scholarly journals In vitro model of inflammatory, hypoxia, and cancer stem cell signaling in pancreatic cancer using heterocellular 3-dimensional spheroids

2018 ◽  
Author(s):  
Megha Suresh ◽  
George Mattheolabakis ◽  
Amit Singh ◽  
Mansoor Amiji
Keyword(s):  
Pancreatic Cancer ◽  
Stem Cell ◽  
Tumor Microenvironment ◽  
Cancer Stem Cell ◽  
Cancer Cells ◽  
Cell Lines ◽  
Pancreatic Tumor ◽  
Stem Cell Markers ◽  
3 Dimensional

AbstractIntroductionAs one of the most aggressive cancers worldwide, pancreatic cancer is associated with an extremely poor prognosis. The pancreatic tumor microenvironment consists of cancer cells and other tumor associated cells. Cross-talk between these different cell types through various signaling molecules results in the development of a more aggressive and malignant phenotype. Additionally, due to the highly dysregulated vasculature of tumors, the inner tumor core becomes hypoxic and eventually necrotic. Therefore, there is a need for the development of a physiologically relevant in vitro model that recapitulates these dynamic cell-cell interactions and the 3-dimensional (3D) structure of pancreatic tumors.MethodsFour different 3D co-culture spheroid models using different combinations of Panc-1 tumor cells, J774.A1 macrophages, and NIH-3T3 fibroblast cell lines were reproducibly developed using the hanging drop technique in order to mimic the tumor microenvironment and to evaluate the differences in expression of various inflammatory, hypoxia, and cancer stem cell markers, including IL-8, TNF-α, TGF-β, HIF-1α HIF-2α, SCF, and LDH-A. Additionally, immunofluorescence studies were employed to investigate whether these spheroids tested positive for a cancer stem cell population.ResultsPronounced differences in morphology as well as expression of signalling markers were observed using qPCR, indicative of strong influences of co-culturing different cell lines. These models also tested positive for cancer stem cell (CSCs) markers based on immunofluorescence and qPCR analysis.ConclusionOur results demonstrate the potential of 3D co-culture spheroid models to capture the inflammatory and hypoxic markers of pancreatic tumor microenvironment. We further demonstrate the presence of cancer cells with stem cell markers, similar to actual pancreatic cancer tumor. These spheroids present excellent in vitro system to study tumor-immune-stromal cell interactions as well as test deliverability of potential therapeutics in the tumor microenvironment with accurate physical and physiological barriers.

scholarly journals PEPT1 is essential for the growth of pancreatic cancer cells: A viable drug target

2021 ◽  
Author(s):  
Bradley Schniers ◽  
Devaraja Rajasekaran ◽  
Ksenija Korac ◽  
Tyler Sniegowski ◽  
Vadivel Ganapathy ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Tumor Microenvironment ◽  
Cancer Cells ◽  
Cell Lines ◽  
Intracellular Localization ◽  
Peptide Transporter ◽  
Pdac Cell ◽  
Functional Link ◽  
Pancreatic Cancer Cells

PEPT1 is a proton-coupled peptide transporter that is upregulated in PDAC cell lines and PDXs, with little expression in normal pancreas. However, the relevance of this upregulation to cancer progression and the mechanism of upregulation have not been investigated. Herein, we show that PEPT1 is not just upregulated in a large panel of PDAC cell lines and PDXs but is also functional and transport-competent. PEPT2, another proton-coupled peptide transporter, is also overexpressed in PDAC cell lines and PDXs, but is not functional due to its intracellular localization. Using glibenclamide as a pharmacological inhibitor of PEPT1, we demonstrate in cell lines in vitro and mouse xenografts in vivothat inh­­ibition of PEPT1 reduces the proliferation of the cancer cells. These findings are supported by genetic knockdown of PEPT1 with shRNA, wherein the absence of the transporter significantly attenuates the growth of cancer cells, both in vitro and in vivo, suggesting that PEPT1 is critical for the survival of cancer cells. We also establish that the tumor-derived lactic acid (Warburg effect) in the tumor microenvironment supports the transport function of PEPT1 in the maintenance of amino acid nutrition in cancer cells by inducing MMPs and DPPIV to generate peptide substrates for PEPT1 and by generating a H+ gradient across the plasma membrane to energize PEPT1. Taken collectively, these studies demonstrate a functional link between PEPT1 and extracellular protein breakdown in the tumor microenvironment as a key determinant of pancreatic cancer growth, thus identifying PEPT1 as a potential therapeutic target for PDAC.

scholarly journals The IL-17A/IL-17RA Axis Is Not Related to Overall Survival and Cancer Stem Cell Modulation in Pancreatic Cancer

2020 ◽  
Vol 21 (6) ◽  
pp. 2215
Author(s):  
Jiahui Li ◽  
Christopher Betzler ◽  
Philipp Lohneis ◽  
Marie Christine Popp ◽  
Jiwei Qin ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Stem Cell ◽  
Cancer Stem Cell ◽  
Intraepithelial Neoplasia ◽  
Ductal Adenocarcinoma ◽  
Pancreatic Tumors ◽  
Stem Cell Markers ◽  
Sphere Formation

(1) Background: IL-17A accelerates pancreatic intraepithelial neoplasia (PanIN) progression. In this study, we examined whether IL-17A/IL-17RA promotes pancreatic ductal adenocarcinoma (PDAC) aggressiveness in terms of survival and cancer stem cell modulation. (2) Methods: In vitro, the wound-healing assay, the sphere formation assay, and flow cytometry were applied to assess cancer stem cell features. In vivo, pancreatic tumors were induced in C57BL/6 mice using electroporation with oncogenic plasmids (P53-/- R172H; KrasG12V). Anti-IL-17 antibodies were administered as immunotherapy. We analyzed IL-17A/IL-17RA related survival using publicly available transcriptomic data (n = 903). (3) Results: IL-17A/IL-17RA expression was not related to survival in PDAC patients. IL-17A neither induces stem cell markers nor increases sphere formation and cell motility in vitro. Blocking the IL-17A/IL-17RA axis in a murine pancreatic cancer model did not improve the survival of mice, but reduced the tumor burden slightly. (4) Conclusions: IL-17A does not promote stem cell expansion in PDAC cell lines. Blocking IL-17A/IL-17RA signaling does not interfere with pancreatic cancer development and progression and may not be considered as a promising monotherapy for PDAC.

scholarly journals Towards understanding cancer stem cell heterogeneity in the tumor microenvironment

2018 ◽  
Author(s):  
Federico Bocci ◽  
Larisa Gearhart-Serna ◽  
Marcelo Boareto ◽  
Mariana Ribeiro ◽  
Eshel Ben-Jacob ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Stem Cell ◽  
Tumor Microenvironment ◽  
Cancer Stem Cell ◽  
Tumor Progression ◽  
Cancer Cells ◽  
Inflammatory Cytokines ◽  
Primary Tumor ◽  
Breast Cancer Cells

AbstractThe Epithelial-Mesenchymal Transition (EMT) and Cancer Stem Cell (CSC) formation are two paramount processes driving tumor progression, therapy resistance and cancer metastasis. Some recent experiments show that cells with varying EMT and CSC phenotypes are spatially segregated in the primary tumor. The underlying mechanisms generating such spatiotemporal dynamics and heterogeneity in the tumor micro-environment, however, remain largely unexplored. Here, we show through a mechanism-based dynamical model that the diffusion of EMT-inducing signals such as TGF-β in a tumor tissue, together with non-cell autonomous control of EMT and CSC decision-making via juxtacrine signaling mediated via the Notch signaling pathway, can explain experimentally observed disparate localization of subsets of CSCs with varying EMT states in the tumor. Our simulations show that the more mesenchymal CSCs lie at the invasive edge, while the hybrid epithelial/mesenchymal (E/M) CSCs reside in the tumor interior. Further, motivated by the role of Notch-Jagged signaling in mediating EMT and stemness, we investigated the microenvironmental factors that promote Notch-Jagged signaling. We show that many inflammatory cytokines that can promote Notch-Jagged signaling such as IL-6 can (a) stabilize a hybrid E/M phenotype, (b) increase the likelihood of spatial proximity of hybrid E/M cells, and (c) expand the fraction of CSCs. To validate the predicted connection between Notch-Jagged signaling and stemness, we knocked down JAG1 in hybrid E/M SUM149 human breast cancer cellsin vitro. JAG1 knockdown significantly restricted organoid formation, confirming the key role that Notch-Jagged signaling can play in tumor progression. Together, our integrated computational-experimental framework reveals the underlying principles of spatiotemporal dynamics of EMT and CSCs in the tumor microenvironment.Significance statementThe presence of heterogeneous subsets of cancer stem cells (CSCs) remains a clinical challenge. These subsets often occupy different regions in the primary tumor and have varied epithelial-mesenchymal phenotypes. Here, we device a theoretical framework to investigate how the tumor microenvironment (TME) modulates this spatial patterning. We find that a spatial gradient of EMT-inducing signal, coupled with juxtacrine Notch-JAG1 signaling triggered by inflammatory cytokines in TME, explains this spatial heterogeneity. Finally,in vitroJAG1 knockdown experiments in triple negative breast cancer cells severely restricts the growth of tumor organoid, hence validating the association between JAG1 and CSC fraction. Our results offer insights into principles of spatiotemporal patterning in TME, and identifies a relevant target to alleviate multiple CSC subsets – JAG1.

658 CD44, Pancreatic Cancer Stem Cell Marker, Positive Cells Are Responsible for the Acquiring Gemcitabine Induced Chemoresistance In Vitro

2008 ◽  
Vol 134 (4) ◽  
pp. A-96
Author(s):  
Sung Pil Hong ◽  
Jeong Youp Park ◽  
Jing Wen ◽  
Jin Wook Yoon ◽  
Kyung Hwa Park ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Stem Cell ◽  
Cancer Stem Cell ◽  
Stem Cell Marker ◽  
Cancer Stem Cell Marker ◽  
Cell Marker

Identification and characterization of CCK-B/gastrin receptors in human pancreatic cancer cell lines

1994 ◽  
Vol 266 (1) ◽  
pp. R277-R283 ◽  
Author(s):  
J. P. Smith ◽  
G. Liu ◽  
V. Soundararajan ◽  
P. J. McLaughlin ◽  
I. S. Zagon
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Pancreatic Cancer Cell ◽  
Cancer Cell Lines ◽  
Human Pancreatic Cancer ◽  
Human Pancreatic Cancer Cell ◽  
Pancreatic Cancer Cells

The gastrointestinal peptide cholecystokinin (CCK) is known to stimulate growth of human pancreatic cancer in a receptor-mediated fashion. The purpose of this study was to characterize the receptor responsible for the trophic effects of CCK in cancer cells. With the use of homogenates of PANC-1 human pancreatic cancer cells grown in vitro, the binding characteristics and optimal conditions of radiolabeled selective CCK-receptor antagonists ([3H]L-365,260 and [3H]L-364,718) were examined. Specific and saturable binding was detected with [3H]L-365,260, and Scatchard analysis revealed that the data were consistent for a single site of binding with a binding affinity of 4.3 +/- 0.6 nM and a binding capacity (Bmax) of 283 +/- 68 fmol/mg protein in log phase cells. Binding was dependent on protein concentration, time, temperature, and pH and was sensitive to Na+, K+, Mg2+, and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. In contrast to log phase cells, Bmax decreased by 80 and 92% in confluent and postconfluent cultures, respectively. Subcellular fractionation studies revealed that binding was in the membrane fraction. Competition experiments indicated that L-365,260 and gastrin were more effective at displacing the radiolabeled L-365,260 than CCK. No binding was detected with the CCK-A antagonist [3H]L-364,718. Assays performed with [3H]L-365,260 on five additional human pancreatic cancer cell lines in vitro and tumor tissue from xenografts in nude mice also revealed specific and saturable binding. These results provide the first identification of a CCK-B/gastrin receptor in human pancreatic cancer cells and tumors and explain the effects of CCK on the growth of this malignancy.

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