The highly selective and oral phosphoinositide 3-kinase delta (PI3K-δ) inhibitor roginolisib induces apoptosis in mesothelioma cells and increases immune effector cell composition.

Targeting aberrantly expressed kinases in malignant pleural mesothelioma (MPM) is a promising therapeutic strategy. We here investigated the effect of the novel and highly selective Phosphoinositide 3-kinase delta (PI3K-δ) inhibitor roginolisib (IOA-244) on MPM cells and on the immune cells in MPM microenvironment. To this aim, we analyzed the expression of PI3K-δ by immunohistochemistry in specimens from primary MPM, cell viability and death in three different MPM cell lines treated with roginolisib alone and in combination with ipatasertib (AKT inhibitor) and sapanisertib (mTOR inhibitor). In a co-culture model of patient-derived MPM cells, autologous peripheral blood mononuclear cells and fibroblasts, the tumor cell viability and changes in immune cell composition were investigated after treatment of roginolisib with nivolumab and cisplatin. PI3K-δ was detected in 66/89 (74%) MPM tumors and was associated with reduced overall survival (12 vs. 25 months, P=0.0452). Roginolisib induced apoptosis in MPM cells and enhanced the anti-tumor efficacy of AKT and mTOR kinase inhibitors by suppressing PI3K-δ/AKT/mTOR and ERK1/2 signaling. Furthermore, the combination of roginolisib with chemotherapy and immunotherapy re-balanced the immune cell composition, increasing effector T-cells and reducing immune suppressive cells. Overall, roginolisib induces apoptosis in MPM cells and increases the antitumor immune cell effector function when combined with nivolumab and cisplatin. These results provide first insights on the potential of roginolisib as a therapeutic agent in patients with MPM and its potential in combination with established immunotherapy regimen.

Indolyl-chalcone derivatives trigger apoptosis in cisplatin-resistant mesothelioma cells through aberrant tubulin polymerization and deregulation of microtubule-associated proteins.

Introduction: Malignant pleural mesothelioma (MPM) is a neoplasm with dismal prognosis and notorious resistance to the standard therapeutics cisplatin and pemetrexed. Chalcone derivatives are efficacious anti-cancer agents with minimal toxicity and have, therefore, gained pharmaceutical interest. Here, we investigated the efficacy of CIT-026 and CIT-223, two indolyl-chalcones (CITs), to inhibit growth and viability of MPM cells and defined the mechanism by which the compounds induce cell death. Methods: The effects of CIT-026 and CIT-223 were analyzed in five MPM cell lines, using viability, immunofluorescence, real-time cell death monitoring, and tubulin polymerization assays, along with siRNA knockdown. Phospho-kinase arrays and immunoblotting were used to identify signaling molecules that contribute to cell death. Results: CIT-026 and CIT-223 were toxic in all cell lines at sub-micromolar concentrations, in particular in MPM cells resistant to cisplatin and pemetrexed, while normal fibroblasts were only modestly affected. Both CITs targeted tubulin polymerization via (1) direct interaction with tubulin and (2) phosphorylation of microtubule regulators STMN1, CRMP2 and WNK1. Formation of aberrant tubulin fibers caused abnormal spindle morphology, mitotic arrest and apoptosis. CIT activity was not reduced in CRMP2-negative and STMN1-silenced MPM cells, indicating that direct tubulin targeting is sufficient for toxic effects of CITs. Discussion: CIT-026 and CIT-223 are highly effective inducers of tumor cell apoptosis by disrupting microtubule assembly, with only modest effects on non-malignant cells. CITs are potent anti-tumor agents against MPM cells, in particular cells resistant to standard therapeutics, and thus warrant further evaluation as potential small-molecule therapeutics in MPM.

A Novel Hydrogel-Based 3D In Vitro Tumor Panel of 30 PDX Models Incorporates Tumor, Stromal and Immune Cell Compartments of the TME for the Screening of Oncology and Immuno-Therapies.

Human-relevant systems that mimic the 3D tumor microenvironment (TME), particularly the complex mechanisms of immuno-modulation in the tumor stroma, in a reproducible and scalable format are of high interest for the drug discovery industry. Here, we describe a novel 3D in vitro tumor panel comprising 30 distinct PDX models covering a range of histotypes and molecular subtypes and cocultured with fibroblasts and PBMCs in planar (flat) extracellular matrix hydrogels to reflect the three compartments of the TME-tumor, stroma, and immune cells. The panel was constructed in a 96-well plate format and assayed tumor size, tumor killing, and T-cell infiltration using high-content image analysis after 4 days of treatment. We screened the panel first against the chemotherapy drug Cisplatin to demonstrate feasibility and robustness, and subsequently assayed immuno-oncology agents Solitomab (CD3/EpCAM bispecific T-cell engager) and the immune checkpoint inhibitors (ICIs) Atezolizumab (anti-PDL1), Nivolumab (anti-PD1) and Ipilimumab (anti-CTLA4). Solitomab displayed a strong response across many PDX models in terms of tumor reduction and killing, allowing for its subsequent use as a positive control for ICIs. Interestingly, Atezolizumab and Nivolumab demonstrated a mild response compared to Ipilimumab in a subset of models from the panel. We later determined that PBMC spatial proximity in the assay setup was important for the PD1 inhibitor, hypothesizing that both duration and concentration of antigen exposure may be critical. The described 30-model panel represents a significant advancement toward screening in vitro models of the tumor microenvironment that include tumor, fibroblast, and immune cell populations in an extracellular matrix hydrogel, with robust and standardized high content image analysis in a planar hydrogel. The platform is aimed at rapidly screening various combinations and novel agents and forming a critical conduit to the clinic, thus accelerating drug discovery for the next generation of therapeutics.

PI3Kß inhibition enhances ALK-inhibitor sensitivity in ALK-rearranged lung cancer.

Treatment with anaplastic lymphoma kinase (ALK) inhibitors significantly improves outcome for non-small-cell lung cancer (NSCLC) patients with ALK-rearranged tumors. However, clinical resistance typically develops over time and, in the majority of cases, resistance mechanisms are ALK-independent. We generated tumor cell cultures from multiple regions of an ALK-rearranged clinical tumor specimen and deployed functional drug screens to identify modulators of ALK-inhibitor response. This identified a role for PI3Kß and EGFR inhibition in sensitizing the response regulating resistance to ALK inhibition. Inhibition of ALK elicited activation of EGFR, and subsequent MAPK and PI3K-AKT pathway reactivation. Sensitivity to ALK targeting was enhanced by inhibition or knockdown of PI3Kß. In ALK-rearranged primary cultures, the combined inhibition of ALK and PI3Kß prevented the EGFR-mediated ALK-inhibitor resistance, and selectively targeted the cancer cells. The combinatorial effect was seen also in the background of TP53 mutations and in epithelial-to-mesenchymal transformed cells. In conclusion, combinatorial ALK- and PI3Kß-inhibitor treatment carries promise as a treatment for ALK-rearranged NSCLC.

EMT, Stemness, and Drug Resistance in Biological Context: A 3D Tumor Tissue/In Silico Platform for Analysis of Combinatorial Treatment in NSCLC with Aggressive KRAS-Biomarker Signatures.

Epithelial-to-mesenchymal transition (EMT) is discussed to be centrally involved in invasion, stemness, and drug resistance. Experimental models to evaluate this process in its biological complexity are limited. To shed light on EMT impact and test drug response more reliably, we use a lung tumor test system based on a decellularized intestinal matrix showing more in vivo-like proliferation levels and enhanced expression of clinical markers and carcinogenesis-related genes. In our models, we found evidence for a correlation of EMT with drug resistance in primary and secondary resistant cells harboring KRASG12C or EGFR mutations, which was simulated in silico based on an optimized signaling network topology. Notably, drug resistance did not correlate with EMT status in KRAS-mutated patient-derived xenograft (PDX) cell lines, and drug efficacy was not affected by EMT induction via TGF-ß. To investigate further determinants of drug response, we tested several drugs in combination with a KRASG12C inhibitor in KRASG12C mutant HCC44 models, which, besides EMT, display mutations in P53, LKB1, KEAP1, and high c-MYC expression. We identified an aurora-kinase A (AURKA) inhibitor as the most promising candidate. In our network, AURKA is a centrally linked hub to EMT, proliferation, apoptosis, LKB1, and c-MYC. This exemplifies our systemic analysis approach for clinical translation of biomarker signatures.

Fibroblast growth factor receptor 4 promotes glioblastoma progression: a central role of integrin-mediated cell invasiveness.

Glioblastoma (GBM) is characterized by a particularly invasive phenotype, supported by oncogenic signals from the fibroblast growth factor (FGF)/ FGF receptor (FGFR) network. However, a possible role of FGFR4 remained elusive so far. Several transcriptomic glioma datasets were analyzed. An extended panel of primary surgical specimen-derived and immortalized GBM (stem)cell models and original tumor tissues were screened for FGFR4 expression. GBM models engineered for wild-type and dominant-negative FGFR4 overexpression were investigated regarding aggressiveness and xenograft formation. Gene set enrichment analyses of FGFR4-modulated GBM models were compared to patient-derived datasets. Despite widely absent in adult brain, FGFR4 mRNA was distinctly expressed in embryonic neural stem cells and significantly upregulated in glioblastoma. Pronounced FGFR4 overexpression defined a distinct GBM patient subgroup with dismal prognosis. Expression levels of FGFR4 and its specific ligands FGF19/FGF23 correlated both in vitro and in vivo and were progressively upregulated in the vast majority of recurrent tumors. Based on overexpression/blockade experiments in respective GBM models, a central pro-oncogenic function of FGFR4 concerning viability, adhesion, migration, and clonogenicity was identified. Expression of dominant-negative FGFR4 resulted in diminished (subcutaneous) or blocked (orthotopic) GBM xenograft formation in the mouse and reduced invasiveness in zebrafish xenotransplantation models. In vitro and in vivo data consistently revealed distinct FGFR4 and integrin/extracellular matrix interactions. Accordingly, FGFR4 blockade profoundly sensitized FGFR4-overexpressing GBM models towards integrin/focal adhesion kinase inhibitors. Collectively, FGFR4 overexpression contributes to the malignant phenotype of a highly aggressive GBM subgroup and is associated with integrin-related therapeutic vulnerabilities.

Drug screening and genome editing in human pancreatic cancer organoids identifies drug-gene interactions and candidates for off-label treatment.

Pancreatic cancer (PDAC) is a highly aggressive malignancy for which the identification of novel therapies is urgently needed. Here, we establish a human PDAC organoid biobank from 31 genetically distinct lines, covering a representative range of tumor subtypes, and demonstrate that these reflect the molecular and phenotypic heterogeneity of primary PDAC tissue. We use CRISPR-Cas9 genome editing and drug screening to characterize drug-gene interactions with ARID1A and BRCA2. We find that missense- but not frameshift mutations in the PDAC driver gene ARID1A are associated with increased sensitivity to the kinase inhibitors dasatinib (p < 0.0001) and VE-821 (p < 0.0001). We conduct an automated drug-repurposing screen with 1,172 FDA-approved compounds, identifying 26 compounds that effectively kill PDAC organoids, including 19 chemotherapy drugs currently approved for other cancer types. We validate the activity of these compounds in vitro and in vivo. The in vivo validated hits include emetine and ouabain, compounds which are approved for non-cancer indications and which perturb the ability of PDAC organoids to respond to hypoxia. Our study provides proof-of-concept for advancing precision oncology and identifying candidates for drug repurposing via genome editing and drug screening in tumor organoid biobanks.

Targeting mitotic exit in solid tumors.

Targeting mitosis by taxanes is one of the most common chemotherapeutic approaches in various malignant solid tumors, but cancer cells may survive antimitotic treatment with attainable in vivo concentrations due to mitotic slippage with a residual activity of the ubiquitin ligase anaphase-promoting complex (APC/C) and a continuous slow ubiquitin-proteasome-dependent cyclin B-degradation leading to mitotic exit. Therefore, blocking cyclin B-proteolysis via additional proteasome (PI) or APC/C-inhibition may have the potential to enhance tumor cell eradication by inducing a more robust mitotic block and mitotic cell death. Here, we analyzed this approach in different cell lines and more physiological patient-derived xenografts (PDX) from lung and breast cancer. The sequential combination of paclitaxel with the PI bortezomib enhanced cell death, but in contrast to the hypothesis during interphase and not in mitosis in both lung and breast cancer. APC/C-inhibition alone or in sequential combination with paclitaxel led to strong mitotic cell death in lung cancer. But in breast cancer, with high expression of the anti-apoptotic regulator Mcl-1, cell death in interphase was induced. Here, combined APC/C- and Mcl-1-inhibition with or without paclitaxel was highly lethal but still resulted in interphase cell death. Taken together, the combination of antimitotic agents with a clinically approved PI or inhibitors of the APC/C and Mcl-1 is a promising approach to improve treatment response in different solid tumors, even though they act entity-dependent at different cell cycle phases.

Stromal fibroblasts shape the myeloid phenotype in normal colon and colorectal cancer and induce CD163 and CCL2 expression in macrophages.

Colorectal cancer (CRC) accounts for about 10% of cancer deaths worldwide. Colon carcinogenesis is critically influenced by the tumor microenvironment. Cancer associated fibroblasts (CAFs) and tumor associated macrophages (TAMs) represent the major components of the tumor microenvironment. TAMs promote tumor progression, angiogenesis and tissue remodeling. However, the impact of the molecular crosstalk of tumor cells (TCs) with CAFs and macrophages on monocyte recruitment and their phenotypic conversion is not known in detail so far. In a 3D human organotypic CRC model, we show that CAFs and normal colonic fibroblasts are critically involved in monocyte recruitment and for the establishment of a macrophage phenotype, characterized by high CD163 expression. This is in line with the steady recruitment and differentiation of monocytes to immunosuppressive macrophages in the normal colon. Cytokine profiling revealed that CAFs produce M-CSF, and IL6, IL8, HGF and CCL2 secretion was specifically induced by CAFs in co-cultures with macrophages. Moreover, macrophage/CAF/TCs co-cultures increased TC invasion. We demonstrate that CAFs and macrophages are the major producers of CCL2 and, upon co-culture, increase their CCL2 production twofold and 40-fold, respectively. CAFs and macrophages expressing high CCL2 were also found in vivo in CRC, strongly supporting our findings. CCL2, CCR2, CSF1R and CD163 expression in macrophages was dependent on active MCSFR signaling as shown by M-CSFR inhibition. These results indicate that colon fibroblasts and not TCs are the major cellular component, recruiting and dictating the fate of infiltrated monocytes towards a specific macrophage population, characterized by high CD163 expression and CCL2 production.

Targeting the Proteasome in Advanced Renal Cell Carcinoma: Complexity and Limitations of Patient-Individualized Preclinical Drug Discovery.

BACKGROUND: Systemic treatment options for metastatic renal cell carcinoma (RCC) have significantly expanded in recent years. However, patients refractory to tyrosine kinase and immune checkpoint inhibitors still have limited treatment options and patient-individualized approaches are largely missing. PATIENTS AND METHODS: In vitro drug screening of tumor-derived short-term cultures obtained from seven patients with clear cell RCC was performed. For one patient, a patient-derived xenograft (PDX) mouse model was established for in vivo validation experiments. Drug effects were further investigated in established RCC cell lines. RESULTS: The proteasome inhibitor carfilzomib was among the top hits identified in three of four patients in which an in vitro drug screening could be performed successfully. Carfilzomib also showed significant acute and long-term cytotoxicity in established RCC cell lines. The in vivo antitumoral activity of carfilzomib was confirmed in a same-patient PDX model. The cytotoxicity of carfilzomib was found to correlate with the level of accumulation of ubiquitinated proteins. CONCLUSIONS: In this proof-of-concept study, we show that patient-individualized in vitro drug screening and preclinical validation is feasible. However, the fact that carfilzomib failed to deliver a clinical benefit in RCC patients in a recent phase II trial unrelated to the present study underscores the complexities and limitations of this strategy.

International Consensus on Minimum Preclinical Testing Requirements for the Development of Innovative Therapies For Children and Adolescents with Cancer.

Cancer remains the leading cause of disease-related death in children. For the many children who experience relapses of their malignant solid tumors, usually after very intensive first-line therapy, curative treatment options are scarce. Preclinical drug testing to identify promising treatment elements that match the molecular make-up of the tumor is hampered by the fact that (i) molecular genetic data on pediatric solid tumors from relapsed patients and thus our understanding of tumor evolution and therapy resistance are very limited to date and (ii) for many of the high-risk entities, no appropriate and molecularly well-characterized patient-derived models and/or genetic mouse models are currently available. However, recent regulatory changes enacted by the European Medicines Agency (class waiver changes) and the maturation of the RACE for Children act with the FDA, will require a significant increase in preclinical pediatric cancer research and clinical development must occur. We detail the outcome of a pediatric cancer international multistakeholder meeting whose output aims at defining an international consensus on minimum preclinical testing requirements for the development of innovative therapies for children and adolescents with cancer. Recommendations based on the experience of the NCI funded PPTP/C (www.ncipptc.org) and the EU funded ITCC-P4 public private partnership (www.itccp4.eu) are provided for the use of cell-based and mouse models for pediatric solid malignancies, as well as guidance on the scope and content of preclinical proof-of-concept data packages to inform clinical development dependent on clinical urgency. These recommendations can serve as a minimal guidance necessary to jumpstart preclinical pediatric research globally.

Identification of CD318, TSPAN8 and CD66c as target candidates for CAR T cell based immunotherapy of pancreatic adenocarcinoma.

A major roadblock prohibiting effective cellular immunotherapy of pancreatic ductal adenocarcinoma (PDAC) is the lack of suitable tumor-specific antigens. To address this challenge, here we combine flow cytometry screenings, bioinformatic expression analyses and a cyclic immunofluorescence platform. We identify CLA, CD66c, CD318 and TSPAN8 as target candidates among 371 antigens and generate 32 CARs specific for these molecules. CAR T cell activity is evaluated in vitro based on target cell lysis, T cell activation and cytokine release. Promising constructs are evaluated in vivo. CAR T cells specific for CD66c, CD318 and TSPAN8 demonstrate efficacies ranging from stabilized disease to complete tumor eradication with CD318 followed by TSPAN8 being the most promising candidates for clinical translation based on functionality and predicted safety profiles. This study reveals potential target candidates for CAR T cell based immunotherapy of PDAC together with a functional set of CAR constructs specific for these molecules.

Decitabine Induces Gene Derepression on Monosomic Chromosomes: In Vitro and In Vivo Effects in Adverse-Risk Cytogenetics AML.

Hypomethylating agents (HMA) have become the backbone of nonintensive acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) treatment, also by virtue of their activity in patients with adverse genetics, for example, monosomal karyotypes, often with losses on chromosome 7, 5, or 17. No comparable activity is observed with cytarabine, a cytidine analogue without DNA-hypomethylating properties. As evidence exists for compounding hypermethylation and gene silencing of hemizygous tumor suppressor genes (TSG), we thus hypothesized that this effect may preferentially be reversed by the HMAs decitabine and azacitidine. An unbiased RNA-sequencing approach was developed to interrogate decitabine-induced transcriptome changes in AML cell lines with or without a deletion of chromosomes 7q, 5q or 17p. HMA treatment preferentially upregulated several hemizygous TSG in this genomic region, significantly derepressing endogenous retrovirus (ERV)3-1, with promoter demethylation, enhanced chromatin accessibility, and increased H3K4me3 levels. Decitabine globally reactivated multiple transposable elements, with activation of the dsRNA sensor RIG-I and interferon regulatory factor (IRF)7. Induction of ERV3-1 and RIG-I mRNA was also observed during decitabine treatment in vivo in serially sorted peripheral blood AML blasts. In patient-derived monosomal karyotype AML murine xenografts, decitabine treatment resulted in superior survival rates compared with cytarabine. Collectively, these data demonstrate preferential gene derepression and ERV reactivation in AML with chromosomal deletions, providing a mechanistic explanation that supports the clinical observation of superiority of HMA over cytarabine in this difficult-to-treat patient group. SIGNIFICANCE: These findings unravel the molecular mechanism underlying the intriguing clinical activity of HMAs in AML/MDS patients with chromosome 7 deletions and other monosomal karyotypes.See related commentary by O'Hagan et al., p. 813.

Semi-automated analysis of digital whole slides from humanized lung-cancer xenograft models for checkpoint inhibitor response prediction.

We propose a deep learning workflow for the classification of hematoxylin and eosin stained histological whole-slide images of non-small-cell lung cancer. The workflow includes automatic extraction of meta-features for the characterization of the tumor. We show that the tissue-classification produces state-of-the-art results with an average F1-score of 83%. Manual supervision indicates that experts, in practice, accept a far higher percentage of predictions. Furthermore, the extracted meta-features are validated via visualization revealing relevant biomedical relations between the different tissue classes. In a hypothetical decision-support scenario, these meta-features can be used to discriminate the tumor response with regard to available treatment options with an estimated accuracy of 84%. This workflow supports large-scale analysis of tissue obtained in preclinical animal experiments, enables reproducible quantification of tissue classes and immune system markers, and paves the way towards discovery of novel features predicting response in translational immune-oncology research.

Tumor-Localized Costimulatory T-Cell Engagement by the 4-1BB/HER2 Bispecific Antibody-Anticalin Fusion PRS-343.

PURPOSE: 4-1BB (CD137) is a key costimulatory immunoreceptor and promising therapeutic target in cancer. To overcome limitations of current 4-1BB-targeting antibodies, we have developed PRS-343, a 4-1BB/HER2 bispecific molecule. PRS-343 is designed to facilitate T-cell costimulation by tumor-localized, HER2-dependent 4-1BB clustering and activation. EXPERIMENTAL DESIGN: PRS-343 was generated by the genetic fusion of 4-1BB-specific Anticalin proteins to a variant of trastuzumab with an engineered IgG4 isotype. Its activity was characterized using a panel of in vitro assays and humanized mouse models. The safety was assessed using ex vivo human cell assays and a toxicity study in cynomolgus monkeys. RESULTS: PRS-343 targets 4-1BB and HER2 with high affinity and binds both targets simultaneously. 4-1BB-expressing T cells are efficiently costimulated when incubated with PRS-343 in the presence of cancer cells expressing HER2, as evidenced by increased production of proinflammatory cytokines (IL2, GM-CSF, TNFα, and IFNγ). In a humanized mouse model engrafted with HER2-positive SK-OV-3 tumor cells and human peripheral blood mononuclear cells, PRS-343 leads to tumor growth inhibition and a dose-dependent increase of tumor-infiltrating lymphocytes. In IND-enabling studies, PRS-343 was found to be well tolerated, with no overt toxicity and no relevant drug-related toxicologic findings. CONCLUSIONS: PRS-343 facilitates tumor-localized targeting of T cells by bispecific engagement of HER2 and 4-1BB. This approach has the potential to provide a more localized activation of the immune system with higher efficacy and reduced peripheral toxicity compared with current monospecific approaches. The reported data led to initiation of a phase I clinical trial with this first-in-class molecule.See related commentary by Su et al., p. 5732.

Pharmacological reactivation of MYC-dependent apoptosis induces susceptibility to anti-PD-1 immunotherapy.

Elevated MYC expression sensitizes tumor cells to apoptosis but the therapeutic potential of this mechanism remains unclear. We find, in a model of MYC-driven breast cancer, that pharmacological activation of AMPK strongly synergizes with BCL-2/BCL-XL inhibitors to activate apoptosis. We demonstrate the translational potential of an AMPK and BCL-2/BCL-XL co-targeting strategy in ex vivo and in vivo models of MYC-high breast cancer. Metformin combined with navitoclax or venetoclax efficiently inhibited tumor growth, conferred survival benefits and induced tumor infiltration by immune cells. However, withdrawal of the drugs allowed tumor re-growth with presentation of PD-1+/CD8+ T cell infiltrates, suggesting immune escape. A two-step treatment regimen, beginning with neoadjuvant metformin+venetoclax to induce apoptosis and followed by adjuvant metformin+venetoclax+anti-PD-1 treatment to overcome immune escape, led to durable antitumor responses even after drug withdrawal. We demonstrate that pharmacological reactivation of MYC-dependent apoptosis is a powerful antitumor strategy involving both tumor cell depletion and immunosurveillance.

APC/CCdh1 regulates the balance between maintenance and differentiation of hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) represent the lifelong source of all blood cells and continuously regenerate the hematopoietic system through differentiation and self-renewal. The process of differentiation is initiated in the G1 phase of the cell cycle, when stem cells leave their quiescent state. During G1, the anaphase-promoting complex or cyclosome associated with the coactivator Cdh1 is highly active and marks proteins for proteasomal degradation to regulate cell proliferation. Following Cdh1 knockdown in HSPCs, we analyzed human and mouse hematopoiesis in vitro and in vivo in competitive transplantation assays. We found that Cdh1 is highly expressed in human CD34+ HSPCs and downregulated in differentiated subsets; whereas, loss of Cdh1 restricts myeloid differentiation, supports B cell development and preserves immature short-term HSPCs without affecting proliferation or viability. Our data highlight a role of Cdh1 as a regulator of balancing the maintenance of HSPCs and differentiation into mature blood cells.

Growth of Epithelial Organoids in a Defined Hydrogel.

Epithelial organoids are simplified models of organs grown in vitro from embryonic and adult stem cells. They are widely used to study organ development and disease, and enable drug screening in patient-derived primary tissues. Current protocols, however, rely on animal- and tumor-derived basement membrane extract (BME) as a 3D scaffold, which limits possible applications in regenerative medicine. This prompted us to study how organoids interact with their matrix, and to develop a well-defined hydrogel that supports organoid generation and growth. It is found that soft fibrin matrices provide suitable physical support, and that naturally occurring Arg-Gly-Asp (RGD) adhesion domains on the scaffold, as well as supplementation with laminin-111, are key parameters required for robust organoid formation and expansion. The possibility to functionalize fibrin via factor XIII-mediated anchoring also allows to covalently link fluorescent nanoparticles to the matrix for 3D traction force microscopy. These measurements suggest that the morphogenesis of budding intestinal organoids results from internal pressure combined with higher cell contractility in the regions containing differentiated cells compared to the regions containing stem cells. Since the fibrin/laminin matrix supports long-term expansion of all tested murine and human epithelial organoids, this hydrogel can be widely used as a defined equivalent to BME.

A high-content image analysis approach for quantitative measurements of chemosensitivity in patient-derived tumor microtissues.

Organotypic, three-dimensional (3D) cancer models have enabled investigations of complex microtissues in increasingly realistic conditions. However, a drawback of these advanced models remains the poor biological relevance of cancer cell lines, while higher clinical significance would be obtainable with patient-derived cell cultures. Here, we describe the generation and data analysis of 3D microtissue models from patient-derived xenografts (PDX) of non-small cell lung carcinoma (NSCLC). Standard of care anti-cancer drugs were applied and the altered multicellular morphologies were captured by confocal microscopy, followed by automated image analyses to quantitatively measure phenotypic features for high-content chemosensitivity tests. The obtained image data were thresholded using a local entropy filter after which the image foreground was split into local regions, for a supervised classification into tumor or fibroblast cell types. Robust statistical methods were applied to evaluate treatment effects on growth and morphology. Both novel and existing computational approaches were compared at each step, while prioritizing high experimental throughput. Docetaxel was found to be the most effective drug that blocked both tumor growth and invasion. These effects were also validated in PDX tumors in vivo. Our research opens new avenues for high-content drug screening based on patient-derived cell cultures, and for personalized chemosensitivity testing.