Enrichment Of Cancer Stem-like Cells Research Articles

Abstract 6373: Spheroid culture of ER+ breast cancer patient-derived tumor cells enriches cancer stem-likecells with EpCAM-/CD49f+and high ALDH activity and poor prognostic gene signatures

Abstract Background: Cancer stem-like cells (CSCs) represent a critical subset of the tumor population, which contributes to tumor recurrence, metastasis and therapy resistance. Spheroid culture is unique method for CSC enrichment. Patient-derived breast cancer cells (PDBCCs) has provided an invaluable tool in preclinical and translational research. The objective of this study is to characterize the immunophenotype and transcriptomes of PDBCCs grown in the adherent monolayer and spheroid condition. Methods: PDBCCs were harvested from residual tumor of fresh surgical specimens of patients with ER-positive subtype (8 cases). The adherent monolayer(M) and spheroid(S) culture of PDBCCs were applied on collagen I-coated plate and an ultra-low attachment polymer-X coated plate. Flow cytometry and immunocytochemistry was performed by using fluorescent-dye conjugated antibodies for EpCAM, CD49f, CD24, CD44, and cytokeratin. ALDEFLUOR® assay to detect ALDH activity was used. Total RNA-sequencing was applied to investigate differentially expressed genes (DEGs), followed by GO and KEGG pathway enrichment analysis. Real-time RT-PCR was performed to evaluate BCSC-related RNA levels. The Kaplan-Meier Plotter online database was used to evaluate the prognostic value of DEGs (2-fold change, p<0.05). Results: Under adherent monolayer condition, more than 95% of PDBCCs passaged for up to 5 passages on collagen I-coated plate sustained EpCAM+/cytokeratin+/fibroblast marker_ phenotype. On polymer-X coated plates, PDBCCs formed compact multicellular spheroids with a diameter of less than 300 µm. As compared with monolayer, CSCs with EpCAM-/CD49f+ phenotype (M vs S; 0.16±0.06 vs 2.34±0.65, p=0.015) and high ALDH activity (M vs S; 0.73±0.43 vs 7.05±1.48, p=0.038) were significantly increased by spheroid culture. In whole-transcriptome analysis (3 cases) between spheroid and monolayer, a total of 561 DEGs were identified, including 290 upregulated and 271 downregulated DEGs (2- fold change, p<0.05) in spheroid. The upregulated and downregulated DEGs in spheroids were enriched in 8 and 19 KEGG pathways, respectively. In recurrence-free survival analysis based on the Kaplan-Meier Plotter database of the genes corresponding to Oncotype Dx, Mammaprint, and Prosigna and top 30 ranked DEGs identified in spheroids, 15 upregulated and 14 downregulated DEGs were associated with poor prognosis of breast cancer patients. Conclusion: Spheroid culture of PDBCCs with ER+ subtype enriches CSCs and poor prognostic gene signatures. Future studies to explore the specific molecular mechanisms underlying these observations may provide new molecular targets for the development of therapies for targeting CSCs in breast cancer patient. Citation Format: Hoe Suk Kim, Seungyeon Ryu, So-Hyun Yoon, Sangeun Lee, Junhyuk Song, Yoonjung Choi, Moonjou Baek, Han-Byoel Lee, Sangyong Jon, Daeyoup Lee, Wonshik Han. Spheroid culture of ER+ breast cancer patient-derived tumor cells enriches cancer stem-likecells with EpCAM-/CD49f+and high ALDH activity and poor prognostic gene signatures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6373.

Enrichment of cancer stem-like cells by controlling oxygen, glucose and fluid shear stress in a microfluidic spheroid culture device

Current chemotherapies can often kill fast-growing cancer cells but are ineffective in destroying cancer stem cells (CSCs). This study aimed to investigate the effect of in vitro cell culture conditions on the features of lung adenocarcinoma cells (represented by A549, non-small cell type (NSCLC), cell lines) and the induction of cancer stem-like cells. For this purpose, a microfluidic system containing U-shaped arrays was designed and numerically optimized. This system was used for the three-dimensional culture of cells under a continuous laminar flow of culture medium. Numerical simulations were also performed to estimate the quiescent and necrotic zones inside the spheroids and the fluid shear stress on the cultured spheroids. Moreover, the effects of culture medium flow rate, oxygen and glucose concentrations and anoikis phenomenon on the number of CD90+ cells and expression of stemness, epithelial-mesenchymal transition (EMT) and ATP-binding cassette (ABC)-transporters genes were investigated. The results showed that all these parameters substantially affected the enrichment of A549 cancer stem-like cells. We also investigated the effect of the CSC enrichment method, i.e., shear stress and oxygen and glucose concentrations, on resistance to cisplatin treatment. Increasing shear stress and glucose concentration and decreasing oxygen concentration led to a sharp increase in chemoresistance of cells. Interestingly, changing oxygen concentration was more significant than changing the other factors. The results of this paper can be helpful for the effective enrichment of CSCs by adjusting in vitro culture conditions.

M1- and M2-Polarized Macrophages Modified Inflammatory Environment and Affects Malignant Behaviors of Cancer Stem-like Cells (CSCs) Derived from Human Melanoma Cells A375

Tumors contain a sub-population of cells characterized by self-renewal and expanding capacities which were known as cancer stem-like cells (CSCs). Sphere formation in conditioned serum-free medium supplemented with epidermal growth factor and basic fibroblast growth factor is a well-accepted manner for enriching CSCs from several kinds of tumor cells, including melanoma cells A375. The effects of polarized macrophages on malignant behaviors of melanoma cells were well studied, however, little was known about the effects of inflammatory environment modified by polarized macrophages on melanoma CSCs. In this study, tied to figure out these effects, we firstly stimulated the polarization of M1- and M2-state macrophages and confirmed the successful stimulation via detecting hallmarkers. Then the successful enrichment of A375 CSCs was confirmed by performing self-renewal capacity. Via detecting the malignant behaviors, it is observed that inflammatory environment modified by M1-polarized macrophages inhibited proliferation, blocked cell cycle phases at G1/G0, decreased invasive, tumor formatting abilities and induced cisplatin-sensitivity by promoting apoptosis. Contrarily, M2-conditioned medium desensitized A375 CSCs to cisplatin without affecting other malignant behaviors including proliferation, cell cycle, invasion and tumor formation. Meanwhile, M2- conditioned medium protected stemness of CSCs under oxidative stress. How the modified inflammatory microenvironment affects CSCs was performed by adding macrophage-specific cytokines and the results indicate that M1-polarized and M2-polarized macrophages partially affected CSCs via increased secretion of cytokines, including IL-1β, TNF-α and TGF-β. Taken together, we provide evidence that different macrophage polarization affected malignant behaviors of A375 CSCs partially via secretion of cytokines.

The Role of the Tumor Microenvironment in CSC Enrichment and Chemoresistance: 3D Co-culture Methods.

Cancer stem-like cells (CSC) are responsible for tumor progression, chemoresistance, recurrence, and poor outcomes in many cancers, making them critical research and therapeutic targets. One of the critical components potentiating CSC chemoresistance is the interactions between CSC and the surrounding cells in the tumor microenvironment. Our lab has developed several 3D co-culture models to study ovarian CSC interactions with stromal or immune cells found in ovarian tumor microenvironments. In this chapter, we use ovarian cancer as a model to describe the methodologies developed in our lab; however, these techniques are applicable to a wide range of cancers. First, we discuss our method for isolating CSC from heterogeneous tumors and for creating 3D self-assembled tumoroids in hanging drop plates, in either monoculture or co-culture with mesenchymal stem cells or monocytes/macrophages. We then discuss methods for analyzing these models with a focus on isolating cell-type-specific changes and mechanism investigation. Specifically, we describe lentiviral transduction and flow cytometry as established and robust methods to identify and separate each cell type for downstream analysis. We then describe methods to examine CSC functionality with transwell migration assays and colorimetric MTS-based proliferation assays. Finally, we demonstrate enzyme-linked immunosorbent assays (ELISA ) and quantitative polymerase chain reaction (qPCR) methods as mechanistic investigation tools to decouple paracrine and juxtacrine interactions. These methods have wide-reaching applications in cancer research from basic biological investigations, to drug discovery, and personalized drug screening for precision medicine.

Identification and Characterization of Cancer Stem-Like Cells in ALK-Positive Anaplastic Large Cell Lymphoma Using the SORE6 Reporter.

Transcription factors Sox2 and Oct4 are essential in maintaining the pluripotency of embryonic stem cells and conferring stemness in cancer stem-like (CSL) cells. SORE6, an in-vitro reporter system, was designed to quantify the transcription activity of Sox2/Oct4 and identify CSL cells in non-hematologic cancers. Using SORE6, we identified and enriched CSL cells in ALK-positive anaplastic large cell lymphoma (ALK + ALCL). Two ALK + ALCL cell lines, SupM2 and UCONN-L2, contained approximately 20% of SORE6+ cells, which were purified based on their expression of green fluorescent protein. We then performed functional studies using single-cell clones derived from SORE6− and SORE6+ cells. Compared to SORE6− cells, SORE6+ cells were significantly more chemoresistant and clonogenic in colony-formation assays. Sox2/Oct4 are directly involved in conferring these CSL properties, since the shRNA knockdown of Sox2 in SORE6+ significantly lowered their chemoresistance, while enforced expression of Sox2/Oct4 in SORE6− cells produced opposite effects. Using Western blots, we found that the expression and subcellular localization of Sox2/Oct4 were similar between SORE6− and SORE6+ cells. However, in SORE6+ but not SORE6− cells, Sox2 and Oct4 abundantly bound to a probe containing the SORE6 consensus sequence. c-Myc, previously shown to regulate cancer stemness in ALK + ALCL, regulated the SORE6 activity. In conclusion, SORE6 is useful in identifying/enriching CSL cells in ALK + ALCL.

Morphological and molecular characteristics of spheroid formation in HT-29 and Caco-2 colorectal cancer cell lines

BackgroundRelapse and metastasis in colorectal cancer (CRC) are often attributed to cancer stem-like cells (CSCs), as small sub-population of tumor cells with ability of drug resistance. Accordingly, development of appropriate models to investigate CSCs biology and establishment of effective therapeutic strategies is warranted. Hence, we aimed to assess the capability of two widely used and important colorectal cancer cell lines, HT-29 and Caco-2, in generating spheroids and their detailed morphological and molecular characteristics.MethodsCRC spheroids were developed using hanging drop and forced floating in serum-free and non-attachment conditions and their morphological features were evaluated by scanning electron microscopy (SEM). Then, the potential of CSCs enrichment in spheroids was compared to their adherent counterparts by analysis of serial sphere formation capacity, real-time PCR of key stemness genes (KLF4, OCT4, SOX2, NANOG, C-MYC) and the expression of potential CRC-CSCs surface markers (CD166, CD44, and CD133) by flow cytometry. Finally, the expression level of some EMT-related (Vimentin, SNAIL1, TWIST1, N-cadherin, E-cadherin, ZEB1) and multi-drug resistant (ABCB1, ABCC1, ABCG2) genes was evaluated.ResultsAlthough with different morphological features, both cell lines were formed CSCs-enriched spheroids, indicated by ability to serial sphere formation, significant up-regulation of stemness genes, SOX2, C-MYC, NANOG and OCT4 in HT-29 and SOX2, C-MYC and KLF4 in Caco-2 spheroids (p-value < 0.05) and increased expression of CRC-CSC markers compared to parental cells (p-value < 0.05). Additionally, HT-29 spheroids exhibited a significant higher expression of both ABCB1 and ABCG2 (p-value = 0.02). The significant up-regulation of promoting EMT genes, ZEB1, TWIST1, E-cadherin and SNAIL1 in HT-29 spheroids (p-value = 0.03), SNAIL1 and Vimentin in Caco-2 spheroids (p-value < 0.05) and N-cadherin down-regulation in both spheroids were observed.ConclusionEnrichment of CSC-related features in HT-29 and Caco-2 (for the first time without applying special scaffold/biochemical) spheroids, suggests spheroid culture as robust, reproducible, simple and cost-effective model to imitate the complexity of in vivo tumors including self-renewal, drug resistance and invasion for in vitro research of CRC-CSCs.

Reversal of cisplatin sensitization and abrogation of cisplatin-enriched cancer stem cells in 5-8F nasopharyngeal carcinoma cell line through a suppression of Wnt/β-catenin-signaling pathway.

Nasopharyngeal carcinoma (NPC) is one of the rare cancers in western countries but predominant in Southeast Asian countries including Thailand. One major cause for failure of NPC chemotherapeutic treatments is reportedly correlated with the elevation of cancer stem cell (CSC) fractions. Thus, this present study aims to investigate the effect of cisplatin (CDDP) treatment on the enrichment of cancer stem-like cells (CSCs) and its associated signaling pathway in EBV-negative NPC cells. Cisplatin-pretreated 5-8F NPC cells (5-8F CDDP) were first generated by treating the cells with 0.5μM cisplatin for 48h. After the instant treatment, 5-8F CDDP showed increased IC50 values, demonstrating a decrease in CDDP sensitization. Besides, the proportion of NPC cells with cancer stem-like phenotypes comprising side population (SP), key stemness-related gene expressions including SOX2, ALDH1, CD24 was significantly enhanced. Additionally, 5-8F CDDP displayed the upregulation of β-catenin gene, suggesting its association with the CSC-initiating mechanism. Furthermore, a tankyrase inhibitor for Wnt/β-catenin pathway, XAV939, substantially reduced CSCs and retrieved the cisplatin sensitivity in 5-8F CDDP. This confirms that the Wnt/β-catenin signaling is accountable for rising of the CSC population in EBV-negative NPC. Finally, the combined treatment of CDDP and XAV939 exhibited lower 5-8F CDDP cell viability compared to the treatment of CDDP alone, suggesting the reversal of cisplatin sensitization. In conclusion, the enhancement of CSCs in 5-8F NPC cells caused by the instant cisplatin treatment is initially mediated through the upregulation of β-catenin and activation of Wnt/β-catenin signaling pathway. As a result, a primary chemotherapeutic treatment with closely monitoring the targeted Wnt/β-catenin signaling pathway could potentially prevent the development of CSCs and improve the treatment efficiency in NPC.

Carcinoma-Associated Mesenchymal Stem Cells Promote Chemoresistance in Ovarian Cancer Stem Cells via PDGF Signaling.

Within the ovarian cancer tumor microenvironment, cancer stem-like cells (CSC) interact with carcinoma associated mesenchymal stem/stromal cells (CA-MSC) through multiple secreted cytokines and growth factors. These paracrine interactions have been revealed to cause enrichment of CSC and their chemoprotection; however, it is still not known if platelet-derived growth factor (PDGF) signaling is involved in facilitating these responses. In order to probe this undiscovered bidirectional communication, we created a model of ovarian malignant ascites in the three-dimensional (3D) hanging drop heterospheroid array, with CSC and CA-MSC. We hypothesized that PDGF secretion by CA-MSC increases self-renewal, migration, epithelial to mesenchymal transition (EMT) and chemoresistance in ovarian CSC. Our results indicate that PDGF signaling in the CSC-MSC heterospheroids significantly increased stemness, metastatic potential and chemoresistance of CSC. Knockdown of PDGFB in MSC resulted in abrogation of these phenotypes in the heterospheroids. Our studies also reveal a cross-talk between PDGF and Hedgehog signaling in ovarian cancer. Overall, our data suggest that when the stromal signaling via PDGF to ovarian CSC is blocked in addition to chemotherapy pressure, the tumor cells are significantly more sensitive to chemotherapy. Our results emphasize the importance of disrupting the signals from the microenvironment to the tumor cells, in order to improve response rates. These findings may lead to the development of combination therapies targeting stromal signaling (such as PDGF and Hedgehog) that can abrogate the tumorigenic, metastatic and platinum resistant phenotypes of ovarian CSC through additional investigations.

Abstract A52: Serially passaging ovarian cancer spheroids as an in situ model for emergence of chemoresistance and enrichment of cancer stem cells

Abstract Introduction: Epithelial ovarian cancer comprises approximately 90% of all cases of ovarian cancer and is the leading gynecologic cause of death in Western societies. Ovarian cancer patients typically respond well to first-line platinum and taxane-based chemotherapies; however, approximately 80% of these patients relapse and succumb to more chemoresistant disease. Relapse and development of chemoresistance is linked to the presence of a rare population of chemoresistant cells, termed cancer stem-like cells (CSCs), which are enriched in spheroids in the malignant ascites. Despite the high levels of chemoresistance and relapse observed in ovarian cancers, there are no in vitro models to understand the development of chemoresistance in situ. Here we implement a 3D model of the development of chemoresistance in ovarian cancer to investigate the functional and genetic changes that occur as chemoresistance develops. Using this model to gain insight into the development of chemoresistance in ovarian cancer will facilitate the development of more effective treatments. Methods: To investigate the development of chemoresistance in ovarian cancer, we utilized an in vitro model using the 3D hanging drop spheroid platform with an ovarian cancer cell line, two primary patient samples. In our model, spheroids were serially passaged every 7 days and evaluated for proliferation and response to treatment with cisplatin and a novel ALDH1A inhibitor. Concomitantly, the expression CSC markers ALDH and CD133 as well as tumor initiating capacity were analyzed. RNA-sequencing and qRT-PCR was performed on spheroids from early (P0), middle (P3), and late (P6) passages for two patient samples to establish gene signatures associated with the evolution of stemness, tumorigenicity, and chemoresistance. Lastly, a mathematical model was developed to predict the emergence of CSCs during serial passaging of ovarian cancer spheroids. Results: Our serial passage model demonstrated increased cell proliferation, enriched CSCs, and emergence of a platinum-resistant phenotype with passaging. Contrarily, serial passaged spheroids were enriched for ALDH and consequently exhibited greater sensitivity to ALDH1A inhibitor. Furthermore, in vivo tumor xenograft assays indicated that later passage spheroids were significantly more tumorigenic with higher CSC proportions, compared to early passage spheroids, validating the increased proliferative capacity predicted by our in vitro serial passage platform. RNA-sequencing revealed several gene signatures supporting the emergence of CSCs, chemoresistance, and malignant phenotypes, with links to poor clinical prognosis. Finally, our mathematical model predicted the emergence of CSC populations within serially passaged spheroids, concurring with experimental data. Conclusions: Our integrated approach illustrates the utility of the serial passage spheroid model for examining the emergence of chemoresistance in ovarian cancer in a controllable and reproducible format. Citation Format: Pooja Mehta, Micheal Bregenzer, Maria Ward Rashidi, Shreya Raghavan, Elyse Fleck, Eric Horst, Zainab Harissa, Visweswaran Ravikumar, Samuel Brady, Andrea Bild, Arvind Rao, Ronald Buckanovich, Geeta Mehta. Serially passaging ovarian cancer spheroids as an in situ model for emergence of chemoresistance and enrichment of cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A52.

BIK drives an aggressive breast cancer phenotype through sublethal apoptosis and predicts poor prognosis of ER-positive breast cancer

Apoptosis is fundamental to normal animal development and is the target for many anticancer therapies. Recent studies have explored the consequences of “failed apoptosis” where the apoptotic program is initiated but does not go to completion and does not cause cell death. Nevertheless, this failed apoptosis induces DNA double-strand breaks generating mutations that facilitate tumorigenesis. Whether failed apoptosis is relevant to clinical disease is unknown. BCL-2 interacting killer (BIK) is a stress-induced BH3-only protein that stimulates apoptosis in response to hormone and growth factor deprivation, hypoxia, and genomic stress. It was unclear whether BIK promotes or suppresses tumor survival within the context of breast cancer. We investigated this and show that BIK induces failed apoptosis with limited caspase activation and genomic damage in the absence of extensive cell death. Surviving cells acquire aggressive phenotypes characterized by enrichment of cancer stem-like cells, increased motility and increased clonogenic survival. Furthermore, by examining six independent cohorts of patients (total n = 969), we discovered that high BIK mRNA and protein levels predicted clinical relapse of Estrogen receptor (ER)-positive cancers, which account for almost 70% of all breast cancers diagnosed but had no predictive value for hormone receptor-negative (triple-negative) patients. Thus, this study identifies BIK as a biomarker for tumor recurrence of ER-positive patients and provides a potential mechanism whereby failed apoptosis contributes to cancer aggression.

PIN1 Inhibition Sensitizes Chemotherapy in Gastric Cancer Cells by Targeting Stem Cell-like Traits and Multiple Biomarkers.

Gastric cancer is the third leading cause of cancer-related death worldwide. Diffuse type gastric cancer has the worst prognosis due to notorious resistance to chemotherapy and enrichment of cancer stem-like cells (CSC) associated with the epithelial-to-mesenchymal transition (EMT). The unique proline isomerase PIN1 is a common regulator of oncogenic signaling networks and is important for gastric cancer development. However, little is known about its roles in CSCs and drug resistance in gastric cancer. In this article, we demonstrate that PIN1 overexpression is closely correlated with advanced tumor stages, poor chemo-response and shorter recurrence-free survival in diffuse type gastric cancer in human patients. Furthermore, shRNA-mediated genetic or all-trans retinoic acid-mediated pharmaceutical inhibition of PIN1 in multiple human gastric cancer cells potently suppresses the EMT, cell migration and invasion, and lung metastasis. Moreover, PIN1 genetic or pharmaceutical inhibition potently eliminates gastric CSCs and suppresses their self-renewal and tumorigenicity in vitro and in vivo Consistent with these phenotypes, are that PIN1 biochemically targets multiple signaling molecules and biomarkers in EMT and CSCs and that genetic and pharmaceutical PIN1 inhibition functionally and drastically enhances the sensitivity of gastric cancer to multiple chemotherapy drugs in vitro and in vivo These results demonstrate that PIN1 inhibition sensitizes chemotherapy in gastric cancer cells by targeting CSCs, and suggest that PIN1 inhibitors may be used to overcome drug resistance in gastric cancer.

Role of Oct4-Sox2 complex decoy oligodeoxynucleotides strategy on reverse epithelial to mesenchymal transition (EMT) induction in HT29-ShE encompassing enriched cancer stem-like cells.

Cancer stem cells are commonly tolerant toward chemotherapy and radiotherapy. Oct4 and Sox2 transcription factors are shown to be overexpressed in various cancers. At the current research, inhibition of Oct4 and Sox2 transcription factors was performed through application of decoy oligodeoxynucleotides (ODNs) strategy via repressing stemness properties in HT29-ShE cells encompassing enriched cancer stem-like cells. Designed Oct4-Sox2 complex decoy ODNs were transfected into HT29-ShE cells with Lipofectamine reagent. At the next step, ODNs efficiency transfection and subcellular localization were determined via flow cytometry and fluorescence microscopy, respectively. Further investigations such as cell proliferation and apoptosis analysis, colonosphere formation, invasion and migration, and real-time PCR assays were also carried out. Obtained results shed light on the fact that the designed complex decoys were effectively transfected into HT29-ShE cells, and they were found to be localized in subcellular compartments. Oct4-Sox2 decoy ODNs led to decreased cell viability, arresting the cell cycle in G0/G1 phases,increasing apoptosis, inhibition of migration/invasion and colonosphere formation ability of HT29-ShE cells in comparison with control and scramble groups. Furthermore, Oct4-Sox2 complex decoy could modulate the MET process via alteration of mRNA expression of downstream genes. It could be concluded that application of Oct4-Sox2 transcription factor decoy strategy in cells with stemness potential could lead to inhibiting the cell growth and triggering differentiation. Therefore, this technique could be applied along with usual remedies(chemotherapy and radiotherapy) as high potential method for treating cancer.

Mesenchymal Stem Cells in the Tumor Microenvironment.

The interactions between tumor cells and the non-malignant stromal and immune cells that make up the tumor microenvironment (TME) are critical to the pathophysiology of cancer. Mesenchymal stem cells (MSCs) are multipotent stromal stem cells found within most cancers and play a critical role influencing the formation and function of the TME. MSCs have been reported to support tumor growth through a variety of mechanisms including (i) differentiation into other pro-tumorigenic stromal components, (ii) suppression of the immune response, (iii) promotion of angiogenesis, (iv) enhancement of an epithelial-mesenchymal transition (EMT), (v) enrichment of cancer stem-like cells (CSC), (vi) increase in tumor cell survival, and (vii) promotion of tumor metastasis. In contrast, MSCs have also been reported to have antitumorigenic functions including (i) enhancement of the immune response, (ii) inhibition of angiogenesis, (iii) regulation of cellular signaling, and (iv) induction of tumor cell apoptosis. Although literature supporting both arguments exists, most studies point to MSCs acting in a cancer supporting role within the confines of the TME. Tumor-suppressive effects are observed when MSCs are used in higher ratios to tumor cells. Additionally, MSC function appears to be tissue type dependent and may rely on cancer education to reprogram a naïve MSC with antitumor effects into a cancer-educated or cancer-associated MSC (CA-MSC) which develops pro-tumorigenic function. Further work is required to delineate the complex crosstalk between MSCs and other components of the TME to accurately assess the impact of MSCs on cancer initiation, growth, and spread.

Co-culture of functionally enriched cancer stem-like cells and cancer-associated fibroblasts for single-cell whole transcriptome analysis

Considerable evidence suggests that breast cancer development and metastasis are driven by cancer stem-like cells (CSCs). Due to their unique role in tumor initiation, the interaction between CSCs and stromal cells is especially critical. In this work, we developed a platform to reliably isolate single cells in suspension and grow single-cell-derived spheres for functional enrichment of CSCs. The platform also allows adherent culture of stromal cells for cancer-stromal interaction. As a proof of concept, we grew SUM149 breast cancer cells and successfully formed single-cell-derived spheres. Cancer-associated fibroblasts (CAFs) as stromal cells were found to significantly enhance the formation and growth of cancer spheres, indicating elevated tumor-initiation potential. After on-chip culture for 14days, we retrieved single-cell derived spheres with and without CAF co-culture for single-cell transcriptome sequencing. Whole transcriptome analysis highlights that CAF co-culture can boost cancer stemness especially ALDHhigh CSCs and alter epithelial/mesenchymal status. Single-cell resolution allows identification of individual CSCs and investigation of cancer cellular heterogeneity. Incorporating whole transcriptome sequencing data with public patient database, we discovered novel genes associated with cancer-CAF interaction and critical to patient survival. The preliminary works demonstrated a reliable platform for enrichment of CSCs and studies of cancer-stromal interaction.

1443P - Characterization of lung tumourspheres reveals cancer stem-like cells potential targets and prognostic markers in non-small cell lung cancer

BackgroundNon-small cell lung cancer (NSCLC) is the first cause of death cancer-related worldwide mainly due to high therapeutic resistance. This resistance is related to cancer stem-like cells (CSCs), for which the identification of targets and markers is still ongoing. MethodsPrimary cultures from 8 NSCLC patients were established as tumorspheres and as monolayers. CSCs properties were tested for both conditions in vitro and in vivo. The expression of 50 CSCs-related genes was assessed by RTqPCR and proteins of significantly overexpressed genes were examined by immunoblot and immunofluorescence. The prognostic role of these genes was analyzed in a cohort of 661 NSCLC patients from TCGA and validated in an independent cohort of 114 lung adenocarcinoma (ADC) patients. ResultsTumorspheres exhibited self-renewal, unlimited exponential growth, drug resistance, great invasion and differentiation capacities in vitro and higher tumorigenic potential than monolayers in vivo. 17 genes were significantly overexpressed in tumorspheres, being NANOG, NOTCH3, CD44, CDKN1A, SNAI1, and ITGA6 the major contributors to distinguish them from adherent cells. Proteins encoded by these genes showed differential localization and expression patterns in ADC tumorspheres. The expression of CDKN1A, SNAI1 and ITGA6 was associated to prognosis, so a score was built based on their regression coefficients from a multivariate model. TCGA patients with high CSCs score show shorter OS in the entire cohort [37.7 vs. 60.4mo., p=0.001] and the ADC subcohort [36.6 vs. 53.5mo., p=0.003]. Multivariate analysis indicated that this score is an independent biomarker of prognosis for OS in the entire cohort from TCGA [HR: 1.498; 95% CI, 1.167-1.922; p=0.001] and the ADC subcohort [HR: 1.869; 95% CI, 1.275-2.738; p=0.001]. The prognostic value is confirmed in an independent cohort of 114 lung adenocarcinoma patients OS (42.90 vs. NR mo, p=0.020). ConclusionsLung tumorspheres are a useful method for CSCs enrichment. Elevated expression of CDKN1A, SNAI1 and ITGA6 genes is associated with worse prognosis in NSCLC. Funded by CB16/12/00350 from CIBEROnc, PI12-02838, and PI15-00753 from ISCIII, Fundacion Arnal Planelles and Domingo Martínez. Legal entity responsible for the studyFundación de Investigación Hospital General de Valencia. FundingCIBEROnc, Instituto de Salud Carlos III; Fundacion Arnal Planelles and Domingo Martínez. DisclosureAll authors have declared no conflicts of interest.

Lung tumorspheres reveal cancer stem cell-like properties and a score with prognostic impact in resected non-small-cell lung cancer

The high resistance against current therapies found in non-small-cell lung cancer (NSCLC) has been associated to cancer stem-like cells (CSCs), a population for which the identification of targets and biomarkers is still under development. In this study, primary cultures from early-stage NSCLC patients were established, using sphere-forming assays for CSC enrichment and adherent conditions for the control counterparts. Patient-derived tumorspheres showed self-renewal and unlimited exponential growth potentials, resistance against chemotherapeutic agents, invasion and differentiation capacities in vitro, and superior tumorigenic potential in vivo. Using quantitative PCR, gene expression profiles were analyzed and NANOG, NOTCH3, CD44, CDKN1A, SNAI1, and ITGA6 were selected to distinguish tumorspheres from adherent cells. Immunoblot and immunofluorescence analyses confirmed that proteins encoded by these genes were consistently increased in tumorspheres from adenocarcinoma patients and showed differential localization and expression patterns. The prognostic role of genes significantly overexpressed in tumorspheres was evaluated in a NSCLC cohort (N = 661) from The Cancer Genome Atlas. Based on a Cox regression analysis, CDKN1A, SNAI1, and ITGA6 were found to be associated with prognosis and used to calculate a gene expression score, named CSC score. Kaplan–Meier survival analysis showed that patients with high CSC score have shorter overall survival (OS) in the entire cohort [37.7 vs. 60.4 months (mo), p = 0.001] and the adenocarcinoma subcohort [36.6 vs. 53.5 mo, p = 0.003], but not in the squamous cell carcinoma one. Multivariate analysis indicated that this gene expression score is an independent biomarker of prognosis for OS in both the entire cohort [hazard ratio (HR): 1.498; 95% confidence interval (CI), 1.167–1.922; p = 0.001] and the adenocarcinoma subcohort [HR: 1.869; 95% CI, 1.275–2.738; p = 0.001]. This score was also analyzed in an independent cohort of 114 adenocarcinoma patients, confirming its prognostic value [42.90 vs. not reached (NR) mo, p = 0.020]. In conclusion, our findings provide relevant prognostic information for lung adenocarcinoma patients and the basis for developing novel therapies. Further studies are required to identify suitable markers and targets for lung squamous cell carcinoma patients.

Tumorsphere assay provides a better in vitro method for cancer stem-like cells enrichment in A549 lung adenocarcinoma cells

Cancer stem cells (CSCs) have been implicated in growth, metastasis, recurrence and chemo-/radio-resistance in several cancer types. Despite a plenty of literature about different in vitro techniques to enrich/isolate CSCs, their comparative characterization for stemness is not well established. In the present study, cells obtained following three in vitro assays [clonogenic assay, tumorsphere assay (TSA) and single cell assay (SCA)] were compared for their cancer stem-like cell characteristics in human lung adenocarcinoma (A549) cells. Expression of the pluripotent (OCT4, NANOG) and lung cancer stem cell marker (CD166) genes were studied in these cells. Results showed that in comparison to cells obtained from routine culture (CC), the cells obtained from TSA showed significantly higher expression of OCT-4 and NANOG. These results were further validated with quantification of cell surface cancer stem cell markers i.e. CD44+/CD24− in the cells obtained from different methods, which were higher in TSA and SCA. Additionally, functional characterization of cancer stem-like cells (CSLCs) using ALDH assay showed the highest % of ALDH+ cells in TSA. These results were in agreement with higher resistance of these cells against 5-Fluorouracil suggesting higher fraction of CSLCs in TSA than the other assays. These results showed that TSA provides a better method to enrich CSLCs in A549 lung adenocarcinoma cells.

Engineered 3D Model of Cancer Stem Cell Enrichment and Chemoresistance

Intraperitoneal dissemination of ovarian cancers is preceded by the development of chemoresistant tumors with malignant ascites. Despite the high levels of chemoresistance and relapse observed in ovarian cancers, there are no in vitro models to understand the development of chemoresistance in situ. Method: We describe a highly integrated approach to establish an in vitro model of chemoresistance and stemness in ovarian cancer, using the 3D hanging drop spheroid platform. The model was established by serially passaging non-adherent spheroids. At each passage, the effectiveness of the model was evaluated via measures of proliferation, response to treatment with cisplatin and a novel ALDH1A inhibitor. Concomitantly, the expression and tumor initiating capacity of cancer stem-like cells (CSCs) was analyzed. RNA-seq was used to establish gene signatures associated with the evolution of tumorigenicity, and chemoresistance. Lastly, a mathematical model was developed to predict the emergence of CSCs during serial passaging of ovarian cancer spheroids. Results: Our serial passage model demonstrated increased cellular proliferation, enriched CSCs, and emergence of a platinum resistant phenotype. In vivo tumor xenograft assays indicated that later passage spheroids were significantly more tumorigenic with higher CSCs, compared to early passage spheroids. RNA-seq revealed several gene signatures supporting the emergence of CSCs, chemoresistance, and malignant phenotypes, with links to poor clinical prognosis. Our mathematical model predicted the emergence of CSC populations within serially passaged spheroids, concurring with experimentally observed data. Conclusion: Our integrated approach illustrates the utility of the serial passage spheroid model for examining the emergence and development of chemoresistance in ovarian cancer in a controllable and reproducible format.

Hormonal Regulation of Patient-Derived Endometrial Cancer Stem-like Cells Generated by Three-Dimensional Culture.

Low-grade and early-stage endometrial cancer usually has a favorable prognosis, whereas recurrent or metastatic disease is often difficult to cure. Thus, the molecular mechanisms underlying advanced pathophysiology remain to be elucidated. From the perspective of the origin of advanced endometrial cancer, the characterization of cancer stem-like cells (CSCs) will be the first step toward the development of clinical management. We established long-term culturable patient-derived cancer cells (PDCs) from patient endometrial tumors by spheroid cell culture, which is favorable for the enrichment of CSCs. PDC-derived xenograft tumors were generated in immunodeficient NOD/Shi-scid, IL-2RγKO Jic mice. Morphologically, PDCs derived from three distinct patient samples and their xenograft tumors recapitulated the corresponding original patient tumors. Of note, CSC-related genes including ALDH1A1 were upregulated in all of these PDCs, and the therapeutic potentiality of aldehyde dehydrogenase inhibitors was demonstrated. In addition, these PDCs and their patient-derived xenograft (PDX) models exhibited distinct characteristics on the basis of their hormone responsiveness and metastatic features. Interestingly, genes associated with inflammation and tumor immunity were upregulated by 17β-estradiol in PDC lines with high estrogen receptor expression and were also overexpressed in secondary PDCs obtained from metastatic tumor models. These results suggest that PDC and PDX models from endometrial cancer specimens would be useful to elucidate CSC traits and to develop alternative diagnostic and therapeutic options for advanced disease.

ALDH1A1 in patient-derived bladder cancer spheroids activates retinoic acid signaling leading to TUBB3 overexpression and tumor progression.

Acquired chemoresistance is a critical issue for advanced bladder cancer patients during long-term treatment. Recent studies reveal that a fraction of tumor cells with enhanced tumor-initiating potential, or cancer stem-like cells (CSCs), may particularly contribute to acquired chemoresistance and recurrence. Thus, CSC characterization will be the first step towards understanding the mechanisms underlying advanced disease. Here we generated long-term patient-derived cancer cells (PDCs) from bladder cancer patient specimens in spheroid culture, which is favorable for CSC enrichment. Pathological features of bladder cancer PDCs and PDC-dependent patient-derived xenografts (PDXs) were basically similar to those of their corresponding patients' specimens. Notably, CSC marker aldehyde dehydrogenase 1A1 (ALDH1A1), a critical enzyme that synthesizes retinoic acid (RA), was abundantly expressed in PDCs. ALDH1A1 inhibitors and shRNAs repressed both PDC proliferation and spheroid formation, whereas all-trans RA could rescue ALDH1A1 shRNA-suppressed spheroid formation. ALDH inhibitor also reduced the in vivo growth of PDC-derived xenografts. ALDH1A1 knockdown study showed that tubulin beta III (TUBB3) was one of the downregulated genes in PDCs. We identified functional RA response elements in TUBB3 promoter, whose transcriptional activities were substantially activated by RA. Clinical survival database reveals that TUBB3 expression may associate with poor prognosis in bladder cancer patients. Moreover, TUBB3 knockdown was sufficient to suppress PDC proliferation and spheroid formation. Taken together, our results indicate that ALDH1A1 and its putative downstream target TUBB3 are overexpressed in bladder cancer, and those molecules could be applied to alternative diagnostic and therapeutic options for advanced disease.