived 3D organoids was associated significantly with poor response to presurgical neoadjuvant chemotherapy or chemoradiation therapy in informative patients (P = 0.0357, progressive and stable diseases, n = 10 vs. partial response, n = 6). The 3D organoid formation capability and 5-fluorouracil resistance were accounted for by cancer cells with high CD44 expression and autophagy, respectively. Such cancer cells were found to be enriched in patient-derived 3D organoids surviving 5-fluorouracil treatment.ConclusionsThe single cell-based 3D organoid system may serve as a highly efficient platform to explore cancer therapeutics and therapy resistance mechanisms in conjunction with morphological and functional assays with implications for translation in personalized medicine.

Emergence of Intrahepatic Cholangiocarcinoma: How High-Throughput Technologies Expedite the Solutions for a Rare Cancer Type.

Intrahepatic cholangiocarcinoma (ICC) is the cancer of the intrahepatic bile ducts, and together with hepatocellular carcinoma (HCC), constitute the majority of primary liver cancers. ICC is a rare disorder as its overall incidence is < 1/100,000 in the United States and Europe. However, it shows much higher incidence in particular geographical regions, such as northeastern Thailand, where liver fluke infection is the most common risk factor of ICC. Since the early stages of ICC are often asymptomatic, the patients are usually diagnosed at advanced stages with no effective treatments available, leading to the high mortality rate. In addition, unclear genetic mechanisms, heterogeneous nature, and various etiologies complicate the development of new efficient treatments. Recently, a number of studies have employed high-throughput approaches, including next-generation sequencing and mass spectrometry, in order to understand ICC in different biological aspects. In general, the majority of recurrent genetic alterations identified in ICC are enriched in known tumor suppressor genes and oncogenes, such as mutations in TP53, KRAS, BAP1, ARID1A, IDH1, IDH2, and novel FGFR2 fusion genes. Yet, there are no major driver genes with immediate clinical solutions characterized. Interestingly, recent studies utilized multi-omics data to classify ICC into two main subgroups, one with immune response genes as the main driving factor, while another is enriched with driver mutations in the genes associated with epigenetic regulations, such as IDH1 and IDH2. The two subgroups also show different hypermethylation patterns in the promoter regions. Additionally, the immune response induced by host-pathogen interactions, i.e., liver fluke infection, may further stimulate tumor growth through alterations of the tumor microenvironment. For in-depth functional studies, although many ICC cell lines have been globally established, these homogeneous cell lines may not fully explain the highly heterogeneous genetic contents of this disorder. Therefore, the advent of patient-derived xenograft and 3D patient-derived organoids as new disease models together with the understanding of evolution and genetic alterations of tumor cells at the single-cell resolution will likely become the main focus to fill the current translational research gaps of ICC in the future.

Abstract 4099: Novel patient-derived 3D (PD3D) cell models and matched patient-derived xenografts (PDX) from peritoneal metastasis of colorectal cancer for drug testing and biomarker analysis

Abstract For patients with colorectal cancer (CRC) peritoneal metastases (PM) represent a terminal tumor stage with limited therapeutic options. To increase therapeutic efficacy and overall survival, availability of appropriate patient-derived 3D cell culture (PD3D) and patient-derived xenografts (PDX) models of PM could help improving the predictability of drug response for a specific tumor, but also foster the identification of novel biomarkers and therapeutic targets for CRC-PM patients. In this context, we generated the first scaffold-based PD3D and matching PDX models of CRC-PM as platform to test for chemotherapy response and to identify novel biomarkers. For model establishment, surgical specimens were processed either for PD3D cell culture or transplanted subcutaneously (s.c.) onto immunocompromized NOD scid gamma (NSG) mice. For 3D cell culture models, tissue was dissected, digested and filtered before embedding into a scaffold matrix. For PDX models engrafted tumors were transferred to NMRI nu/nu mice for further passaging. They were characterized by histopathology, immunohistochemistry and gene expression analyses using real-time RT-PCR. Chemosensitivity of both sibling models was evaluated on a panel of conventional chemotherapeutic and of targeted drugs. The same panel of drugs was used in 15 matched PD3D/PDX models and revealed individual response patterns both in PD3D and PDX. Most interestingly, different drug response pattern was observed in models derived from tumor tissue of the omentum vs. tissue from the peritoneum of the same patient. Our results demonstrate, that matched models maintain basic characteristics such as the morphology of the patient tumor in early passages, reflect heterogeneous response rates, and can be used as preclinical platform for translational studies of potential clinical use. Citation Format: Christian RA Regenbrecht, Guido Gambara, Eva Pachmayr, Alessandra Silvestri, Mathias Dahlmann, Bernadette Brzezicha, Britta Buettner, Beate Rau, Ulrich Keilholz, Urike Stein, Wolfgang Walther. Novel patient-derived 3D (PD3D) cell models and matched patient-derived xenografts (PDX) from peritoneal metastasis of colorectal cancer for drug testing and biomarker analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4099.

Abstract 5013: Rapid generation of phenomic and functional profiles of patient-derived 3D cell culture models for identification of treatment vulnerabilities of breast cancer: Early results of the EFRE-PoP project

Abstract Targeted treatment for breast cancer subsets currently relies on the occurrence of estrogen, progesterone and Her2/neu receptors. For triple negative breast cancer (TNBC) there is no identified targeted therapy. The mutational landscape for breast cancer subsets has been characterized, but drug development has been limited due to the lack of appropriate preclinical models. Development of patient-derived xenograft models (PDX) has been difficult with take rates of below 30%.To establish a series of patient-derived 3D (PD3D) cell culture models as a versatile resource for ex vivo drug sensitivity screens as well as secondary establishment of PDX. We obtained breast cancer specimens either by biopsy or surgical resection. Upon arrival tumor tissue was minced and enzymatically digested. After subsequent filtering, respective tissue size fractions were seeded as Matrix-droplets into 24-well plates and incubated under standard conditions. Growth of PD3Ds was monitored daily by microscopy. PD3Ds were splitted when their diameter reached appropriate size. Organoids were than fixed and embedded. FFPE sections of donor-tissues and derived PD3D models were than used for IHC and inspected by a pathologist. In parallel, mutational profiling of snap-frozen tumor tissue and cell cultures was performed. All models were subjected to a semi-automated multi-drug response assay in a 384-well format to assess individual compound sensitivities.We established a scalable workflow for culturing and screening of PD3D cell cultures from limited quantities of breast cancer tissue from true-cut and vacuum biopsies as well as surgical specimen. Tissue fragments of ca. 3mm in diameter were sufficient to successfully establish PD3D cell cultures at a high yield. The time needed to expand PD3D cell cultures to obtain a sufficient amount of cells for subsequent molecular analyses varies from 2-6 weeks depending on amount and quality of samples as well as grade and stage of the donor tumor tissue. At the time, we can report a rate of culture establishment rate of 75% (11 models from 15 patient samples) for various clinical relevant subgroups, including TNBC, HR+, Her 2 pos. BC and one DCIS sample. The immunohistology and mutational profiles of these samples are currently being confirmed. The established workflow for culturing PD3D cell cultures offers high yield rates within a potentially clinically relevant time-frame for HTP cytotoxicity screenings. We are encouraged that this method is suitable for various molecular subtypes of breast cancer. Once adequately validated in co-clinical trials, PD3D models would make for an intriguing tool in supporting clinical decision-making for individual patients. Particularly patients with breast cancer refractory to standard of care compounds could benefit from this approach. Citation Format: Verena I. Kiver, Guido Gambara, Olga Gorea, Jens-Uwe Blohmer, Philipp Jurmeister, Carsten Denkert, Alessandra Silvestri, Caroline M. Schweiger, Maxine Silvestrov, Ulrich Keilholz, Christian R. Regenbrecht. Rapid generation of phenomic and functional profiles of patient-derived 3D cell culture models for identification of treatment vulnerabilities of breast cancer: Early results of the EFRE-PoP project [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5013.

Abstract 5034: Time-lapse analysis of drug response and invasive capacity using patient-derived CRC microfluidic 3D cancer models under oxygen-controlled conditions

Abstract The development of relevant, reliable, human-derived, and cost-effective cellular models is a present need to bring precision medicine to fruition and to accelerate drug discovery. 3D cultures show robust genomic stability and tissue-specific gene expression with high fidelity to their tissue of origin, making them ideal for preserving healthy and cancer tissue molecular and morphological traits. Our objective is to expand the number of relevant human cancer models available and implement methods that allow cellular response quantification of 3D cultures. We sought to establish and implement 3D cell-based assays suitable for detection of known molecular biomarkers in cancer using spherical cancer models (multicellular tumor spheroids and tissue-derived tumor spheres). We validate our procedures by detecting colorectal cancer (CRC) biomarkers both in patient-derived cultures and cell-lines (HCT116, HT29, LS174T). We expanded the study's scope to the analysis of specific CRC transcriptional and proteomic signatures in 3D. To this end, we employ primary cultures derived from CRC patients, and cell lines. Our methodology takes advantage of multiplex antibody microarrays to detect proteomic levels of 10 metalloproteinases of which MMP-13 and MMP- 9, as well as of surface proteins known to be involved in colorectal cancer invasion. Alterations in oxygen tension are milestones in tumor progression, and low oxygen tension (hypoxia) has been demonstrated in CRC. The Hypoxia Inducible Factor (HIF) transcription factor family is central to the response to hypoxic stress and is correlated with CRC carcinogenesis, invasion, VEGF expression, and poorer diagnosis. To increase the relevance of our model we use low oxygen conditions (3-5% physioxia conditions), and atmospheric oxygen levels (normoxia conditions) finding relevant differences at the proteomic, transcriptional and miRNA levels (also verified by qRT-PCR). We test the spherical cancer models on microfluidics chip and assess their vessel invasion capacity in normoxia and hypoxia, finding a correlation between hypoxia stress response and augmented invasiveness. Additionally, we test cellular responses to standard drugs and vasculature invasive capacity by time-lapse microscopy on-chip. We design, develop and validate cellular assays for human cancer 3D culture generation, long-term maintenance and analysis. We analyze and quantify cellular responses to cytotoxic drugs, image and quantify invasion in fluidic chambers, determine levels of apoptosis, cell proliferation. We demonstrate that a combination of controllable assays can successfully be used for identification of biomarker expression and compound screening in preclinical studies. 3D models are, therefore, a cost-effective in vitro model capable of recapitulating reliably physiological responses. Citation Format: Dora Sabino, Isabelle Fixe, Alexandra Foucher, Eric Mennesson, Nadia Normand. Time-lapse analysis of drug response and invasive capacity using patient-derived CRC microfluidic 3D cancer models under oxygen-controlled conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5034.

Abstract LB-037: 3D organoids from milligrams of genitourinary cancer patients tissue retain key features of original tumors

Abstract 3D organoid culture, especially that generated from patient samples, is becoming prevalent as 2D cell culture models fail to provide clinical relevance. However, a relatively large amount of starting tissue is still required for this process, and the turnover rate and overall success rate vary greatly if relying solely on spontaneous aggregation. Here, we report the successful use of magnetic 3D bioprinting to drive organoid formation derived from fresh tumor samples of patients with prostate cancer, renal cell cancer, and urothelial cancer. Only milligrams to tens of milligrams of starting tissues (from fine-needle biopsies, core biopsies, or surgical excision) were used. Most organoids formed overnight, with a very high overall success rate (&amp;gt95%, n = 25). We have demonstrated that these cultures can be maintained, passed, and cryopreserved like conventional 2D cultures. More importantly, we characterized the organoids derived from prostatic adenocarcinomas and neuroendocrine tumors by the means of immunostaining and showed that the organoids expressed the same signature proteins as the originating tumors. This clinical concordance is crucial for downstream translational and biological research. We also showed that these adenocarcinoma cells responded to enzalutamide, an androgen receptor inhibitor that is used clinically to treat prostatic adenocarcinoma. In summary, we have established an efficient and robust system to create patient-derived 3D organoids; molecular characterization shows clinical concordance with original patient tumors; and our system shows great potential in drug screening with patient-derived 3D cultures. Citation Format: Yue Wang, Glauco Souza, Robert J. Amato. 3D organoids from milligrams of genitourinary cancer patients tissue retain key features of original tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-037.

PO-439 Establishment and characterisation of prostate cancer patient-derived organoids and cells

IntroductionThe currently available cancer models fail to recapitulate the heterogeneity of Prostate cancer (PCa), its metastasis, and progression to castration-resistant states. Many drug candidates that succeed in preclinical models fail to deliver a good outcome in clinical trials, resulting in ineffective patient treatment and misused resources. In this aspect, the development of three-dimensional (3D) organoid culture systems render it feasible to recap the convolution of organogenesis in vitro, promoting the generation of novel and more representative cancer models. Thus, the aim of this study is to generate patient-specific 3D organoids and two-dimensional (2D) cell lines, then characterise these models to identify potential prognostic biomarkers and treatments for PCa while correlating the outcome with the collected clinical parameters.Material and methodsWe are employing the R-spondin-1 based organoids technology to generate PCa organoids and primary cell lines derived from fresh unaffected and tumour tissues of treatment-naïve patients undergoing radical prostatectomy. Consequently, molecular characterisation of the different patient-derived PCa organoids and cell lines will be performed, followed by assessing different classical and in-clinical trial drugs.Results and discussionsWe were successful in generating 26 unaffected and 24 tumour patient-derived organoids, out of a total of 30 patients, and capable of culturing organoids for up to 6 generations. Interestingly, 2D cells were derived from these organoids using the same culture media and were continuously passaged for up to 30 passages (more than 3 months). Our preliminary data indicates that our patient-derived organoids and cell lines show a typical epithelial phenotype and express Cytokeratin 5, Cytokeratin 8, Androgen Receptor and Prostate Specific Antigen. These models were further used to assess clinically used drugs and treatment approaches including chemotherapeutic agents (Docetaxel), Androgen-deprivation therapy (Bicalutamide and Enzalutamide). Our results show differential drug effects from one patient to the other.ConclusionIn this study, we are employing a novel methodology to establish patient-derived 3D organoids and cells to be used as a model for drug screening and to further characterise the mutational landscape of PCa. Consequently, the molecular characterisation of this model when combined with pharmacological profiles can aid in personalising the treatment of PCa.

PO-503 HDAC inhibitor resistance in colorectal cancer: RAS and AMP; MYC – the partners in crime

IntroductionOncogenic KRAS is widely acknowledged as a critical determinant of the therapeutic response of colorectal cancer (CRC) – a fact that to date is pivotal for defining an appropriate treatment strategy. Whether this also extends beyond RTK- and MAPK- targeted molecular cancer therapies, to a novel and promising class of anti-cancer drugs, namely HDAC inhibitors (HDACi), is an aspect that until now has remained largely undefined. Our aim is, therefore, to establish whether RAS is an effective predictor of response to HDACi in CRC. Ultimately, we intend to shed light on the cause underlying the limited clinical benefits of HDACi as a treatment option for solid tumours, including CRC and thereby identify opportunities to improve the prospects of HDACi treatment.Material and methodsWe investigated the presence of an oncogenic-RAS dependency against a wide range of different HDAC inhibitors using model systems with intrinsic and conditionally active RAS oncogenes.Results and discussionsWe uncovered an oncogenic RAS-dependent ‘safeguard’ mechanism imposed in order to evade the cytotoxic effect of HDACi and thereby apoptosis. Cells harbouring oncogenic RAS were observed to undergo a reversible senescence-like growth arrest in G2 phase, allowing for re-entry into cell cycle following a withdrawal of the inhibitor. This mechanism is implemented as a consequence of the inhibition of the RAS deacetylase, namely HDAC2, which in turn result in the generation of (hyper)acetylated RAS with increased binding affinity to BRAF and CRAF. This translates to a further amplification in MAPK-signalling and thus an increase in the priming of c-MYC for ubiquitin-mediated proteasomal degradation, thereby enabling the cells to exit the cell cycle and enter the defined protective state of G2 arrest. The prospect of HDACi treatment was effectively improved using current MAPK-targeted therapy and senolytic drugs by effectively preventing the observed pro-oncogenic effect of the HDACi treatment alone.ConclusionOur study reveals an oncogenic RAS-dependent resistance mechanism, enabling cells harbouring oncogenic RAS, to establish a favourable cellular state of prolonged pharmacological hideout - a phenomenon that is replicated in patient-derived 3D cell culture models of CRC. This highlights the potential clinical relevance of our findings and thus the importance of a rational mechanism-based combinatorial therapeutic design in order to realise the true therapeutic potential of HDACi.

3D Organoids from Milligrams of Genitourinary Cancer Patients Tissue Retain Key Features of Original Tumors.

268 Background: 3D organoid culture, especially that generated from patient samples, is becoming prevalent as 2D cell culture models fail to provide clinical relevance. However, a relatively large amount of starting tissue is still required for this process, and the turnover rate and overall success rate vary greatly if relying solely on spontaneous aggregation. Methods: Here, we report the successful use of magnetic 3D bioprinting to drive organoid formation derived from fresh tumor samples of patients with prostate cancer, renal cell cancer, and urothelial cancer. Results: Only milligrams to tens of milligrams of starting tissues (from fine-needle biopsies, core biopsies, or surgical excision) were used. Most organoids formed overnight, with a very high overall success rate ( &gt; 95%, n = 25). We have demonstrated that these cultures can be maintained, passed, and cryopreserved like conventional 2D cultures. More importantly, we characterized the organoids derived from prostatic adenocarcinomas and neuroendocrine tumors by the means of immunostaining and showed that the organoids expressed the same signature proteins as the originating tumors. This clinical concordance is crucial for downstream translational and biological research. We also showed that these adenocarcinoma cells responded to enzalutamide, an androgen receptor inhibitor that is used clinically to treat prostatic adenocarcinoma. Conclusions: In summary, we have established an efficient and robust system to create patient-derived 3D organoids; molecular characterization shows clinical concordance with original patient tumors; and our system shows great potential in drug screening with patient-derived 3D cultures.

From Chemotherapy to Combined Targeted Therapeutics: In Vitro and in Vivo Models to Decipher Intra-tumor Heterogeneity.

Recent advances in next-generation sequencing and other omics technologies capable to map cell fate provide increasing evidence on the crucial role of intra-tumor heterogeneity (ITH) for cancer progression. The different facets of ITH, from genomic to microenvironmental heterogeneity and the hierarchical cellular architecture originating from the cancer stem cell compartment, contribute to the range of tumor phenotypes. Decoding these complex data resulting from the analysis of tumor tissue complexity poses a challenge for developing novel therapeutic strategies that can counteract tumor evolution and cellular plasticity. To achieve this aim, the development of in vitro and in vivo cancer models that resemble the complexity of ITH is crucial in understanding the interplay of cells and their (micro)environment and, consequently, in testing the efficacy of new targeted treatments and novel strategies of tailoring combinations of treatments to the individual composition of the tumor. This challenging approach may be an important cornerstone in overcoming the development of pharmaco-resistances during multiple lines of treatment. In this paper, we report the latest advances in patient-derived 3D (PD3D) cell cultures and patient-derived tumor xenografts (PDX) as in vitro and in vivo models that can retain the genetic and phenotypic heterogeneity of the tumor tissue.

In vitro patient-derived 3D mesothelioma tumor organoids facilitate patient-centric therapeutic screening

Variability in patient response to anti-cancer drugs is currently addressed by relating genetic mutations to chemotherapy through precision medicine. However, practical benefits of precision medicine to therapy design are less clear. Even after identification of mutations, oncologists are often left with several drug options, and for some patients there is no definitive treatment solution. There is a need for model systems to help predict personalized responses to chemotherapeutics. We have microengineered 3D tumor organoids directly from fresh tumor biopsies to provide patient-specific models with which treatment optimization can be performed before initiation of therapy. We demonstrate the initial implementation of this platform using tumor biospecimens surgically removed from two mesothelioma patients. First, we show the ability to biofabricate and maintain viable 3D tumor constructs within a tumor-on-a-chip microfluidic device. Second, we demonstrate that results of on-chip chemotherapy screening mimic those observed in subjects themselves. Finally, we demonstrate mutation-specific drug testing by considering the results of precision medicine genetic screening and confirming the effectiveness of the non-standard compound 3-deazaneplanocin A for an identified mutation. This patient-derived tumor organoid strategy is adaptable to a wide variety of cancers and may provide a framework with which to improve efforts in precision medicine oncology.

The Esophageal Organoid System Reveals Functional Interplay Between Notch and Cytokines in Reactive EpithelialChanges.

Background & AimsAberrations in the esophageal proliferation-differentiation gradient are histologic hallmarks in eosinophilic esophagitis (EoE) and gastroesophageal reflux disease. A reliable protocol to grow 3-dimensional (3D) esophageal organoids is needed to study esophageal epithelial homeostasis under physiological and pathologic conditions.MethodsWe modified keratinocyte-serum free medium to grow 3D organoids from endoscopic esophageal biopsies, immortalized human esophageal epithelial cells, and murine esophagi. Morphologic and functional characterization of 3D organoids was performed following genetic and pharmacologic modifications or exposure to EoE-relevant cytokines. The Notch pathway was evaluated by transfection assays and by gene expression analyses in vitro and in biopsies.ResultsBoth murine and human esophageal 3D organoids displayed an explicit proliferation-differentiation gradient. Notch inhibition accumulated undifferentiated basal keratinocytes with deregulated squamous cell differentiation in organoids. EoE patient-derived 3D organoids displayed normal epithelial structure ex vivo in the absence of the EoE inflammatory milieu. Stimulation of esophageal 3D organoids with EoE-relevant cytokines resulted in a phenocopy of Notch inhibition in organoid 3D structures with recapitulation of reactive epithelial changes in EoE biopsies, where Notch3 expression was significantly decreased in EoE compared with control subjects.ConclusionsEsophageal 3D organoids serve as a novel platform to investigate regulatory mechanisms in squamous epithelial homeostasis in the context of EoE and other diseases. Notch-mediated squamous cell differentiation is suppressed by cytokines known to be involved in EoE, suggesting that this may contribute to epithelial phenotypes associated with disease. Genetic and pharmacologic manipulations establish proof of concept for the utility of organoids for future studies and personalized medicine in EoE and other esophageal diseases.

New tools for old drugs: Functional genetic screens to optimize current chemotherapy.

Despite substantial advances in the treatment of various cancers, many patients still receive anti-cancer therapies that hardly eradicate tumor cells but inflict considerable side effects. To provide the best treatment regimen for an individual patient, a major goal in molecular oncology is to identify predictive markers for a personalized therapeutic strategy. Regarding novel targeted anti-cancer therapies, there are usually good markers available. Unfortunately, however, targeted therapies alone often result in rather short remissions and little cytotoxic effect on the cancer cells. Therefore, classical chemotherapy with frequent long remissions, cures, and a clear effect on cancer cell eradication remains a corner stone in current anti-cancer therapy. Reliable biomarkers which predict the response of tumors to classical chemotherapy are rare, in contrast to the situation for targeted therapy. For the bulk of cytotoxic therapeutic agents, including DNA-damaging drugs, drugs targeting microtubules or antimetabolites, there are still no reliable biomarkers used in the clinic to predict tumor response. To make progress in this direction, meticulous studies of classical chemotherapeutic drug action and resistance mechanisms are required. For this purpose, novel functional screening technologies have emerged as successful technologies to study chemotherapeutic drug response in a variety of models. They allow a systematic analysis of genetic contributions to a drug-responsive or −sensitive phenotype and facilitate a better understanding of the mode of action of these drugs. These functional genomic approaches are not only useful for the development of novel targeted anti-cancer drugs but may also guide the use of classical chemotherapeutic drugs by deciphering novel mechanisms influencing a tumor’s drug response. Moreover, due to the advances of 3D organoid cultures from patient tumors and in vivo screens in mice, these genetic screens can be applied using conditions that are more representative of the clinical setting. Patient-derived 3D organoid lines furthermore allow the characterization of the “essentialome”, the specific set of genes required for survival of these cells, of an individual tumor, which could be monitored over the course of treatment and help understanding how drug resistance evolves in clinical tumors. Thus, we expect that these functional screens will enable the discovery of novel cancer-specific vulnerabilities, and through clinical validation, move the field of predictive biomarkers forward. This review focuses on novel advanced techniques to decipher the interplay between genetic alterations and drug response.

Machine Learning Enables Live Label-Free Phenotypic Screening in Three Dimensions.

There is a large amount of information in brightfield images that was previously inaccessible by using traditional microscopy techniques. This information can now be exploited by using machine-learning approaches for both image segmentation and the classification of objects. We have combined these approaches with a label-free assay for growth and differentiation of leukemic colonies, to generate a novel platform for phenotypic drug discovery. Initially, a supervised machine-learning algorithm was used to identify in-focus colonies growing in a three-dimensional (3D) methylcellulose gel. Once identified, unsupervised clustering and principle component analysis of texture-based phenotypic profiles were applied to group similar phenotypes. In a proof-of-concept study, we successfully identified a novel phenotype induced by a compound that is currently in clinical trials for the treatment of leukemia. We believe that our platform will be of great benefit for the utilization of patient-derived 3D cell culture systems for both drug discovery and diagnostic applications.

Highlights of This Issue

With the recent FDA-approval of pembrolizumab for mismatch repair (MMR)-deficient cancers, interest has emerged in identifying rapid and inexpensive methods to screen for MMR deficiency, especially in tumor types where this is uncommon. Guedes and colleagues employed an immunohistochemical assay to screen 1176 primary prostate cancers for loss of MSH2, the most commonly inactivated MMR protein in prostate cancer. While the prevalence of MSH2 deficiency was rare in the entire cohort (1%), MSH2 loss was enriched in patients with primary Gleason pattern 5 cancers (8%) and small-cell prostate cancers (5%). If validated, screening for MSH2 inactivation in high-risk patients could facilitate selection of patients for pembrolizumab.Most patients with recurrent glioblastoma (GBM) are treated with bevacizumab, an anti-angiogenic IgG monoclonal antibody. Bevacizumab prolongs survival but approximately 20% of patients show no anti-tumor response. Its ability to mitigate edema has further promoted its widespread use. In this study, Müller-Greven and colleagues demonstrate that anti-VEGF-A IgG is internalized by Sox2+/CD44+ glioblastoma stem-like tumor cells residing in the perivascular niche in patient-derived tumor and syngeneic models of GBM. Survival of CD133+ cancer stem-like cells is promoted by macropinocytosis of bevacizumab and its trafficking to the lysosome and by autophagy induced by bevacizumab depletion of VEGF-A within the microenvironment.Ovarian cancer is the most lethal gynecologic malignancy, with nearly 70% relapse within 24 months. Raghavan and colleagues have developed a patient-derived 3D hanging drop spheroid platform to screen chemotherapeutics and biologics and to improve clinical outcomes by rapidly identifying optimal personalized treatment routes. The authors show that spheroids formed with ovarian cancer stem cells from different patients exhibited distinct responses to chemotherapeutics, which corresponded with responses observed in primary tumor xenografts. Drug resistance was related to levels of stem cell markers CD133 and ALDH. This platform can be used to further study cancer stem cell biology and tumor re-emergence, leading to improved clinical outcomes.The ability to distinguish primary prostate cancer (PCa) with metastatic potential has not been achieved. Labbé and colleagues interrogated the impact of concurrent TOP2A and EZH2 expression in multiple PCa patient cohorts. Concurrent expression of both marks identified patients more susceptible to progress to metastatic disease and PCa-specific death. Importantly, TOP2A and EZH2 in PCa cells act as key driving oncogenes, highlighted by sensitivity to combination-targeted therapy. This work provides substantial evidence for the utility of TOP2A and EZH2 as prognostic biomarkers and therapeutic targets to identify and intercept aggressive PCa progressing to metastatic disease.