OrganoID: a versatile deep learning platform for organoid image analysis

Organoids are three-dimensional in vitro tissue models that closely represent the native heterogeneity, microanatomy, and functionality of an organ or diseased tissue. Analysis of organoid morphology, growth, and drug response is challenging due to the diversity in shape and size of organoids, movement through focal planes, and limited options for live-cell staining. Here, we present OrganoID, an open-source image analysis platform that automatically recognizes, labels, and tracks single organoids in brightfield and phase-contrast microscopy. The platform identifies organoid morphology pixel by pixel without the need for fluorescence or transgenic labeling and accurately analyzes a wide range of organoid types in time-lapse microscopy experiments. OrganoID uses a modified u-net neural network with minimal feature depth to encourage model generalization and allow fast execution. The network was trained on images of human pancreatic cancer organoids and was validated on images from pancreatic, lung, colon, and adenoid cystic carcinoma organoids with a mean intersection-over-union of 0.76. OrganoID measurements of organoid count and individual area concurred with manual measurements at 96% and 95% agreement respectively. Tracking accuracy remained above 89% over the duration of a four-day validation experiment. Automated single-organoid morphology analysis of a dose-response experiment identified significantly different organoid circularity after exposure to different concentrations of gemcitabine. The OrganoID platform enables straightforward, detailed, and accurate analysis of organoid images to accelerate the use of organoids as physiologically relevant models in high-throughput research.

Restriction of extracellular lipids renders pancreatic cancer dependent on autophagy

Background KRAS is the predominant oncogene mutated in pancreatic ductal adenocarcinoma (PDAC), the fourth cause of cancer-related deaths worldwide. Mutant KRAS-driven tumors are metabolically programmed to support their growth and survival, which can be used to identify metabolic vulnerabilities. In the present study, we aimed to understand the role of extracellularly derived fatty acids in KRAS-driven pancreatic cancer. Methods To assess the dependence of PDAC cells on extracellular fatty acids we employed delipidated serum or RNAi-mediated suppression of ACSL3 (to inhibit the activation and cellular retention of extracellular fatty acids) followed by cell proliferation assays, qPCR, apoptosis assays, immunoblots and fluorescence microscopy experiments. To assess autophagy in vivo, we employed the KrasG12D/+;p53flox/flox;Pdx1-CreERT2 (KPC) mice crossed with Acsl3 knockout mice, and to assess the efficacy of the combination therapy of ACSL3 and autophagy inhibition we used xenografted human cancer cell-derived tumors in immunocompromised mice. Results Here we show that depletion of extracellularly derived lipids either by serum lipid restriction or suppression of ACSL3, triggers autophagy, a process that protects PDAC cells from the reduction of bioenergetic intermediates. Combined extracellular lipid deprivation and autophagy inhibition exhibits anti-proliferative and pro-apoptotic effects against PDAC cell lines in vitro and promotes suppression of xenografted human pancreatic cancer cell-derived tumors in mice. Therefore, we propose lipid deprivation and autophagy blockade as a potential co-targeting strategy for PDAC treatment. Conclusions Our work unravels a central role of extracellular lipid supply in ensuring fatty acid provision in cancer cells, unmasking a previously unappreciated metabolic vulnerability of PDAC cells.

Two Laser Treatments Can Improve Tumor Ablation Efficiency of Chemophototherapy

Chemophototherapy is an emerging tumor ablation modality that can improve local delivery of chemotherapeutic agents. Long circulating doxorubicin (Dox) in porphyrin-phospholipid (PoP) liposomes (LC-Dox-PoP) has previously been developed as an effective chemophototherapy agent. In the present study, we observed that in mice, LC-Dox-PoP showed enhanced accumulation in human pancreatic tumor xenografts even with suboptimal light doses, as assessed by fluorometric analysis of tissue homogenates and microscopic imaging of Dox and PoP in tumor slices. A second laser treatment, at a time point in which tumors had greater drug accumulation as a result of the first laser treatment, induced potent tumor ablation. Efficacy studies were carried out in two human pancreatic cancer subcutaneous mouse tumor models; MIA PaCa-2 or low-passage patient derived pancreatic cancer xenografts. A single treatment of 3 mg/kg LC-Dox-PoP and an initial 150 J/cm2 laser treatment 1 h after drug administration, followed by second laser treatment of 50 J/cm2 8 h after drug administration, was more effective than a single laser treatment of 200 J/cm2 at either of those time points. Thus, this study presents proof-of-principle and rationale for using two discrete laser treatments to enhance the efficacy of chemophototherapy.

Dynamic interplay between structural variations and 3D chromosome organization in pancreatic cancer

Structural variations (SVs) are the greatest source of variation in the genome and can lead to oncogenesis. However, the identification and interpretation of SVs in human pancreatic cancer remain largely undefined due to technological limitations. Here, we investigate the spectrum of SVs and three-dimensional (3D) chromatin architecture in human pancreatic ductal epithelial cell carcinogenesis by using state-of-the-art long-read single-molecule real-time (SMRT) and high-throughput chromosome conformation capture (Hi-C) sequencing techniques. We find that the 3D genome organization is remodeled and correlated with gene expressional change. The bulk remodeling effect of cross-boundary SVs in the 3D genome partly depends on intercellular genomic heterogeneity. Meanwhile, contact domains tend to minimize these disrupting effects of SVs within local adjacent genomic regions to maintain overall stability of 3D genome organization. Moreover, our data also demonstrates complex genomic rearrangements involving two key driver genes CDKN2A and SMAD4, and elucidates their influence on cancer-related gene expression from both linear view and 3D perspective. Overall, this study provides a valuable resource and highlights the impact, complexity and dynamicity of the interplay between SVs and 3D genome organization, which further expands our understanding of pathogenesis of SVs in human pancreatic cancer.

Development of an Icariin-Loaded Bilosome-Melittin Formulation with Improved Anticancer Activity against Cancerous Pancreatic Cells

Pancreatic cancer currently represents a severe issue for the entire world. Therefore, much effort has been made to develop an effective treatment against it. Emerging evidence has shown that icariin, a flavonoid glycoside, is an effective anti-pancreatic cancer drug. Melittin, as a natural active biomolecule, has also shown to possess anticancer activities. In the present study, with the aim to increase its effectiveness against cancerous cells, icariin-loaded bilosome-melittin (ICA-BM) was developed. For the selection of an optimized ICA-BM, an experimental design was implemented, which provided an optimized formulation with a particle size equal to 158.4 nm. After estimation of the release pattern, the anti-pancreatic cancer efficacy of this new formulation was evaluated. The MTT assay was employed for the determination of half maximal inhibitory concentration (IC50), providing smaller IC50 for ICA-BM (2.79 ± 0.2 µM) compared to blank-BM and ICA-Raw (free drug) against PNAC1, a human pancreatic cancer cell line isolated from a pancreatic carcinoma of ductal cell origin. Additionally, cell cycle analysis for ICA-BM demonstrated cell arrest at the S-phase and pre-G1 phase, which indicated a pro-apoptotic behavior of the new developed formulation. The pro-apoptotic and anti-proliferative activity of the optimized ICA-BM against PNAC1 cells was also demonstrated through annexin V staining as well as estimation of caspase-3 and p53 protein levels. It can be concluded that the optimized ICA-BM formulation significantly improved the efficacy of icariin against cancerous pancreatic cells.

Design, Synthesis and Biological Evaluation of Coumarin Derivatives as NEDD8 Activating Enzyme Inhibitors in Pancreatic Cancer Cells

Background: NEDD8 (neural precursor cell expressed developmentally downregulated protein 8) is one of the ubiquitin-like proteins which is activated by the NEDD8 activating enzyme (NAE). The overexpressed NAE can cause a variety of diseases such as numerous cancer types and inflammatory diseases. The selective inhibition of NAE could mediate the rate of ubiquitination and the subsequent degradation of proteins associated with cancer so as to achieve the purpose of treatment. Objective: In this article, we decided to study the synthesis and screening of coumarin scaffold derivatives against cancer cell lines, specifically the human pancreatic cancer cell line BxPC-3. Methods: Twenty-four targeted compounds were synthesized, and their anti-proliferative activity against three cancer cell lines, cytotoxicity against three normal cell lines through CCK-8 and MTT assay were evaluated to screen out the candidate compound. Then the target was further confirmed by both enzyme and cell-based experiments, as well as cell apoptosis research. Results: Several new 4-position substituted coumarin derivatives (12a~x) were synthesized and most of them exhibit antiproliferative activity in three cancer cell lines. A series of experiments were performed to identify the best candidate compound 12v. This compound displayed the highest potency against BxPC-3 with an IC50 value of 0.28 µM. It can also inhibit NAE activity in enzyme and cellbased assay, and induce CRLs-mediated accumulation of the substrate and apoptosis in BxPC-3 cells. Meanwhile, it exhibited relatively low toxicity in three normal cells. Conclusion: Based on these results, we found that compound 12v inhibited NAE activity in enzyme and cell-based systems and induced apoptosis in BxPC-3 cells. Additionally, it also had a low toxicity. These results suggested that 12v may be promising lead compounds for the development of new anticancer drugs.

Romidepsin and Tamoxifen Cooperatively Induce Senescence of Pancreatic Cancer Cells Through Downregulation of FOXM1 Expression and Induction of ROS/Lipid Peroxidation

Although progress has been made in chemotherapeutic strategies against pancreatic cancer, overall survival has not significantly improved over the past decade. Thus, the development of better therapeutic regimens remains a high priority. Pancreatic cancer cell lines were treated with romidepsin, an inhibitor of histone deacetylase, and tamoxifen, and their effects on cell growth, signaling and gene expression were determined. Xenografts of human pancreatic cancer CFPAC1 cells were treated with romidepsin and tamoxifen to determine their effects on tumor growth. The inhibition of the growth of pancreatic cancer cells induced by romidepsin and tamoxifen was effectively reduced by N-acetyl cysteine and α-tocopherol, respectively. The combined treatment greatly induced reactive oxygen species production and mitochondrial lipid peroxidation, and these effects were prevented by N-acetyl cysteine and α-tocopherol. Tamoxifen enhanced romidepsin-induced cell senescence. FOXM1 expression was markedly downregulated in pancreatic cancer cells treated with romidepsin, and tamoxifen further reduced FOXM1 expression in cells treated with romidepsin. Siomycin A, an inhibitor of FOXM1, induced senescence in pancreatic cancer cells. Similar results were obtained in knockdown of FOXM1 expression by siRNA. Since FOXM1 is used as a prognostic marker and therapeutic target for pancreatic cancer, a combination of the clinically available drugs romidepsin and tamoxifen might be considered for the treatment of patients with pancreatic cancer.