Spheroid-forming Cells Research Articles

The use of hyaluronic acid in a 3D biomimetic scaffold supports spheroid formation and the culture of cancer stem cells

Three-dimensional (3D) bioprinting culture models capable of reproducing the pathological architecture of diseases are increasingly advancing. In this study, 3D scaffolds were created using extrusion-based bioprinting method with alginate, gelatin, and hyaluronic acid to investigate the effects of hyaluronic acid on the physical properties of the bioscaffold as well as on the formation of liver cancer spheroids. Conformational analysis, rheological characterization, and swelling-degradation tests were performed to characterize the scaffolds. After generating spheroids from hepatocellular carcinoma cells on the 3D scaffolds, cell viability and proliferation assays were performed. Flow cytometry and immunofluorescence microscopy were used into examine the expression of albumin, CD44, and E-cadherin to demonstrate functional capability and maturation levels of the spheroid-forming cells. The results show that hyaluronic acid in the scaffolds correlates with spheroid formation and provides high survival rates. It is also associated with an increase in CD44 expression and a decrease in E-cadherin, while there is no significant change in the albumin expression in the cells. Overall, the findings demonstrate that hyaluronic acid in a 3D hydrogel scaffold supports spheroid formation and may induce stemness. We present a promising 3D scaffold model for enhancing liver cancer spheroid formation and mimicking solid tumors. This model also has the potential for further studies to examine stem cell properties in 3D models.

Selective Accumulation of Poly(Lactic-Co-Glycolic Acid) Nanoparticles in Endotheliocytes and Mesenchymal Stromal Cells Cultured as Mixed-Cell Spheroids.

The use of drug-loaded nanoparticles is an actively developed approach in targeted cancer therapy. Prevascularized spheroids generated from mesenchymal stem cells and endotheliocytes are considered as a model to evaluate the tropism of therapeutic nanoparticles to a specific tissue. Nanoparticles based on co-polymer of lactic and glycolic acids (poly(lactic-co-glycolic acid; PLGA) labeled with cyanine dye (Cy5) were incubated with prevascularized spheroids, and the rate of their penetration and their distribution in the spheroid-forming cells were evaluated. Endotheliocytes more intensively accumulated nanoparticles than mesenchymal stem cells: the number of nanoparticles in mixed-cell spheroids of mesenchymal stem cells and endotheliocytes was greater than in spheroids built solely of mesenchymal stem cells by 5±1.2 times. The developed 3D in vitro cell model provides a low-cost way to assess tissue tropism of therapeutic nanoparticles under conditions closer to natural in comparison with 2D culture.

TWIST1 induces proteasomal degradation of β-catenin during the differentiation of ovarian cancer stem-like cells

Ovarian cancer (OC) is one of the leading gynecologic cancers worldwide. Cancer stem-like cells are correlated with relapse and resistance to chemotherapy. Twist1, which is involved in ovarian cancer stem-like cell differentiation, is positively correlated with CTNNB1 in different differentiation stages of ovarian cancer cells: primary epithelial ovarian cancer cells (primary EOC cells), mesenchymal spheroid-forming cells (MSFCs) and secondary epithelial ovarian cancer cells (sEOC cells). However, the expression of β-catenin is inversed compared to CTNNB1 in these 3 cell states. We further demonstrated that β-catenin is regulated by the protein degradation system in MSFCs and secondary EOC but not in primary EOC cells. The differentiation process from primary EOC cells to MSFCs and sEOC cells might be due to the downregulation of β-catenin protein levels. Finally, we found that TWIST1 can enhance β-catenin degradation by upregulating Axin2.

Changes of cell membrane fluidity for mesenchymal stem cell spheroids on biomaterial surfaces.

BACKGROUNDThe therapeutic potential of mesenchymal stem cells (MSCs) in the form of three-dimensional spheroids has been extensively demonstrated. The underlying mechanisms for the altered cellular behavior of spheroids have also been investigated. Cell membrane fluidity is a critically important physical property for the regulation of cell behavior, but it has not been studied for the spheroid-forming cells to date.AIMTo explore the association between cell membrane fluidity and the morphological changes of MSC spheroids on the surface of biomaterials to elucidate the role of membrane fluidity during the spheroid-forming process of MSCs.METHODSWe generated three-dimensional (3D) MSC spheroids on the surface of various culture substrates including chitosan (CS), CS-hyaluronan (CS-HA), and polyvinyl alcohol (PVA) substrates. The cell membrane fluidity and cell morphological change were examined by a time-lapse recording system as well as a high-resolution 3D cellular image explorer. MSCs and normal/cancer cells were pre-stained with fluorescent dyes and co-cultured on the biomaterials to investigate the exchange of cell membrane during the formation of heterogeneous cellular spheroids.RESULTSWe discovered that vesicle-like bubbles randomly appeared on the outer layer of MSC spheroids cultured on different biomaterial surfaces. The average diameter of the vesicle-like bubbles of MSC spheroids on CS-HA at 37 °C was approximately 10 μm, smaller than that on PVA substrates (approximately 27 μm). Based on time-lapse images, these unique bubbles originated from the dynamic movement of the cell membrane during spheroid formation, which indicated an increment of membrane fluidity for MSCs cultured on these substrates. Moreover, the membrane interaction in two different types of cells with similar membrane fluidity may further induce a higher level of membrane translocation during the formation of heterogeneous spheroids. CONCLUSIONChanges in cell membrane fluidity may be a novel path to elucidate the complicated physiological alterations in 3D spheroid-forming cells.

Single-Cell Proteomic Profiling Identifies Nanoparticle Enhanced Therapy for Triple Negative Breast Cancer Stem Cells.

Breast cancer remains a major cause of cancer-related deaths in women worldwide. Chemotherapy-promoted stemness and enhanced stem cell plasticity in breast cancer is a cause for great concern. The discovery of drugs targeting BCSCs was suggested to be an important advancement in the establishment of therapy that improves the efficacy of chemotherapy. In this work, by using single-cell mass cytometry, we observed that stemness in spheroid-forming cells derived from MDA-MB-231 cells was significantly increased after doxorubicin administration and up-regulated integrin αvβ3 expression was also observed. An RGD-included nanoparticle (CS-V) was designed, and it was found that it could promote doxorubicin’s efficacy against MDA-MB-231 spheroid cells. The above observations suggested that the combination of RGD-included nanoparticles (CS-V) with the chemo-drug doxorubicin could be developed as a potential therapy for breast cancer.

RETRACTED ARTICLE: PI3K/Akt pathway and Nanog maintain cancer stem cells insarcomas

The self-renewal transcription factor Nanog and the phosphoinositide3-kinase (PI3K)–Akt pathway are known to be essential for maintenance ofmesenchymal stem cells. We evaluated their contribution to the maintenance ofCD133(+) cancer stem-like cells (CSCs) and spheroid-forming cells inpatient-derived cell lines from three human sarcoma subtypes: HT1080 fibrosarcoma,SK-LMS-1 leiomyosarcoma, and DDLS8817 dedifferentiated liposarcoma. Levels of Nanogand activated Akt were significantly higher in sarcoma cells grown as spheroids orsorted for CD133 expression to enrich for CSCs. shRNA knockdown of Nanog decreasedspheroid formation 10- to 14-fold, and reversed resistance to both doxorubicin andradiation in vitro and in H1080 flank xenografts. In the HT1080 xenograft model,doxorubicin and Nanog knockdown reduced tumor growth by 34% and 45%, respectively,and the combination reduced tumor growth by 74%. Using a human phospho-kinaseantibody array, Akt1/2 signaling, known to regulate Nanog, was found to be highlyactivated in sarcoma spheroid cells compared with monolayer cells. Pharmacologicinhibition of Akt using LY294002 and Akt1/2 knockdown using shRNA in sarcoma CSCsdecreased Nanog expression and spheroid formation and reversed chemotherapyresistance. Akt1/2 inhibition combined with doxorubicin treatment of HT1080 flankxenografts reduced tumor growth by 73%. Finally, in a human sarcoma tumormicroarray, expression of CD133, Nanog, and phospho-Akt were 1.8- to 6.8-fold higherin tumor tissue compared with normal tissue. Together, these results indicate thatthe Akt1/2–Nanog pathway is critical for maintenance of sarcoma CSCs andspheroid-forming cells, supporting further exploration of this pathway as atherapeutic target in sarcoma.

Extracellular vesicle-associated miR-135b and -135a regulate stemness in Group 4 medulloblastoma cells by targeting angiomotin-like 2

BackgroundExtracellular vesicles (EVs) secreted by tumours, including exosomes, are important factors that regulate cell–cell interactions in oncogenesis. Although EV studies are ongoing, the biological understanding of EV-miRNAs derived from brain tumour spheroid-forming cells (BTSCs) of medulloblastoma is poor.PurposesWe explored the specific cellular miRNAs and EV-miRNAs in medulloblastoma BTSCs to determine their potential biological function.MethodsBulk tumor cells (BTCs) and BTSCs were cultured under different conditions from medulloblastoma tissues (N = 10).ResultsTwenty-four miRNAs were simultaneously increased in both cells and EVs derived from BTSCs in comparison to BTCs. After inhibition of miR-135b or miR135a which were the most significantly increased in BTSCs, cell viability, self-renewal and stem cell marker expression decreased remarkably. Through integrated analysis of mRNAs and miRNAs data, we found that angiomotin-like 2 (AMOTL2), which was significantly decreased, was targeted by both miR-135b and miR-135a. STAT6 and GPX8 were targeted only by miR-135a. Importantly, low expression of AMOTL2 was significantly associated with overall poor survival in paediatric Group 3 and Group 4 medulloblastoma patients.ConclusionOur results indicated that inhibition of miR-135b or miR-135a leads to suppress stemness of BTSC through modulation of AMOTL2.

Pyrvinium Pamoate Induces Death of Triple-Negative Breast Cancer Stem-Like Cells and Reduces Metastases through Effects on Lipid Anabolism.

Cancer stem-like cells (CSC) induce aggressive tumor phenotypes such as metastasis formation, which is associated with poor prognosis in triple-negative breast cancer (TNBC). Repurposing of FDA-approved drugs that can eradicate the CSC subcompartment in primary tumors may prevent metastatic disease, thus representing an effective strategy to improve the prognosis of TNBC. Here, we investigated spheroid-forming cells in a metastatic TNBC model. This strategy enabled us to specifically study a population of long-lived tumor cells enriched in CSCs, which show stem-like characteristics and induce metastases. To repurpose FDA-approved drugs potentially toxic for CSCs, we focused on pyrvinium pamoate (PP), an anthelmintic drug with documented anticancer activity in preclinical models. PP induced cytotoxic effects in CSCs and prevented metastasis formation. Mechanistically, the cell killing effects of PP were a result of inhibition of lipid anabolism and, more specifically, the impairment of anabolic flux from glucose to cholesterol and fatty acids. CSCs were strongly dependent upon activation of lipid biosynthetic pathways; activation of these pathways exhibited an unfavorable prognostic value in a cohort of breast cancer patients, where it predicted high probability of metastatic dissemination and tumor relapse. Overall, this work describes a new approach to target aggressive CSCs that may substantially improve clinical outcomes for patients with TNBC, who currently lack effective targeted therapeutic options. SIGNIFICANCE: These findings provide preclinical evidence that a drug repurposing approach to prevent metastatic disease in TNBC exploits lipid anabolism as a metabolic vulnerability against CSCs in primary tumors.

Differences in drug sensitivity between two-dimensional and three-dimensional culture systems in triple-negative breast cancer cell lines

Three-dimensional (3D) culture reflects tumor biology complexities compared with two-dimensional (2D) culture. Thus, 3D culture has attracted attention in cell biology studies including drug sensitivity tests. Herein, we investigated differences in anticancer drug sensitivities between 2D and 3D culture systems in triple-negative breast cancer (TNBC) cell lines. Thirteen TNBC cell lines were maintained in 2D and 3D cultures for 3 days before drug exposure. Cell morphology in the 3D culture was examined by phase-contrast microscopy. Sensitivities to epirubicin (EPI), cisplatin (CDDP), and docetaxel (DTX) were investigated by cell viability assay in both cultures and compared. The IC50s of all 3 drugs were significantly higher in the 3D culture than in the 2D culture in most cell lines. Those were correlated between the 2D and 3D cultures in EPI (R = 0.555) and CDDP (R = 0.955), but not in DTX (R = 0.221). Round spheroid-forming cells were more resistant to agents than grape-like types. In conclusion, 3D culture was more resistant to all 3 drugs than 2D culture in most TNBC cell lines. Sensitivity to CDDP was highly correlated between the 2D and 3D cultures, but not to DTX. 2D culture may be acceptable for sensitivity test for DNA-damaging agents.

Integrative analysis of transcription factors and microRNAs in ovarian cancer cell spheroids

BackgroundCancer stem cells (CSCs) can self-renew, proliferate into differentiated cells, or enter a quiescent state and are regarded to cause chemoresistance and recurrence. An integrative analysis of transcription factors (TF) and miRNAs was performed in ovarian CSC-enriched spheroid-forming cells (SFCs) to identify factors relevant to ovarian CSCs.MethodsFresh tumor cells from three ovarian cancer patients were cultured in standard and in selective medium. The mRNAs and miRNAs that exhibited significant differential expression between SFCs and adherent cells were identified using mRNA and miRNAs microarrays. Target genes of miRNAs were further selected if predicted with TargetScan by half of the miRNAs or more. Gene enrichment analysis was performed on over- or under-expressed mRNAs and target genes of miRNAs using DAVID tools. Complex regulatory networks were combined from TF-genes and miRNA-genes interactions using the MAGIA webtool.ResultsA total of 1245 mRNA and 55 miRNAs were differentially expressed (p-value< 0.05, paired t-test). Elevation of transcription-related processes and suppression of focal adhesion pathway were noted in SFCs, according to the enrichment analyses. Transcriptional hyperactivity is a known characteristic of the stem cell transcriptome. The integrative network suggested that cell cycle was arrested in SFCs where over-expressed EGR1 and under-expressed MYC and miR-130a-3p had multiple connections with target genes.ConclusionsMYC, EGR1, and miR-130a-3p were hubs in our integrative analysis of ovarian CSC-enriched SFCs, suggesting that ovarian cancer SFCs display a stem cell identity with the quiescent phenotype where adhesion- and cell cycle-related genes were suppressed.

Characterization of spontaneous spheroids from oral mucosa-derived cells and their direct comparison with spheroids from skin-derived cells

BackgroundOur group has developed a novel method for spontaneous spheroid formation using a specific low-adherence culture plate with around 90° water contact angle. In this study, this method was applied for oral mucosa-derived cells. First, the feasibility of spontaneous spheroid formation was tested. Next, the characteristics of spontaneous spheroids from oral mucosa- and skin-derived cells were compared with special focus on the stemness and neuronal differentiation capability.MethodsOral mucosal cells were obtained from the palate and buccal mucosa of C57BL/6J mice. Similarly, skin cells were obtained from the back of the same mouse strain. Passage 2–3 cells were inoculated into the specific low-adherence culture plates to form spontaneous spheroids. The effect of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and B27 supplement on spheroid formation and maintenance was assessed. Immunofluorescence and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were performed to investigate the expression of pluripotency markers, cell proliferation and apoptosis markers, and neurogenic differentiation markers.ResultsUsing this culture plate, spontaneous spheroid formation was feasible. This process depended on the presence of serum but was independent of the additives such as bFGF, EGF, and B27 supplement, although they improved the efficiency and were essential for spheroid maintenance. This result was confirmed by the higher expression of Caspase7 in the spheroids cultured without the additives than that with the additives. The spheroids from oral mucosa-derived cells expressed stem cell markers, such as Sox2, SSEA1, Oct4, Nanog, and Nestin. The expression of Sox2 in spheroids from oral mucosal cells was higher than that in spheroids from skin-derived cells. Both spheroid-forming cell types had the ability to differentiate into neural and Schwann cells after neurogenic induction, although significantly higher MAP 2, MBP, Nestin, and Nurr1 gene expression was noted in the cells from oral mucosa-derived spheroids.ConclusionsThe results showed that spontaneous spheroids from oral mucosa-derived cells contain highly potent stem cells, which were as good as skin-derived stem cells. The high expression of certain neuronal marker genes suggests an advantage of these cells for regeneration therapy for neuronal disorders.

Molecular carcinogenesis of gastric cancer: Lauren classification, mucin phenotype expression, and cancer stem cells.

Gastric cancer (GC), one of the most common human cancers, is a heterogeneous disease with different phenotypes, prognoses, and responses to treatment. Understanding the pathogenesis of GC at the molecular level is important for prognosis prediction and determining treatments. Microsatellite instability (MSI), silencing of MLH1, MGMT, and CDKN2A genes by DNA hypermethylation, KRAS mutation, APC mutation, and ERBB2 amplification are frequently found in intestinal type GC. Inactivation of CDH1 and RARB by DNA hypermethylation, and amplification of FGFR and MET, are frequently detected in diffuse type GC. In addition, BST2 and PCDHB9 genes are overexpressed in intestinal type GC. Both genes are associated with GC progression. GC can be divided into gastric/intestinal mucin phenotypes according to mucin expression. MSI, alterations of TP73, CDH1 mutation, and DNA methylation of MLH are detected frequently in the gastric mucin phenotype. TP53 mutation, deletion of APC, and DNA methylation of MGMT are detected frequently in the intestinal mucin phenotype. FKTN is overexpressed in the intestinal mucin phenotype, and IQGAP3 is overexpressed in the gastric mucin phenotype. These genes are involved in GC progression. To characterize cancer stem cells, a useful method is spheroid colony formation. KIFC1 and KIF11 genes show more than twofold higher expression in spheroid-forming cells than that in parental cells. Both KIF genes are overexpressed in GC, and knockdown of these genes inhibits spheroid formation. Alterations of these molecules may be useful to understand gastric carcinogenesis. Specific inhibitors of these molecules may also be promising anticancer drugs.

Identification of different gene expressions between diffuse- and intestinal-type spheroid-forming gastric cancer cells.

Three-dimensional in vitro spheroid models are unique because they are considered for enrichment of specific cell populations with self-renewal ability. In this study, we explored the different mechanisms of gastric cancer spheroid-forming cells according to the Lauren classification. We isolated and enriched cells with self-renewal ability using spheroid-forming methods from gastric cancer cell lines. The expression of candidate target genes was investigated using western blot and qRT-PCR analysis. Lentiviral shRNA knockdown of target gene expression was performed and the effects on spheroid, colony forming, and tumorigenic ability were analyzed. The SNU-638, SNU-484, MKN-28, and NCI-N87 successfully formed spheroid from single cell and enriched for self-renewal ability from 11 gastric cancer cell lines, including diffuse and intestinal types. The expression of SOX2 and E-cadherin increased in spheroid-forming cells in a diffuse-type cell line (SNU-638 and SNU-484), but not in the intestinal type (MKN-28 and NCI-N87). In contrast, ERBB3 expression was only increased in intestinal-type spheroid cells. The depletion of each candidate target gene expression suppressed self-renewal ability to grow as spheroids and colonies in a soft agar assay. In particular, down-regulated ERBB3 in the intestinal-type cell lines inhibited tumor growth in a mouse xenograft model. We found that high ERBB3 gene expression correlates with decreased survival in the intestinal type of gastric cancer. Our results suggest that diffuse- and intestinal-type spheroid-forming cells express genes differently. Our data suggest that these candidate genes from spheroid-forming cells can be used in applications in targeted therapy.

The oncogene KRAS promotes cancer cell dissemination by stabilizing spheroid formation via the MEK pathway

BackgroundPeritoneal dissemination is a critical prognostic factor in ovarian cancer. Although stabilized spheroid formation promotes cancer cell peritoneal dissemination in ovarian cancer, the associated oncogenes are unknown. In this study, we assessed the role of the KRAS oncogene in ovarian cancer cell dissemination, focusing on the stability of cells in spheroid condition, as well as the modulation of intracellular signaling following spheroid transformation.MethodsWe used ID8, a murine ovarian cancer cell line, and ID8-KRAS, an oncogenic KRAS (G12 V)-transduced ID8 cell line in this study. Spheroid-forming (3D) culture and cell proliferation assays were performed to evaluate the growth characteristics of these cells. cDNA microarray analysis was performed to identify genes involved in KRAS-associated signal transduction in floating condition. A MEK inhibitor was used to evaluate the effect on cancer peritoneal dissemination.ResultsCell viability and proliferation in monolayer (2D) cultures did not differ between ID8 and ID8-KRAS cells. However, the proportions of viable and proliferating ID8-KRAS cells in 3D culture were approximately 2-fold and 5-fold higher than that of ID8, respectively. Spheroid-formation was increased in ID8-KRAS cells. Analysis of peritoneal floating cells obtained from mice intra-peritoneally injected with cancer cells revealed that the proportion of proliferating cancer cells was approximately 2-fold higher with ID8-KRAS than with ID8 cells. Comprehensive cDNA microarray analysis revealed that pathways related to cell proliferation, and cell cycle checkpoint and regulation were upregulated specifically in ID8-KRAS cells in 3D culture, and that some genes partially regulated by the MEK-ERK pathway were upregulated only in ID8-KRAS cells in 3D culture. Furthermore, a MEK inhibitor, trametinib, suppressed spheroid formation in 3D culture of ID8-KRAS cells, although trametinib did not affect 2D-culture cell proliferation. Finally, we demonstrated that trametinib dramatically improved the prognosis for mice with ID8-KRAS tumors in an in vivo mouse model.ConclusionsOur data indicated that KRAS promoted ovarian cancer dissemination by stabilizing spheroid formation and that the MEK pathway is important for stabilized spheroid formation. Disruption of spheroid formation by a MEK inhibitor could be a therapeutic target for cancer peritoneal dissemination.

P3.03-14 Downregulation of FOXM1 Inhibits Tumor Proliferation, Colony Formation and Spheroid Formation of Non-Small Cell Lung Cancer

Forkhead box protein M1 (FOXM1) is a member of the forkhead superfamily of transcription factors. It plays numerous critical roles in cancer development and progression, such as the regulation of G2/M transitions of cell cycle, anti-apoptosis, DNA damage repair, invasion, and drug resistance. In a pan-cancer meta-analysis of mRNA expression signatures from ∼18,000 human tumors with overall survival outcomes across 39 malignancies, over expression of the FOXM1 was a major predictor of adverse outcomes and appeared anti-correlated with tumor immune cell populations (Nat Med. 2015;21(8):938-945). Thus, we examined the prognostic and biological role of FOXM1 in non-small cell lung cancer (NSCLC). We examined the prognostic impact of FOXM1 expression in lung cancer patients using a genomic database (UTSW – “Lung Cancer Explorer” and KM-Plotter). We also assessed the expression of FOXM1 in NSCLC lines, and human bronchial epithelial cells (HBECs), and assessed the association between FOXM1 and cell cycle related genes in RNA-seq data. We examined the effect of the downregulation of FOXM1 on tumor proliferation and colony formation of lung adenocarcinoma cell lines by short hairpin RNA (Tet-pLKO-FOXM1-shRNA), and the effect of the downregulation of FOXM1 on tumor 3D spheroid formation in spheroid-forming cell lines using Nunclon Sphera. High FOXM1 expression correlated with poor prognosis in NSCLC patients who underwent surgical resection, especially adenocarcinoma. FOXM1 expression in lung cancer cell lines was also much higher than in HBECs. The expression level of FOXM1 was significantly correlated with cell cycle regulator genes such as CCNB1, CCNA2, and PLK1 in both tissue samples (Lung cancer explorer) and cell lines (our data). shRNA mediated reduction of FOXM1 expression significantly inhibited tumor cell proliferation and colony formation of NSCLC cells. Additionally, in the 3D spheroid formation assay, FOXM1 knockdown altered spheroid morphology. FOXM1 was over expressed in NSCLC compared to normal lung epithelial cells, and high tumor FOXM1 expression was prognostic of poor survival in patients with NSCLC who underwent surgical resection, especially in patients with adenocarcinoma. FOXM1 expression correlated with the expression of regulators of the G2/M transition, and functional knockdown studies demonstrate an important role in tumor proliferation, colony formation, and 3D spheroid formation. Overall, our results suggest that FOXM1 has important roles in NSCLC growth, while data from other groups using “CIBERSORT” analyses suggest a possible role for FOXM1 in tumor immune cells infiltration, collectively indicating that FOXM1 is a potential target for the treatment of lung cancer adenocarcinoma.

Isolation of spheroid-forming single cells from gastric cancer cell lines: enrichment of cancer stem-like cells.

Isolation of spheroid-forming cells is important to investigate cancer stem cell (CSC) characteristics. However, conventional tumor spheroid culture methods have not proven suitable because the aggregated spheroids generally maintain their original heterogeneity and harbor multiple cells with various characteristics. Here we cultured spheroids using a polydimethylsiloxane microwell-based method and a limiting dilution protocol. We then isolated and enriched for CSCs that formed single cell-derived spheroids from gastric cancer cell lines. Cells from the microwell demonstrated higher self-renewal, increased expression of stem cell markers and resistance to apoptosis compared with spheroid cells made by the traditional method. This novel approach allows efficient cancer stem cell isolation and represents a step forward in cancer stem cell studies.

Exclusion from spheroid formation identifies loss of essential cell-cell adhesion molecules in colon cancer cells

Many cell lines derived from solid cancers can form spheroids, which recapitulate tumor cell clusters and are more representative of the in vivo situation than 2D cultures. During spheroid formation, a small proportion of a variety of different colon cancer cell lines did not integrate into the sphere and lost cell-cell adhesion properties. An enrichment protocol was developed to augment the proportion of these cells to 100% purity. The basis for the separation of spheroids from non-spheroid forming (NSF) cells is simple gravity-sedimentation. This protocol gives rise to sub-populations of colon cancer cells with stable loss of cell-cell adhesion. SW620 cells lacked E-cadherin, DLD-1 cells lost α-catenin and HCT116 cells lacked P-cadherin in the NSF state. Knockdown of these molecules in the corresponding spheroid-forming cells demonstrated that loss of the respective proteins were indeed responsible for the NSF phenotypes. Loss of the spheroid forming phenotype was associated with increased migration and invasion properties in all cell lines tested. Hence, we identified critical molecules involved in spheroid formation in different cancer cell lines. We present here a simple, powerful and broadly applicable method to generate new sublines of tumor cell lines to study loss of cell-cell adhesion in cancer progression.

Osteopontin and vasculogenic mimicry formation are associated with response to neoadjuvant chemotherapy in advanced breast cancer.

IntroductionOsteopontin (OPN), a multifunctional phosphoprotein, has been implicated in a series of important physiologic and pathophysiologic processes. In breast cancer, OPN functionally contributes to the tumorigenicity of spheroid-forming cells. It also plays a critical role in enhancing the proliferation, tumorigenicity, and ability to display vasculogenic mimicry (VM) of spheroid-forming cells in breast cancer. However, the role of OPN in breast cancer is not clear.Patients and methodsThis study investigated OPN expression and VM in breast cancer patients before neoadjuvant chemotherapy (NACT). Their association with clinicopathologic factors was first analyzed by immunohistochemistry. Then, the response of breast cancer patients to NACT was evaluated. The correlation between the clinicopathologic factors, including the molecular subtype, and the response to NACT was analyzed.ResultsImmunohistochemical analysis showed positive staining of OPN in 40% of the breast cancer patients, whereas VM, which was related to tumor stage, was observed in 30% of cases. OPN expression was found to have a significant correlation with VM (P<0.05). The results also indicated that the clinicopathologic factors were not related to the response to NACT, including the molecular subtype. The multivariate analysis of clinicopathologic features correlated with pathological complete response (pCR) indicated that OPN(+)VM(+) was correlated with pCR (P<0.001).ConclusionOur findings underlined that the concurrence of OPN-positive expression and VM can predict the pCR to NACT in breast cancer. The efficiency of NACT in certain patients can be easily predicted by detecting the expression of OPN and VM.

Abstract 3835: High-purity patient-derived cells: An advanced cell culture model for precision cancer medicine

Abstract Purpose: Patient-derived cells (PDCs) established from malignant effusions can represent an excellent tool for the study of non-small cell lung cancer (NSCLC) with oncogenic driver mutations. However, because of cellular paucity and low tumor purity, PDCs have a limited utility for in-depth molecular analysis. In this study, we developed a high-purity patient-derived cell (HP-PDC) method for primary culture of malignant effusions. Experimental design: HP-PDCs were established by a multistep tumor cell isolation protocol as follows. 1) Initial cultures of malignant effusions were observed under a light microscope to identify spheroid-forming cells. 2) Tumor purity of PDCs was analyzed by Immunofluorescence (IF) and flow cytometry. 3) Mutation status of EGFR or ALK in PDCs was screened by direct or PCR-based sequencing and RT-PCR and then was compared with driver mutations detected in matching patient tumor samples. 4) HP-PDCs were tested for drug efficacies by a cell viability assay, a colony-forming assay, and western blot. 5) HP-PDCs were screened with a panel of 84 kinase inhibitors to identify effective drugs. Results: A consecutive series of 45 malignant effusion samples was collected from 36 patients with advanced NSCLC. Spheroid-forming cells were detected in 19 samples (42.2%) which were cultured in AR5 media to establish PDCs. IF analysis revealed that TTF-1 was upregulated in 9 PDCs. These 9 PDCs with high TTF-1 expression also showed EpCAM-positive cell population in flow cytometry with high purity of over 70%. Using Sanger sequencing, EGFR exon 19 deletion was detected 3 cases, EGFR T790M with L858R in 2 cases, and ALK rearrangements in 4 cases. Two of PDCs established from patients, who carried EGFR mutations and progressed on gefitinib, showed resistance to gefitinib in vitro. One of gefitinib-resistant PDCs was sensitive to AZD9291 with IC50 value of 47 nM. Three of PDCs established from patients, who carried ALK fusion genes and progressed on ALK inhibitors (crizotinib, alectinib, and ceritinib respectively), also recapitulated patient’s response to the ALK inhibitors. In an alectinib-resistant PDC, crizotinib and ceritinib exhibited potency with IC50 values of 321 nM and 215 nM, respectively. Colony-forming assay and western blot results were correlated with the cell viability assay data. Time spent for establishment of HP-PDCs ranged from 26 days to 132 days. Conclusions: Nine HP-PDCs established by multistep tumor cell isolation protocol had high tumor purity, and recapitulated patient’s genomic profile and drug responses. HP-PDC is a powerful preclinical model which predicts responses to targeted drugs in a short period of time and may provide an excellent platform for personalized therapies in patients with advanced NSCLC. Citation Format: Seok-Young Kim, Hwan Kim, Sung-Moo Kim, Hye Ryun Kim, Mi Ran Yun, Ji Min Lee, Hun Mi Choi, Byoung Chul Cho. High-purity patient-derived cells: An advanced cell culture model for precision cancer medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3835. doi:10.1158/1538-7445.AM2017-3835

The STAT3-miRNA-92-Wnt Signaling Pathway Regulates Spheroid Formation and Malignant Progression in Ovarian Cancer.

Ovarian cancer spheroids constitute a metastatic niche for transcoelomic spread that also engenders drug resistance. Spheroid-forming cells express active STAT3 signaling and display stem cell-like properties that may contribute to ovarian tumor progression. In this study, we show that STAT3 is hyperactivated in ovarian cancer spheroids and that STAT3 disruption in this setting is sufficient to relieve chemoresistance. In an NSG murine model of human ovarian cancer, STAT3 signaling regulated spheroid formation and self-renewal properties, whereas STAT3 attenuation reduced tumorigenicity. Mechanistic investigations revealed that Wnt signaling was required for STAT3-mediated spheroid formation. Notably, the Wnt antagonist DKK1 was the most strikingly upregulated gene in response to STAT3 attenuation in ovarian cancer cells. STAT3 signaling maintained stemness and interconnected Wnt/β-catenin signaling via the miR-92a/DKK1-regulatory pathways. Targeting STAT3 in combination with paclitaxel synergistically reduced peritoneal seeding and prolonged survival in a murine model of intraperitoneal ovarian cancer. Overall, our findings define a STAT3-miR-92a-DKK1 pathway in the generation of cancer stem-like cells in ovarian tumors, with potential therapeutic applications in blocking their progression. Cancer Res; 77(8); 1955-67. ©2017 AACR.