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In a screen of compounds to selectively suppress the growth of cancer spheroids, which contained mutant (mt) KRAS, NPD10621 was discovered and associated derivatives were investigated.Spheroid areas from HCT116-derived HKe3 spheroids expressing wild type (wt) KRAS (HKe3-wtKRAS) and mtKRAS (HKe3-mtKRAS) were treated with 12 NPD10621 derivatives and measured in three-dimensional floating (3DF) cultures. Several cancers were treated with NPD1018 (pyra-metho-carnil: PMC) in 3DF cultures. In a nude mouse assay, 50% cell growth inhibition (GIFrom these 12 derivatives, PMC was the most effective inhibitor of HKe3-mtKRAS spheroid growth with the least toxicity. Furthermore, PMC-mediated growth suppression was observed in all tested cancer cell lines, independent of tissue context, driver gene mutations, and drug resistance, suggesting that the PMC target(s) was crucial for cancer growth in a context-independent manner. The GIPMC is a low-toxicity compound that inhibits the growth of different tumor cell types.

Multicellular tumor spheroids are rapidly emerging as an improved in vitro model with respect to more traditional 2D culturing. Microwell culturing is a simple and accessible method for generating a large number of uniformly sized spheroids, but commercially available systems often do not enable researchers to perform complete culturing and analysis pipelines and the mechanical properties of their culture environment are not commonly matching those of the target tissue. We herein report a simple method to obtain custom-designed self-built microwell arrays made of polydimethylsiloxane or agarose for uniform 3D cell structure generation. Such materials can provide an environment of tunable mechanical flexibility. We developed protocols to culture a variety of cancer and non-cancer cell lines in such devices and to perform molecular and imaging characterizations of the spheroid growth, viability, and response to pharmacological treatments. Hundreds of tumor spheroids grow (in scaffolded or scaffold-free conditions) at homogeneous rates and can be harvested at will. Microscopy imaging can be performed in situ during or at the end of the culture. Fluorescence (confocal) microscopy can be performed after in situ staining while retaining the geographic arrangement of spheroids in the plate wells. This platform can enable statistically robust investigations on cancer biology and screening of drug treatments. ispartof: Cell Mol Life Sci vol:80 issue:4 pages:93- ispartof: location:Switzerland status: Published online

Tumour spheroid experiments are routinely used to study cancer progression and treatment. Various and inconsistent experimental designs are used, leading to challenges in interpretation and reproducibility. Using multiple experimental designs, live-dead cell staining, and real-time cell cycle imaging, we measure necrotic and proliferation-inhibited regions in over 1000 4D tumour spheroids (3D space plus cell cycle status). By intentionally varying the initial spheroid size and temporal sampling frequencies across multiple cell lines, we collect an abundance of measurements of internal spheroid structure. These data are difficult to compare and interpret. However, using an objective mathematical modelling framework and statistical identifiability analysis we quantitatively compare experimental designs and identify design choices that produce reliable biological insight. Measurements of internal spheroid structure provide the most insight, whereas varying initial spheroid size and temporal measurement frequency is less important. Our general framework applies to spheroids grown in different conditions and with different cell types., Tumour spheroid experiments with real-time fluorescent ubiquitination-based cell cycle imaging are interpreted using mathematical modelling and statistical identifiability analysis. A framework is provided to compare data obtained across different experimental designs and the experimental design choices that provide reliable biological insight are revealed.

Treatments for advanced and recurrent ovarian cancer remain a challenge due to a lack of potent, selective, and effective therapeutics. Here, we developed the basis for a transformative anticancer strategy based on anthrax toxin that has been engineered to be selectively activated by the catalytic power of zymogen-activating proteases on the surface of malignant tumor cells to induce cell death. Exposure to the engineered toxin is cytotoxic to ovarian tumor cell lines and ovarian tumor spheroids derived from patient ascites. Preclinical studies demonstrate that toxin treatment induces tumor regression in several in vivo ovarian cancer models, including patient-derived xenografts, without adverse side effects, supportive of progression toward clinical evaluation. These data lay the groundwork for developing therapeutics for treating women with late-stage and recurrent ovarian cancers, utilizing a mechanism distinct from current anticancer therapies.

The aryl hydrocarbon receptor (AHR) pathway modulates the immune system in response to kynurenine, an endogenous tryptophan metabolite. IDO1 and TDO2 catalyze kynurenine production, which promotes cancer progression by compromising host immunosurveillance. However, it is unclear whether the AHR activation regulates the malignant traits of cancer such as metastatic capability or cancer stemness. Here, we carried out systematic analyses of metabolites in patient‐derived colorectal cancer spheroids and identified high levels of kynurenine and TDO2 that were positively associated with liver metastasis. In a mouse colon cancer model, TDO2 expression substantially enhanced liver metastasis, induced AHR‐mediated PD‐L1 transactivation, and dampened immune responses; these changes were all abolished by PD‐L1 knockout. In patient‐derived cancer spheroids, TDO2 or AHR activity was required for not only the expression of PD‐L1, but also for cancer stem cell (CSC)‐related characteristics and Wnt signaling. TDO2 was coexpressed with both PD‐L1 and nuclear β‐catenin in colon xenograft tumors, and the coexpression of TDO2 and PD‐L1 was observed in clinical colon cancer specimens. Thus, our data indicate that the activation of the TDO2‐kynurenine‐AHR pathway facilitates liver metastasis of colon cancer via PD‐L1–mediated immune evasion and maintenance of stemness., We carried out systematic analyses of metabolites in patient‐derived colorectal cancer spheroids, and identified high levels of kynurenine and TDO2 that were positively associated with liver metastasis. Our data indicate that the activation of the TDO2‐kynurenine‐AHR pathway may lead to emergence of immune‐evasive cancer stem cells (CSCs) promoting liver metastasis.

Recent studies have shown that circular RNA circFLNA is abnormally expressed in a variety of malignant tumors, but its role and mechanism in bladder carcinoma (BCa) are still unclear. The present paper aims to contribute to research on the effects and mechanism of circFLNA on the malignant phenotype of BCa. In this study, the expressions of circFLNA, miR-216a-3p and BTG2 in BCa and BCa cells (EJ, T24, 5637, TCC-SUP) were detected by qRT-PCR. EdU staining, colony formation, Transwell assay, wound healing assays, and sphere formation assay were used to measure the cell proliferation, viability, invasion, migration, and cell stemness of BCa cells after circFLNA overexpression. In addition, the correlation existed between miR-216a-3p and circFLNA or BTG2 was confirmed by Dual-Luciferase Reporter assay and RNA pull-down. Western blot was utilized to determine the expression of BTG2, MMP2, epithelial-mesenchymal transition (EMT)-related proteins (vimentin, E-cadherin) and stem cell-specific proteins (CD34, OCT4, SOX2). Our study confirmed that downregulated circFLNA and BTG2 expression and upregulated miR-216a-3p were found in both BCa tissues and cell lines. Meanwhile, upregulated circFLNA inhibited proliferation, invasion and migration, EMT and stemness of BCa cells. MiR-216a-3p was a target gene of circFLNA and could target BTG2. Further analysis finally demonstrated that circFLNA sponged miR-216a-3p and indirectly promoted BTG2 expression, ultimately regulating proliferation, migration, invasion and EMT of BCa cells. In conclusion, circFLNA inhibits the malignant phenotype of BCa cells and their stemness through miR-216a-3p/BTG2, thus suppressing BCa progression.

Malignant tumors are complex tissues composed of malignant cells, vascular cells, structural mesenchymal cells including pericytes and carcinoma-associated fibroblasts, infiltrating immune cells, and others, collectively called the tumor stroma. The number of stromal cells in a tumor is often much greater than the number of malignant cells. The physical associations among all these cell types are critical to tumor growth, survival, and response to therapy. Most cell-based screens for cancer drug discovery and precision medicine validation use malignant cells in isolation as monolayers, embedded in a matrix, or as spheroids in suspension. Medium- and high-throughput screening with multiple cell lines requires a scalable, reproducible, robust cell-based assay. Complex spheroids include malignant cells and two normal cell types, human umbilical vein endothelial cells and highly plastic mesenchymal stem cells, which rapidly adapt to the malignant cell microenvironment. The patient-derived pancreatic adenocarcinoma cell line, K24384-001-R, was used to explore complex spheroid structure and response to anticancer agents in a 96-well format. We describe the development of the complex spheroid assay as well as the growth and structure of complex spheroids over time. Subsequently, we demonstrate successful assay miniaturization to a 384-well format and robust performance in a high-throughput screen. Implementation of the complex spheroid assay was further demonstrated with 10 well-established pancreatic cell lines. By incorporating both human stromal and tumor components, complex spheroids might provide an improved model for tumor response in vivo.

Galaxolide and tonalide are well-known polycyclic musks whose intensive use without limitations in numerous cleaning, hygiene, and personal care products has resulted in widespread direct human exposure via absorption, inhalation, and oral ingestion. Latest data shows that long-term, low-dose exposure to toxic chemicals can induce unpredictable harmful effects in a variety of living systems, however, interactions between synthetic musks and brain tumours remain largely unexplored. Glioblastoma (GB) accounts for nearly half of all tumours of the central nervous system and is characterized by very poor prognosis. The aims of this study were (1) to investigate the potential effect of long-term (20-generation) single and combined application of galaxolide and tonalide at sub-lethal doses (5–2.5 u M) on the angiogenesis, invasion, and migration of human U87 cells or tumour spheroids, and (2) to explore the underlying molecular mechanisms. Random amplified polymorphic DNA assays revealed significant DNA damage and increased total mutation load in galaxolide- and/or tonalide-treated U87 cells. In those same groups, we also detected remarkable tumour spheroid invasion and up-regulation of both HIF1-α/VEGF/MMP9 and IL6/JAK2/STAT3 signals, known to have important roles in hypoxia-related angiogenesis and/or proliferation. Prolonged musk treatment further altered angio-miRNA expression in a manner consistent with poor prognosis in GB. We also detected significant over-expression of the genes Slug, Snail, ZEB1, and Vimentin, which are biomarkers of epithelial to mesenchymal transition. In addition, matrigel, transwell, and wound healing assays clearly showed that long-term sub-lethal exposure to galaxolide and/or tonalide induced invasion and migration proposing a high metastatic potential. Our results suggest that assessing expression of HIF-1a, VEGF, STAT3, and the miR-17-92 cluster in biopsy samples of GB patients who have a history of possible long-term exposure to galaxolide or tonalide could be beneficial for deciding a therapy regime. Additionally, we recommend that extensively-used hygiene and cleaning materials be selected from synthetic musk-free products, especially when used in palliative care processes for GB patients.

Electrochemotherapy (ECT) is the combination of transient pore formation following electric pulse application with the administration of cytotoxic drugs, which enhances the cytotoxic effect of the applied agent due to membrane changes. In vitro 3D culture systems simulate the in vivo tumor growth and preserve the biological characteristics of tumors more accurately than conventional monolayer cell cultures. We describe a protocol for the development of 3D tumor organoids using conjunctival melanoma (CM) and uveal melanoma (UM) cell lines as well as the use of hand-held customized electrodes, suitable for in vitro ECT in the culture well without destruction of the tumor environment. This protocol analyzes the culture and growth of 3D CM and UM spheroids and their reaction to bleomycin (2.5 µg/mL) alone, electroporation (EP) (750 Volts/cm, 8 pulses, 100 µs, 5 Hz) alone, and ECT as a combination of EP and bleomycin. The drug concentration and the EP settings used in this protocol were established as preferred ECT conditions according to previous experiments. The assay used to determine the spheroid viability was conducted 3-7 days following treatment. The effect on viability and growth of the 3D tumor spheroids was significant only after ECT. The customized electrodes are described in detail in order to facilitate the application of pulses in the culture well. This novel treatment of 3D UM and CM spheroids sets a steppingstone for future clinical application.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy in the developed world. EOC metastasis is unique since malignant cells detach directly from the primary tumor site into the abdominal fluid and form multicellular aggregates, called spheroids, that possess enhanced survival mechanisms while in suspension. As such, altered cell adhesion properties are paramount to EOC metastasis with cell detachment from the primary tumor, dissemination as spheroids, and reattachment to peritoneal surfaces for secondary tumor formation. The ability for EOC cells to establish and maintain cell–cell contacts in spheroids is critical for cell survival in suspension. Integrins are a family of cell adhesion receptors that play a crucial role in cell–cell and cell-extracellular matrix interactions. These glycoprotein receptors regulate diverse functions in tumor cells and are implicated in multiple steps of cancer progression. Altered integrin expression is detected in numerous carcinomas, where they play a role in cell migration, invasion, and anchorage-independent survival. Like that observed for other carcinomas, epithelial-mesenchymal transition (EMT) occurs during metastasis and integrins can function in this process as well. Herein, we provide a review of the evidence for integrin-mediated cell adhesion mechanisms impacting steps of EOC metastasis. Taken together, targeting integrin function may represent a potential therapeutic strategy to inhibit progression of advanced EOC.

The occurrence of epithelial-mesenchymal transition (EMT) within tumors, which enables invasion and metastasis, is linked to cancer stem cells (CSCs) with drug and radiation resistance. We used two specific peptides, F7 and SP peptides, to detect EMT derived cells or CSCs. Human tongue squamous carcinoma cell line-SAS transfected with reporter genes was generated and followed by spheroid culture. A small molecule inhibitor-Unc0642 and low-dose ionizing radiation (IR) were used for induction of EMT. Confocal microscopic imaging and fluorescence-activated cell sorting analysis were performed to evaluate the binding ability and specificity of peptides. A SAS xenograft mouse model with EMT induction was established for assessing the binding affinity of peptides. The results showed that F7 and SP peptides not only specifically penetrated into cytoplasm of SAS cells but also bound to EMT derived cells and CSCs with high nucleolin and vimentin expression. In addition, the expression of CSC marker and the binding of peptides were increased in tumors isolated from Unc0642/IR-treated groups. Our study demonstrates the potential of these peptides for detecting EMT derived cells or CSCs and might provide an alternative isolation method for these subpopulations within the tumor in the future.

STEAP3 (Six-transmembrane epithelial antigen of the prostate 3, TSAP6, dudulin-2) has been reported to be involved in tumor progression in human malignancies. Nevertheless, how it participates in the progression of human cancers, especially HCC, is still unknown. In the present study, we found that STEAP3 was aberrantly overexpressed in the nuclei of HCC cells. In a large cohort of clinical HCC tissues, high expression level of nuclear STEAP3 was positively associated with tumor differentiation and poor prognosis (p

Chemotherapy for glioblastoma multiforme (GBM) remains ineffective due to insufficient penetration of therapeutic agents across the blood–brain barrier (BBB) and into the GBM tumor. Herein, is described, the optimization of the lipid composition and fabrication conditions for a BBB- and tumor penetrating terpolymer-lipid-hybrid nanoparticle (TPLN) for delivering doxorubicin (DOX) to GBM. The composition of TPLNs was first screened using different lipids based on nanoparticle properties and in vitro cytotoxicity by using 23 full factorial experimental design. The leading DOX loaded TPLNs (DOX-TPLN) were prepared by further optimization of conditions and used to study cellular uptake mechanisms, in vitro cytotoxicity, three-dimensional (3D) glioma spheroid penetration, and in vivo biodistribution in a murine orthotopic GBM model. Among various lipids studied, ethyl arachidate (EA) was found to provide excellent nanoparticle properties e.g., size, polydispersity index (PDI), zeta potential, encapsulation efficiency, drug loading, and colloidal stability, and highest anticancer efficacy for DOX-TPLN. Further optimized EA-based TPLNs were prepared with an optimal particle size (103.8 ± 33.4 nm) and PDI (0.208 ± 0.02). The resultant DOX-TPLNs showed ~ sevenfold higher efficacy than free DOX against human GBM U87-MG-RED-FLuc cells in vitro. The interaction between the TPLNs and the low-density lipoprotein receptors also facilitated cellular uptake, deep penetration into 3D glioma spheroids, and accumulation into the in vivo brain tumor regions of DOX-TPLNs. This work demonstrated that the TPLN system can be optimized by rational selection of lipid type, lipid content, and preparation conditions to obtain DOX-TPLN with enhanced anticancer efficacy and GBM penetration and accumulation.

Funder: WD Armstrong studentship, Glioblastoma (GBM) angiogenesis is critical for tumor growth and recurrence, making it a compelling therapeutic target. Here, a disease-relevant, vascularized tumoroid in vitro model with stem-like features and stromal surrounds is reported. The model is used to recapitulate how individual components of the GBM’s complex brain microenvironment such as hypoxia, vasculature-related stromal cells and growth factors support GBM angiogenesis. It is scalable, tractable, cost-effective and can be used with biologically-derived or biomimetic matrices. Patient-derived primary GBM cells are found to closely participate in blood vessel formation in contrast to a GBM cell line containing differentiated cells. Exogenous growth factors amplify this effect under normoxia but not at hypoxia suggesting that a significant amount of growth factors is already being produced under hypoxic conditions. Under hypoxia, primary GBM cells strongly co-localize with umbilical vein endothelial cells to form sprouting vascular networks, which has been reported to occur in vivo. These findings demonstrate that our 3D tumoroid in vitro model exhibits biomimetic attributes that may permit its use as a preclinical model in studying microenvironment cues of tumor angiogenesis.

Multicellular spheroids are expected to be used for in vivo-like tissue models and cell transplantation. Microwell devices are useful for the fabrication of multicellular spheroids to improve productivity and regulate their size. However, the high cell density in microwell devices leads to accelerated cell death. In this study, we developed O2-generating microwells by incorporating calcium peroxide (CaO2) into polydimethylsiloxane (PDMS)-based microwells. The CaO2-containing PDMS was shown to generate O2 for 3 d. Then, CaO2-containing PDMS was used to fabricate O2-generating microwells using a micro-molding technique. When human hepatocellular carcinoma (HepG2) spheroids were prepared using the conventional microwells, the O2 concentration in the culture medium reduced to approx. 67% of the cell-free level. In contrast, the O2-generating microwells maintained O2 at constant levels. The HepG2 spheroids prepared using the O2-generating microwells had a larger number of live cells than those prepared using the conventional microwells. In addition, the O2-generating microwells rescued hypoxia in the HepG2 spheroids and increased cell viability. Lastly, the O2-generating microwells were also useful for the preparation of multicellular spheroids of other cell types (i.e., MIN6, B16-BL6, and adipose-derived stem cells) with high cell viability. These results showed that the O2-generating microwells are useful for preparing multicellular spheroids with high cell viability.

Neuroblastoma, the most common extracranial solid tumor of childhood, is thought to arise from neural crest‐derived immature cells. The prognosis of patients with high‐risk or recurrent/refractory neuroblastoma remains quite poor despite intensive multimodality therapy; therefore, novel therapeutic interventions are required. We examined the expression of a cell adhesion molecule CD146 (melanoma cell adhesion molecule [MCAM]) by neuroblastoma cell lines and in clinical samples and investigated the anti‐tumor effects of CD146‐targeting treatment for neuroblastoma cells both in vitro and in vivo. CD146 is expressed by 4 cell lines and by most of primary tumors at any stage. Short hairpin RNA‐mediated knockdown of CD146, or treatment with an anti‐CD146 polyclonal antibody, effectively inhibited growth of neuroblastoma cells both in vitro and in vivo, principally due to increased apoptosis via the focal adhesion kinase and/or nuclear factor‐kappa B signaling pathway. Furthermore, the anti‐CD146 polyclonal antibody markedly inhibited tumor growth in immunodeficient mice inoculated with primary neuroblastoma cells. In conclusion, CD146 represents a promising therapeutic target for neuroblastoma., Targeting CD146 inhibits in vitro proliferation of neuroblastoma (NB) cells and in vivo growth of NB tumors. Exposure of NB cells to the anti‐CD146 antibody led to a significant reduction in survival and anchorage‐independent growth. Intraperitoneal injection of the anti‐CD146 antibody into immunodeficient mice for ~50 d led to a significant inhibition of tumor growth.

Background Neuroblastoma (NB) patients with MYCN amplification or overexpression respond poorly to current therapies and exhibit extremely poor clinical outcomes. PI3K-mTOR signaling-driven deregulation of protein synthesis is very common in NB and various other cancers that promote MYCN stabilization. In addition, both the MYCN and mTOR signaling axes can directly regulate a common translation pathway that leads to increased protein synthesis and cell proliferation. However, a strategy of concurrently targeting MYCN and mTOR signaling in NB remains unexplored. This study aimed to investigate the therapeutic potential of targeting dysregulated protein synthesis pathways by inhibiting the MYCN and mTOR pathways together in NB. Methods Using small molecule/pharmacologic approaches, we evaluated the effects of combined inhibition of MYCN transcription and mTOR signaling on NB cell growth/survival and associated molecular mechanism(s) in NB cell lines. We used two well-established BET (bromodomain extra-terminal) protein inhibitors (JQ1, OTX-015), and a clinically relevant mTOR inhibitor, temsirolimus, to target MYCN transcription and mTOR signaling, respectively. The single agent and combined efficacies of these inhibitors on NB cell growth, apoptosis, cell cycle and neurospheres were assessed using MTT, Annexin-V, propidium-iodide staining and sphere assays, respectively. Effects of inhibitors on global protein synthesis were quantified using a fluorescence-based (FamAzide)-based protein synthesis assay. Further, we investigated the specificities of these inhibitors in targeting the associated pathways/molecules using western blot analyses. Results Co-treatment of JQ1 or OTX-015 with temsirolimus synergistically suppressed NB cell growth/survival by inducing G1 cell cycle arrest and apoptosis with greatest efficacy in MYCN-amplified NB cells. Mechanistically, the co-treatment of JQ1 or OTX-015 with temsirolimus significantly downregulated the expression levels of phosphorylated 4EBP1/p70-S6K/eIF4E (mTOR components) and BRD4 (BET protein)/MYCN proteins. Further, this combination significantly inhibited global protein synthesis, compared to single agents. Our findings also demonstrated that both JQ1 and temsirolimus chemosensitized NB cells when tested in combination with cisplatin chemotherapy. Conclusions Together, our findings demonstrate synergistic efficacy of JQ1 or OTX-015 and temsirolimus against MYCN-driven NB, by dual-inhibition of MYCN (targeting transcription) and mTOR (targeting translation). Additional preclinical evaluation is warranted to determine the clinical utility of targeted therapy for high-risk NB patients.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates various toxicological and biological functions. We reported previously that 3-methylcholanthrene (3MC), an exogenous AhR agonist, inhibited tumorsphere (mammosphere) formation from breast cancer cell lines, while the endogenous AhR agonist, indirubin, very weakly inhibited this process. However, the difference in inhibition mechanism of mammosphere formation by 3MC or indirubin is still unknown. In this study, we established AhR-re-expressing (KOTR-AhR) cells from AhR knockout MCF-7 cells using the tetracycline (Tet)-inducible gene expression systems. To identify any difference in inhibition of mammosphere formation by 3MC or indirubin, RNA-sequencing (RNA-seq) experiments were performed using KOTR-AhR cells. RNA-seq experiments revealed that cell division cycle 20 (CDC20), which regulates the cell cycle and mitosis, was decreased by 3MC, but not by indirubin, in the presence of AhR expression. Furthermore, the mRNA and protein levels of CDC20 were decreased by 3MC in MCF-7 cells via the AhR. In addition, mammosphere formation was suppressed by small interfering RNA-mediated CDC20 knockdown compared to the negative control in MCF-7 cells. These results suggest that AhR activation by 3MC suppresses mammosphere formation via downregulation of CDC20 expression in breast cancer cells. This study provides useful information for the development of AhR-targeted anti-cancer drugs.

Funding Information: This work was supported by AbbVie and by iNOVA4Health – UIDB/04462/2020, a program financially supported by Fundação para a Ciência e Tecnologia (FCT) / Ministério da Educação e Ciência, through national funds. RM and TFM were recipients of PhD fellowships funded by FCT (SFRH/BD/132163/2017 and PD/BD/128377/2017, respectively) and CB was funded by “The Discoveries Centre for Regenerative and Precision Medicine” (European Commission Horizon 2020 Research and Innovation programme, under the Grant Agreement 739572). Publisher Copyright: © 2021, The Author(s). The current standard preclinical oncology models are not able to fully recapitulate therapeutic targets and clinically relevant disease biology, evidenced by the 90% attrition rate of new therapies in clinical trials. Three-dimensional (3D) culture systems have the potential to enhance the relevance of preclinical models. However, the limitations of currently available cellular assays to accurately evaluate therapeutic efficacy in these models are hindering their widespread adoption. We assessed the compatibility of the lactate dehydrogenase (LDH) assay in 3D spheroid cultures against other commercially available readout methods. We developed a standardized protocol to apply the LDH assay to ex vivo cultures, considering the impact of culture growth dynamics. We show that accounting for growth rates and background release levels of LDH are sufficient to make the LDH assay a suitable methodology for longitudinal monitoring and endpoint assessment of therapeutic efficacy in both cell line-derived xenografts (xenospheres) and patient-derived explant cultures. This method has the added value of being non-destructive and not dependent on reagent penetration or manipulation of the parent material. The establishment of reliable readout methods for complex 3D culture systems will further the utility of these tumor models in preclinical and co-clinical drug development studies. publishersversion published

Cancer stem cells (CSCs) are responsible for therapy resistance and share several properties with normal stem cells. Here, we show that brain‐expressed X‐linked gene 2 (BEX2), which is essential for dormant CSCs in cholangiocarcinoma, is highly expressed in human hepatocellular carcinoma (HCC) lesions compared with the adjacent normal lesions and that in 41 HCC cases the BEX2high expression group is correlated with a poor prognosis. BEX2 localizes to Ki67‐negative (nonproliferative) cancer cells in HCC tissues and is highly expressed in the dormant fraction of HCC cell lines. Knockdown of BEX2 attenuates CSC phenotypes, including sphere formation ability and aldefluor activity, and BEX2 overexpression enhances these phenotypes. Moreover, BEX2 knockdown increases cisplatin sensitivity, and BEX2 expression is induced by cisplatin treatment. Taken together, these data suggest that BEX2 induces dormant CSC properties and affects the prognosis of patients with HCC., BEX2 is essential for dormant cancer stem cell and affects patients’ prognosis in hepatocellular carcinoma. BEX2 could be a promising therapeutic target.

Various three-dimensional (3D) cell culture methods have been developed to implement tumor models similar to in vivo. However, the conventional 3D cell culture method has limitations such as difficulty in using an extracellular matrix (ECM), low experimental reproducibility, complex 3D cell culture protocol, and difficulty in applying to high array plates such as 96- or 384-plates. Therefore, detailed protocols related to robust 3D-aggregated spheroid model (3D-ASM) production were optimized and proposed. A specially designed wet chamber was used to implement 3D-ASM using the hepatocellular carcinoma (HCC) cell lines, and the conditions were established for the icing step to aggregate the cells in one place and optimized ECM gelation step. Immunofluorescence (IF) staining is mainly used to simultaneously analyze drug efficacy and changes in drug-target biomarkers. By applying the IF staining method to the 3D-ASM model, confocal microscopy imaging and 3D deconvolution image analysis were also successfully performed. Through a comparative study of drug response with conventional 2D-high throughput screening (HTS), the 3D-HTS showed a more comprehensive range of drug efficacy analyses for HCC cell lines and enabled selective drug efficacy analysis for the FDA-approved drug sorafenib. This suggests that increased drug resistance under 3D-HTS conditions does not reduce the analytical discrimination of drug efficacy, also drug efficacy can be analyzed more selectively compared to the conventional 2D-HTS assay. Therefore, the 3D-HTS-based drug efficacy analysis method using an automated 3D-cell spotter/scanner, 384-pillar plate/wet chamber, and the proposed 3D-ASM fabrication protocol is a very suitable platform for analyzing target drug efficacy in HCC cells.

Multicellular 3D tumor models are becoming a powerful tool for testing of novel drug products and personalized anticancer therapy. Tumor spheroids, a commonly used 3D multicellular tumor model, more closely reproduce the tumor microenvironment than conventional 2D cell cultures. It should be noted that spheroids can be produced using different techniques, which can be subdivided into scaffold-free (SF) and scaffold-based (SB) methods. However, it remains unclear, to what extent spheroid properties depend on the method of their generation. In this study, we aimed to carry out a head-to-head comparison of drug sensitivity and molecular expression profile in SF and SB spheroids along with a monolayer (2D) cell culture. Here, we produced non-small cell lung cancer (NSCLC) spheroids based on human lung adenocarcinoma cell line A549. Drug sensitivity analysis of the tested cell cultures to five different chemotherapeutics resulted in IC50 (A549-SB) > IC50 (A549-SF) > IC50 (A549-2D) trend. It was found that SF and SB A549 spheroids displayed elevated expression levels of epithelial-to-mesenchymal transition (EMT) markers and proteins associated with drug resistance compared with the monolayer A549 cell culture. Enhanced drug resistance of A549-SB spheroids can be a result of larger diameters and elevated deposition of extracellular matrix (ECM) that impairs drug penetration into spheroids. Thus, the choice of the spheroid production method can influence the properties of the generated 3D cell culture and their drug resistance. This fact should be considered for correct interpretation of drug testing results.

Reprogramming of energy metabolism is regarded as one of the hallmarks of cancer; in particular, oncogenic RAS has been shown to be a critical regulator of cancer metabolism. Recently, asparagine metabolism has been heavily investigated as a novel target for cancer treatment. For example, Knott et al. showed that asparagine bioavailability governs metastasis in a breast cancer model. Gwinn et al. reported the therapeutic vulnerability of asparagine biosynthesis in KRAS-driven non-small cell lung cancer. We previously reported that KRAS-mutated CRC cells can adapt to glutamine depletion through upregulation of asparagine synthetase (ASNS), an enzyme that synthesizes asparagine from aspartate. In our previous study, we assessed the efficacy of asparagine depletion using human cancer cell lines. In the present study, we evaluated the clinical relevance of asparagine depletion using a novel patient-derived spheroid xenograft (PDSX) mouse model. First, we examined ASNS expression in 38 spheroid lines and found that 12 lines (12/37, 32.4%) displayed high ASNS expression, whereas 26 lines (25/37, 67.6%) showed no ASNS expression. Next, to determine the role of asparagine metabolism in tumor growth, we established ASNS-knockdown spheroid lines using lentiviral short hairpin RNA constructs targeting ASNS. An in vitro cell proliferation assay demonstrated a significant decrease in cell proliferation upon asparagine depletion in the ASNS-knockdown spheroid lines, and this was not observed in the control spheroids lines. In addition, we examined asparagine inhibition with the anti-leukemia drug L-asparaginase (L-Asp) and observed a considerable reduction in cell proliferation at a low concentration (0.1 U/mL) in the ASNS-knockdown spheroid lines, whereas it exhibited limited inhibition of control spheroid lines at the same concentration. Finally, we used the PDSX model to assess the effects of asparagine depletion on tumor growth in vivo. The nude mice injected with ASNS-knockdown or control spheroid lines were administered with L-Asp once a day for 28 days. Surprisingly, in mice injected with ASNS-knockdown spheroids, the administration of L-Asp dramatically inhibited tumor engraftment. On the other hands, in mice injected with control spheroids, the administration of L-Asp had no effect on tumor growth inhibition at all. These results suggest that ASNS inhibition could be critical in targeting asparagine metabolism in cancers.

Tumor spheroid models are widely used for drug screening as in vitro models of the tumor microenvironment. There are various ways in which tumor spheroid models can be prepared, including the self-assembly of cells using low-adherent plates, micro-patterned plates, or hanging-drop plates. Recently, drug high-throughput screening (HTS) approaches have incorporated the use of these culture systems. These HTS culture systems, however, require complicated equipment, such as robot arms, detectors, and software for handling solutions and data processing. Here, we describe protocols that allow tumor spheroids to be tested with different concentrations of a drug in a parallel fashion using a microfluidic device that generates a gradient of anti-cancer drugs. This microfluidic spheroid culture device with a concentration gradient generator (μFSCD-CGG) enables the formation of 50 tumor spheroids and the testing of drugs at five different concentrations. First, we provide a protocol for the fabrication of the μFSCD-CGG, which has both a culture array in which tumor cells are injected and aggregate to form spheroids and a concentration gradient generator for drug testing. Second, we provide a protocol for tumor spheroid formation and HTS of anti-cancer drugs using the device. Finally, we provide a protocol for assessing the response of tumor spheroids at different drug concentrations. To address the needs of the pharmaceutical industry, this protocol can be used for various cell types, including stem cells, and the number of tumor spheroids and drug concentration ranges that can be tested in the μFSCD-CGG can be increased. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Fabrication of a microfluidic spheroid culture device with a concentration gradient generator (μFSCD-CGG) Basic Protocol 2: Seeding cells and formation of spheroids in the μFSCD-CGG Basic Protocol 3: Drug treatment and assessment of cell viability in the μFSCD-CGG.

Rational design of a drug delivery system with enhanced therapeutic potency is critical for efficient tumor chemotherapy. Many protein-based drug delivery platforms have been designed to deliver drugs to target sites and improve the therapeutic efficacy. In this study, paclitaxel (PTX) molecules were encapsulated within an apoferritin nanocage-based drug delivery system with the modification of an extracellular-signal-regulated kinase (ERK) peptide inhibitor at the C-terminus of ferritin (HERK). Apoferritin is an endogenous nano-sized spherical protein which has the ability to specially bind to a majority of tumor cells via interacting with transferrin receptor 1. The ERK peptide inhibitor is a peptide which can disrupt the interaction of MEK with ERK in the mitogen-activated protein kinase/ERK pathway. By combining the targeted delivery effect of ferritin and the inhibitory effect of the ERK peptide inhibitor, the newly fabricated ferritin carrier nanoparticle HERK could still be taken up by tumor cells, and it displayed higher cell cytotoxicity than the parent ferritin. After loading with PTX, HERK-PTX displayed a favorable anticancer effect in human breast cancer cells MDA-MB-231 and lung carcinoma cells A549. The remarkable inhibitory effect on MDA-MB-231 tumor spheroids was also identified. These results indicated that the constructed HERK nanocarrier is a promising multi-functional drug delivery vehicle to enhance the therapeutic effect of drugs in cancer therapy.

Multicellular tumor spheroids (MCTSs) can serve as in vitro models for solid tumors and have become widely used in basic cancer research and drug screening applications. The major challenges when studying MCTSs by optical microscopy are imaging and analysis due to light scattering within the 3-dimensional structure. Herein, we used an ultrasound-based MCTS culture platform, where A498 renal carcinoma MCTSs were cultured, DAPI stained, optically cleared and imaged, to connect nuclear segmentation to biological information at the single cell level. We show that DNA-content analysis can be used to classify the cell cycle state as a function of position within the MCTSs. We also used nuclear volumetric characterization to show that cells were more densely organized and perpendicularly aligned to the MCTS radius in MCTSs cultured for 96 h compared to 24 h. The method presented herein can in principle be used with any stochiometric DNA staining protocol and nuclear segmentation strategy. Since it is based on a single counter stain a large part of the fluorescence spectrum is free for other probes, allowing measurements that correlate cell cycle state and nuclear organization with e.g., protein expression or drug distribution within MCTSs.

Patient‐derived cancer models that reconstitute the characteristics of the tumor microenvironment may facilitate efforts in precision immune‐oncology and the discovery of effective anticancer therapies. Organoids that have recently emerged as robust preclinical models typically contain tumor epithelial cells and lack the native tumor immune microenvironment. A patient‐derived organotypic tumor spheroid (PDOTS) is a novel and innovative ex vivo system that retains key features of the native tumor immune microenvironment. Here, we established and characterized a series of colorectal cancer PDOTS models for use as a preclinical platform for testing effective immunotherapy and its combinations with other drugs. Partially dissociated (> 100 μm in diameter) tumor tissues were embedded in Matrigel‐containing organoid media and subsequently formed into organoid structures within 3 to 7 days of culture. The success rate of growing PDOTS from fresh tissues was ~86%. Morphological analysis showed that the PDOTSs varied in size and structure. Immunofluorescence and flow cytometry analysis revealed that the PDOTSs retained autologous tumor‐infiltrating lymphoid cells and tumor‐infiltrating lymphoid cells were continually decreased through serial passages. Notably, PDOTSs from tumors from a high‐level microsatellite instability‐harboring patient were sensitive to anti‐PD‐1 or anti‐PD‐L1 antibodies. Our results demonstrate that the PDOTS model in which the tumor immune microenvironment is preserved may represent an advantageous ex vivo system to develop effective immune therapeutics., As stated in the manuscript, we established and characterized a series of colorectal cancer PDOTS models for use as a preclinical platform for testing effective immunotherapy and its combinations with other drugs. PDOTSs from tumors from a high‐level microsatellite instability‐harboring patient were sensitive to anti‐PD‐1 or anti‐PD‐L1 antibodies. Our results demonstrate that the PDOTS model in which the tumor immune microenvironment is preserved may represent an advantageous ex vivo system to develop effective immune therapeutics.

Giant cell tumor of bone (GCTB) is a locally aggressive and rarely metastasizing tumor. GCTB is characterized by the presence of unique giant cells and a recurrent mutation in the histone tail of the histone variant H3.3, which is encoded by H3F3A on chromosome 1. GCTB accounts for ~ 5% of primary bone tumors. Although GCTB exhibits an indolent course, it has the potential to develop aggressive behaviors associated with local recurrence and distant metastasis. Currently, complete surgical resection is the only curative treatment, and novel therapeutic strategies are required. Patient-derived cancer cell lines are critical tools for basic and pre-clinical research. However, only a few GCTB cell lines have been reported, and none of them are available from public cell banks. Therefore, we aimed to establish novel GCTB cell lines in the present study. Using curetted tumor tissues of GCTB, we established two cell lines and named them NCC-GCTB2-C1 and NCC-GCTB3-C1. These cells harbored a typical mutation in histones and exhibited slow but constant growth, formed spheroids, and had invasive capabilities. We demonstrated the utility of these cell lines for high-throughput drug screening using 214 anticancer agents. We concluded that NCC-GCTB2-C1 and NCC-GCTB3-C1 cell lines were useful for the in vitro study of GCTB.

The large‐conductance Ca2+‐activated K+ channel KCa1.1 plays a pivotal role in tumor development and progression in several solid cancers. The three‐dimensional (3D) in vitro cell culture system is a powerful tool for cancer spheroid formation, and mimics in vivo solid tumor resistance to chemotherapy in the tumor microenvironment (TME). KCa1.1 is functionally expressed in osteosarcoma and chondrosarcoma cell lines. KCa1.1 activator‐induced hyperpolarizing responses were significantly larger in human osteosarcoma MG‐63 cells isolated from 3D spheroid models compared with in those from adherent 2D monolayer cells. The present study investigated the mechanisms underlying the upregulation of KCa1.1 and its role in chemoresistance using a 3D spheroid model. KCa1.1 protein expression levels were significantly elevated in the lipid‐raft‐enriched compartments of MG‐63 spheroids without changes in its transcriptional level. 3D spheroid formation downregulated the expression of the ubiquitin E3 ligase FBXW7, which is an essential contributor to KCa1.1 protein degradation in breast cancer. The siRNA‐mediated inhibition of FBXW7 in MG‐63 cells from 2D monolayers upregulated KCa1.1 protein expression. Furthermore, a treatment with a potent and selective KCa1.1 inhibitor overcame the chemoresistance of the MG‐63 and human chondrosarcoma SW‐1353 spheroid models to paclitaxel, doxorubicin, and cisplatin. Among several multidrug resistance ATP‐binding cassette transporters, the expression of the multidrug resistance‐associated protein MRP1 was upregulated in both spheroids and restored by the inhibition of KCa1.1. Therefore, the pharmacological inhibition of KCa1.1 may be an attractive new strategy for acquiring resistance to chemotherapeutic drugs in the TME of KCa1.1‐positive sarcomas., The inhibition of large‐conductance Ca2+‐activated K+ channel KCa1.1 may be a promising therapeutic strategy for reversing chemoresistance in human sarcomas.

In vitro 3D culture models have emerged in the cancer field due to their ability to recapitulate characteristics of the in vivo tumor. Herein, we described the establishment and characterization of 3D multicellular spheroids using ovarian cancer cells (SKOV-3) in co-culture with mesenchymal cells (MUC-9) or fibroblasts (CCD27-Sk). We demonstrated that SKOV-3 cells in co-culture were able to form regular and compact spheroids with diameters ranging from 300 to 400 µm and with a roundness close to 1.0 regardless of the type of stromal cell used. In the 3D culture an increase was not observed in spheroid diameter nor was there significant cell growth. What is more, the 3D co-cultures presented an up regulation of genes related to tumorigenesis, angiogenesis and metastases (MMP2, VEGFA, SNAI1, ZEB1 and VIM) when compared with 2D and 3D monoculture. As expected, both 3D cultures (mono and co-cultures) exhibited a higher Paclitaxel chemoresistance when compared to 2D condition. Although we did not observe differences in the Paclitaxel resistance between the 3D mono and co-cultures, the gene expression results indicate that the presence of mesenchymal cells and fibroblasts better recapitulate the in vivo tumor microenvironment, being able, therefore, to more accurately evaluate drug efficacy for ovarian cancer therapy.

Background Organ culture models have been used over the past few decades to study development and disease. The in vitro three-dimensional (3D) culture system of organoids is well known, however, these 3D systems are both costly and difficult to culture and maintain. As such, less expensive, faster and less complex methods to maintain 3D cell culture models would complement the use of organoids. Chick embryos have been used as a model to study human biology for centuries, with many fundamental discoveries as a result. These include cell type induction, cell competence, plasticity and contact inhibition, which indicates the relevance of using chick embryos when studying developmental biology and disease mechanisms. Results Here, we present an updated protocol that enables time efficient, cost effective and long-term expansion of fetal organ spheroids (FOSs) from chick embryos. Utilizing this protocol, we generated FOSs in an anchorage-independent growth pattern from seven different organs, including brain, lung, heart, liver, stomach, intestine and epidermis. These three-dimensional (3D) structures recapitulate many cellular and structural aspects of their in vivo counterpart organs and serve as a useful developmental model. In addition, we show a functional application of FOSs to analyze cell-cell interaction and cell invasion patterns as observed in cancer. Conclusion The establishment of a broad ranging and highly effective method to generate FOSs from different organs was successful in terms of the formation of healthy, proliferating 3D organ spheroids that exhibited organ-like characteristics. Potential applications of chick FOSs are their use in studies of cell-to-cell contact, cell fusion and tumor invasion under defined conditions. Future studies will reveal whether chick FOSs also can be applicable in scientific areas such as viral infections, drug screening, cancer diagnostics and/or tissue engineering.

Cisplatin resistance is one of the major concerns in the treatment of oral squamous cell carcinoma (OSCC). Accumulating evidence suggests microRNA (miRNA) dysregulation as one of the mediators of chemoresistance. Toward this, our previous study revealed the role of exosomal microRNA-155 (miR-155) in cisplatin resistance via downregulation of FOXO3a, a direct target of miR-155, and induction of epithelial-to-mesenchymal transition in OSCC. In the present study, we demonstrate the therapeutic potential of miR-155 inhibitor-laden exosomes in the sensitization of a cisplatin-resistant (cisRes) OSCC 3D tumor spheroid and xenograft mouse model. The cisRes OSSC 3D tumor spheroid model recapitulated the hallmarks of solid tumors such as enhanced hypoxia, reactive oxygen species, and secretory vascular endothelial growth factor. Further treatment with miR-155 inhibitor-loaded exosomes showed the upregulation of FOXO3a and induction of the mesenchymal-to-epithelial transition with improved sensitization to cisplatin in cisRes tumor spheroids and xenograft mouse model. Moreover, the exosomal miR-155 inhibitor suppressed the stem-cell-like property as well as drug efflux transporter protein expression in cisplatin-resistant tumors. Taken together, our findings, for the first time, established that the miR-155 inhibitor-loaded exosomes reverse chemoresistance in oral cancer, thereby providing an alternative therapeutic strategy for the management of refractory oral cancer patients.

Due to the absence of curative treatments for glioblastoma (GBM), we assessed the efficacy of single and combination treatments with a translationally relevant 2nd generation TRAIL-receptor agonist (IZI1551) and the blood–brain barrier (BBB) permeant proteasome inhibitor marizomib in a panel of patient-derived glioblastoma cell lines. These cells were cultured using protocols that maintain the characteristics of primary tumor cells. IZI1551+marizomib combination treatments synergistically induced apoptotic cell death in the majority of cases, both in 2D, as well as in 3D spheroid cultures. In contrast, single-drug treatments largely failed to induce noticeable amounts of cell death. Kinetic analyses suggested that time-shifted drug exposure might further increase responsiveness, with marizomib pre-treatments indeed strongly enhancing cell death. Cell death responses upon the addition of IZI1551 could also be observed in GBM cells that were kept in a medium collected from the basolateral side of a human hCMEC/D3 BBB model that had been exposed to marizomib. Interestingly, the subset of GBM cell lines resistant to IZI1551+marizomib treatments expressed lower surface amounts of TRAIL death receptors, substantially lower amounts of procaspase-8, and increased amounts of cFLIP, suggesting that apoptosis initiation was likely too weak to initiate downstream apoptosis execution. Indeed, experiments in which the mitochondrial apoptosis threshold was lowered by antagonizing Mcl-1 re-established sensitivity to IZI1551+marizomib in otherwise resistant cells. Overall, our study demonstrates a high efficacy of combination treatments with a latest-generation TRAIL receptor agonist and the BBB permeant proteasome inhibitor marizomib in relevant GBM cell models, as well as strategies to further enhance responsiveness and to sensitize subgroups of otherwise resistant GBM cases., European Union’s Horizon 2020 research and innovation program, Deutsche Forschungsgemeinschaft, Projekt DEAL

Glioblastoma brain tumors form in brains’ white matter and remains one of the most lethal cancers despite intensive therapy and surgery. The complex morphology of these tumors includes infiltrative growth and gain of cell motility. Therefore, various brain-mimetic model systems have been developed to investigate invasion dynamics. Despite this, exactly how gradients of cell density, chemical signals and metabolites influence individual cells’ migratory behavior remains elusive. Here we show that the gradient field – induced by the spheroid – accelerates cells’ invasion of the extracellular matrix. We show that cells are pushed away from the spheroid along a radial gradient, as predicted by a biased persistent random walk (BPRW). Thus, our results grasp in a simple model the complex behavior of metastasizing cells. We anticipate that this well-defined and quantitative assay could be instrumental in the development of new anti-cancer strategies.

Hypoxia has been noted as a key factor for induction and maintenance of cancer stemness thereby leading to therapy resistance. Three-dimensional (3D) spheroid models demonstrate a heterogeneity of hypoxic regions replicating the in vivo situation within tumors. Utilizing an established 3D spheroid model, we investigated whether extrinsic hypoxia reinforced chemoresistance in malignant pleural mesothelioma (MPM) spheroids.Tumor spheres were generated from Meso-1 (a typical human MPM cell line) cells having high spheroid-forming ability. To induce hypoxia condition, we utilized a hypoxia chamber with regulation of O2 and CO2 levels. Cell viability was estimated by a WST-8 assay. Real-time polymerase chain reaction and Western blot were performed to evaluate the expression at mRNA and protein levels.Compared with cells cultured in the two-dimensional monolayer model, tumor sphere cells showed elevated mRNA levels of cancer stemness markers (CD26, CD44 and ABCG2) and protein levels of the stemness and hypoxia adaptation markers (ABCG2, ALDH1A1 and HIFs). Correlating with this, 3D spheroid cells were more resistant to permetrexed and topotecan than the two-dimensional cells, indicative of their potential for hypoxic adaptation. Furthermore, significantly stronger resistance to both chemotherapeutic agents was observed in spheroid cells upon hypoxic challenge compared to spheroid cells under normoxia.From the present data, it is concluded that hypoxia adaptation of MPM cells from tumor spheres could enhance their chemoresistance.

Altered lipid metabolism is one of the hallmarks of cancer. Cellular proliferation and de novo synthesis of lipids are related to cancer progression. In this study, we evaluated the lipidomic profile of two-dimensional (2D) monolayer and multicellular tumor spheroids from the HCT 116 colon carcinoma cell line. We utilized serial trypsinization on the spheroid samples to generate three cellular populations representing the proliferative, quiescent, and necrotic regions of the spheroid. This analysis enabled a comprehensive identification and quantification of lipids produced in each of the spheroid layer and 2D cultures. We show that lipid subclasses associated with lipid droplets form in oxygen-restricted and acidic regions of spheroids and are produced at higher levels than in 2D cultures. Additionally, sphingolipid production, which is implicated in cell death and survival pathways, is higher in spheroids relative to 2D cells. Finally, we show that increased numbers of lipids comprised of polyunsaturated fatty acids (PUFAs) are produced in the quiescent and necrotic regions of the spheroid. The lipidomic signature for each region and cell culture type highlights the importance of understanding the spatial aspects of cancer biology. These results provide additional lipid biomarkers in colon cancer cells that can be further studied to target pivotal lipid production pathways.

Three-dimensional multicellular spheroids (MCSs) are complex structure of cellular aggregates and cell-to-matrix interaction that emulates the in-vivo microenvironment. This research field has grown to develop and improve spheroid generation techniques. Here, we present a new platform for spheroid generation using Layer-by-Layer (LbL) technology. Layer-by-Layer (LbL) containing cellulose nanofibrils (CNF) assemble on a standard 96 well plate. Various bi-layer numbers, multiple cell seeding concentration, and two tumor cell lines (HEK 293 T, HCT 116) are utilized to generate and characterize spheroids. The number and proliferation of generated spheroids, the viability, and the response to the anti-cancer drug are examined. The spheroids are formed and proliferated on the LbL-CNF coated wells with no significant difference in connection to the number of LbL-CNF bi-layers; however, the number of formed spheroids correlates positively with the cell seeding concentration (122 ± 17) and (42 ± 8) for HCT 116 and HEK 293T respectively at 700 cells mlsup-1/sup. The spheroids proliferate progressively up to (309, 663) µm of HCT 116 and HEK 293T respectively on 5 bi-layers coated wells with maintaining viability. The (HCT 116) spheroids react to the anti-cancer drug. We demonstrate a new (LbL-CNF) coating strategy for spheroids generation, with high performance and efficiency to test anti-cancer drugs.

Glioblastoma multiforme (GBM) is a highly malignant brain tumor with a poor prognosis. The GBM microenvironment is highly heterogeneous and is composed of many cell types including astrocytes and endothelial cells (ECs) along with tumor cells, which are responsible for heightened resistance to standard chemotherapeutic drugs such as Temozolomide (TMZ). Here, we investigated how drug treatments impact stemness marker expression of GBM cells in multicellular tumor spheroid (MCTS) models. Co- and tri-culture MCTS constructed using U87-MG GBM cells, astrocytes, and/or ECs were cultured for 7 days. At Day 7, 5 μM lonafarnib (LNF), 100 μM TMZ, or combination of 5 μM LNF + 100 μM TMZ was added and the MCTS were cultured for an additional 48 h. We assessed the spheroid sizes and expression of stemness markers- NESTIN, SOX2, CD133, NANOG, and OCT4- through qRT-PCR and immunostaining. Following 48 h treatment with LNF, TMZ or their combination (LNF + TMZ), the spheroid sizes decreased compared to the untreated control. We also observed that the expression of most of the stemness markers significantly increased in the LNF + TMZ treated condition as compared to the untreated condition. These results indicate that drug treatment can influence the stemness marker expression of GBM cells in MCTS models and these aspects must be considered while evaluating therapies. In future, by incorporating other relevant cell types, we can further our understanding of their crosstalk, eventually leading to the development of new therapeutic strategies.

Multicellular tumor spheroid (MCTSs) models have demonstrated increasing utility for in vitro study of cancer progression and drug discovery. These relatively simple avascular constructs mimic key aspects of in vivo tumors, such as 3D structure and pathophysiological gradients. MCTSs models can provide insights into cancer cell behavior during spheroid development and in response to drugs; however, their requisite size drastically limits the tools used for non-destructive assessment. Optical Coherence Tomography structural imaging and Imaris 3D analysis software are explored for rapid, non-destructive, and label-free measurement of regional cell density within MCTSs. This approach is utilized to assess MCTSs over a 4-day maturation period and throughout an extended 5-day treatment with Trastuzumab, a clinically relevant anti-HER2 drug. Briefly, AU565 HER2+ breast cancer MCTSs were created via liquid overlay with or without the addition of Matrigel (a basement membrane matrix) to explore aggregates of different morphologies (thicker, disk-like 2.5D aggregates or flat 2D aggregates, respectively). Cell density within the outer region, transitional region, and inner core was characterized in matured MCTSs, revealing a cell-density gradient with higher cell densities in core regions compared to outer layers. The matrix addition redistributed cell density and enhanced this gradient, decreasing outer zone density and increasing cell compaction in the cores. Cell density was quantified following drug treatment (0 h, 24 h, 5 days) within progressively deeper 100 µm zones to assess potential regional differences in drug response. By the final timepoint, nearly all cell death appeared to be constrained to the outer 200 µm of each aggregate, while cells deeper in the aggregate appeared largely unaffected, illustrating regional differences in the drug response, possibly due to limitations in drug penetration. The current protocol provides a unique technique to non-destructively quantify regional cell density within dense cellular tissues and measure it longitudinally.

Myxofibrosarcoma (MFS) is one of the most aggressive sarcomas with highly complex karyotypes and genomic profiles. Although a complete resection is required in the treatment of MFS, it is often not achieved due to its strong invasive nature. Additionally, MFS is refractory to conventional chemotherapy, leading to poor prognosis. Therefore, it is necessary to develop novel treatment modalities for MFS. Patient-derived cell lines are important tools in basic research and preclinical studies. However, only 10 MFS cell lines have been reported to date. Furthermore, among these cell lines, merely two MFS cell lines are publicly available. Hence, we established a novel MFS cell line named NCC-MFS3-C1, using a surgically resected tumor specimen from a patient with MFS. NCC-MFS3-C1 cells had copy number alterations corresponding to the original tumor. NCC-MFS3-C1 cells demonstrate constant proliferation, spheroid formation, and aggressive invasion. In drug screening tests, the proteasome inhibitor bortezomib and the histone deacetylase inhibitor romidepsin demonstrated significant antiproliferative effects on NCC-MFS3-C1 cells. Thus, the NCC-MFS3-C1 cell line is a useful tool in both basic and preclinical studies for MFS.

Increasing proofs have declared that liver cancer stem cells (CSCs) are the main contributors to tumor initiation, metastasis, therapy resistance, and recurrence of hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying CSCs regulation remain largely unclear. Recently, PCNA-associated factor (PAF) was identified to play a key role in maintaining breast cancer cell stemness, but its role in liver cancer stem cells has not been declared yet. Herein, we found that both mRNA and protein expression levels of PAF were significantly higher in HCC tissues and cell lines than normal controls. CSC-enriched hepatoma spheres displayed an increase in PAF expression compared to monolayer-cultured cells. Both loss-of-function and gain-of-function experiments revealed that PAF enhanced sphere formation and the percentage of CD133(+) or EpCAM(+) cells in HCCLM3 and Huh7 cells. In the xenograft HCC tumor model, tumor initiation rates and tumor growth were suppressed by knockdown of PAF. Mechanistically, PAF can amplify the self-renewal of liver CSCs by activating β-catenin signaling. Taken together, our results demonstrate that PAF plays a crucial role in maintaining the hepatoma cell stemness by β-catenin signaling. Abbreviations: CSCs: cancer stem cells; HCC: hepatocellular carcinoma; PAF: pCNA-associated factor.

Previously, we investigated gene expression in a high aldehyde dehydrogenase 1 expression (ALDH1high) population of urothelial carcinoma (UC) cells as UC cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) and found that NRG1 expression was upregulated in ALDH1high cells. NRG1 is a trophic factor that contains an epidermal growth factor (EGF)-like domain that signals by stimulating ERBB receptor tyrosine kinases and the cytoplasmic domain. NRG1 has been determined to be involved in frequent gene fusions with other partners in several malignancies and has a role in carcinogenesis through the NRG1 EGF-like domain and its cognitive receptor ERBBs. We thus aimed to elucidate the function of NRG1 in UC CSCs/CICs in this study. Both NRG1α and NRG1-β1 were preferentially expressed in ALDH1high cells compared with ALDH1low cells; however, siRNA experiments revealed that NRG1-β1 but not NRG1-α has a role in sphere formation. The EGF-like domain of NRG1 had a role in sphere formation of UC cells to some extent but was not essential. The intracellular domain of NRG1 did not have a role in sphere-formation. Inhibition of γ-secretase suppressed sphere formation. These findings indicate that cleavage of NRG1-β1 by γ-secretase plays an important role in UC CSC/CIC proliferation; however, the downstream targets of NRG1-β1 remain elusive.

Despite the development of new targeted and immune therapies, the prognosis of metastatic melanoma remains bleak. Therefore, it is critical to better understand the mechanisms controlling advanced melanoma to develop more effective treatment regimens. Hedgehog/GLI (HH/GLI) signaling inhibitors targeting the central pathway transducer Smoothened (SMO) have shown to be clinical efficacious in skin cancer; however, several mechanisms of non-canonical HH/GLI pathway activation limit their efficacy. Here, we identify a novel SOX2-BRD4 transcriptional complex driving the expression of GLI1, the final effector of the HH/GLI pathway, providing a novel mechanism of non-canonical SMO-independent activation of HH/GLI signaling in melanoma. Consistently, we find a positive correlation between the expression of GLI1 and SOX2 in human melanoma samples and cell lines. Further, we show that combined targeting of canonical HH/GLI pathway with the SMO inhibitor MRT-92 and of the SOX2-BRD4 complex using a potent Proteolysis Targeted Chimeras (PROTACs)-derived BRD4 degrader (MZ1), yields a synergistic anti-proliferative effect in melanoma cells independently of their BRAF, NRAS, and NF1 mutational status, with complete abrogation of GLI1 expression. Combination of MRT-92 and MZ1 strongly potentiates the antitumor effect of either drug as single agents in an orthotopic melanoma model. Together, our data provide evidence of a novel mechanism of non-canonical activation of GLI1 by the SOX2-BRD4 transcriptional complex, and describe the efficacy of a new combinatorial treatment for a subset of melanomas with an active SOX2-BRD4-GLI1 axis.

A chronic, local inflammatory milieu can cause tissue fibrosis that results in epithelial-to-mesenchymal transition (EMT), endothelial-to-mesenchymal transition (EndMT), increased abundance of fibroblasts, and further acceleration of fibrosis. In this study, we aimed to identify potential mechanisms and inhibitors of fibrosis using 3D model-based phenotypic screening. We established liver fibrosis models using multicellular tumor spheroids (MCTSs) composed of hepatocellular carcinoma (HCC) and stromal cells such as fibroblasts (WI38), hepatic stellate cells (LX2), and endothelial cells (HUVEC) seeded at constant ratios. Through high-throughput screening of FDA-approved drugs, we identified retinoic acid and forskolin as candidates to attenuate the compactness of MCTSs as well as inhibit the expression of ECM-related proteins. Additionally, retinoic acid and forskolin induced reprogramming of fibroblast and cancer stem cells in the HCC microenvironment. Of interest, retinoic acid and forskolin had anti-fibrosis effects by decreasing expression of α-SMA and F-actin in LX2 cells and HUVEC cells. Moreover, when sorafenib was added along with retinoic acid and forskolin, apoptosis was increased, suggesting that anti-fibrosis drugs may improve tissue penetration to support the efficacy of anti-cancer drugs. Collectively, these findings support the potential utility of morphometric analyses of hepatic multicellular spheroid models in the development of new drugs with novel mechanisms for the treatment of hepatic fibrosis and HCCs.

The flavone apigenin (APG), alone as well as in combination with other chemotherapeutic agents, is known to exhibit potential anticancer effects in various tumors and inhibit growth and metastasis of melanoma. However, the potential of apigenin nanoparticles (APG-NPs) to prevent lung colonization of malignant melanoma has not been well investigated. APG-loaded PLGA-NPs were surface-functionalized with meso-2,3-dimercaptosuccinic acid (DMSA) for the treatment of melanoma lung metastasis. DMSA-conjugated APG-loaded NPs (DMSA-APG-NPs) administered by an oral route exhibited sustained APG release and showed considerable enhancement of plasma half-life, Cmax value, and bioavailability compared to APG-NPs both in plasma and the lungs. DMSA-conjugated APG-NPs showed comparably higher cellular internalization in B16F10 and A549 cell lines compared to that of plain NPs. Increased cytotoxicity was observed for DMSA-APG-NPs compared to APG-NPs in A549 cells. This difference between the two formulations was lower in B16F10 cells. Significant depolarization of mitochondrial transmembrane potential and an enhanced level of caspase activity were observed in B16F10 cells treated with DMSA-APG-NPs compared to APG-NPs as well. Western blot analysis of various proteins was performed to understand the mechanism of apoptosis as well as prevention of melanoma cell migration and invasion. DMSA conjugation substantially increased accumulation of DMSA-APG-NPs given by an intravenous route in the lungs compared to APG-NPs at 6 and 8 h. This was also corroborated by scintigraphic imaging studies with radiolabeled formulations administered by an intravenous route. Conjugation also allowed comparatively higher penetration as evident from an in vitro three-dimensional tumor spheroid model study. Finally, the potential therapeutic efficacy of the formulation was established in experimental B16F10 lung metastases, which suggested an improved bioavailability with enhanced antitumor and antimetastasis efficacy of DMSA-conjugated APG-NPs following oral administration.