Co-culture Of Cancer Cells Research Articles

Abstract 3202: Pharmacologic modulation of Sigma1 induces autophagic degradation of programmed death-ligand 1 in cancer cells

Abstract Emerging evidence suggests that Sigma1 (also known as sigma1 receptor) is a unique ligand-operated integral membrane chaperone or scaffolding protein that contributes to cellular protein homeostasis. Previously, we found that treatment of various cancer cell lines with some prototypic small molecule modulators of Sigma1 can engage endoplasmic reticulum (ER) associated protein homeostasis pathways including the unfolded protein response and autophagy. Programmed death-ligand 1 (PD-L1) is a type 1 integral membrane glycoprotein that is processed and transported through the ER and secretory pathway of tumor cells. PD-L1 expressed at the surface of tumor cells can act as a T-cell inhibitory checkpoint molecule that inactivates tumor infiltrating immune cells that express PD-1, its cognate receptor. Here, we show that Sigma1 physically associates with PD-L1. In triple negative breast and androgen-independent prostate cancer cells, PD-L1 protein levels are suppressed by both RNAi mediated knockdown of Sigma1 and pharmacological modulation of Sigma1. We observe decreased cell surface and intracellular levels of PD-L1 by flow cytometry and biochemical subcellular fractionation respectively, which corresponds with a dose-responsive decrease in functional PD-L1/PD-1 interaction in a co-culture of cancer cells and T-cells. Inhibitors of autophagy block this suppression of PD-L1 protein levels, suggesting PD-L1 is degraded away by autophagy after Sigma1 modulation. Through confocal microscopy, we show that Sigma1 modulation results in colocalization of PD-L1 and GFP-LC3, a marker of autophagosomes. From these conclusions, we hypothesize that autophagic degradation of nascent PD-L1 after Sigma1 modulation plays a key role in preventing the transport of functional PD-L1 to the plasma membrane. Together, these data demonstrate that Sigma1 modulators have the potential to act as novel therapeutic agents in PD-1/PD-L1 blockade strategies. Citation Format: Christina M. Maher, Jeffrey D. Thomas, Charles G. Longen, Derick A. Haas, Halley M. Oyer, Jane Y. Tong, Felix J. Kim. Pharmacologic modulation of Sigma1 induces autophagic degradation of programmed death-ligand 1 in cancer cells [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 3202. doi:10.1158/1538-7445.AM2017-3202

Abstract 4513: Investigation of bystander effects of hypoxia activated prodrugs using three dimensional cell cultures

Abstract Background: Hypoxia in solid tumors acts as the major barrier to anti-cancer therapies and promotes tumor progression. Hypoxia-activated prodrugs (HAPs) are designed to not only eliminate these resistant cells, but also to exploit hypoxia as a basis of tumor selectivity. A key concept in exploiting hypoxia with HAPs is that active drug metabolite should be able to diffuse from hypoxic zones to maximize effect in surrounding aerobic tumor tissue. There is some evidence that these ‘bystander effects’ contribute to the antitumor activity of HAPs, but currently there is a lack of robust methods for their detection. Objectives: To investigate bystander effects from HAPs in a novel spheroid co-culture system using PR104A which releases a diffusible DNA crosslinking mustard and the tirapazamine analogue SN30000 which is converted to a short lived radical that is not expected to diffuse out of cells. Methods: A new method for measuring the bystander effect with multicellular spheroids under hypoxic conditions was developed using co-cultures of HCT116 colon cancer cells with high metabolic capacity for HAP metabolism (activators, transfected to overexpress cytochrome P450 reductase [POR]) and cells that cannot activate HAPs efficiently (targets, with POR knocked out by zinc finger nuclease gene targeting) along with differential antibiotic resistance and fluorescent protein markers to enable their quantitation in mixed cultures. Sensitivity of activators and targets to PR104A and SN30000 was determined in spheroid co-cultures by dissociation and clonogenic cell survival assay. Results were compared to output of a novel agent-based mathematical model (ABM) of spheroid co-cultures. Results: In monolayer cultures, activator cells showed markedly higher sensitivity to the HAPs than did targets. Tagging of activators and targets with fluorescent proteins allowed visualization of their spatial distribution in spheroid co-cultures. When spheroids were treated with PR104A, killing of targets markedly increased with increasing proportion of activators, confirming a bystander effect. However, increasing proportions of activators reduced cell killing of targets after SN30000 treatment, suggesting that rapid metabolism by activators inhibits its penetration, which was confirmed by flux experiments in multicellular layer co-cultures. ABM simulations of cell killing by PR104A and SN30000 in spheroids using our published transport parameters agreed with experimental results indicating diffusion of active metabolites of PR104A but not SN30000. Discussion: Using this novel spheroid co-culture model, a bystander effect of PR104A was demonstrated while SN30000 showed no bystander effect. This is the most direct demonstration of a hypoxic bystander effect for PR104A to date, and demonstrates the utility of the novel spheroid ABM in simulating active metabolite diffusion in 3D co-cultures. Citation Format: Cho Rong Hong, Gib Bogle, William R. Wilson, Kevin O. Hicks. Investigation of bystander effects of hypoxia activated prodrugs using three dimensional cell cultures [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 4513. doi:10.1158/1538-7445.AM2017-4513

Abstract 877: Real-time imaging of adherent and non-adherent cell interactions: utility of an automated microfluidic trap platform to recapitulate in vivo cell culture microenvironment

Abstract The study of dynamic cell processes and their interactions is of crucial importance to understand the complexities of tissue microenvironments. Simulation of in vivo cell culture microenvironments can greatly improve biologic relevance of cell-cell interaction studies, and so there is an ongoing demand for heuristic optimization of cell co-culture devices and protocols. One particularly challenging task is establishing real-time image analysis while co-culturing non-adherent cells in contact with adherent cell layers under precisely controlled environmental parameters. To address this challenge, we present a novel microfluidic approach to trap two different cell types in precise locations in order to image and study their interactions over time. The platform utilizes a well plate format that contains multiple microfluidic units. Each unit consists of a 3 mm x 3 mm x 40 µm (L x W x H) chamber for culture, and an array of microscopic trap areas tailored to the dimensions of the cells of interest, typically ~15 µm. The micro-scale geometries of the traps physically confine non-adherent cells, and co-cultures can be achieved by trapping the non-adherent cells over a monolayer of cells. The microfluidic plate is integrated with a system which enables perfusion-based nutrient supply along with gas and temperature control for long-term cell culture. Each chamber within the plate can be addressed by programmable and on-demand perturbation of up to 6 reagents, enabling uninterrupted real-time live cell imaging assays. We present here the use of this microfluidic platform to replicate tumor microenvironment by maintaining adjacent co-cultures of cancer and immune cells. We have successfully imaged and cultured monolayers of tumor cell lines for 7 days followed by subsequent loading and trapping of immune (non-adherent) cells. In conclusion, the microfluidic platform enables unique co-cultures with environmental control and real-time imaging to facilitate the investigation of cell-cell interactions in a wide range of applications such as drug response and screening in tumor microenvironments, invasion, evasion pathways and other mechanisms governing cell-cell interactions. Citation Format: Rekha Kannan, Victor Yeh, James Helton, Amedeo Cappione. Real-time imaging of adherent and non-adherent cell interactions: utility of an automated microfluidic trap platform to recapitulate in vivo cell culture microenvironment [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 877. doi:10.1158/1538-7445.AM2017-877

Abstract 4191: RXDX-106, a novel, selective and potent small molecule TAM (TYRO3, AXL, MER) inhibitor, demonstrates efficacy in TAM-driven tumors

Abstract In recent years, the TAM (TYRO3, AXL, and MER) family of receptor tyrosine kinases (RTKs) has emerged as attractive targets for oncology therapeutics. Under homeostatic conditions, the TAM RTKs are expressed by a number of immune cells where they play key roles in the negative regulation of the immune response. However, their expression on cancer cells has been specifically associated with epithelial-to-mesenchymal transition, an invasive phenotype, and more generally, with a poor prognostic outcome in human cancers. Here, we sought (1) to establish whether expression of the TAM was sufficient to drive cellular transformation, (2) to demonstrate that RXDX-106, a small molecule inhibitor of TAM RTKs, could inhibit tumors harboring activating TAM gene fusions, and furthermore, (3) to decipher how pharmacological inhibition of TAM signaling pathways both on cancer cells and immune cells would be beneficial, given their complex regulation and intimate relationship in the tumor microenvironment. TAM expression on cancer cells has emerged as one of the key mechanisms of resistance to targeted therapies, particularly to EGFR tyrosine kinase inhibitors (TKI). In addition, novel gene rearrangements (fusions) have been identified in the TCGA database for both Mertk (TMEM87B-Mertk) and Axl (Axl-MBIP) that retain the functionality of the tyrosine kinase domain, but have fusion partners that alter the expression profile or dimerization frequency, respectively. Here, we demonstrate both in vitro and in vivo that expression of either wild type receptors or TMEM87B-MER and AXL-MBIP fusion proteins is sufficient to drive oncogenic transformation by both ligand dependent and independent mechanisms. Treatment of these TAM-expressing cellular populations with RXDX-106 completely inhibits TAM phosphorylation and downstream signaling and, consistent with our signaling data, RXDX-106 completely inhibits cellular proliferation and viability at sub-nanomolar concentrations. Furthermore, we demonstrate that, in the co-culture of TAM-expressing cancer cells with immune cells such as macrophages, TAM ligands such as Gas6 are secreted to drive ligand dependent activation of TAM on cancer cells, leading to their survival and proliferation. In summary, our data suggest that TAM RTKs can act as traditional oncodrivers when activated either in a ligand dependent or ligand independent manner. In addition, we demonstrate that TAM fusions are tractable therapeutic targets and that patients with tumors harboring such molecular alterations may derive clinical benefit from RXDX-106. Finally, we show that RXDX-106 not only has the potential to inhibit cellular proliferation and survival on the cancer cell itself, but also affect the TAM-expressing tumor microenvironment to result in a global anti-cancer environment. Citation Format: Erin D. Lew, Elizabeth A. Tindall, Joanne Oh, Colin Walsh, Maria Barrera, Heather Ely, Amy Diliberto, Yumi Yokoyama, Gary Li, Amanda Albert. RXDX-106, a novel, selective and potent small molecule TAM (TYRO3, AXL, MER) inhibitor, demonstrates efficacy in TAM-driven tumors [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 4191. doi:10.1158/1538-7445.AM2017-4191

Abstract 2627: A study of dynamic changes in PD-L1 expression in KRAS mutant adenocarcinoma of the lung exposed to signal transduction inhibitors

Abstract Aim MEK and AKT inhibitors inhibit signaling down steam of KRAS and are being evaluated as single agents or in combination with other anticancer drugs for the treatment of KRAS mutant adenocarcinoma of the lung (adeno-NSCLC). We investigated whether increased PD-L1 expression with functional T cell inhibitory consequences were a common mechanism of resistance to these drugs in this setting. Material and Methods A panel of 10 KRAS mutant adeno-NSCLC cell lines were studied. Cells were exposed to GI50 concentrations of a MEK (trametinib) and AKT (AZD5363) inhibitor for 6hrs, 24hrs and 3 weeks. PD-L1 expression on cell lines was studied by immunofluorescence. Immunofluorescence at various time points were expressed as a ratio of values in drug treated cells compared to untreated control. Functional consequences of PD-L1 expression were studied using a T cell cancer cell line (Jurkat) transfected with a luciferase reporter where co-culture of cancer cells expressing PD-L1 led to reduction in luminescence. Luminescence at various time points were expressed as ratio of luminescence values of drug treated cells compared to untreated controls. Results Following characterization of expression of PD-L1 in 10 KRAS mutant adeno-NSCLC cell lines, 5 cell lines with the highest expression of PD-L1 was chosen for functional assays (H441, H2291, H23, H2030 and A549). When exposed to trametinib for 3 weeks, 3/5 cell lines (H23, H2030 and A549) showed an statistically significant increase in expression of PD-L1 (range 1.1-2.4) however significant reduction of luciferase activity was only observed in H23 and H2030, 0.93 and 0.75, p= 0.018 and p=0.01 respectively. When exposed to AZD5363 for 3 weeks, 3/5 cell lines (H441, H23 and H2030) showed a statistically significant increase in PD-L1 expression (range 1.3-1.9) however significant reduction in luciferase reduction was seen in only H441 and H23, 0.86 and 0.76, p= 0.03 and p= 0.04 respectively. Conclusion Chronic exposure of KRAS mutant cell lines to MEK and AKT inhibitors cause minor increases in PD-L1 expression but this does not result in functional inhibition of T cells in all instances. Consequently, a functional increase in PD-L1 expression is unlikely to be a common mechanism of resistance to MEK and AKT inhibitors in KRAS mutant adeno-NSCLC. 1 Citation Format: Anna R. Minchom, Parames Thavasu, Zai Ahmad, Adam Stewart, Alexandros Georgiou, Mary ER O'Brien, Sanjay Popat, Jaishree Bhosle, Timothy A. Yap, Johann de Bono, Udai Banerji. A study of dynamic changes in PD-L1 expression in KRAS mutant adenocarcinoma of the lung exposed to signal transduction inhibitors [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 2627. doi:10.1158/1538-7445.AM2017-2627

Macrophage Polarization Contributes to the Anti-Tumoral Efficacy of Mesoporous Nanovectors Loaded with Albumin-Bound Paclitaxel.

Therapies targeted to the immune system, such as immunotherapy, are currently shaping a new, rapidly developing branch of promising cancer treatments, offering the potential to change the prognosis of previously non-responding patients. Macrophages comprise the most abundant population of immune cells in the tumor microenvironment (TME) and can undergo differentiation into functional phenotypes depending on the local tissue environment. Based on these functional phenotypes, tumor-associated macrophages (TAMs) can either aid tumor progression (M2 phenotype) or inhibit it (M1 phenotype). Presence of M2 macrophages and a high ratio of M2/M1 macrophages in the TME are clinically associated with poor prognosis in many types of cancers. Herein, we evaluate the effect of macrophage phenotype on the transport and anti-cancer efficacy of albumin-bound paclitaxel (nAb-PTX) loaded into porous silicon multistage nanovectors (MSV). Studies in a coculture of breast cancer cells (3D-spheroid) with macrophages and in vivo models were conducted to evaluate the therapeutic efficacy of MSV-nAb-PTX as a function of macrophage phenotype. Association with MSV increased drug accumulation within the macrophages and the tumor spheroids, shifting the inflammation state of the TME toward the pro-inflammatory, anti-tumorigenic milieu. Additionally, the treatment increased macrophage motility toward cancer cells, promoting the active transport of therapeutic nanovectors into the tumor lesion. Consequently, apoptosis of cancer cells was increased and proliferation decreased in the MSV-nAb-PTX-treated group as compared to controls. The results also confirmed that the tested system shifts the macrophage differentiation toward an M1 phenotype, possessing an anti-proliferative effect toward the breast cancer cells. These factors were further incorporated into a mathematical model to help analyze the synergistic effect of the macrophage polarization state on the efficacy of MSV-nAb-PTX in alleviating hypovascularized tumor lesions. In conclusion, the ability of MSV-nAb-PTX to polarize TAM to the M1 phenotype, causing (1) enhanced penetration of the drug-carrying macrophages to the center of the tumor lesion and (2) increased toxicity to tumor cells may explain the increased anti-cancer efficacy of the system in comparison to nAb-PTX and other controls.

Interleukin (IL) 10 and Pyridone 6 (P6) Modify the Migration of Pancreatic Cancer Cells in Co-Cultures with Macrophages

This paper examines Chinese mutual fund managers' abilities to time market, market volatility, and market-wide liquidity. Using a sample of Chinese mutual funds, we employ both cross-sectional and bootstrap analyses and find strong evidence that, during 2001–2011, Chinese mutual fund managers demonstrated the ability to time market returns, volatility, and market liquidity. We also find top timers outperform bottom timers by 6–7% annually in out-of-sample tests, manifesting the practical meaning of timing ability. We then conduct robustness checks of our findings and the results are the same.

The influence of stromal cells and tumor-microenvironment-derived cytokines and chemokines on CD3+CD8+ tumor infiltrating lymphocyte subpopulations

ABSTRACT The tumor microenvironment has been identified as a major mediator of immunological processes in solid tumors. In particular, tumor-associated fibroblasts are known to interact with tumor infiltrating immune cells. We describe the influence of fibroblasts and tumor-microenvironment-derived cytokines on the infiltration capacity of CD3+CD8+ cytotoxic T lymphocyte subpopulations using a multicellular 3D co-culture system. 3D tumor microtissues were cultivated using a hanging drop system. Human A549 and Calu-6 cancer cell lines were incubated alone or together with the human fibroblast cell line SV80 for 10 d to form microtissues. On day 10, peripheral blood mononuclear cells (PBMC) were added with or without cytokine stimulation for 24 h. Infiltrating PBMC subpopulations were investigated by flow cytometry. Aggregation of the microtissues and the infiltration of the PBMCs were analyzed by immunohistochemistry, and endogenous cytokine and chemokine expression was analyzed with a multi-cytokine immunoassay. Secretion of chemokines is increased in microtissues consisting of cancer cells and fibroblasts. PBMC infiltrate the whole spheroid in cancer cell monocultures, whereas in co-cultures of cancer cells and fibroblasts, PBMCs are rather localized at the margin. Activated CD69+ and CD49d+ T lymphocytes show an increased microtissue infiltration in the presence of fibroblasts. We demonstrate that the stromal component of cancer microtissues significantly influences immune cell infiltration. The presence of fibroblasts in cancer microtissues induces a shift of T lymphocyte infiltration toward activated T lymphocytes.

Bioengineered three-dimensional co-culture of cancer cells and endothelial cells: A model system for dual analysis of tumor growth and angiogenesis.

Angiogenesis marks the transformation of a benign local tumor into a life-threatening disease. Many in vitro assays are available on two-dimensional (2D) platforms, however, limited research has been conducted to investigate the behavior of tumors and endothelial cells (ECs) grown on three-dimensional (3D) platforms. This study provides a 3D co-culture spheroid of tumor cells with ECs to study the interplay between ECs and tumor cells. In a 3D co-culture with HepG2 hepatocellular carcinoma (HCC) cells, ECs differentiate to form tubule networks when in co-culture. Addition of angiogenic factors or angiogenesis inhibitors to the model system enhanced or inhibited endothelial differentiation in the 3D model, enabling investigations of the cellular signaling pathways utilized in HCC development. The 3D model demonstrated similar protein expression levels as a HCC xenograft, as well as exhibited upregulation of essential signaling proteins such as Akt/mTor in the 3D model, which is not reflected in the 2D model. The effects of several anti-angiogenic agents, such as sorafenib, sunitinib, and axitinib were analyzed in the 3D co-culture model by utilizing fluorescent proteins and a fluorescence resonance energy transfer (FRET)-based caspase-3 sensor in the ECs, which can detect apoptosis in real time. The apoptotic capability of a drug to inhibit angiogenesis in the 3D model can be easily distinguished via the FRET sensor, and dual screening of anti-angiogenesis and anti-tumor drugs can be achieved in a single step via the 3D co-culture model. In summary, a 3D co-culture model is constructed, where a HCC tumor microenvironment with a hypoxic core and true gradient penetration of drugs is achieved for drug screening purposes and in vitro studies utilizing a small HCC tumor. Biotechnol. Bioeng. 2017;114: 1865-1877. © 2017 Wiley Periodicals, Inc.

Tumour-associated macrophages activate migration and STAT3 in pancreatic ductal adenocarcinoma cells in co-cultures

ObjectivesTumour-associated macrophages participate in tumour development and progression. The aim of this study was to assess the interactions of pancreatic cancer cells and pro-inflammatory M1 and anti-inflammatory M2 macrophages, specifically their effect on pancreatic cancer cell migration and the changes in STAT-signalling. MethodsMonocytes were isolated from healthy subjects and differentiated into macrophages with M-CSF. The macrophages were polarized towards M1 by IL-12 and towards M2 by IL-10. We studied also the effect of pan-JAK/STAT-inhibitor P6. Macrophage polarization and STAT and NFkB-activation in both MiaPaCa-2 and macrophages were assessed by flow cytometry. We recorded the effect of co-culture on migration rate of pancreatic cancer cells MiaPaCa-2. ResultsMacrophages increased the migration rate of pancreatic cancer cells. Co-culture activated STAT1, STAT3, STAT5, AKT, and NFkB in macrophages and STAT3 in MiaPaCa-2 cells. IL-12 polarized macrophages towards M1 and decreased the migration rate of pancreatic cancer cells in co-cultures as well as P6. IL-10 skewed macrophage polarization towards M2 and induced increase of pancreatic cancer cells in co-cultures. ConclusionCo-culture with macrophages increased pancreatic cancer cell migration and activated STAT3. It is possible to activate and deactivate migration of pancreatic cancer cells trough macrophage polarization.

Oroxylin A inhibits the generation of Tregs in non-small cell lung cancer.

Oroxylin A (OA), a naturally occurring monoflavonoid isolated from Scutellariae radix, has previously been reported to inhibit the proliferation of several cancer cell lines. CD4+CD25+Foxp3+ regulatory T cells (Tregs) play an important role in maintenance of immunologic self-tolerance. Tregs also increase in cancer and take part in suppressing antitumor immune responses. Here, we explored how OA affected the Tregs in lung cancer environment and the involved underlying mechanism. It is found that OA reversed the generation of Tregs induced by H460 lung cancer cells co-culture. Furthermore, in vivo, OA reduced tumor formation rate and attenuated Foxp3 expression in tumor-infiltrating lymphocytes. We also found that transforming growth factor-β1 (TGF-β1) neutralizing antibody reversed the enhancement of Treg number and expression of p-Smad3ˎ p-p38ˎp-JNKˎp-ERK1/2 in the co-culture model. Moreover, OA reduced the secretion of TGF-β1 and down-regulated the activation of NF-κB signaling in H460 cells. OA also inhibited Treg activity by a direct inhibition of the T cells' response to TGF-β1. In conclusion, our study demonstrated that OA inhibits the generation of Tregs in lung cancer environment by inhibiting the T cells' response to TGF-β1 and decreasing the secretion of TGF-β1 in lung cancer cells via NF-κB signaling.

MP87-02 TUMOR-ASSOCIATED MACROPHAGES PROMOTE PROSTATE CANCER METASTASIS VIA CCL2-CCR2 AXIS-INDUCED CCL22-CCR4 AXIS

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2017MP87-02 TUMOR-ASSOCIATED MACROPHAGES PROMOTE PROSTATE CANCER METASTASIS VIA CCL2-CCR2 AXIS-INDUCED CCL22-CCR4 AXIS Kouji Izumi, Aerken Maolake, Ariunbold Natsagdorj, Kazutaka Narimoto, Yoshifumi Kadono, and Atsushi Mizokami Kouji IzumiKouji Izumi More articles by this author , Aerken MaolakeAerken Maolake More articles by this author , Ariunbold NatsagdorjAriunbold Natsagdorj More articles by this author , Kazutaka NarimotoKazutaka Narimoto More articles by this author , Yoshifumi KadonoYoshifumi Kadono More articles by this author , and Atsushi MizokamiAtsushi Mizokami More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2706AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Early studies have found that tumor-associated macrophages (TAMs) promote cancer progression. We previously reported that TAMs promote prostate cancer metastasis via activation of the CCL2-CCR2 axis. Recently, it was reported that the CCR4 (receptor of CCL17 and CCL22) expression level in breast cancer was associated with lung metastasis. However, the role of CCR4 and the relationship between CCR2 and CCR4 in prostate cancer is unclear. The aim of this study was to elucidate the role of CCR4 and the relationship between CCL2-CCR2 axis and CCL17/CCL22-CCR4 axis in prostate cancer progression. METHODS Human prostate cancer cell line cells and monocyte-lineage cells were used. Transwell migration and invasion assays co-cultured with or without macrophages were performed. Chemokines and their receptors in prostate cancer cells were measured. CCR2 and CCR4 in prostate cancer tissue were immunohistochemically analyzed. RESULTS Co-culture of macrophages and prostate cancer cells increased prostate cancer cell migration and invasion and induced secretion of CCL2. CCL2 promoted prostate cancer cell migration in an autocrine manner and induced CCR2, CCR4 expressions, and CCL22 secretion of prostate cancer cells. RT-PCR, western blotting, and immunocytochemical staining revealed both CCR2 and CCR4 expressions in prostate cancer cells. CCL22 also promoted prostate cancer cell migration. Blockade of the CCL2-CCR2 or CCL17/22-CCR4 axis with receptor antagonist inhibited the migration of prostate cancer cells. The CCL2-CCR2 and CCL22-CCR4 axes increased phosphorylation of Akt and Erk1/2. Although both CCR2 and CCR4 antagonists could inhibit phosphorylation of Akt and Erk1/2, the CCR4 antagonist, compared with the CCR2 antagonist, strongly inhibited phosphorylation of Akt. CCR4 may have contributed more to prostate cancer cell migration than did CCR2. CCR4 and CCR2 were increased in prostate cancer tissues by IHC staining. Interestingly, the staining intensities of CCR2 and CCR4 in each specimen were significantly correlated. Moreover, the staining intensity of CCR4 was correlated with the progression of TNM stage. CONCLUSIONS This is the first study to show that CCR4 was expressed in prostate cancer cell lines and human prostate cancer tissues and that the CCL22-CCR4 axis contributed to prostate cancer migration and invasion. Targeting of the CCL22-CCR4 axis, which is activated by TAMs, may be a novel therapeutic target and a potential biomarker for prostate cancer. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1166 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Kouji Izumi More articles by this author Aerken Maolake More articles by this author Ariunbold Natsagdorj More articles by this author Kazutaka Narimoto More articles by this author Yoshifumi Kadono More articles by this author Atsushi Mizokami More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Modification of cytokine-induced killer cells with folate receptor alpha (FRα)-specific chimeric antigen receptors enhances their antitumor immunity toward FRα-positive ovarian cancers.

Folate receptor alpha (FRα) is aberrantly expressed in ovarian cancers but largely absent in normal tissues, and therefore represents an attractive target for immunotherapy. In recent years, modification of T cells with chimeric antigen receptor (CAR) targeting FRα has been reported to improve antitumor immunity of T cells. However, there are limited data regarding CAR-modified cytokine-induced killer (CAR-CIK) cells. In the present study, we modified CIK cells with FRα-specific CARs and investigated their antitumor immunity against ovarian cancers. We found that both non-transduced and mock CAR-transduced CIK cells showed only low antitumor activity against either FRα-positive (FRα+) or FRα-negative (FRα-) targets. However, all three generations of CAR-modified CIK cells showed enhanced antitumor activity against FRα+ targets, but not FRα- targets. First-generation ζ-CAR-CIK cells increased production of IFN-γ, enhanced short-term cytotoxicity against FRα+ ovarian cancer cells, and showed modest and short-term suppression of established tumors; while second-generation 28ζ- and third-generation 28BBζ-CAR-CIK cells showed significant proliferation, enhanced secretion of IL-2, eliminated the FRα+ ovarian cancer cells in long-term co-culture, and showed dramatic and long-term inhibition of tumor growth and prolonged survival of xenograft-bearing mice. It is noteworthy that the 28BBζ-CAR was more potent in the modification of CIK cells than 28ζ-CAR both in vitro and in vivo. Moreover, CAR-CIK cells showed more efficient anticancer activity compared with CAR-T cells in vitro, but less efficient than CAR-T cells in vivo. According to these results, we conclude that modification of CIK cells with FRα-specific CARs enhances their antitumor immunity to FRα+ ovarian cancers. The third-generation 28BB-ζ CAR containing 4-1BB co-stimulation was more efficient in modification of CIK cells than either first-generation ζ-CAR or second-generation CD28-ζ-CAR.

Selectively Inducing Cancer Cell Death by Intracellular Enzyme-Instructed Self-Assembly (EISA) of Dipeptide Derivatives.

Tight ligand-receptor binding, paradoxically, is a major root of drug resistance in cancer chemotherapy. To address this problem, instead of using conventional inhibitors or ligands, this paper focuses on the development of a novel process-enzyme-instructed self-assembly (EISA)-to kill cancer cells selectively. Here it is demonstrated that EISA as an intracellular process to generate nanofibrils of short peptides for selectively inhibiting cancer cell proliferation, including drug resistant ones. As the process that turns the non-self-assembling precursors into the self-assembling peptides upon the catalysis of carboxylesterases (CES), EISA occurs intracellularly to selectively inhibit a range of cancer cells that exhibit relatively high CES activities. More importantly, EISA inhibits drug resistant cancer cells (e.g., triple negative breast cancer cells (HCC1937) and platinum-resistant ovarian cells (SKOV3, A2780cis)). With the IC50 values of 28-80 and 25-44 µg mL-1 of l- and d-dipeptide precursors against cancer cells, respectively, EISA is innocuous to normal cells. Moreover, using coculture of cancer and normal cells, the selectivity of EISA is validated against cancer cells. Besides revealing that intracellular EISA cause apoptosis or necroptosis to kill the cancer cells, this work illustrates a new approach to amplify the enzymatic difference between cancer and normal cells and to expand the pool of drug candidates for potentially overcoming drug resistance in cancer therapy.

Influence of TGF-β1 on tumor transition in oral cancer cell and BMSC co-cultures

ObjectivesTGF-β1 signaling modulates epithelial mesenchymal transitions (EMT) of head and neck squamous cell carcinoma (HNSCC). Bone marrow mesenchymal stromal cells (BMSC) are able to exert a regulating influence on the expression of markers of EMT in HNSCC cells. It was thus the aim of this study to test the hypothesis that TGF-β1 modulates the interactions of tumor transition between BMSCs and HNSCC, affecting the expression of E-cadherin, Vimentin, Snail, Twist, MMP14 and beta-catenin. Furthermore, we analyzed alterations in the AKT-signaling of tumor and stroma cells. Materials and methodsBMSCs were isolated from iliac bone marrow aspirates and co-cultured in trans-well permeable membrane wells with tumor cells of the established HNSCC cell line PCI-13. Following the induction with TGF-β1 under serum free conditions the expression of Vimentin and E-Cadherin was assessed via immunofluorescence. A quantitative RT-PCR analysis of tumor transition markers E-cadherin, Vimentin, Snail, Twist, MMP14 and beta-catenin was performed. Changes in AKT-Signaling were identified via protein analysis. ResultsIn non-induced co-cultures, BMSC were able to suppress Vimentin in PCI-13 as a marker of tumor transition. In TGF-β1 induced co-cultures PCI-13 significantly increased the expression of Vimentin, Twist, Snail, MMP14, GSK3a, PRAS40, 4E-BP1, and AMPKa compared to monolayer controls. TGF-β1 co-cultured BMSC demonstrated a significant increase of Snail, PRAS40, mTOR, GSK3a/b, Bad, PDK1 and 4E-BP1. ConclusionsTGF-β1 was able to attenuate the modulating influence of BMSC in co-culture and drive the co-culture towards a progressive tumor transition, affecting the expression of markers of EMT, AKT-Signaling and proliferative checkpoints.

Abstract P6-08-10: Osteoprotegerin mediates tumor-promoting effects of Interleukin-1beta in breast cancer cells

Abstract It is widely recognized that inflammation promotes breast cancer invasion and metastasis. Given the complex nature of the breast tumor inflammatory microenvironment, much remains to be understood of the molecular mechanisms that govern these effects. We have previously shown that osteoprotegerin (OPG) knockdown in breast cancer cells resulted in reduced invasion and metastasis. Here we present novel insight into the role of OPG in inflammation-driven tumor progression in breast cancer cells by investigating the link between OPG and the potent pro-inflammatory cytokine IL1B (IL1B). We used human breast cancer cell lines to investigate the effects of IL1B treatment on OPG expression. We analyzed public datasets containing human breast cancer genome-wide mRNA expression data to reveal a significant and positive correlation between OPG mRNA expression and the mRNA expression of IL1B as well as CCL2 (a monocyte chemoattractant protein). We determined the effect of macrophages (which produce IL1B) on OPG expression by co-culturing breast cancer cells and differentiated THP-1 macrophages. In order to demonstrate that OPG mediated functional effects of IL1B we performed cell invasion studies with control and OPG siRNA knockdown breast cancer cells treated with IL1B. We report that OPG expression is induced by IL1B, independent of breast cancer subtype and basal OPG levels. Co-culture of breast cancer cells with IL1B-secreting macrophages resulted in a similar increase in OPG expression in breast cancer cells as IL1B treatment. We show that OPG expression is regulated by IL1B in a p38-dependent manner. We also demonstrate that OPG knockdown represses IL1B expression, IL1B-mediated breast cancer cell invasion and MMP3 expression.It is widely recognized that inflammation promotes breast cancer invasion and metastasis. Given the complex nature of the breast tumor inflammatory microenvironment, much remains to be understood of the molecular mechanisms that govern these effects. We have previously shown that osteoprotegerin (OPG) knockdown in breast cancer cells resulted in reduced invasion and metastasis. Here we present novel insight into the role of OPG in inflammation-driven tumor progression in breast cancer cells by investigating the link between OPG and the potent pro-inflammatory cytokine IL1B (IL1B). We used human breast cancer cell lines to investigate the effects of IL1B treatment on OPG expression. We analyzed public datasets containing human breast cancer genome-wide mRNA expression data to reveal a significant and positive correlation between OPG mRNA expression and the mRNA expression of IL1B as well as CCL2 (a monocyte chemoattractant protein). We determined the effect of macrophages (which produce IL1B) on OPG expression by co-culturing breast cancer cells and differentiated THP-1 macrophages. In order to demonstrate that OPG mediated functional effects of IL1B we performed cell invasion studies with control and OPG siRNA knockdown breast cancer cells treated with IL1B. We report that OPG expression is induced by IL1B, independent of breast cancer subtype and basal OPG levels. Co-culture of breast cancer cells with IL1B-secreting macrophages resulted in a similar increase in OPG expression in breast cancer cells as IL1B treatment. We show that OPG expression is regulated by IL1B in a p38-dependent manner. We also demonstrate that OPG knockdown represses IL1B expression, IL1B-mediated breast cancer cell invasion and MMP3 expression. Citation Format: Tsang Mui Chung S, Geerts D, Roseman K, Renaud A, Connelly L. Osteoprotegerin mediates tumor-promoting effects of Interleukin-1beta in breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-08-10.

Mechanical Properties of Normal Breast Cells and Metastatic Cancer Cells in Co-Culture

The mechanical properties of cells play an important role in cellular processes such as cell migration, division, and mechanotransduction. Stiffness is an important mechanical property and can be measured using an atomic force microscope (AFM) (to determine the Young's modulus). Our previous studies showed that normal breast cells (human mammary epithelial cells, HMEC) are stiffer than highly metastatic breast cancer cells (MDA-MB-231). Also, normal breast cells in the center of a monolayer micro-colony are stiffer than cells on the periphery and isolated cells which means that the microenvironment significantly affects the mechanical properties of cells. However, the effect on stiffness when normal cells and cancer cells grow together has not been well established. In this study, we investigate how the mechanical properties of HMEC and MDA-MB-231 cells change in co-culture. Our preliminary results show that both HMEC and MDA-MB-231 cells get significantly stiffer in co-culture (HMEC 1.3 kPa -> 15 kPa; MDA-MB-231 0.59 kPa -> 1.48 kPa). This study will offer an understanding of normal-to-cancer cell interactions affect stiffness, and consequently how it might be correlated to metastasis.

Osteoprotegerin mediates tumor-promoting effects of Interleukin-1beta in breast cancer cells

BackgroundIt is widely recognized that inflammation promotes breast cancer invasion and metastasis. Given the complex nature of the breast tumor inflammatory microenvironment, much remains to be understood of the molecular mechanisms that govern these effects. We have previously shown that osteoprotegerin knockdown in breast cancer cells resulted in reduced invasion and metastasis. Here we present novel insight into the role of osteoprotegerin in inflammation-driven tumor progression in breast cancer by investigating the link between osteoprotegerin, macrophages and the potent pro-inflammatory cytokine Interleukin-1beta.MethodsWe used human breast cancer cell lines to investigate the effects of Interleukin-1beta treatment on osteoprotegerin secretion as measured by ELISA. We analyzed public datasets containing human breast cancer genome-wide mRNA expression data to reveal a significant and positive correlation between osteoprotegerin mRNA expression and the mRNA expression of Interleukin-1beta and of monocyte chemoattractant protein CC-chemokine ligand 2. Osteoprotegerin, Interleukin-1beta and CC-chemokine ligand 2 mRNA levels were also examined by qPCR on cDNA from normal and cancerous human breast tissue. We determined the effect of Interleukin-1beta–producing macrophages on osteoprotegerin expression by co-culturing breast cancer cells and differentiated THP-1 macrophages. Immunohistochemistry was performed on human breast tumor tissue microarrays to assess macrophage infiltration and osteoprotegerin expression. To demonstrate that osteoprotegerin mediated functional effects of Interleukin-1beta we performed cell invasion studies with control and OPG siRNA knockdown on Interleukin-1beta-treated breast cancer cells.ResultsWe report that Interleukin-1beta induces osteoprotegerin secretion, independent of breast cancer subtype and basal osteoprotegerin levels. Co-culture of breast cancer cells with Interleukin-1beta-secreting macrophages resulted in a similar increase in osteoprotegerin secretion in breast cancer cells as Interleukin-1beta treatment. Macrophage infiltration correlates with osteoprotegerin secretion in human breast tumor tissue samples. We show that osteoprotegerin secretion is regulated by Interleukin-1beta in a p38- and p42/44-dependent manner. We also demonstrate that osteoprotegerin knockdown represses Interleukin-1beta expression, Interleukin-1beta-mediated breast cancer cell invasion and MMP3 expression.ConclusionsThese data indicate a novel role for osteoprotegerin as a mediator of inflammation- promoted breast cancer progression.

Selective sensitiveness of mesenchymal stem cells to shock waves leads to anticancer effect in human cancer cell co-cultures

AimMesenchymal stem cells (MSC) possess the distinctive feature of homing in on and engrafting into the tumor stroma making their therapeutic applications in cancer treatment very promising. Research into new effectors and external stimuli, which can selectively trigger the release of cytotoxic species from MSC toward the cancer cells, significantly raises their potential. Main methodsShock waves (SW) have recently gained recognition for their ability to induce specific biological effects, such as the local generation of cytotoxic reactive oxygen species (ROS) in a non-invasive and tunable manner. We thus investigate whether MSC are able to generate ROS and, in turn, affect cancer cell growth when in co-culture with human glioblastoma (U87) or osteosarcoma (U2OS) cells and exposed to SW. Key findingsMSC were found to be the cell line that was most sensitive to SW treatment as shown by SW-induced ROS production and cytotoxicity. Notably, U87 and U2OS cancer cell growth was unaffected by SW exposure. However, significant decreases in cancer cell growth, 1.8 fold for U87 and 2.3 fold for U2OS, were observed 24h after the SW treatment of MSC co-cultures with cancer cells. The ROS production induced in MSC by SW exposure was then responsible for lipid peroxidation and cell death in U87 and U2OS cells co-cultured with MSC. SignificanceThis experiment highlights the unique ability of MSC to generate ROS upon SW treatment and induce the cell death of co-cultured cancer cells. SW might therefore be proposed as an innovative tool for MSC-mediated cancer treatment.

Adipocyte lipolysis links obesity to breast cancer growth: adipocyte-derived fatty acids drive breast cancer cell proliferation and migration

BackgroundObesity is associated with increased recurrence and reduced survival of breast cancer. Adipocytes constitute a significant component of breast tissue, yet their role in provisioning metabolic substrates to support breast cancer progression is poorly understood.ResultsHere, we show that co-culture of breast cancer cells with adipocytes revealed cancer cell-stimulated depletion of adipocyte triacylglycerol. Adipocyte-derived free fatty acids were transferred to breast cancer cells, driving fatty acid metabolism via increased CPT1A and electron transport chain complex protein levels, resulting in increased proliferation and migration. Notably, fatty acid transfer to breast cancer cells was enhanced from “obese” adipocytes, concomitant with increased stimulation of cancer cell proliferation and migration. This adipocyte-stimulated breast cancer cell proliferation was dependent on lipolytic processes since HSL/ATGL knockdown attenuated cancer cell responses.ConclusionsThese findings highlight a novel and potentially important role for adipocyte lipolysis in the provision of metabolic substrates to breast cancer cells, thereby supporting cancer progression.