Tumor-derived Epithelial Cells Research Articles

The Inhibitory Activity of Lactobacillus plantarum Supernatant against Enterobacteria, Campylobacter, and Tumor Cells.

Cell-free supernatant of Lactobacillus plantarum exhibit a strong antimicrobial effect against a number of pathogenic enterobacteria (E. coli, Shigella flexneri, Salmonella typhimurium, Proteus mirabilis, and Campylobacter jejuni). The degree of growth inhibition in broth culture reached a high level for all tested bacteria. The highest rates were noted for P. mirabilis (by 13 times) and the lowest for S. flexneri (by 5 times) and C. jejuni (by 4.5 times). Significant antiproliferative effect of the supernatant on cells of tumor-derived epithelial cell lines was shown. The highest degree of inhibition (by 22 times) was observed for HT-29 cells (colon carcinoma). Thus, inclusion of probiotics in traditional treatment schemes can increase the effectiveness of antibacterial and antitumor drug therapy.

The Emerging Role of Tissue-Resident Memory CD8+ T Lymphocytes in Human Digestive Tract Cancers.

Malignant digestive tract tumors are a great threat to human public health. In addition to surgery, immunotherapy brings hope for the treatment of these tumors. Tissue-resident memory CD8+ T (Trm) cells are a focus of tumor immunology research and treatment due to their powerful cytotoxic effects, ability to directly kill epithelial-derived tumor cells, and overall impact on maintaining mucosal homeostasis and antitumor function in the digestive tract. They are a group of noncirculating immune cells expressing adhesion and migration molecules such as CD69, CD103, and CD49a that primarily reside on the barrier epithelium of nonlymphoid organs and respond rapidly to both viral and bacterial infection and tumorigenesis. This review highlights new research exploring the role of CD8+ Trm cells in a variety of digestive tract malignant tumors, including esophageal cancer, gastric cancer, colorectal cancer, and hepatocellular carcinoma. A summary of CD8+ Trm cell phenotypes and characteristics, tissue distribution, and antitumor functions in different tumor environments is provided, illustrating how these cells may be used in immunotherapies against digestive tract tumors.

Decreased invariant natural killer T-cell-mediated antitumor immune response in patients with gastric cancer.

Gastric cancer (GC) is the third most common cause of cancer-related death worldwide. Invariant natural killer T (iNKT) cells are innate-like cytotoxic T lymphocytes involved in tumor immune surveillance. They can be activated either through CD1d-presented glycolipid antigens recognized by their invariant T-cell receptor, cytokines or by sensing tumor-associated stress-induced ligands through the natural killer group 2, member D (NKG2D) receptor. Although the number and functionality of iNKT cells may be decreased in several types of cancer, here we show that GC patients presented a mild increase in iNKT cell frequencies and numbers in the blood compared with healthy donors. In GC patients, iNKT cells, expanded in vitro with α-galactosyl ceramide and stimulated with phorbol 12-myristate 13-acetate and ionomycin, produced higher levels of interleukin-2 and transforming growth factor-beta, while their capacity to degranulate remained preserved. Because tumor-derived epithelial cell adhesion molecule-positive epithelial cells did not display surface CD1d, and NKG2D ligands (NKG2DLs) were detected in the gastric tumor milieu, we envisioned a role for NKG2D in iNKT cell functions. Peripheral iNKT cells from GC patients and controls presented similar levels of NKG2D; nevertheless, the percentages of interferon-γ-producing and CD107a-positive iNKT cells from patients were reduced upon challenge with CD1d-negative, NKG2DL-positive K562 cells, suggesting a compromised response by iNKT cells in GC patients, which may not result from impaired NKG2D/NKG2DL signaling. The decreased response of iNKT cells may explain the fact that higher frequencies of circulating iNKT cells did not confer a survival benefit for GC patients. Therefore, functional impairment of iNKT cells in GC may contribute to tumor immune escape and favor disease progression.

MEF2 plays a significant role in the tumor inhibitory mechanism of encapsulated RENCA cells via EGF receptor signaling in target tumor cells

BackgroundAgarose encapsulated murine renal adenocarcinoma cells (RENCA macrobeads) are currently being investigated in clinical trials as a treatment for therapy-resistant metastatic colorectal cancer. We have previously demonstrated the capacity of RENCA macrobeads to produce diffusible substances that markedly inhibit the proliferation of epithelial-derived tumor cells outside the macrobead environment. This study examined the molecular mechanisms underlying the observed inhibition in targeted tumor cells exposed to RENCA macrobeads.MethodsWe evaluated changes in transcription factor responses, participating intracellular signaling pathways and the involvement of specific cellular receptors in targeted tumor cells exposed to RENCA macrobeads.ResultsFactors secreted by RENCA macrobeads significantly up-regulated the activity of the MEF2 transcription factor as well as altered the transcription of MEF2b and MEF2d isoforms in targeted tumor cells. Suppression of individual or multiple MEF2 isoforms in target tumor cells markedly reduced the growth inhibitory effects of RENCA macrobeads. Furthermore, these effects were linked to the activation of the EGF receptor as attenuation of EGFR resulted in a substantial reduction of the cancer cell growth-inhibitory effect.ConclusionsSince interruption of the EGFR signaling cascade did not eliminate RENCA macrobead-induced growth control, our data suggests that RENCA macrobeads exert their full growth inhibitory effects through the simultaneous activation of multiple signaling pathways. In contrast to a precision medicine approach targeting single molecular abnormalities, the RENCA macrobead functions as a biological-systems therapy to re-establish regulation in a highly dysfunctional and dysregulated cancer system.

Functional impairment triggered by altertoxin II (ATXII) in intestinal cells in vitro: cross-talk between cytotoxicity and mechanotransduction

Intestinal cells are able to continuously integrate response to multiple stimuli/stressors; these include the concomitant activation of “chemically driven” pathways, of paramount importance in the response to toxicants, as well as physical stimulation derived from motility. Altertoxin II (ATXII, 0.1, 1 and 10 µM), a mycotoxin produced by the food contaminant fungus Alternaria alternata was studied in HT-29 intestinal adenocarcinoma cells and in non-transformed intestinal epithelial cells, HCEC. One-hour incubation with ATXII was sufficient to trigger irreversible cytotoxicity in both cell types, as well as to modify cellular responses to concomitant pro-oxidant challenge (H2O2, 100–500 µM, DCF-DA assay) suggesting that even relatively short-time exposure of the intestinal cells could be sufficient to alter their functionality. Combination of ATXII (1 µM) with physical stimulation typical of the intestinal compartment (shear stress) revealed differential response of tumor-derived epithelial cells HT-29 in comparison to HCEC, in particular in the localization of the transcription factor Nrf2 (NF-E2-related factor 2). Moreover, ATXII reduced the migratory potential of HCEC as well as their membrane fluidity, but had no respective impact on HT-29 cells. Taken together, ATXII appeared to alter predominantly membrane functionality in HCEC thus hampering crucial functions for cellular motility/turnover, as well as barrier function of healthy intestinal cells and had very limited activity on the tumor counterparts.

The metal-nonoate Ni(SalPipNONO) inhibits in vitro tumor growth, invasiveness and angiogenesis.

Nitric oxide (NO) exerts conflicting effect on tumor growth and progression, depending on its concentration. We aimed to characterize the anti-cancer activity of a new NO donor, Ni(SalPipNONO) belonging to the class of metal-nonoates, in epithelial derived tumor cells, finally exploring its antiangiogenic properties. Tumor epithelial cells were screened to evaluate the cytotoxic effect of Ni(SalPipNONO), which was able to inhibit cell proliferation in a dose dependent manner, being more effective than the commercial DETA/NO. The human lung carcinoma cells A549 were chosen as model to study the anti-cancer mechanisms exerted by the compound. In these cells, Ni(SalPipNONO) inhibited clonogenicity and cell invasion, while promoting apoptosis. The antitumor activity was partly due to NO-cGMP dependent pathway, contributing to reduced cell number and apoptosis, and partly to the salicylaldehyde moiety and reactive oxygen species (ROS) activated ERK1/2 signaling converging on p53 dependent caspase-3 cleavage. An additional contribution by downstream cycloxygenase-2 (COX-2) derived cyclopentenones may explain the tumor inhibitory activities. As NO has been described to affect tumor angiogenesis, we checked this activity both on tumor and endothelial cell co-cultures and in Matrigel in vivo assay. Our data document that Ni(SalPipNONO) was able to both reduce angiogenic factor expression by tumor cells acting on hypoxia inducible factor-1α (HIF-1 α) level, and endothelial cell functions related to angiogenesis. Collectively, these data confirm the potential use of NO donors and in particular Ni(SalPipNONO) acting through multiple mechanisms, as an agent to be further developed to be used alone or in combination with conventional therapy.

Fibronectin Modulates Cell Adhesion and Signaling to Promote Single Cell Migration of Highly Invasive Oral Squamous Cell Carcinoma.

Cell migration is regulated by adhesion to the extracellular matrix (ECM) through integrins and activation of small RhoGTPases, such as RhoA and Rac1, resulting in changes to actomyosin organization. During invasion, epithelial-derived tumor cells switch from laminin-enriched basal membrane to collagen and fibronectin-enriched connective tissue. How this switch affects the tumor migration is still unclear. We tested the hypothesis that ECM dictates the invasiveness of Oral Squamous Cell Carcinoma (OSCC). We analyzed the migratory properties of two OSCC lines, a low invasive cell line with high e-cadherin levels (Linv/HE-cad) or a highly invasive cell line with low e-cadherin levels (Hinv/LE-cad), plated on different ECM components. Compared to laminin, fibronectin induced non-directional collective migration and decreased RhoA activity in Linv/HE-cad OSCC. For Hinv/LE-cad OSCC, fibronectin increased Rac1 activity and induced smaller adhesions, resulting in a fast single cell migration in both 2D and 3D environments. Consistent with these observations, human OSCC biopsies exhibited similar changes in cell-ECM adhesion distribution at the invasive front of the tumor, where cells encounter fibronectin. Our results indicate that ECM composition might induce a switch from collective to single cell migration according to tumor invasiveness due to changes in cell-ECM adhesion and the resulting signaling pathways that alter actomyosin organization.

Noncoding RNAs in Tumor Epithelial-to-Mesenchymal Transition.

Epithelial-derived tumor cells acquire the capacity for epithelial-to-mesenchymal transition (EMT), which enables them to invade adjacent tissues and/or metastasize to distant organs. Cancer metastasis is the main cause of cancer-related death. Molecular mechanisms involved in the switch from an epithelial phenotype to mesenchymal status are complicated and are controlled by a variety of signaling pathways. Recently, a set of noncoding RNAs (ncRNAs), including miRNAs and long noncoding RNAs (lncRNAs), were found to modulate gene expressions at either transcriptional or posttranscriptional levels. These ncRNAs are involved in EMT through their interplay with EMT-related transcription factors (EMT-TFs) and EMT-associated signaling. Reciprocal regulatory interactions between lncRNAs and miRNAs further increase the complexity of the regulation of gene expression and protein translation. In this review, we discuss recent findings regarding EMT-regulating ncRNAs and their associated signaling pathways involved in cancer progression.

Abstract 4609: MEF2 plays a critical role in RENCA macrobead-induced tumor cell growth inhibition

Abstract Regulation of cell proliferation and cell death is often dependent on the coupling of extracellular signals to the activation or repression of specific intracellular signaling pathways. We have previously demonstrated the capacity of mouse renal adenocarcinoma (RENCA) cells encapsulated in agarose macrobeads to form numerous small tumor colonies within the macrobeads over a period of several months. As the encapsulated tumor colonies mature and reach a maximum size, the macrobeads themselves, or conditioned media from cultured macrobeads, markedly inhibit the in vitro and in vivo growth of epithelial-derived tumor cells outside the macrobead microenvironment. Myocyte enhancer factor 2 (MEF2) is a family of four transcription factors that have been shown to play a central role in linking signaling pathways to the genes responsible for cell division, differentiation and death. In this study, we evaluated the expression and role of MEF2 in regulating cell proliferation in mouse (RENCA) and human cell lines (J82, MCF7) in response to RENCA macrobeads. Freely growing RENCA target cells transiently transfected with a MEF2 transcriptional response element and exposed to conditioned media from variously aged macrobeads demonstrated increased response element activation in parallel with the increasing age of the macrobeads. Similarly, growth inhibition of the freely growing RENCA cells increased with older macrobeads. Suppression of the expressed MEF2 isoforms in target RENCA cells, either individually (MEF2a, MEF2b, and MEF2d) or in combination (MEF2pool) using synthetic small interfering RNA (siRNA) markedly reduced the growth inhibitory effects of RENCA macrobeads. In J82 and MCF7 cell lines, MEF2D expression was responsive to replete media from RENCA macrobeads, increasing 2.0-fold and 2.7-fold respectively. Degradation of MEF2D mRNA transcripts in the human cell lines studied resulted in abatement of the growth inhibitory effect of the RENCA macrobeads. These findings reveal an essential role for MEF2 in RENCA macrobead-induced cancer cell growth inhibition and raise interesting questions about the molecular basis of this response. Citation Format: Prithy C. Martis, Atira Dudley, Melissa A. Laramore, Barry H. Smith, Lawrence S. Gazda. MEF2 plays a critical role in RENCA macrobead-induced tumor cell growth inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4609. doi:10.1158/1538-7445.AM2014-4609

The acetylenic tricyclic bis(cyano enone), TBE-31 inhibits non-small cell lung cancer cell migration through direct binding with actin.

The migratory and invasive potential of the epithelial-derived tumor cells depends on epithelial-to-mesenchymal transition (EMT) as well as the reorganization of the cell cytoskeleton. Here, we show that the tricyclic compound acetylenic tricyclic bis(cyano enone), TBE-31, directly binds to actin and inhibits linear and branched actin polymerization in vitro. Furthermore, we observed that TBE-31 inhibits stress fiber formation in fibroblasts as well as in non-small cell lung cancer cells during TGFβ-dependent EMT. Interestingly, TBE-31 does not interfere with TGFβ-dependent signaling or changes in E-cadherin and N-cadherin protein levels during EMT. Finally, we observed that TBE-31 inhibits fibroblast and non-small cell lung tumor cell migration with an IC50 of 1.0 and 2.5 μmol/L, respectively. Taken together, our results suggest that TBE-31 targets linear actin polymerization to alter cell morphology and inhibit cell migration.

The adenocarcinoma cell surface mucin receptor for alpha-fetoprotein: is the same receptor present on circulating monocytes and macrophages? A commentary.

The mucin family of proteins is largely expressed on sedentary epithelial cells lining the gastrointestinal, pulmonary, and reproductive tracts and their associated organs and malignant tumors. It is less well-known that mucins are also expressed on circulatory cells of the immune and inflammatory systems, such as monocytes, macrophages, leukemic, and lymphoma cells. The epithelial mucins function in (a) protection and lubrication of mucosal linings, (b) cell adhesion and cell-to-cell contact, (c) cell migration and metastasis, and (d) signal transduction. It would be logical to presume that mucins expressed on circulating mononuclear cells could perform similar functions. Recently, it was proposed that the alpha-fetoprotein (AFP) receptor, known to be present on solid epithelial-derived malignant tumor cells, can be identified as a mucin glycoprotein. Interestingly, it was also reported that AFP binds to a receptor on circulating cells and sedentary tumor cells of lymphoreticular origin, especially monocytes associated with lymphomas and leukemias. The primary objective of the present commentary is to present literature-based evidence that some of the cell surface mucins on sedentary epithelial tumor cells and certain mucins expressed on circulating monocytes/macrophages are identical to the AFP receptor. The secondary objective is to discuss the role of AFP and its derived peptides in the growth suppression of adenocarcinomas and lymphomas using the AFP-mucin receptor concept as a key to the mechanism of tumor growth inhibition.

HPV16 E2-mediated potentiation of NF-κB activation induced by TNF-α involves parallel activation of STAT3 with a reduction in E2-induced apoptosis

Human papilloma virus is associated with cervical and other tumors, and several cellular conditions also play an important role in carcinogenesis. Human papilloma virus (HPV)-infected cells exhibit activation of NF-κB and STAT3 (mediators of inflammation), but little is known about their regulation by HPV. This study attempts to understand the role of HPV16 E2, an important early protein of HPV16, in the regulation of NF-κB and STAT3 by reporter assays, quantitative reverse transcriptase-polymerase chain reaction, and immunoblotting. We demonstrate that E2 enhances NF-κB activation induced by TNF-α, a proinflammatory cytokine, in both non-tumor- and tumor-derived epithelial cell lines besides potentiating STAT3 transcriptional activity induced by TNF-α in HEK293 cells. E2 increases the expression of RelA and its transcriptional activation, and retention of E2 was observed in the nucleus with significant interaction with RelA (immunoprecipitation) upon TNF-α treatment. Transfection with shRNA-RelA or pretreatment with a STAT3 inhibitor had a negative effect on the ability of E2 to enhance TNF-α-induced NF-κB activation. Experiments with co-expression of a mutant of STAT3 with E2 also suggested that the activation of STAT3 is indispensible for TNF-α-induced NF-κB activation. Inhibition of STAT3 activation enhanced E2-induced apoptosis, whereas parallel activation of NF-κB and STAT3 by the combined action of E2 and TNF-α increased the expression of their common targets, cyclin-D1, c-Myc, survivin, and Bcl-2, leading to a decrease in E2-induced apoptosis (viability and cell cycle). Our results reveal novel mechanisms by which E2 may regulate NF-κB and STAT3 activation in the presence of TNF-α with implications on the survival of HPV-infected cells.

MLK3 regulates fMLP-stimulated neutrophil motility

IntroductionMixed lineage kinase 3 (MLK3) is part of the intracellular regulatory system that connects extracellular cytokine or mitogen signals received through G-protein coupled receptors to changes in gene expression. MLK3 activation stimulates motility of epithelial cells and epithelial-derived tumor cells, but its role in mediating the migration of other cell types remains unknown. Since neutrophils play a crucial role in innate immunity and contribute to the pathogenesis of several diseases, we therefore examined whether MLK3 might regulate the motility of mouse neutrophils responding to a chemotactic stimulus, the model bacterial chemoattractant fMLP. MethodsThe expression of Mlk3 in mouse neutrophils was determined by immunocytochemistry and by RT-PCR. In vitro chemotaxis in a gradient of fMLP, fMLP-stimulated random motility, fMLP-stimulated F-actin formation were measured by direct microscopic observation using neutrophils pre-treated with a novel small molecule inhibitor of MLK3 (URMC099) or neutrophils obtained from Mlk3−/− mice. In vivo effects of MLK3 inhibition were measured by counting the fMLP-induced accumulation of neutrophils in the peritoneum following pre-treatment with URMC099 in wild-type C57Bl/6 or mutant Mlk3−/− mice. ResultsThe expression of Mlk3 mRNA and protein was observed in neutrophils purified from wild-type C57Bl/6 mice but not in neutrophils from mutant Mlk3−/− mice. Chemotaxis by wild-type neutrophils induced by a gradient of fMLP was reduced by pre-treatment with URMC099. Neutrophils from C57Bl/6 mice pretreated with URMC099 and neutrophils from Mlk3−/− mice moved far less upon fMLP-stimulation and did not form F-actin as readily as untreated neutrophils from C57Bl/6 controls. In vivo recruitment of neutrophils into the peritoneum by fMLP was significantly reduced in wild-type mice treated with URMC099, as well as in untreated Mlk3−/− mice—thereby confirming the role of MLK3 in neutrophil migration. ConclusionsMlk3 mRNA is expressed in murine neutrophils. Genetic or pharmacologic inhibition of MLK3 blocks fMLP-mediated motility of neutrophils both in vitro and in vivo, suggesting that MLK3 may be a therapeutic target in human diseases characterized by exuberant neutrophil migration.

Wnt5a signaling promotes apical and basolateral polarization of single epithelial cells

Single epithelial-derived tumor cells have been shown to induce apical and basolateral (AB) polarity by expression of polarization-related proteins. However, physiological cues and molecular mechanisms for AB polarization of single normal epithelial cells are unclear. When intestinal epithelial cells 6 (IEC6 cells) were seeded on basement membrane proteins (Matrigel), single cells formed an F-actin cap on the upper cell surface, where apical markers accumulated, and a basolateral marker was localized to the rest of the cell surface region, in a Wnt5a signaling-dependent manner. However, these phenotypes were not induced by type I collagen. Rac1 activity in the noncap region was higher than that in the cap region, whereas Rho activity increased toward the cap region. Wnt5a signaling activated and inhibited Rac1 and RhoA, respectively, independently through Tiam1 and p190RhoGAP-A, which formed a tertiary complex with Dishevelled. Furthermore, Wnt5a signaling through Rac1 and RhoA was required for cystogenesis of IEC6 cells. These results suggest that Wnt5a promotes the AB polarization of IEC6 cells through regulation of Rac and Rho activities in a manner dependent on adhesion to specific extracellular matrix proteins.

Genetic reconstitution of tumorigenesis in primary intestinal cells

Animal models for human colorectal cancer recapitulate multistep carcinogenesis that is typically initiated by activation of the Wnt pathway. Although potential roles of both genetic and environmental modifiers have been extensively investigated in vivo, it remains elusive whether epithelial cells definitely require interaction with stromal cells or microflora for tumor development. Here we show that tumor development could be simply induced independently of intestinal microenvironment, even with WT murine primary intestinal cells alone. We developed an efficient method for lentiviral transduction of intestinal organoids in 3D culture. Despite seemingly antiproliferative effects by knockdown of adenomatous polyposis coli (APC), we managed to reproducibly induce APC-inactivated intestinal organoids. As predicted, these organoids were constitutively active in the Wnt signaling pathway and proved tumorigenic when injected into nude mice, yielding highly proliferative tubular epithelial glands accompanied by prominent stromal tissue. Consistent with cellular transformation, tumor-derived epithelial cells acquired sphere formation potential, gave rise to secondary tumors on retransplantation, and highly expressed cancer stem cell markers. Inactivation of p53 or phosphatase and tensin homolog deleted from chromosome 10, or activation of Kras, promoted tumor development only in the context of APC suppression, consistent with earlier genetic studies. These findings clearly indicated that genetic cooperation for intestinal tumorigenesis could be essentially recapitulated in intestinal organoids without generating gene-modified mice. Taken together, this in vitro model for colon cancer described herein could potentially provide unique opportunities for carcinogenesis studies by serving as a substitute or complement to the currently standard approaches.

Abstract P1-07-16: Liver derived epithelial cells as source of false positive circulating tumor cells in early breast cancer

Abstract The MAINTRAC technique as introduced by our coworkers from Jena (RBC lysis, fluorometric detection and analysis on Olympius ScanR) detects more circulating epithelial cells than techniques using enrichment. Also cells with a low EPCAM expression are detected and not only the typical cells with bright expression found after immunomagnetic enrichment. The relative cheapness and reproducibility allows frequent monitoring during and after therapy Using 3 colour detection (EPCAMfitc, DAPI, Vimentin PE) living and dead circulating epithelial cells in EMT, or cells in EMT with stemcell markers (EPCAMfitc, Vimentin-PE, CD44PacBlue) can be detected. In early breast cancer (n = 135) cells can be found in 60% of patients and in 40% higher cell counts (>100 ml are detectable. A control population(n = 100) showed low numbers in 98% (e.g (<100 CECin 1 ml blood). Expression of the mesenchymal marker (vimentin) ranges between 10 and 40% with different expression. CD44 shows also a wide range of expression. Two main cell types can be distinguished: type 1 shows generalized but weaker expression patterns and a second type with very bright dotted expression. The clinical relevance of these subsets is not known and their behavior under therapy has not been analysed in depth yet. In advanced breast cancers high cell counts were detectable in most patients with a less agressive disease course. In the rapidly progressing unfavorable subtypes (TN and Her2+. HRneg) less or none cells were found. During crossvalidation in non cancer patients we found high cell numbers in several forms of liver affections (n = 108). The expression patterns of markers on these cells were not differing from those in cancer patients. So this same cell type merging EMT, stemcell an hypoxic stress markers is detectable in advanced and early breast cancer (n = 40) and in benign disease. These cells disappear or decrease after response to chemo or anti-hormonal therapy in cancer or antioxidant therapy in NAFLD. We believe that the evasion of these cells is driven by the same force in cancer as in non cancer conditions. We suggest that this are the wellknown hypoxic and hyperacidic conditions causing epithelial mesenchymal transition. Cancer cell hijack this functions occurring normally under these conditions to survive and to facilitate evasion. More comprehensive analysis (four colour analysis on the AMNIS Flowsight) is needed and should show differences in expression patterns of liver derived epithelial cells (LDEC) and real tumor derived epithelial cells (TDEC). Further clarification of these phenomena should give new insights of the biological events in early disease and the possibilities and reliability of “fluid biopsy”. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-16.

At the crossroads: EGFR and PTHrP signaling in cancer-mediated diseases of bone

The epidermal growth factor receptor is a well-established cancer therapeutic target due to its stimulation of proliferation, motility, and resistance to apoptosis. Recently, additional roles for the receptor have been identified in growth of metastases. Similar to development, metastatic spread requires signaling interactions between epithelial-derived tumor cells and mesenchymal derivatives of the microenvironment. This necessitates reactivation of developmental signaling molecules, including the hypercalcemia factor parathyroid hormone-related protein. This review covers the variations of epidermal growth factor receptor signaling in cancers that produce bone metastases, regulation of parathyroid hormone-related protein, and evidence that the two molecules drive cancer-mediated diseases of bone.

Diagnostic microchip to assay 3D colony-growth potential of captured circulating tumor cells

Microfluidic technology has been successfully applied to isolate very rare tumor-derived epithelial cells (circulating tumor cells, CTCs) from blood with relatively high yield and purity, opening up exciting prospects for early detection of cancer. However, a major limitation of state-of-the-art CTC-chips is their inability to characterize the behavior and function of captured CTCs, for example to obtain information on proliferative and invasive properties or, ultimately, tumor re-initiating potential. Although CTCs can be efficiently immunostained with markers reporting phenotype or fate (e.g. apoptosis, proliferation), it has not yet been possible to reliably grow captured CTCs over long periods of time and at single cell level. It is challenging to remove CTCs from a microchip after capture, therefore such analyses should ideally be performed directly on-chip. To address this challenge, we merged CTC capture with three-dimensional (3D) tumor cell culture on the same microfluidic platform. PC3 prostate cancer cells were isolated from spiked blood on a transparent PDMS CTC-chip, encapsulated on-chip in a biomimetic hydrogel matrix (QGel™) that was formed in situ, and their clonal 3D spheroid growth potential was assessed by microscopy over one week in culture. The possibility to clonally expand a subset of captured CTCs in a near-physiological in vitro model adds an important element to the expanding CTC-chip toolbox that ultimately should improve prediction of treatment responses and disease progression.

In vivo gene manipulations of epithelial cell sheets: A novel model to study epithelial‐to‐mesenchymal transition

Embryonic cells are classified into two types of cells by their morphology, epithelial and mesenchymal cells. During dynamic morphogenesis in development, epithelial cells often switch to mesenchymal by the process known as epithelial-to-mesenchymal transition (EMT). EMT is a central issue in cancer metastasis where epithelial-derived tumor cells are converted to mesenchymal with high mobility. Although many molecules have been identified to be involved in the EMT mostly by in vitro studies, in vivo model systems have been limited. We here established a novel model with which EMT can be analyzed directly in the living body. By an electroporation technique, we targeted a portion of the lateral plate mesoderm that forms epithelial cell sheets delineating the kidney region, called nephric coelomic epithelium (Neph-CE). Enhanced green fluorescent protein-electroporated Neph-CE retained the epithelial integrity without invading into the underling stroma (mesonephros). The Neph-CE transgenesis further allowed us to explore EMT inducers in vivo, and to find that Ras-Raf and RhoA signals were potent inducers. Live-imaging confocal microscopy revealed that during EMT processes cells started extending cellular protrusions toward the stroma, followed by translocation of their cell bodies. Furthermore, we established a long-term tracing of EMT-induced cells, which were dynamically relocated within the kidney stroma. The Neph-CE-transgenesis will open a way to study cellular and molecular mechanisms underlying EMT directly in actual body.

Hydrophilic Agarose Macrobead Cultures Select for Outgrowth of Carcinoma Cell Populations That Can Restrict Tumor Growth

Cancer cells and their associated tumors have long been considered to exhibit unregulated proliferation or growth. However, a substantial body of evidence indicates that tumor growth is subject to both positive and negative regulatory controls. Here, we describe a novel property of tumor growth regulation that is neither species nor tumor-type specific. This property, functionally a type of feedback control, is triggered by the encapsulation of neoplastic cells in a growth-restricting hydrogel composed of an agarose matrix with a second coating of agarose to form 6- to 8-mm diameter macrobeads. In a mouse cell model of renal adenocarcinoma (RENCA cells), this process resulted in selection for a stem cell-like subpopulation which together with at least one other cell subpopulation drove colony formation in the macrobeads. Cells in these colonies produced diffusible substances that markedly inhibited in vitro and in vivo proliferation of epithelial-derived tumor cells outside the macrobeads. RENCA cells in monolayer culture that were exposed to RENCA macrobead-conditioned media exhibited cell-cycle accumulation in S phase due to activation of a G(2)/M checkpoint. At least 10 proteins with known tumor suppression functions were identified by analysis of RENCA macrobead-conditioned media, the properties of which offer opportunities to further dissect the molecular basis for tumor growth control. More generally, macrobead culture may permit the isolation of cancer stem cells and other cells of the stem cell niche, perhaps providing strategies to define more effective biologically based clinical approaches to treat neoplastic disease.