Co-culture Of Cancer Cells Research Articles

The selective effect of plasma activated medium in an in vitro co-culture of liver cancer and normal cells

In this work, a co-culture system with liver cancer cell line HepG2 and normal cell line L02 is used to investigate the selective effect on cancer and normal cells by plasma activated medium (PAM), which is closer to the real environment where cancer cells develop. Besides, the co-culture system is a better model to study the selective effect than the widely used separate culture systems, where the cancer cell line and normal cell line are cultured independently. By using the co-culture system, it is found that there is an optimum dose of PAM to induce significant cancer cell apoptosis while keeping minimum damage to normal cells.

Tumor-associated macrophages promote prostate cancer migration through activation of the CCL22-CCR4 axis.

Previous 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. The CCR4 (receptor of CCL17 and CCL22) expression level in breast cancer was reported to be associated with lung metastasis. The aim of this study was to elucidate the role of CCR2 and CCR4 in prostate cancer progression. CCR2 and CCR4 were expressed in human prostate cancer cell lines and prostate cancer tissues. In vitro co-culture of prostate cancer cells and macrophages resulted in increased CCL2 and CCR2 levels in prostate cancer cells. The addition of CCL2 induced CCL22 and CCR4 production in prostate cancer cells. The migration and invasion of prostate cancer cells via enhanced phosphorylation of Akt were promoted by CCL17 and CCL22. CCR4 may be a potential candidate for molecular-targeted therapy.

Cancer-associated fibroblasts promote cancer cell growth through a miR-7-RASSF2-PAR-4 axis in the tumor microenvironment.

Cancer-associated fibroblasts (CAFs), a major component of cancer stroma, play an important role in cancer progression but little is known about how CAFs affect tumorigenesis and development. MicroRNAs (miRNAs) are small non-coding RNAs that can negatively regulate target mRNA expression at post-transcriptional levels. In head and neck cancer (HNC), our analysis of miRNA arrays showed that miR-7, miR-196 and miR-335 were significantly up-regulated in CAFs when compared with their paired normal fibroblasts (NFs). FAP, α-SMA and FSP, specific markers of CAFs, were significantly expressed in CAFs. Functionally, exogenous expression of miR-7 in NFs induced a functional conversion of NFs into CAFs. In contrast, inhibition of miR-7 expression in CAFs could induce a functional conversion of CAFs into NFs. Our study demonstrated that overexpression of miR-7 in NFs significantly increased the migration activity and growth rates of cancer cells in co-culture experiments. Mechanistically, we confirmed that the RASSF2-PAR-4 axis was mainly responsible for miR-7 functions in CAFs using bioinformatics methods. Overexpression of miR-7 in CAFs led to down-regulation of RASSF2, which dramatically decreased the secretion of PAR-4 from CAFs and then enhanced the proliferation and migration of the co-cultured cancer cells. Thus, these results reveal that the inactivation of the RASSF2-PAR-4 axis controlled by miR-7 may be a novel strategy for gene therapy in HNCs.

Analysis of Proliferation of Melanoma Cells and Mesenchymal Stem Cells in Co-Culture and Contribution of Experimental Conditions into Interpretation of the Results.

A series of experiments on co-culturing of Mel IL melanoma cells and mesenhymal stem cells showed that these cells do not influence proliferation of each other, but we observed weaker adhesion of stromal stem cells to plastic in cocultures where with melanoma cells were grown on mesenhymal stem cells feeder. Cell proliferation was also considerably influenced by experimental conditions, which should be taken into account for correct interpretation of obtained results. The principles of experiments on co-culturing of cancer and stromal cells are formulated that take into account the most important factors influencing cell behavior and minimize the probability of artifact results. It was concluded that co-culturing conditions cells significantly affect the experimental results and can be the source of conflicting conclusions on mutual influence of stromal and cancer cells in vitro.

Potential role of CXCL9 induced by endothelial cells/CD133+ liver cancer cells co-culture system in tumor transendothelial migration.

Transendothelial migration is a pivotal step before the dissemination of tumor cells into the blood circulation. Related researches about the crosstalk between tumor cells and endothelial cells could contribute to understanding the mechanism of transendothelial migration. Cumulative studies showed that CD133 was an important marker for cancer stem cells. In our research, a co-culture system was developed to study the interaction between CD133+ liver cancer cells and human umbilical vein endothelial cells. The results showed that the direct co-cultured supernatants promoted the migration and invasion of CD133+ liver cancer cells. It was further investigated that the expression level of chemokine CXCL9 was significantly elevated in the culture supernatants of direct co-culture system by activating the NF-kB, rather than in the indirect co-culture system or mono-culture system. High expression of CXCL9 in the direct co-cultured supernatants played a significant role in enhancing the migration and invasion of CD133+ liver cancer cells. Collectively, these findings suggest that chemokine CXCL9 may function as a potential target during the process of transendothelial migration.

Ultrastructural investigation of the time-dependent relationship between breast cancer cells and thrombosis induction

Thromboembolic complications are a common cause of death in breast cancer patients. The in vivo relationship between coagulation factors and circulating tumours is a multifaceted one, with tumour cells implicated in thrombocytosis and platelets associated with coagulation-mediated metastasis. Platelets and fibrin networks are known to be morphologically altered in patients with cancer, and their relationship with breast cancer cells themselves may increase thrombosis susceptibility. This was investigated in vitro, assessing such morphological alterations through the establishment of a MCF-7 breast cancer cell co-culture system with blood plasma. Co-culture duration ranged from zero to thirty minutes, with five-minute intervals, in order to assess the time-dependent ultrastructural conformations of platelet and fibrin networks, using scanning electron microscopy. It was found that enhanced coagulability was related to co-culture duration. Changes in platelet and fibrin network morphology from normal were visible as early as five minutes in co-culture with MCF-7 cells. With longer co-culture duration thrombosis-linked variation in structural configuration was intensified, including advanced platelet aggregation and adherence characteristics, compression of fibrin networks into plaques of increased density, and upsurge of microparticulate extrusion implicated in amplifying procoagulant events during the metastatic process. These results confirm that cancer cells are stimulators of coagulation even in an in vitro system and breast cancer patients may become increasingly susceptible to thrombotic-related consequences with increased exposure to tumour cells, especially during metastasis.

Proteolytic fragments of fibronectin function as matrikines driving the chemotactic affinity of prostate cancer cells to human bone marrow mesenchymal stromal cells via the α5β1 integrin

ABSTRACTThe haematopoietic niche is contributed to by bone marrow-resident mesenchymal stromal cells (BM-MSCs) and subverted by prostate cancer cells. To study mechanisms by which BM-MSCs and prostate cancer cells may interact, we assessed the migration, invasion, adhesion and proliferation of bone-derived prostate cancer cells (PC-3) in co-culture with pluripotent human BM-MSCs. We observed a strong adhesive, migratory and invasive phenotype of PC-3 cells with BM- MSC-co-culture and set out to isolate and characterize the bioactive principle. Initial studies indicated that chemotaxis was secondary to a protein residing in the >100kDa fraction. Size-exclusion chromatography (SEC) recovered peak activity in a high-molecular weight fraction containing thrombospondin-1 (TSP1). While TSP1 immunodepletion decreased activity, put-back with purified TSP1 did not reproduce bioactivity. Further purification of the TSP1-containing high-molecular weight fraction of the BM-MSC secretome with heparin-affinity chromatography recovered bioactivity with highly restricted bands on polyacrylamide gel electrophoresis, determined by mass spectroscopy to be proteolytic fragments of fibronectin (FN). Put-back experiments with full-length FN permitted adhesion but failed to induce migration. Monospecific antibodies to FN blocked adhesion. Proteolytic cleavage of FN generated FN fragments which now induced migration. Neutralizing monoclonal antibodies to FN receptors α5 and β1 integrins, and α5 knockdown specifically blocked migration and adhesion. Conclusion: Fibronectin fragments (FNFr) function as matrikines driving the chemotactic affinity of prostate cancer cells via the α5β1 integrin. Taken together with the high-frequency of α5β1 expression in disseminated prostate cancer cells in bone marrow aspirates from patients, the FNFr/FN-α5β1 interaction warrants further study as a therapeutic target.

Abstract A33: Physical intimacy of breast cancer cells with mesenchymal stem cells elicits trastuzumab resistance through Src activation

Abstract The development of resistance to trastuzumab is a major obstacle for lasting effective treatment of patients with ErbB2-overexpressing tumors. Here, we demonstrate that the physical contact of breast cancer cells with mesenchymal stem cells (MSCs) is a potential modulator of trastuzumab response by activation of nonreceptor tyrosine kinase c-SRC (Src) and down regulation of phosphatase and tensin homolog (PTEN). In this study we demonstrate a method for controlling breast cancer cells adhesion on polyelectrolyte multilayer (PEM) films without the aid of adhesive proteins/ligands to study the role of tumor and stromal cell interaction on cancer biology. Numerous studies have explored engineering co-culture of tumor and stromal cells predominantly using transwell co-culture of stromal cells cultured onto cover slips that were subsequently added to tumor cell cultures. However, these systems imposed an artificial boundary that precluded cell-cell interactions. To our knowledge this is the first demonstration of patterned co-culture of tumor cells and stromal cells that captures the temporal changes in the miRNA signature as the breast tumor develops through various stages. In this study we utilized cancer cells derived from two different tumor stages and two different stromal cells to effectively model heterogeneous tumor microenvironment and emulate various tumor stages. The co-culture model mimics the proliferative index (Ki67 expression) and tumor aggressiveness (HER-2 expression) akin to those observed in clinical tumor samples. We also demonstrated that our patterned co-culture model captures the temporal changes in the miRNA-21 and miRNA-34 signature as the breast tumor develops through various stages. Using our patterned breast cancer/MSC co-culture model, we find that the presence of MSCs results in Src activation that is missing in cancer cells monoculture, transwell co-culture, and cells treated with MSCs conditioned media. Interestingly, the co-culture model also results in PTEN loss and activation of PI3K/AKT pathway that has been demonstrated as fundamental proliferative and survival pathways in clinical settings. In addition, breast cancer cells in co-culture with MSCs conferred trastuzumab resistance in vitro as observed in the lack of inhibition of proliferative and migrative properties of the cancer cells. Our findings show that MSCs are potent mediators of resistance to trastuzumab and might reveal targets to enhance trastuzumab efficacy in patients. The engineered co-culture platform lays groundwork towards precision medicine wherein patient-derived tumor cells can be incorporated within our in vitro models to identify potential pathways and drug treatment regimens for individual patients. Citation Format: Srivatsan Kidambi. Physical intimacy of breast cancer cells with mesenchymal stem cells elicits trastuzumab resistance through Src activation. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A33.

Abstract 5059: Cancer cell invasion dynamics in microchannels during stromal cell coculture

Abstract Metastasizing cancer cells must escape their tumor microenvironment and invade into surrounding healthy stromal tissue. During this process, they must balance intercellular crosstalk with ECM degradation, physical force generation, and cytoskeletal rearrangement. To mimic this process in vitro, PDMS microchannels fabricated with photolithography and replica molding were used to examine the interaction of cancer cells with healthy stromal cells during physical confinement in three dimensional environments. A focus was placed on understanding the variations in invasion behavior between healthy cells and cancer cells, then attempting to explain these differences by cell mechanical properties and gene expression. Distinct differences in cancer cell invasion characteristics were observed between wide 10 μm channels and narrow 3 μm channels, with the latter exhibiting smooth velocity profiles and increased channel permeation. Live cell actin staining, ECM protein alteration, and chemical inhibition of both Rac1 and RhoA mechanical pathways were all used to better understand these differences, leading to the conclusion that the actin cytoskeleton is greatly altered during invasion into very confined channels. To better understand the role of stromal cells in this process, both indirect and direct coculture of MDA MB-231 basal breast cancer cells and stromal cells were performed in microchannel chips with the aid of live cell permeable tracking dyes. To reflect a diverse range of stromal cell types, cocultures were performed with MCF 10A non-tumorigenic breast epithelial cells, macrophage-differentiated THP-1 monocytes, and HN-CAFs (head and neck carcinoma-associated fibroblasts). Both the cancer cells and the stromal cells exhibited increases in invasion speed upon direct co-culture of the two cancer cell lines, both in wide and narrow channels. The use of stromal cell-conditioned media as well as observed differences in migration speed and persistence when unconfined provided further understanding of the complex processes underlying cancer cell and stromal cell invasion. Citation Format: Andrew W. Holle, Verena Kast, Ralf Kemkemer, Joachim Spatz. Cancer cell invasion dynamics in microchannels during stromal cell coculture. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5059.

Abstract 620: Microfluidic cancer-on-a-chip platform for assessing anti-cancer drug efficacies

Abstract Introduction: The tumor microenvironment is known to play an influential role in the angiogenic and metastatic progression of cancer and is regulated by different factors (stromal fibroblasts, extracellular matrix (ECM) proteins and endothelial cells) present in the complex milieu. Recapitulation of this complexity in three-dimensional (3D) tumor models is critical in understanding the processes involved in cancer progression and to provide clinically relevant efficacy data for cancer drugs. To address this challenge, we developed a microfluidic oncomimetic platform where breast cancer cells are co-cultured with fibroblasts along with a complex, intricate vascular network. We further investigated the effect of mechanical stiffness of the tumor stroma on the growth and morphology of cancer cells, migration of cancer cells into surrounding vasculature and the ability of standard cancer drugs to perfuse through the vasculature and target cancer cells. Materials and Methods: Poly(dimethyl siloxane) (PDMS)-based microfluidic devices, containing vascular networks in communication with tumor chamber, were fabricated using photolithography as described earlier1. Poly(ethylene glycol)-fibrinogen (PEG-Fb), used for 3D co-culture of cancer cells and fibroblasts, was prepared using established protocols2. Human breast tumor associated endothelial cells (hBTECs) were seeded within fibronectin-coated vascular channels and maintained under flow to develop endothelial networks. To obtain 3D cancer-fibroblast co-culture system, MCF7 or MDA-MB-231 breast cancer cells were co-encapsulated with BJ-5ta human fibroblasts in PEG-Fb hydrogel in the tumor chamber. Immunostaining with standard endothelial and cancer markers was conducted to confirm maturity and functionality of seeded cells. GFP-labelled cancer cells were co-cultured with hBTECs to visualize tumor cell migration. Cancer cells were also maintained in culture over several weeks within the devices to demonstrate applicability of this system to perform long-term drug dosing experiments. Finally, the cytotoxic effects of doxorubicin and paclitaxel at two different concentrations on cancer cells were evaluated by perfusing the drugs through the endothelial channels and cell viability quantified via Live/Dead staining. Conclusions: We have developed a novel 3D microfluidic, vascularized cancer-fibroblast co-culture platform, with the ability to predict drug efficacy for breast cancer. This platform can also be extended in future for cancer-immunotherapy based investigations and screening of novel nano-carrier based anti-cancer drugs. Acknowledgements: We gratefully acknowledge financial support from NIH (#HHSN261201400037C) and AURIC.

Abstract 555: Prostate cancer cell-induced differentiation of human monocytes into MDSCs ex vivo is inhibited by targeting STAT3

Abstract Background: The transcription factor Signal Transducer and Activator of Transcription Factor 3 (STAT3) is implicated in acquired drug resistance, metastases and immune suppression in prostate cancer and is a potential target in drug-resistant prostate cancer. Today, the treatment options for metastatic drug-resistant prostate cancer are very limited and existing immunotherapies have so far shown low efficacy. Cancer cells may avoid immune responses by expressing immunosuppressive markers or activating immunosuppressive cells. Myeloid-derived suppressor cells (MDSC) play a major role in the suppression of antitumor immunity and active STAT3 signaling has been shown to be involved in the immunosuppressive functions of these cells. Elevated levels of MDSC have been found in the peripheral blood and at the tumor site of prostate cancer patients and to correlate with disease progression. Aims: In this study we aimed to investigate if human monocytes could be induced to differentiate into immunosuppressive cells in the presence of prostate cancer cells and if this effect could be blocked by the STAT3 inhibitor GPA500 (galiellalactone). Methods: Monocytes were isolated from peripheral blood mononuclear cells from healthy human donors using a magnetic monocyte enrichment kit. Monocytes were co-cultured with the prostate cancer cell lines DU145, LNCaP or longterm IL6 treated LNCaP cells (LNCaP-IL6+) in transwells allowing cancer cells and monocytes to share culture medium. The co-cultures were grown for three days with or without the STAT3 inhibitor GPA500 (5 uM or 10 uM) with subsequent analysis by flow cytometry for cell surface markers of MDSC differentiation (CD14+ HLA-DRlo). The presence of active STAT3 (pSTAT3) in the prostate cancer cell lines was determined by Western blot. Results: Monocytes in the presence of DU145 and LNCaP-IL6+ cells showed a significantly increased population of CD14+ HLA-DRlo cells. In the presence of GPA500 the induction of this population was inhibited in a dose dependent manner compared to untreated cells. Co-culture with LNCaP cells that lack pSTAT3 did not induce this population in monocytes after three days in culture. Furthermore, GPA500 induced a dendritic cell-like population in monocytes in the absence of prostate cancer cell co-culture. This population was also observed in monocytes when co-cultured with LNCaP-IL6+ cells, but not in DU145 co-cultures, with GPA500 present. Conclusion: The prostate cancer cell lines DU145 and LNCaP-IL6+ were able to induce a monocyte population displaying MDSC markers and this induction could be inhibited by the STAT3 inhibitor GPA500. Impact: This study demonstrates that inhibiting the transcription factor STAT3 in prostate cancer could potentially reverse the immunosuppressive mechanisms caused by MDSC activation and that STAT3 inhibitors, alone or in combination with other therapies, could be a new treatment option for prostate cancer. Citation Format: Rebecka Hellsten, Karin Leandersson, Anders Bjartell, Martin E. Johansson. Prostate cancer cell-induced differentiation of human monocytes into MDSCs ex vivo is inhibited by targeting STAT3. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 555.

Selective interaction of PEGylated polyglutamic acid nanocapsules with cancer cells in a 3D model of a metastatic lymph node

BackgroundMetastases are the most common reason of cancer death in patients with solid tumors. Lymph nodes, once invaded by tumor cells, act as reservoirs before cancer cells spread to distant organs. To address the limited access of intravenously infused chemotherapeutics to the lymph nodes, we have developed PEGylated polyglutamic acid nanocapsules (PGA-PEG NCs), which have shown ability to reach and to accumulate in the lymphatic nodes and could therefore act as nanotransporters. Once in the lymphatics, the idea is that these nanocapsules would selectively interact with cancer cells, while avoiding non-specific interactions with immune cells and the appearance of subsequent immunotoxicity.ResultsThe potential of the PGA-PEG NCs, with a mean size of 100 nm and a negative zeta potential, to selectively reach metastatic cancer cells, has been explored in a novel 3D model that mimics an infiltrated lymph node. Our 3D model, a co-culture of cancer cells and lymphocytes, allows performing experiments under dynamic conditions that simulate the lymphatic flow. After perfusion of the nanocarriers, we observe a selective interaction with the tumor cells. Efficacy studies manifest the need to develop specific therapies addressed to treat metastatic cells that can be in a dormant state.ConclusionsWe provide evidence of the ability of PGA-PEG NCs to selectively interact with the tumor cells in presence of lymphocytes, highlighting their potential in cancer therapeutics. We also state the importance of designing precise in vitro models that allow performing mechanistic assays, to efficiently develop and evaluate specific therapies to confront the formation of metastasis.

Functional Interference in the Bone Marrow Microenvironment by Disseminated Breast Cancer Cells.

Skeletal metastasis of breast cancer is associated with a poor prognosis and significant morbidity. Investigations in other solid tumors have revealed an impairment in hematopoietic function upon bone marrow invasion. However, the interaction between disseminated breast cancer cells and the bone marrow microenvironment which harbors them has not been addressed comprehensively. Employing advanced co-culture assays, proteomic studies, organotypic models as well as in vivo xenotransplant models, we define the consequences of this interaction on the stromal compartment of bone marrow, affected molecular pathways and subsequent effects on the hematopoietic stem and progenitor cells (HSPCs). The results showed a basic fibroblast growth factor (bFGF)-mediated, synergistic increase in proliferation of breast cancer cells and mesenchymal stromal cells (MSCs) in co-culture. The stromal induction was associated with elevated phosphoinositide-3 kinase (PI3K) signaling in the stroma, which coupled with elevated bFGF levels resulted in increased migration of breast cancer cells towards the MSCs. The perturbed cytokine profile in the stroma led to reduction in the osteogenic differentiation of MSCs via downregulation of platelet-derived growth factor-BB (PDGF-BB). Long term co-cultures of breast cancer cells, HSPCs, MSCs and in vivo studies in NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl) /SzJ (NSG) mice showed a reduced support for HSPCs in the altered niche. The resultant non- conducive phenotype of the niche for HSPC support emphasizes the importance of the affected molecular pathways in the stroma as clinical targets. These findings can be a platform for further development of therapeutic strategies aiming at the blockade of bone marrow support to disseminated breast cancer cells. Stem Cells 2016;34:2224-2235.

Schwann cells induce cancer cell dispersion and invasion.

Nerves enable cancer progression, as cancers have been shown to extend along nerves through the process of perineural invasion, which carries a poor prognosis. Furthermore, the innervation of some cancers promotes growth and metastases. It remains unclear, however, how nerves mechanistically contribute to cancer progression. Here, we demonstrated that Schwann cells promote cancer invasion through direct cancer cell contact. Histological evaluation of murine and human cancer specimens with perineural invasion uncovered a subpopulation of Schwann cells that associates with cancer cells. Coculture of cancer cells with dorsal root ganglion extracts revealed that Schwann cells direct cancer cells to migrate toward nerves and promote invasion in a contact-dependent manner. Upon contact, Schwann cells induced the formation of cancer cell protrusions in their direction and intercalated between the cancer cells, leading to cancer cell dispersion. The formation of these processes was dependent on Schwann cell expression of neural cell adhesion molecule 1 (NCAM1) and ultimately promoted perineural invasion. Moreover, NCAM1-deficient mice showed decreased neural invasion and less paralysis. Such Schwann cell behavior reflects normal Schwann cell programs that are typically activated in nerve repair but are instead exploited by cancer cells to promote perineural invasion and cancer progression.

Interaction between Esophageal Squamous Cell Carcinoma and Adipose Tissue in Vitro

Esophageal squamous cell carcinoma (ESCC) develops within the squamous epithelial layer and invades the submucosa to the subadventitia that has adipose tissue (AT). AT seems critical to ESCC progression, but the underlying mechanism is unknown. We aimed to address the association between ESCC and AT invitro. ESCC cells were cultured on rat or human subcutaneous AT-embedded or -non-embedded collagen gel. AT promoted the growth of ESCC cells and inhibited their apoptosis. AT promoted the expression of the squamous differentiation marker involucrin in ESCC cells. AT accelerated the expression of invasion-related factors in poorly differentiated ESCC cells only. AT promoted the expression of phosphorylated-insulin-like growth factor-1 receptor in ESCC cells, whereas it inhibited that of the human epidermal growth factor receptor 2. Insulin-like growth factor-1, but not leptin, adiponectin, or resistin, promoted and inhibited the growth and apoptosis of ESCC cells, respectively. In turn, ESCC cells decreased the production of these adipokines in AT and the number of preadipocytes and mesenchymal stem cell-like cells, which developed from AT. These results suggest that i) AT may influence the progression of ESCC with increased growth or invasion and decreased apoptosis through insulin-like growth factor-1/insulin-like growth factor-1 receptor signaling, ii) AT may affect human epidermal growth factor receptor 2-targeted therapy; and iii) the cancer cells may affect adipokine production in AT.

P-166 Macrophage-Cancer Cell Fusion Potentiates Tumor Progression

Inflammatory bowel disease (IBD) afflicts more than one million Americans and although great strides have been made in disease management, insufficient knowledge exists about the increased risk of cancer and the more aggressive nature of colitis associated cancer. Specifically, it is not known how cancer cells acquire altered behaviors that propagate tumor progression. We showed that circulating macrophages (MФs) spontaneously fuse with intestinal cancer cells. This fusion is enhanced in an inflammatory microenvironment and results in cells (fusion hybrids) that retain both MФ and epithelial cell gene expression. While the underlying physiologic function of cell fusion cancer cells is not clear, we hypothesize that it ultimately results in cancer cells that retain macrophage-like behaviors, making them more responsive to cytokine, migratory and more successful in seeding distant metastatic organs. Two models for cell fusion: (1) in vitro co-culture of intestinal colorectal cancer cells and MФs that result in spontaneous fusion, and (2) in vivo fusion with marked MФs were employed to generate fusion hybrids and explore their physiologic behavior and query their expression profiles. We have found that fusion between MФ and cancer cells result in fusion hybrids that have enhanced adhesive and proliferative function. Further, these fusion hybrids have demonstrated an increased ability to migrate towards various cytokines based upon their genetic profile. Finally, fusion hybrids seed and grow in the liver with increased ability relative to unfused cancer cells. Intriguingly, our data suggest that MФ-cancer cell fusion represents an under-appreciated mechanism for reprogramming of the cancer cell genome leading to acquisition of non-epithelial behaviors (e.g., differences in adhesion, migration and seeding in metastatic sites). Our study provides a potential paradigm-shifting mechanism underlying how cancer cells in a highly inflammatory microenvironment more readily gain aggressive metastatic properties associated with lethal disease. We postulate that identification of novel gene expression in these fusion hybrids have promise to provide new biomarkers that could predict risk for cancer and identify new therapeutic options for abating fusion-associated cancer cell behavior.

Three-dimensional culture and interaction of cancer cells and dendritic cells in an electrospun nano-submicron hybrid fibrous scaffold.

An artificial three-dimensional (3D) culture system that mimics the tumor microenvironment in vitro is an essential tool for investigating the cross-talk between immune and cancer cells in tumors. In this study, we developed a 3D culture system using an electrospun poly(ε-caprolactone) (PCL) nanofibrous scaffold (NFS). A hybrid NFS containing an uninterrupted network of nano- and submicron-scale fibers (400 nm to 2 µm) was generated by deposition onto a stainless steel mesh instead of an aluminum plate. The hybrid NFS contained multiplanar pores in a 3D structure. Surface-seeded mouse CT26 colon cancer cells and bone marrow-derived dendritic cells (BM-DCs) were able to infiltrate the hybrid NFS within several hours. BM-DCs cultured on PCL nanofibers showed a baseline inactive form, and lipopolysaccharide (LPS)-activated BM-DCs showed increased expression of CD86 and major histocompatibility complex Class II. Actin and phosphorylated FAK were enriched where unstimulated and LPS-stimulated BM-DCs contacted the fibers in the 3D hybrid NFS. When BM-DCs were cocultured with mitoxantrone-treated CT26 cells in a 3D hybrid NFS, BM-DCs sprouted cytoplasm to, migrated to, synapsed with, and engulfed mitoxantrone-treated CT26 cancer cells, which were similar to the naturally occurring cross-talk between these two types of cells. The 3D hybrid NFS developed here provides a 3D structure for coculture of cancer and immune cells.

Abstract B58: Inflammatory signaling regulates osteoprotegerin expression in breast cancer cells

Abstract Osteoprotegerin (OPG) is a secreted member of the Tumor Necrosis Factor family. In addition to the functional roles of OPG in bone metabolism and apoptosis, there is growing evidence of the tumor promoting role of OPG in breast cancer. We have previously shown OPG can promote the invasion and metastasis of triple negative breast cancer cells. To understand the regulation of OPG, we are investigating the molecular mechanisms that control OPG expression in breast cancer cells. Breast cancer cell lines MDA-MB-231 and MDA-MB-436 (triple negative); SKBR-3 and HCC1954 (Human Epidermal Growth Factor Receptor 2 positive; HER2+); ZR75-1 and MCF-7 (Estrogen Receptor positive; ER+) were treated with Interleukin1beta (IL1-beta) for 24 hours. Levels of OPG mRNA and secreted OPG protein were analyzed by real time RT-PCR and ELISA, respectively. IL1-beta induced OPG expression regardless of basal OPG levels, with the exception of MDA-MB-231 which exhibited relatively moderate OPG induction. To explore the signaling mechanisms downstream of IL1-beta, we treated breast cancer cells with MAP kinase inhibitors in the presence of IL1-beta and examined the effects on OPG protein induction. Both p38 MAPK inhibitors, SB203580 and SB202190, were observed to partially blocked OPG induction by IL1-beta. Western blot analysis confirmed IL1-beta induced p38 phosphorylation. Additionally, we examined the effects of NFkappaB p65 binding to potential NFKappaB binding sites within the OPG promoter upon IL1-beta treatment by ChIP assay in SKBR3 and MDA-MB-436 cells. The highest fold increased binding was observed to occur within the -188 and -4675bp regions upon cytokine treatment in SKBR3 cells. No detectable binding was observed in MDA-MB-436 cells. We also looked at whether OPG expression levels could be altered by incubating breast cancer cells with macrophages, a cell type found in the breast tumor microenvironment. We co-cultured cells from each subtype with THP-1 human macrophages for 8 hours and extracted mRNA from the breast cancer cells or incubated the breast cancer cells for an additional 16 hours alone to measure secreted OPG protein. We found that co-culture with macrophages significantly increased OPG mRNA and protein expression in MCF-7 (ER+) and SKBR-3 (HER2+) cells but there was no increase in the MDA-MB-231 cells (triple negative). Overall, we show that IL1-beta can induce OPG expression in breast cancer cells and this signaling may be mediated by activating p38 MAP kinases. The ChIP analysis indicates signaling may involve activation of classical NFkappaB signaling. The similar effects of cytokine induced OPG induction observed in the co-culture of breast cancer cells with THP-1 suggests a mechanism by which inflammatory signaling could increase OPG expression in vivo. Citation Format: Stephanie Tsang Mui Chung, Ashleigh Renaud, Kim Roseman, Nalani Yadav, Linda Connelly. Inflammatory signaling regulates osteoprotegerin expression in breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B58.

Identification of novel 2-(1H-indol-1-yl)-benzohydrazides CXCR4 ligands impairing breast cancer growth and motility.

Stromal-derived-factor-1 (SDF-1) and the G-protein-coupled receptor CXCR4 are involved in several physiological and pathological processes including breast cancer spread and progression. Several CXCR4 antagonists have currently reached advanced development stages as potential therapeutic agents for different diseases. A small series of novel CXCR4 ligands, based on a 2-(1H-indol-1-yl)-benzohydrazide scaffold, has been designed and synthesized. The interaction with CXCR4-active site was predicted by molecular docking and confirmed by whole cell-based [(125)I]-SDF-1 ligand competition binding assays. One of the synthesized compounds was particularly active in blocking SDF-1-induced breast cancer cell motility, proliferation and downstream signaling activation in different breast cancer cell models and coculture systems. The newly synthesized compounds represent suitable leads for the development of innovative therapeutic agents targeting CXCR4.

How Little Is Too Much? - How Transient Tumor-Stromal Crosstalk Can Control Tumor Progression.

Tumorigenesis is driven by genetic and physiological alterations of tumor cells as well as by the host microenvironment. In a co-culture of breast cancer cells and fibroblasts, short term interactions between tumor cells and stromal fibroblasts increase levels of active, fibroblast derived TGF-β in the extracellular medium, which in turn induces an expanded metastatic pattern of MCF10CA1a cells. These findings suggest that the effects of stromal TGF-β on tumor cell phenotype can be modeled as a dynamical system rather than a continuous linear system. In such a model, small changes of certain parameters of a system that is at a critical point can cause sudden changes of the system, explaining why experimentally and clinically observed small changes in the tumor environment can cause dramatic changes in cell phenotype or disease outcome.