Innervated mouse pancreas organoids as an ex vivo model to study pancreatic neuropathy in pancreatic cancer.

Pancreatic cancer is characterized by bi-directional interactions between pancreatic cancer cells and stromal cells including neural cells. The absence of neural cells in pancreatic organoids limits the investigation of cell- cell interaction and tumor innervation. This protocol describes how to generate innervated wild type (WT) and Kras+/LSLG12D Trp53fl/f lp48+/Cre (KPC) murine pancreatic organoids. To specifically investigate neurogenesis, organoids are co-cultured with iPSCs-derived neural crest cells, while co-culture with dorsal root ganglia explants is used for comparing organoids with mature neurons. For complete details on the use and execution of this protocol, please refer to Huch et al. (2013), Boj et al. (2015), and Demir et al. (2014).

Quantitative analysis of tumour spheroid structure.

Tumour spheroids are common in vitro experimental models of avascular tumour growth. Compared with traditional two-dimensional culture, tumour spheroids more closely mimic the avascular tumour microenvironment where spatial differences in nutrient availability strongly influence growth. We show that spheroids initiated using significantly different numbers of cells grow to similar limiting sizes, suggesting that avascular tumours have a limiting structure; in agreement with untested predictions of classical mathematical models of tumour spheroids. We develop a novel mathematical and statistical framework to study the structure of tumour spheroids seeded from cells transduced with fluorescent cell cycle indicators, enabling us to discriminate between arrested and cycling cells and identify an arrested region. Our analysis shows that transient spheroid structure is independent of initial spheroid size, and the limiting structure can be independent of seeding density. Standard experimental protocols compare spheroid size as a function of time; however, our analysis suggests that comparing spheroid structure as a function of overall size produces results that are relatively insensitive to variability in spheroid size. Our experimental observations are made using two melanoma cell lines, but our modelling framework applies across a wide range of spheroid culture conditions and cell lines.

CombiFlow: Flow cytometry-based identification and characterization of genetically and functionally distinct AML subclones.

Many cancers, including leukemias, are dynamic oligoclonal diseases. Tools to identify and prospectively isolate genetically distinct clones for functional studies are needed. We describe our CombiFlow protocol, which is a combinatorial flow cytometry-based approach to identify and isolate such distinct clones. CombiFlow enables the visualization of clonal evolution during disease progression and the identification of potential relapse-inducing cells at minimal residual disease (MRD) time points. The protocol can be adapted to various research questions and allows functional studies on live sorted cell populations. For complete details on the use and execution of this protocol, please refer to de Boer et al. (2018).

Protocol for photoactivation of YAP in cancer cell spheroids embedded in collagen gels.

This protocol describes the necessary preparations and procedures to photo-activate Yes-associated protein (YAP) with optoYAP in cancer cell spheroids in 3D collagen matrices. We detail steps for immunofluorescent staining of the resulting YAP-activated HeLa spheroids. In addition, we describe handling of optoYAP on 2D substrates. While this protocol focuses on the use of optoYAP in 3D HeLa cell culture, it can be modified for other cell types. For complete details on the use and execution of this protocol, please refer to Illes et al. (2021).

Hydroxyethyl chitosan hydrogels for enhancing breast cancer cell tumorigenesis.

Polysaccharide hydrogels are promising candidate matrices for recapitulating the characteristics of extracellular matrix (ECM) in breast tumors in terms of their structure and composition. Herein, to obtain an ECM-mimetic matrix, hydroxyethyl chitosan (HECS) hydrogels were prepared through Schiff-base crosslinking reaction using dialdehyde hyaluronic acid as crosslinker. The obtained HECS hydrogels displayed a highly porous structure, a stiffness comparable to that of breast tissue, and a fast water-absorption speed. The amount of crosslinker had great effects on the swelling and rheological behaviors of the HECS hydrogels. Preliminary results from in vitro biological assessments confirmed that MCF-7 cells incubated within HECS hydrogels preferred to grow into three-dimensional spheroids. Importantly, the cells displayed enhanced migrative capability and upregulated expression levels of MMP-2, TGF-ß and VEGF in comparison to two-dimension cultured cells. Hence, the HECS hydrogels show great promise as a biomimetic ECM in constructing breast tumor models.

Protocol for derivation of organoids and patient-derived orthotopic xenografts from glioma patient tumors.

Tumor organoids and patient-derived orthotopic xenografts (PDOXs) are some of the most valuable pre-clinical tools in cancer research. In this protocol, we describe efficient derivation of organoids and PDOX models from glioma patient tumors. We provide detailed steps for organoid culture, intracranial implantation, and detection of tumors in the brain. We further present technical adjustments for standardized functional assays and drug testing. For complete details on the use and execution of this protocol, please refer to Golebiewska et al. (2020).

Purification of differentiated tumor cells from medulloblastoma for transplantation into mouse cerebellum.

This protocol provides the procedures for isolating differentiated tumor cells from medulloblastoma (MB) in mice. Procedures for transplantation into cerebella are also included to examine the tumorigenesis of differentiated MB cells. This protocol outlines the detailed steps required for (1) isolation of tumor cells from mouse MB, (2) purification of differentiated tumor cells by fluorescence-activated cell sorting, and (3) transplantation of tumor cells into cerebella. This protocol is useful to purify differentiated tumor cells for investigating mechanisms underlying MB progression. For complete details on the use and execution of this protocol, please refer to Cheng et. al. (2020).

A survey of electrokinetically-driven microfluidics for cancer cells manipulation.

Cancer is one of the leading causes of annual deaths worldwide, accounting for nearly 10 million deaths each year. Metastasis, the process by which cancer spreads across the patient's body, is the main cause of death in cancer patients. Because the rising trend observed in statistics of new cancer cases and cancer-related deaths does not allow for an optimistic viewpoint on the future-in relation to this terrible disease-the scientific community has sought methods to enable early detection of cancer and prevent the apparition of metastatic tumors. One such method is known as liquid biopsy, wherein a sample is taken from a bodily fluid and analyzed for the presence of CTCs or other cancer biomarkers (e.g., growth factors). With this objective, interest is growing by year in electrokinetically-driven microfluidics applied for the concentration, capture, filtration, transportation, and characterization of CTCs. Electrokinetic techniques-electrophoresis, dielectrophoresis, electrorotation, and electrothermal and EOF-have great potential for miniaturization and integration with electronic instrumentation for the development of point-of-care devices, which can become a tool for early cancer diagnostics and for the design of personalized therapeutics. In this contribution, we review the state of the art of electrokinetically-driven microfluidics for cancer cells manipulation.

Generation of Pancreatic Organoid-Derived Isografts.

This protocol is a procedure for generating orthotopic isografts using mouse pancreatic cancer organoids. These isografts can be used to track the evolution of pancreatic ductal adenocarcinoma (PDA) from a preinvasive lesion to a metastatic disease and therefore represent a suitable model for identification of determinants of PDA progression. For complete details on the use and execution of this protocol, please refer to Boj et al. (2015) and Filippini et al. (2019).

Metabolic Profiling of Single Cancer Cells Using Mitochondrial ATP Probes.

The metabolic activity of cells is interrelated with cell signaling, functions, and fate. Uncontrolled cancer cell proliferation requires metabolic adaptations. Research focusing on understanding the characteristics of cell metabolism is crucial for the development of novel diagnostic and therapeutic strategies. Here, we describe protocols for the ATP profiling of single cancer cells by fluorescence live-cell imaging. In response to distinct metabolic inhibitions, we record individual mitochondrial ATP dynamics using established Förster resonance energy transfer-based genetically encoded fluorescent ATP probes. For complete details on the use and execution of this protocol, please refer to Depaoli et al. (2018).

Patient-Derived Tumor Organoid Rings for Histologic Characterization and High-Throughput Screening.

Tumor organoids are promising tools for cancer biology investigations and preclinical drug screenings because they are often representative of the histology and drug responses of patients. Here, we introduce a facile protocol to overcome technical limitations by generating patient-derived tumor organoids using a simplified ring-like geometry. This facilitates media exchange and drug treatment for histopathology characterization and automated high-throughput drug screenings. For complete details on the use and execution of this protocol, please refer to Phan et al. (2019).

Patient-derived malignant pleural mesothelioma cell cultures: a tool to advance biomarker-driven treatments.

Malignant pleural mesothelioma (MPM) is an aggressive cancer, associated with poor prognosis. We assessed the feasibility of patient-derived cell cultures to serve as an ex vivo model of MPM. Patient-derived MPM cell cultures (n=16) exhibited stemness features and reflected intratumour and interpatient heterogeneity. A subset of the cells were subjected to high-throughput drug screening and coculture assays with cancer-specific cytotoxic T cells and showed diverse responses. Some of the biphasic MPM cells were capable of processing and presenting the neoantigen SSX-2 endogenously. In conclusion, patient-derived MPM cell cultures are a promising and faithful ex vivo model of MPM.

Mathematical modelling reveals cellular dynamics within tumour spheroids.

Tumour spheroids are widely used as an in vitro assay for characterising the dynamics and response to treatment of different cancer cell lines. Their popularity is largely due to the reproducible manner in which spheroids grow: the diffusion of nutrients and oxygen from the surrounding culture medium, and their consumption by tumour cells, causes proliferation to be localised at the spheroid boundary. As the spheroid grows, cells at the spheroid centre may become hypoxic and die, forming a necrotic core. The pressure created by the localisation of tumour cell proliferation and death generates an cellular flow of tumour cells from the spheroid rim towards its core. Experiments by Dorie et al. showed that this flow causes inert microspheres to infiltrate into tumour spheroids via advection from the spheroid surface, by adding microbeads to the surface of tumour spheroids and observing the distribution over time. We use an off-lattice hybrid agent-based model to re-assess these experiments and establish the extent to which the spatio-temporal data generated by microspheres can be used to infer kinetic parameters associated with the tumour spheroids that they infiltrate. Variation in these parameters, such as the rate of tumour cell proliferation or sensitivity to hypoxia, can produce spheroids with similar bulk growth dynamics but differing internal compositions (the proportion of the tumour which is proliferating, hypoxic/quiescent and necrotic/nutrient-deficient). We use this model to show that the types of experiment conducted by Dorie et al. could be used to infer spheroid composition and parameters associated with tumour cell lines such as their sensitivity to hypoxia or average rate of proliferation, and note that these observations cannot be conducted within previous continuum models of microbead infiltration into tumour spheroids as they rely on resolving the trajectories of individual microbeads.

Cryopreservation of Viable Human Tissues: Renewable Resource for Viable Tissue, Cell Lines, and Organoid Development.

The availability of viable human tissues is critical to support translational research focused on personalized care. Most studies have relied on fresh frozen or formalin-fixed paraffin-embedded tissues for histopathology, genomics, and proteomics. Yet, basic, translational, and clinical research downstream assays such as tumor progression/invasion, patient-derived xenograft, organoids, immunoprofiling, and vaccine development still require viable tissue, which are time-sensitive and rare commodities. We describe the generation of two-dimensional (2D) and three-dimensional (3D) cultures to validate a viable freeze cryopreservation technique as a standard method of highest quality specimen preservation. After surgical resection, specimens were minced, placed in CryoStor™ media, and frozen using a slow freezing method (-1°C/min in -80°C) for 24 hours and then stored in liquid nitrogen. After 15-18 months, the tissues were thawed, dissociated into single-cell suspensions, and evaluated for cell viability. To generate primary 2D cultures, cells were plated onto Collagen-/Matrigel-coated plates. To develop 3D cultures (organoids), the cells were plated in reduced serum RPMI media on nonadherent plates or in Matrigel matrix. The epithelial nature of the cells was confirmed by using immunohistochemistry for cytokeratins. DNA and RNA isolation was performed using QIAGEN AllPrep kits. We developed primary lines (2D and 3D) of colon, thyroid, lung, renal, and liver cancers that were positive for cytokeratin staining. 3D lines were developed from the same cohort of tumor types in both suspended media and Matrigel matrix. Multiple freeze-thaw cycles did not significantly alter the viability and growth of 2D and 3D lines. DNA/RNA recovery was similar to its fresh frozen cohort. In this study, we validated 2D and 3D tissue cultures as methods to corroborate the feasibility of viable cryopreservation of tumor tissue. This proof-of-principle study, if more widely implemented, should improve accessibility of human viable tumor tissue/cells in a time-independent manner for many basic, preclinical, and translational assays.

A perivascular niche in the bone marrow hosts quiescent and proliferating tumorigenic colorectal cancer cells.

Disseminated tumor cells (dTCs) can frequently be detected in the bone marrow (BM) of colorectal cancer (CRC) patients, raising the possibility that the BM serves as a reservoir for metastatic tumor cells. Identification of dTCs in BM aspirates harbors the potential of assessing therapeutic outcome and directing therapy intensity with limited risk and effort. Still, the functional and prognostic relevance of dTCs is not fully established. We have previously shown that CRC cell clones can be traced to the BM of mice carrying patient-derived xenografts. However, cellular interactions, proliferative state and tumorigenicity of dTCs remain largely unknown. Here, we applied a coculture system modeling the microvascular niche and used immunofluorescence imaging of the murine BM to show that primary CRC cells migrate toward endothelial tubes. dTCs in the BM were rare, but detectable in mice with xenografts from most patient samples (8/10) predominantly at perivascular sites. Comparable to primary tumors, a substantial fraction of proliferating dTCs was detected in the BM. However, most dTCs were found as isolated cells, indicating that dividing dTCs rather separate than aggregate to metastatic clones-a phenomenon frequently observed in the microvascular niche model. Clonal tracking identified subsets of self-renewing tumor-initiating cells in the BM that formed tumors out of BM transplants, including one subset that did not drive primary tumor growth. Our results indicate an important role of the perivascular BM niche for CRC cell dissemination and show that dTCs can be a potential source for tumor relapse and tumor heterogeneity.

Efficient primary culture model of patient­derived tumor cells from colorectal cancer using a Rho­associated protein kinase inhibitor and feeder cells.

In vitro culture of patient­derived tumor cells offers many advantages in the development of novel therapies for colorectal cancer. Although various culture systems have been developed, the long­term expansion of patient­derived tumor cells remains challenging. The present results suggested that tumor cells isolated from colorectal cancer patient­derived xenografts can be efficiently immortalized in conditioned medium from irradiated feeder cells containing Y­27632, a rho­associated coiled­coil containing protein kinase (ROCK) inhibitor. Patient­derived tumor cells proliferated rapidly, reaching 90­95% confluence in ~6 days. Short tandem repeat analysis suggested that these tumor tissues and cultured cells presented 13 identical short tandem repeat loci, including Amelogenin, Penta E, Penta D, D2S1338 and D19S433. Their epithelial phenotype was confirmed by staining for epithelial cell adhesion molecule and cytokeratin 20, whereas vimentin was used as a mesenchymal marker. When cells were transferred to 3D cultures, they continued to proliferate, forming well­defined tumor spheroids. Expression levels of human telomerase reverse transcriptase and C­Myc mRNA were increased in cultured cells. Finally, immortalized cells were used for the screening of 65 anticancer drugs approved by the Food and Drug Administration, allowing the identification of gene­drug associations. In the present study, primary culture models of colorectal cancer were efficiently established using a ROCK inhibitor and feeder cells, and this approach could be used for personalized treatment strategies for patients with colorectal cancer.

Micro-Electrical Impedance Spectroscopy and Identification of Patient-Derived, Dissociated Tumor Cells.

Fine needle aspirate sampling of tumors requires acquisition of sufficient cells to complete a diagnosis. Aspirates through such fine needles are typically composed of small cell clusters in suspension, making them readily amenable to microfluidic analysis. Here we show a microfluidic device with integrated electrodes capable of interrogating and identifying cellular components in a patient-derived sample of dissociated tumor cells using micro-electrical impedance spectroscopy ( µ EIS). We show that the µ EIS system can distinguish dissociated tumor cells in a sample consisting of red blood cell (RBCs) and peripheral blood mononucleated cells (PBMCs). Our µ EIS system can also distinguish dissociated tumor cells from normal cells and we show results for five major cancer types, specifically, lung, thyroid, breast, ovarian, and kidney cancer. Moreover, our µ EIS system can make these distinctions in a label-free manner, thereby opening the possibility of integration into standard clinical workflows at the point of care.

Aberrant Super-Enhancer Landscape in Human Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) cells exploit an aberrant transcriptional program to sustain their infinite growth and progression. Emerging evidence indicates that the continuous and robust transcription of oncogenes in cancer cells is often driven by super-enhancers (SEs). In this study, we systematically compared the SE landscapes between normal liver and HCC cells and revealed that the cis-acting SE landscape was extensively reprogrammed during liver carcinogenesis. HCC cells acquired SEs at multiple prominent oncogenes to drive their vigorous expression. We identified sphingosine kinase 1 (SPHK1) as an SE-associated oncogene, and we used this gene as an example to illustrate the impact of SEs on the activation of oncogenes in HCC. Concurrently, we also showed that the critical components of the trans-acting SE complex, namely, cyclin-dependent kinase 7 (CDK7), bromodomain-containing protein 4 (BRD4), E1A binding protein P300 (EP300), and mediator complex subunit 1 (MED1), were frequently overexpressed in human HCCs and were associated with the poor prognosis of patients with HCC. Using the CRISPR/Cas9 gene-editing system and specific small-molecule inhibitors, we further demonstrated that HCC cells were highly sensitive to perturbations of the SE complex. The inactivation of CDK7, BRD4, EP300, and MED1 selectively repressed the expression of SE-associated oncogenes in HCC. Finally, we demonstrated that THZ1, which is a small-molecule inhibitor of CDK7, exerted a prominent anticancer effect in both in vitro and in vivo HCC models. Conclusion: The SE landscape and machinery were significantly altered in human HCCs. HCC cells are highly susceptible to perturbations of the SE complex due to the resulting selective suppression of SE-associated oncogenes. Our results suggest that targeting SE complex is a promising therapeutic strategy for HCC treatment.

Anions reversibly responsive luminescent nanocellulose hydrogels for cancer spheroids culture and release.

Artificial stimuli-responsive hydrogels that can mimic natural extracellular matrix for growth and release of cancer spheroids (CSs) have attracted much attention. However, such hydrogels still face a challenge in regulating CSs growth and controlled release as well as keeping CSs integrity. Herein, a new class of ClO-/SCN- reversibly responsive nanocellulose hydrogel with fluorescence on-off reporter is developed. Upon addition of ClO-, the gel network of nanocellulose hydrogel was destructed, accompanying by the fluorescent quenching. Notably, when introducing of SCN-, a red fluorescence filamentous hydrogel was recovered by coordination cross-linking. The hydrogel reforms in a completely reversible process through the regulation of ClO-/SCN-. Benefit from the above response features of the hydrogel, the growth of cancer spheroids (CSs) in the hydrogel and on demand release of CSs from the hydrogel could be easily achieved through ClO-/SCN- regulation. Importantly, the growth and release of CSs can be monitored in real time by fluorescence imaging. Overall, such design strategy based on ClO-/SCN--responsive fluorescent hydrogels provided a new type of multi-responsive hydrogels as main scaffolds for cancer research and cancer drug screening.

Three-Dimensional Cell Culture Model Utilization in Renal Carcinoma Cancer Stem Cell Research.

Specific 3D conditions of cancer cell lines have been optimized over last years, with growing significance of serum-free and xeno-free culture variants. The choice of proper culture media enables cancer stem cells proliferation in primary and stable cell lines. To obtain renal cell cancer stem-like phenotype, we employed media dedicated for mesenchymal cells and adult stem cells. Developed RCC cell line 3D culture system enables effective drug testing, including tyrosine kinase inhibitor anti-cancer cell toxicity. To induce formation of 3D spheroids by RCC cell lines, StemXvivo and NutriStem media must be used. Usage of laminin- or poly-D-lysine coated plates enhances also the formation of spheroids in 3D-promoting media. Seeding is optimal with Caki-1 or ACHN cell lines as well as 786-O or HKCSC cells. Our bio-mimic 3D RCC cell culture model promotes cell viability and stem-related gene expression including E-cadherin, N-cadherin, HIF1, HIF2, VEGF, Sox2, Pax2, and Nestin. 3D spheroid formation ability and spheroid volume increase are disturbed upon drug treatment. Untreated 3D structures reach ~100 µm in diameter at the end of 14-day long experiment. Sorter-based cell cycle analysis and Ki-67 staining should be conducted to verify specific toxicity. We suggest that due to the more complex architecture 3D RCC culture is more relevant to investigate the in vivo-like tumor drug response.