The advantages of co-culture over mono cell culture in simulating in vivo environment

Breast cancer tissue consists of both carcinoma cells and stromal cells, and intratumoral stroma is composed of various cell types such as fibroblasts, adipocytes, inflammatory including lymphocytes and macrophage and lymphatic and blood capillaries including pericytes and endothelial cells. Recently, cell-cell communications or interactions among these cells have been considered to play an important role to cancer initiation, promotion, and progression. In particular, intratumoral fibroblasts are well known as cancer-associated fibroblast (CAF). CAF is considered to be different from normal fibroblasts in terms of promoting cancer progression through the cytokine signals. Carcinoma cell lines have contributed to the advancement of our understanding of cancer cell biology. Numerous researches have employed these carcinoma cell lines as a single- or mono-culture. However, it is also true that this mono-culture system cannot evaluate interactions between carcinoma and intratumoral stromal cells. Co-culture compositions of two different cell type of cancer tissues i.e., carcinoma cell lines and fibroblasts, were established in order to evaluate cell-cell interactions in these cancer microenvironment. This co-culture condition has the advantage of evaluating cell-cell interactions of cancer microenvironment. Therefore, in this review, we focused upon co-culture system and its application to understanding of various biological phenomenon as an ex vivo evaluation method of cancer microenvironment in breast cancer.     

Enhancement of Apoptotic and Autophagic Induction by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin in Human Breast Adenocarcinoma and Neuroblastoma Cells with Tamoxifen

Breast cancer is one of the most common cancers amongst women in North America. Many current anti-cancer treatments, including ionizing radiation, induce apoptosis via DNA damage. Unfortunately, such treatments are non-selective to cancer cells and produce similar toxicity in normal cells. We have reported selective induction of apoptosis in cancer cells by the natural compound pancratistatin (PST). Recently, a novel PST analogue, a C-1 acetoxymethyl derivative of 7-deoxypancratistatin (JCTH-4), was produced by de novo synthesis and it exhibits comparable selective apoptosis inducing activity in several cancer cell lines. Recently, autophagy has been implicated in malignancies as both pro-survival and pro-death mechanisms in response to chemotherapy. Tamoxifen (TAM) has invariably demonstrated induction of pro-survival autophagy in numerous cancers. In this study, the efficacy of JCTH-4 alone and in combination with TAM to induce cell death in human breast cancer (MCF7) and neuroblastoma (SH-SY5Y) cells was evaluated. TAM alone induced autophagy, but insignificant cell death whereas JCTH-4 alone caused significant induction of apoptosis with some induction of autophagy. Interestingly, the combinatory treatment yielded a drastic increase in apoptotic and autophagic induction. We monitored time-dependent morphological changes in MCF7 cells undergoing TAM-induced autophagy, JCTH-4-induced apoptosis and autophagy, and accelerated cell death with combinatorial treatment using time-lapse microscopy. We have demonstrated these compounds to induce apoptosis/autophagy by mitochondrial targeting in these cancer cells. Importantly, these treatments did not affect the survival of noncancerous human fibroblasts. Thus, these results indicate that JCTH-4 in combination with TAM could be used as a safe and very potent anti-cancer therapy against breast cancer and neuroblastoma cells.     

Prostate cancer cell survival pathways activated by bone metastasis microenvironment

The development of resistance to anti-cancer therapies in bones is a major hurdle preventing long-lasting clinical responses to anti-cancer therapies in hormone refractory prostate cancer. Herein, we present the major signal transduction pathways, which are activated in prostate cancer cells residing at bone metastasis microenvironment. These intracellular signal transduction pathways can inhibit anti-cancer therapy-induced apoptosis of metastatic prostate cancer cells, thereby optimizing their survival, locally. Employment of this knowledge in a clinical setting provides the conceptual framework for the development of bone-targeted therapies for advanced prostate cancer. Indeed, bone metastasis microenvironment-targeted therapies illustrate a novel paradigm in cancer treatment: anti-tumor treatment strategies may not only aim at directly inducing cancer cell apoptosis, but can also target the tumor metastasis microenvironment, and neutralize the protection it confers on metastatic cancer cells.     

Modulation of host cell mechanics by Trypanosoma cruzi

To investigate the effects of Trypanosoma cruzi on the mechanical properties of infected host cells, cytoskeletal stiffness and remodeling dynamics were measured in parasite-infected fibroblasts. We find that cell stiffness decreases in a time-dependent fashion in T. cruzi-infected human foreskin fibroblasts without a significant change in the dynamics of cytoskeletal remodeling. In contrast, cells exposed to T. cruzi secreted/released components become significantly stiffer within 2 h of exposure and exhibit increased remodeling dynamics. These findings represent the first direct mechanical data to suggest a physical picture in which an intact, stiff, and rapidly remodeling cytoskeleton facilitates early stages of T. cruzi invasion and parasite retention, followed by subsequent softening and disassembly of the cytoskeleton to accommodate intracellular replication of parasites. We further suggest that these changes occur through protein kinase A and inhibition of the Rho/Rho kinase signaling pathway. In the context of tissue infection, changes in host cell mechanics could adversely affect the function of the infected organs, and may play an important role on the pathophysiology of Chagas' disease.     

Intracellular Mechanics and Activity of Breast Cancer Cells Correlate with Metastatic Potential

Mechanics of cancer cells are directly linked to their metastatic potential, or ability to produce a secondary tumor at a distant site. Metastatic cells survive in the circulatory system in a non-adherent state, and can squeeze through barriers in the body. Such considerable structural changes in cells rely on rapid remodeling of internal structure and mechanics. While external mechanical measurements have demonstrated enhanced pliability of cancer cells with increased metastatic potential, little is known about dynamics of their interior and we expect that to change significantly in metastatic cells. We perform a comparative study, using particle-tracking to evaluate the intracellular mechanics of living epithelial breast cells with varying invasiveness. Particles in all examined cell lines exhibit super-diffusion with a scaling exponent of 1.4 at short lag times, likely related to active transport by fluctuating microtubules and their associated molecular motors. Specifics of probe-particle transport differ between the cell types, depending on the cytoskeleton network-structure and interactions with it. Our study shows that the internal microenvironment of the highly metastatic cells evaluated here is more pliable and their cytoskeleton is less dense than the poorly metastatic and benign cells. We thus reveal intracellular structure and mechanics that can support the unique function and invasive capabilities of highly metastatic cells.     

Cytokine production and inflammation drive autophagy in the tumor microenvironment

Recently, we proposed a new paradigm for understanding the role of the tumor microenvironment in breast cancer onset and progression. In this model, cancer cells induce oxidative stress in adjacent fibroblasts. This, in turn, results in the onset of stromal autophagy, which produces recycled nutrients to “feed” anabolic cancer cells. However, it remains unknown how autophagy in the tumor microenvironment relates to inflammation, another key driver of tumorigenesis. To address this issue, here we employed a well-characterized co-culture system in which cancer cells induce autophagy in adjacent fibroblasts via oxidative stress and NFκB-activation. We show, using this co-culture system, that the same experimental conditions that result in an autophagic microenvironment, also drive in the production of numerous inflammatory mediators (including IL-6, IL-8, IL-10, MIP1a, IFNg, RANTES (CCL5) and GMCSF). Furthermore, we demonstrate that most of these inflammatory mediators are individually sufficient to directly induce the onset of autophagy in fibroblasts. To further validate the in vivo relevance of these findings, we assessed the inflammatory status of Cav-1 (-/-) null mammary fat pads, which are a model of a bonafide autophagic microenvironment. Notably, we show that Cav-1 (-/-) mammary fat pads undergo infiltration with numerous inflammatory cell types, including lymphocytes, T-cells, macrophages and mast cells. Taken together, our results suggest that cytokine production and inflammation are key drivers of autophagy in the tumor microenvironment. These results may explain why a loss of stromal Cav-1 is a powerful predictor of poor clinical outcome in breast cancer patients, as it is a marker of both (1) autophagy and (2) inflammation in the tumor microenvironment. Lastly, hypoxia in fibroblasts was not sufficient to induce the full-blown inflammatory response that we observed during the co-culture of fibroblasts with cancer cells, indicating that key reciprocal interactions between cancer cells and fibroblasts may be required.     

Overexpression of monocarboxylate anion transporter 1 and 4 in T24-induced cancer-associated fibroblasts regulates the progression of bladder cancer cells in a 3D microfluidic device

Stromal fibroblasts are essential for tumor proliferation and invasion. Here we presented a 3-dimensional (3D) microfluidic co-culture device to reconstruct an in vivo-like tumor microenvironment for investigation of the interactions of cancer-associated fibroblasts (CAFs) and bladder cancer cells. With this device, we verified that the cytokines secreted by bladder cancer cells T24 effectively transform the fibroblasts into CAFs. Compared to fibroblasts, the CAFs, which undergo the aerobic glycolysis, showed higher ability to produce lactate and provide energy for bladder cancer cell proliferation and invasion. We also demonstrated that this kind of tumor-promoting effect was associated with the upregulation of monocarboxylate anion transporter 1 (MCT1) and MCT4 expression in CAFs. We concluded that MCT1 and MCT4 are involved in bladder cancer cell proliferation and invasiveness. Moreover, this 3D microfluidic co-culture device allows for the assay to characterize various cellular events in a single device sequentially, facilitating a better understanding of the interactions among heterotypic cells in a sophisticated microenvironment.     

Priming cancer cells for drug resistance: role of the fibroblast niche

Conventional and targeted chemotherapies remain integral strategies to treat solid tumors. Despite the large number of anti-cancer drugs available, chemotherapy does not completely eradicate disease. Disease recurrence and the growth of drug resistant tumors remain significant problems in anti-cancer treatment. To develop more effective treatment strategies, it is important to understand the underlying cellular and molecular mechanisms of drug resistance. It is generally accepted that cancer cells do not function alone, but evolve through interactions with the surrounding tumor microenvironment. As key cellular components of the tumor microenvironment, fibroblasts regulate the growth and progression of many solid tumors. Emerging studies demonstrate that fibroblasts secrete a multitude of factors that enable cancer cells to become drug resistant. This review will explore how fibroblast secretion of soluble factors act on cancer cells to enhance cancer cell survival and cancer stem cell renewal, contributing to the development of drug resistant cancer.     

Cytokine production and inflammation drive autophagy in the tumor microenvironment: Role of stromal caveolin-1 as a key regulator

Recently, we proposed a new paradigm for understanding the role of the tumor microenvironment in breast cancer onset and progression. In this model, cancer cells induce oxidative stress in adjacent fibroblasts. This, in turn, results in the onset of stromal autophagy, which produces recycled nutrients to “feed” anabolic cancer cells. However, it remains unknown how autophagy in the tumor microenvironment relates to inflammation, another key driver of tumorigenesis. To address this issue, here we employed a well-characterized co-culture system in which cancer cells induce autophagy in adjacent fibroblasts via oxidative stress and NFκB-activation. We show, using this co-culture system, that the same experimental conditions that result in an autophagic microenvironment, also drive in the production of numerous inflammatory mediators (including IL-6, IL-8, IL-10, MIp1α, IFNγ, RANTES (CCL5) and GMCSF). Furthermore, we demonstrate that most of these inflammatory mediators are individually sufficient to directly induce the onset of autophagy in fibroblasts. To further validate the in vivo relevance of these findings, we assessed the inflammatory status of Cav-1 (−/−) null mammary fat pads, which are a model of a bonafide autophagic microenvironment. Notably, we show that Cav-1 (−/−) mammary fat pads undergo infiltration with numerous inflammatory cell types, including lymphocytes, T-cells, macrophages and mast cells. Taken together, our results suggest that cytokine production and inflammation are key drivers of autophagy in the tumor microenvironment. These results may explain why a loss of stromal Cav-1 is a powerful predictor of poor clinical outcome in breast cancer patients, as it is a marker of both (1) autophagy and (2) inflammation in the tumor microenvironment. Lastly, hypoxia in fibroblasts was not sufficient to induce the full-blown inflammatory response that we observed during the co-culture of fibroblasts with cancer cells, indicating that key reciprocal interactions between cancer cells and fibroblasts may be required.Key words: caveolin-1, oxidative stress, cytokine production, inflammation, tumor microenvironment, autophagy, breast cancer     

Three-dimensional multicellular cell culture for anti-melanoma drug screening: focus on tumor microenvironment

AbstractThe development of new treatments for malignant melanoma, which has the worst prognosis among skin neoplasms, remains a challenge. The tumor microenvironment aids tumor cells to grow and resist to chemotherapeutic treatment. One way to mimic and study the tumor microenvironment is by using three-dimensional (3D) co-culture models (spheroids). In this study, a melanoma heterospheroid model composed of cancer cells, fibroblasts, and macrophages was produced by liquid-overlay technique using the agarose gel. The size, growth, viability, morphology, cancer stem-like cells population and inflammatory profile of tumor heterospheroids and monospheroids were analyzed to evaluate the influence of stromal cells on these parameters. Furthermore, dacarbazine cytotoxicity was evaluated using spheroids and two-dimensional (2D) melanoma model. After finishing the experiments, it was observed the M2 macrophages induced an anti-inflammatory microenvironment in heterospheroids; fibroblasts cells support the formation of the extracellular matrix, and a higher percentage of melanoma CD271 was observed in this model. Additionally, melanoma spheroids responded differently to the dacarbazine than the 2D melanoma culture as a result of their cellular heterogeneity and 3D structure. The 3D model was shown to be a fast and reliable tool for drug screening, which can mimic the in vivo tumor microenvironment regarding interactions and complexity.Graphic abstract      

Ethanol exposure induces the cancer-associated fibroblast phenotype and lethal tumor metabolism

Little is known about how alcohol consumption promotes the onset of human breast cancer(s). One hypothesis is that ethanol induces metabolic changes in the tumor microenvironment, which then enhances epithelial tumor growth. To experimentally test this hypothesis, we used a co-culture system consisting of human breast cancer cells (MCF7) and hTERT-immortalized fibroblasts. Here, we show that ethanol treatment (100 mM) promotes ROS production and oxidative stress in cancer-associated fibroblasts, which is sufficient to induce myofibroblastic differentiation. Oxidative stress in stromal fibroblasts also results in the onset of autophagy/mitophagy, driving the induction of ketone body production in the tumor microenvironment. Interestingly, ethanol has just the opposite effect in epithelial cancer cells, where it confers autophagy resistance, elevates mitochondrial biogenesis and induces key enzymes associated with ketone re-utilization (ACAT1/OXCT1). During co-culture, ethanol treatment also converts MCF7 cells from an ER(+) to an ER(-) status, which is thought to be associated with “stemness,” more aggressive behavior and a worse prognosis. Thus, ethanol treatment induces ketone production in cancer-associated fibroblasts and ketone re-utilization in epithelial cancer cells, fueling tumor cell growth via oxidative mitochondrial metabolism (OXPHOS). This “two-compartment” metabolic model is consistent with previous historical observations that ethanol is first converted to acetaldehyde (which induces oxidative stress) and then ultimately to acetyl-CoA (a high-energy mitochondrial fuel), or can be used to synthesize ketone bodies. As such, our results provide a novel mechanism by which alcohol consumption could metabolically convert “low-risk” breast cancer patients to “high-risk” status, explaining tumor recurrence or disease progression. Hence, our findings have clear implications for both breast cancer prevention and therapy. Remarkably, our results also show that antioxidants [such as N-acetyl cysteine (NAC)] can effectively reverse or prevent ethanol-induced oxidative stress in cancer-associated fibroblasts, suggesting a novel strategy for cancer prevention. We also show that caveolin-1 and MCT4 protein expression can be effectively used as new biomarkers to monitor oxidative stress induced by ethanol.     

Fibroblast EXT1-Levels Influence Tumor Cell Proliferation and Migration in Composite Spheroids

Background

Stromal fibroblasts are important determinants of tumor cell behavior. They act to condition the tumor microenvironment, influence tumor growth, support tumor angiogenesis and affect tumor metastasis. Heparan sulfate proteoglycans, present both on tumor and stromal cells, interact with a large number of ligands including growth factors, their receptors, and structural components of the extracellular matrix. Being ubiquitously expressed in the tumor microenvironment heparan sulfate proteoglycans are candidates for playing central roles in tumor-stroma interactions. The objective of this work was to investigate the role of heparan sulfate expressed by stromal fibroblasts in modulating the growth of tumor cells and in controlling the interstitial fluid pressure in a 3-D model.

Methodology/Principal Findings

We generated spheroids composed of fibroblasts alone, or composite spheroids, composed of fibroblasts and tumor cells. Here we show that stromal fibroblasts with a mutation in the heparan sulfate elongating enzyme Ext1 and thus a low heparan sulfate content, formed composite fibroblast/tumor cell spheroids with a significant lower interstitial fluid pressure than corresponding wild-type fibroblast/tumor cell composite spheroids. Furthermore, immunohistochemistry of composite spheroids revealed that the cells segregated, so that after 6 days in culture, the wild-type fibroblasts formed an inner core and the tumor cells an outer layer of cells. For composite spheroids containing Ext1-mutated fibroblasts this segregation was less obvious, indicating impaired cell migration. Analysis of tumor cells expressing the firefly luciferase gene revealed that the changes in tumor cell migration in mutant fibroblast/tumor cell composite spheroids coincided with a lower proliferation rate.

Conclusions/Significance

This is the first demonstration that stromal Ext1-levels modulate tumor cell proliferation and affect the interstitial fluid pressure in a 3-D spheroid model. Learning how structural changes in stromal heparan sulfate influence tumor cells is essential for our understanding how non-malignant cells of the tumor microenvironment influence tumor cell progression.     

BRCA1 mutations drive oxidative stress and glycolysis in the tumor microenvironment

Mutations in the BRCA1 tumor suppressor gene are commonly found in hereditary breast cancer. Similarly, downregulation of BRCA1 protein expression is observed in the majority of basal-like breast cancers. Here, we set out to study the effects of BRCA1 mutations on oxidative stress in the tumor microenvironment. To mimic the breast tumor microenvironment, we utilized an in vitro co-culture model of human BRCA1-mutated HCC1937 breast cancer cells and hTERT-immortalized human fibroblasts. Notably, HCC1937 cells induce the generation of hydrogen peroxide in the fibroblast compartment during co-culture, which can be inhibited by genetic complementation with the wild-type BRCA1 gene. Importantly, treatment with powerful antioxidants, such as NAC and Tempol, induces apoptosis in HCC1937 cells, suggesting that microenvironmental oxidative stress supports cancer cell survival. In addition, Tempol treatment increases the apoptotic rates of MDA-MB-231 cells, which have wild-type BRCA1, but share a basal-like breast cancer phenotype with HCC1937 cells. MCT4 is the main exporter of L-lactate out of cells and is a marker for oxidative stress and glycolytic metabolism. Co-culture with HCC1937 cells dramatically induces MCT4 protein expression in fibroblasts, and this can be prevented by either BRCA1 overexpression or by pharmacological treatment with NAC. We next evaluated caveolin-1 (Cav-1) expression in stromal fibroblasts. Loss of Cav-1 is a marker of the cancer-associated fibroblast (CAF) phenotype, which is linked to high stromal glycolysis, and is associated with a poor prognosis in numerous types of human cancers, including breast cancers. Remarkably, HCC1937 cells induce a loss of Cav-1 in adjacent stromal cells during co-culture. Conversely, Cav-1 expression in fibroblasts can be rescued by administration of NAC or by overexpression of BRCA1 in HCC1937 cells. Notably, BRCA1-deficient human breast cancer samples (9 out of 10) also showed a glycolytic stromal phenotype, with intense mitochondrial staining specifically in BRCA1-deficient breast cancer cells. In summary, loss of BRCA1 function leads to hydrogen peroxide generation in both epithelial breast cancer cells and neighboring stromal fibroblasts, and promotes the onset of a reactive glycolytic stroma, with increased MCT4 and decreased Cav-1 expression. Importantly, these metabolic changes can be reversed by antioxidants, which potently induce cancer cell death. Thus, antioxidant therapy appears to be synthetically lethal with a BRCA1-deficiency in breast cancer cells and should be considered for future cancer prevention trials. In this regard, immunostaining with Cav-1 and MCT4 could be used as cost-effective biomarkers to monitor the response to antioxidant therapy.     

Curcumin Suppresses Crosstalk between Colon Cancer Stem Cells and Stromal Fibroblasts in the Tumor Microenvironment: Potential Role of EMT

Objective

Interaction of stromal and tumor cells plays a dynamic role in initiating and enhancing carcinogenesis. In this study, we investigated the crosstalk between colorectal cancer (CRC) cells with stromal fibroblasts and the anti-cancer effects of curcumin and 5-Fluorouracil (5-FU), especially on cancer stem cell (CSC) survival in a 3D-co-culture model that mimics in vivo tumor microenvironment.

Methods

Colon carcinoma cells HCT116 and MRC-5 fibroblasts were co-cultured in a monolayer or high density tumor microenvironment model in vitro with/without curcumin and/or 5-FU.

Results

Monolayer tumor microenvironment co-cultures supported intensive crosstalk between cancer cells and fibroblasts and enhanced up-regulation of metastatic active adhesion molecules (β1-integrin, ICAM-1), transforming growth factor-β signaling molecules (TGF-β3, p-Smad2), proliferation associated proteins (cyclin D1, Ki-67) and epithelial-to-mesenchymal transition (EMT) factor (vimentin) in HCT116 compared with tumor mono-cultures. High density tumor microenvironment co-cultures synergistically increased tumor-promoting factors (NF-κB, MMP-13), TGF-β3, favored CSC survival (characterized by up-regulation of CD133, CD44, ALDH1) and EMT-factors (increased vimentin and Slug, decreased E-cadherin) in HCT116 compared with high density HCT116 mono-cultures. Interestingly, this synergistic crosstalk was even more pronounced in the presence of 5-FU, but dramatically decreased in the presence of curcumin, inducing biochemical changes to mesenchymal-epithelial transition (MET), thereby sensitizing CSCs to 5-FU treatment.

Conclusion

Enrichment of CSCs, remarkable activation of tumor-promoting factors and EMT in high density co-culture highlights that the crosstalk in the tumor microenvironment plays an essential role in tumor development and progression, and this interaction appears to be mediated at least in part by TGF-β and EMT. Modulation of this synergistic crosstalk by curcumin might be a potential therapy for CRC and suppress metastasis.     

Tumor cells induce the cancer associated fibroblast phenotype via caveolin-1 degradation: Implications for breast cancer and DCIS therapy with autophagy inhibitors

Loss of stromal caveolin 1 (Cav-1) is a novel biomarker for cancer-associated fibroblasts that predicts poor clinical outcome in breast cancer and DCIS patients. We hypothesized that epithelial cancer cells may have the ability to drive Cav-1 downregulation in adjacent normal fibroblasts, thereby promoting the cancer associated fibroblast phenotype. To test this hypothesis directly, here we developed a novel co-culture model employing (i) human breast cancer cells (MCF7), and (ii) immortalized fibroblasts (hTERT-BJ1), which are grown under defined experimental conditions. Importantly, we show that co-culture of immortalized human fibroblasts with MCF7 breast cancer cells leads to Cav-1 downregulation in fibroblasts. These results were also validated using primary cultures of normal human mammary fibroblasts co-cultured with MCF7 cells. In this system, we show that Cav-1 downregulation is mediated by autophagic/lysosomal degradation, as pre-treatment with lysosome-specific inhibitors rescues Cav-1 expression. Functionally, we demonstrate that fibroblasts co-cultured with MCF7 breast cancer cells acquire a cancer associated fibroblast phenotype, characterized by Cav-1 downregulation, increased expression of myofibroblast markers and extracellular matrix proteins, and constitutive activation of TGFβ/Smad2 signaling. siRNA-mediated Cav-1 downregulation mimics several key changes that occur in co-cultured fibroblasts, clearly indicating that a loss of Cav-1 is a critical initiating factor, driving stromal fibroblast activation during tumorigenesis. As such, this co-culture system can now be used as an experimental model for generating “synthetic” cancer associated fibroblasts (CAFs). More specifically, these “synthetic” CAFs could be used for drug screening to identify novel therapeutics that selectively target the Cav-1-negative tumor micro-environment. Our findings also suggest that chloroquine, or other autophagy/lysosome inhibitors, may be useful as anti-cancer agents, to therapeutically restore the expression of stromal Cav-1 in cancer associated fibroblasts. We discuss this possibility, in light of the launch of a new clinical trial that uses chloroquine to treat DCIS patients: PINC (Preventing Invasive Breast Neoplasia with Cholorquine) [See http://clinicaltrials.gov/show/NCT01023477].     

Fibroblast-derived 3D matrix differentially regulates the growth and drug-responsiveness of human cancer cells

Recent studies have emphasized the importance of cellular microenvironment in modulating cell growth and signaling. In vitro, collagen matrices, Matrigel, and other synthetic support systems have been used to simulate in vivo microenvironments, and epithelial cells grown in these matrices manifest significant differences in proliferation, differentiation, response to drugs, and other parameters. However, these substrates do not closely resemble the mesenchymal microenvironment that is typically associated with advanced carcinomas in vivo, which is produced to a large extent by fibroblasts. In this study, we have evaluated the ability of a fibroblast-derived three-dimensional matrix to regulate the growth of a panel of 11 human tumor epithelial cell lines. Although proliferative and morphological responses to three-dimensional cues segregated independently, general responsiveness to the matrix correlated with the ability of matrix to influence drug responses. Fibroblast-derived three-dimensional matrix increased beta1-integrin-dependent survival of a subset of human cancer cell lines during taxol treatment, while it sensitized or minimally influenced survival of other cells. beta1-integrin-dependent changes in cell resistance to taxol did not correlate with the degree of modulation of FAK and Akt, implying that additional signaling factors are involved. Based on these results, we propose that these matrices potentially have value as in vitro drug screening platforms.     

Functional Investigation of NCI-H460-Inducible Myofibroblasts on the Chemoresistance to VP-16 with a Microfluidic 3D Co-Culture Device

Fibroblasts, the major cell type in tumor stroma, are essential for tumor growth and survival, and represent an important therapeutic target for cancers. Here we presented a microfluidic co-culture device in which the three-dimensional (3D) matrix was employed to reconstruct an in vivo-like fibroblast-tumor cell microenvironment for investigation of the role of myofibroblasts induced by lung cancer cells in the chemoresistance to VP-16. Composed of a double-layer chip and an injection pump, the device houses fibroblasts and lung cancer cells co-cultured in 3D matrix and 2D mode to induce fibroblasts to become myofibroblasts with the supplement of the medium continuously. With this device, we verified that the cytokines secreted by lung cancer cells could effectively transform the fibroblasts into myofibroblasts. Moreover, compared to fibroblasts, the myofibroblasts showed higher resistance to anticancer drug VP-16. We also demonstrated that this kind of acquired resistance in myofibroblasts was associated with the expression of Glucose-regulated protein 78 (GP78). We concluded that this device allows for the assay to characterize various cellular events in a single device sequentially, facilitating a better understanding of the interactions among heterotypic cells in a sophisticated microenvironment.     

Differences in intracellular mobile zinc levels affect susceptibility to plasma-activated medium-induced cytotoxicity

There is growing evidence that plasma-activated medium (PAM), which is prepared by non-thermal plasma (NTP) irradiation of cell-free medium, is a beneficial tool for cancer therapy. PAM has been reported to preferentially kill cancer cells; however, its mechanism is not fully understood. Since PAM contains reactive oxygen species (ROS) and reactive nitrogen species, the anti-cancer effects of PAM are thought to be attributed to oxidative stress induced by these reactive molecules. Oxidative stress has been shown to release zinc (Zn²⁺) from intracellular Zn²⁺ stores and provoke Zn²⁺-dependent cell death. We have previously demonstrated that intracellular free Zn²⁺ plays a critical role in PAM-induced cell death in human neuroblastoma SH-SY5Y cells. In this study, we found that normal human fibroblasts were less susceptible to PAM cytotoxicity compared with SH-SY5Y cells. PAM decreased intracellular NAD⁺ levels in both cells, whereas the depletion of ATP and mitochondrial ROS generation was hardly observed in fibroblasts. Intracellular mobile Zn²⁺ contents of fibroblasts were lower than those of SH-SY5Y cells. PAM suppressed the activity of aconitase, which is a tricarboxylic acid cycle enzyme, only in SH-SY5Y cells, and N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a Zn²⁺ chelator, counteracted the suppression. The combination treatment with PAM and Zn²⁺ augmented PAM-induced ATP depletion, mitochondrial ROS generation, and cytotoxicity in fibroblasts. These findings suggest the possibility that cells with high intracellular mobile Zn²⁺ are susceptible to PAM cytotoxicity. Therefore, we concluded that the differences in mobile Zn²⁺ levels affect PAM-induced cellular responses.     

The gene expression profiles of canine mammary cancer cells grown with carcinoma-associated fibroblasts (CAFs) as a co-culture in vitro

Background

It is supposed that fibroblasts present in tumour microenvironment increase cancer invasiveness and its ability to metastasize but the mechanisms have not been clearly defined yet. Thus, the current study was designed to assess changes in gene expression in five various cancer cell lines grown as a co-culture with the carcinoma-associated fibroblasts (CAFs) in vitro.

Results

A carcinoma-associated fibroblast cell line was isolated from a canine mammary cancer. Then, a co-culture of cancer cells with the CAFs was established and maintained for 72 hrs. Having sorted the cells, a global gene expression in cancer cells using DNA microarrays was examined. The analysis revealed an up-regulation of 100 genes and a down-regulation of 106 genes in the cancer cells grown as a co-culture with the CAFs in comparison to control conditions. The PANTHER binomial statistics tool was applied to determine statistically over-manifested pathways (p < 0.05). Bulk of the up-regulated genes are involved in the adhesion, the angiogenesis, the epithelial-mesenchymal transition (EMT) and generally take part in the developmental processes. These results were further confirmed using real-time qPCR. Moreover, a wound-healing assay and growth characteristics on Matrigel matrix showed that CAFs increase cancer cell migration and matrix invasion.

Conclusion

The results of the current study showed that the co-culturing of cancer cells and the CAFs caused significant changes to the cancer gene expression. The presence of the CAFs in a microenvironment of cancer cells promotes adhesion, angiogenesis and EMT.     

Rho kinase regulates the intracellular micromechanical response of adherent cells to rho activation

Local sol-gel transitions of the cytoskeleton modulate cell shape changes, which are required for essential cellular functions, including motility and adhesion. In vitro studies using purified cytoskeletal proteins have suggested molecular mechanisms of regulation of cytoskeleton mechanics; however, the mechanical behavior of living cells and the signaling pathways by which it is regulated remains largely unknown. To address this issue, we used a nanoscale sensing method, intracellular microrheology, to examine the mechanical response of the cell to activation of the small GTPase Rho. We observe that the cytoplasmic stiffness and viscosity of serum-starved Swiss 3T3 cells transiently and locally enhances upon treatment with lysophosphatidic acid, and this mechanical behavior follows a trend similar to Rho activity. Furthermore, the time-dependent activation of Rho decreases the degree of microheterogeneity of the cytoplasm. Our results reveal fundamental differences between intracellular elasticity and cellular tension and suggest a critical role for Rho kinase in the regulation of intracellular mechanics.