Lung-conditioned Medium Research Articles

Lung-derived soluble factors support stemness/plasticity and metastatic behaviour of breast cancer cells via the FGF2-DACH1 axis

Patients with triple-negative breast cancer (TNBC) have an increased propensity to develop lung metastasis. Our previous studies demonstrated that stem-like ALDHhiCD44+ breast cancer cells interact with lung-derived soluble factors, resulting in enhanced migration and lung metastasis particularly in TNBC models. We have also observed that the presence of a primary TNBC tumor can ‘prime’ the lung microenvironment in preparation for metastasis. In this study, we hypothesized that soluble lung-derived factors secreted in the presence of a primary TNBC tumor can influence stemness/plasticity of breast cancer cells. Using an ex vivo pulmonary metastasis assay (PuMA), we observed that the lung microenvironment supports colonization and growth of ALDHhiCD44+ TNBC cells, potentially via interactions with lung-derived FGF2. Exposure of TNBC cells to lung-conditioned media (LCM) generated from mice bearing TNBC primary tumors (tbLCM) significantly enhanced the proportion of ALDHhiCD44+ cells compared to control or LCM from tumor-naïve mice (tnLCM). Further analysis using a human cancer stem cell qPCR array revealed that, relative to tnLCM or control, exposure of TNBC cells to tbLCM leads to downregulation of the transcription factor and putative tumor suppressor Dachshund homolog 1 (DACH1), a downstream regulator of FGF2. In addition, inhibition of DACH1 using siRNA or treatment with recombinant FGF2 enhanced the ALDHhiCD44+ phenotype. Taken together, our findings suggest that the FGF2-DACH1 signaling axis supports stemness/plasticity of TNBC cells in the lung microenvironment and lays the foundation for future evaluation of FGF2 as a potential novel therapeutic target for treatment or prevention of breast cancer metastasis to the lung.

Human embryonic mesenchymal lung-conditioned medium promotes differentiation to myofibroblast and loss of stemness phenotype in lung adenocarcinoma cell lines

BackgroundWhen genes responsible for normal embryonic development are abnormally expressed in adults, it can lead to tumor development. This can suggest that the same mechanism that controls embryonic differentiation can also control tumor differentiation. We hypothesize that the malignant phenotype of lung cancer cells could acquire benign characteristics when in contact with an embryonic lung microenvironment. We cultured two lung cancer cell lines in embryonic lung mesenchyme-conditioned medium and evaluated morphological, functional and molecular changes.MethodsThe human embryonic mesenchymal lung-conditioned medium (hEML-CM) was obtained by culturing lung cells from embryos in the pseudoglandular stage of development. The NSCLC cell lines A549 and H1299 we cultured in the hEML-CM and in a tumor-conditioned medium. Morphological changes were analyzed with optical and transmission electron microscopy. To evaluate the functional effect of conditioned medium in tumor cells, we analyzed cell proliferation, migration, colony formation capacity in 2D and 3D and in vivo tumor growth capacity. The expression of the pluripotency genes OSKM, the adenocarcinoma marker NKX2-1, the lung surfactant proteins SFTP, the myofibroblast marker MYH and DNMT3A/3B was analyzed with qRT-PCR and the presence of the myofibroblast markers vimentin and α-SMA with immunofluorescence. Transcriptomic analysis was performed using Affymetrix arrays.ResultsThe A549 and H1299 cells cultured in hEML-CM lost their epithelial morphology, acquired mesodermal characteristics, and decreased proliferation, migration, and colony formation capacity in 2D and 3D, as well as reduced its capacity to growth in vivo. The expression of OSKM, NKX2-1 and SFTP decreased, while that of DNMT3A/3B, vimentin, α-SMA and MYH increased. Distant matrix analysis based on transcriptomic profile showed that conditioned cells were closer to myoblast and human lung fibroblast than to normal epithelial immortalized lung cells. A total of 1631 for A549 and 866 for H1299 differentially expressed genes between control and conditioned cells were identified.ConclusionsTo the best of our knowledge, this is the first study to report that stimuli from the embryonic lung can modulate the malignant phenotype of lung cancer cells, control their growth capacity and activate their differentiation into myofibroblasts. These findings could lead to new strategies for lung cancer management.

Triple-Negative Primary Breast Tumors Induce Supportive Premetastatic Changes in the Extracellular Matrix and Soluble Components of the Lung Microenvironment.

The lung is one of the deadliest sites of breast cancer metastasis, particularly in patients with triple-negative (TN) disease. We hypothesized that the presence of a TN primary breast tumor induces changes in the extracellular matrix (ECM) and soluble components of the lung microenvironment that support metastatic behavior. SUM159 (TN) and MCF7 (luminal A) breast cancer cells were injected into mice, and primary breast tumors were established prior to assessing metastatic niche changes. We observed increased CD117+ hematopoietic progenitor cells in the bone marrow of SUM159 mice versus MCF7 or control mice (p < 0.05). Relative to mice bearing MCF7 tumors and non-tumor controls, mice bearing SUM159 tumors demonstrated enhanced expression of ECM proteins in the lung (fibronectin, tenascin-c and periostin), with similar changes observed in lung fibroblasts treated with extracellular vesicles (EVs) from TN breast cancer cells (p < 0.05). Exposure to lung-conditioned media (LCM) from SUM159 tumor-bearing mice resulted in increased migration/proliferation of both SUM159 and MCF7 cells relative to the control (p < 0.05). In contrast, LCM from MCF-7 tumor-bearing mice had no such effect. LCM from SUM159 tumor-bearing mice contained 16 unique proteins relative to other LCM conditions, including the metastasis-associated proteins CCL7, FGFR4, GM-CSF, MMP3, thrombospondin-1 and VEGF. These findings suggest for the first time that the TN breast cancer molecular subtype may be an important determinant of premetastatic changes to both the ECM and soluble components of the lung, potentially mediated via breast cancer-derived EVs.

Abstract 1896: Soluble lung-derived selectins promote breast cancer cell migration

Abstract Ninety percent of all breast cancer-related deaths are attributed to metastasis, as current therapies are largely non-curative in the metastatic setting. One of the most common sites of breast cancer metastasis is the lung. Previous work from our lab has demonstrated that there are over 70 soluble factors produced by murine lung tissue, many of which have been associated with cancer cell growth, migration, invasion and metastasis. However, the relative contribution of each individual factor in enhancing the metastatic behavior of breast cancer cells has not been examined in detail. Three of the identified soluble lung-derived proteins are E-, L- and P-selectin, and these were the focus of the current study. The selectins are a family of glycoproteins with known physiological roles in vascular/immune cell migration, and soluble selectins have been clinically observed to be elevated in the sera of cancer patients. We hypothesized that soluble lung-derived selectins enhance the metastatic behavior of breast cancer cells. We used a 2D ex vivo model of the soluble lung microenvironment, generated by isolating conditioned media from dissociated healthy murine lung. E-, L- and P-selectin were then individually immunodepleted from the lung-conditioned media (lung-CM) and the subsequent functional effect on breast cancer cell migration and proliferation was assessed using transwell migration and BrdU incorporation assays, respectively. MDA-MB-231 and SUM149PT human breast cancer cells demonstrated significantly enhanced cell migration toward lung-CM relative to basal media, and this migration was significantly reduced when E-, L- or P-selectin were depleted (p&amp;lt;0.05). In contrast, although MDA-MB-231 and SUM149PT human breast cancer cells demonstrated significantly enhanced cell proliferation in response to lung-CM relative to basal media (p&amp;lt;0.05), depletion of soluble E-, L- or P-selectin from lung-CM had no influence on reducing proliferation (p&amp;gt;0.05). Ongoing studies are aimed at elucidating the pathways through which soluble lung-derived selectins exert their pro-migratory function. If these results implicate a common receptor and downstream signalling pathways for all three selectins, these ligand-receptor interactions may have value as potential therapeutic targets for the prevention of pulmonary metastasis in breast cancer patients. Citation Format: Sami U. Khan, Jenny E. Chu, Ying Xia, Alison L. Allan. Soluble lung-derived selectins promote breast cancer cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1896. doi:10.1158/1538-7445.AM2017-1896

Metformin attenuates gefitinib-induced exacerbation of pulmonary fibrosis by inhibition of TGF-β signaling pathway.

Interstitial lung disease (ILD) is a serious side-effect of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) treatment. Therefore, it is necessary to study underlying mechanisms for the development of pulmonary fibrosis induced by EGFR-TKI and potential approaches to attenuate it. Metformin is a well-established and widely prescribed oral hypoglycemic drug, and has gained attention for its potential anticancer effects. Recent reports have also demonstrated its role in inhibiting epithelial-mesenchymal transition and fibrosis. However, it is unknown whether metformin attenuates EGFR-TKI-induced pulmonary fibrosis. The effect of metformin on EGFR-TKI-induced exacerbation of pulmonary fibrosis was examined in vitro and in vivo using MTT, Ki67 incorporation assay, flow cytometry, immunostaining, Western blot analysis, and a bleomycin-induced pulmonary fibrosis rat model. We found that in lung HFL-1 fibroblast cells, TGF-β or conditioned medium from TKI-treated lung cancer PC-9 cells or conditioned medium from TKI-resistant PC-9GR cells, induced significant fibrosis, as shown by increased expression of Collegen1a1 and α-actin, while metformin inhibited expression of fibrosis markers. Moreover, metformin decreased activation of TGF-β signaling as shown by decreased expression of pSMAD2 and pSMAD3. In vivo, oral administration of gefitinib exacerbated bleomycin-induced pulmonary fibrosis in rats, as demonstrated by HE staining and Masson staining. Significantly, oral co-administration of metformin suppressed exacerbation of bleomycin-induced pulmonary fibrosis by gefitinib. We have shown that metformin attenuates gefitinib-induced exacerbation of TGF-β or bleomycin-induced pulmonary fibrosis. These observations indicate metformin may be combined with EGFR-TKI to treat NSCLC patients.

Intercellular Communication Between Extracellular Vesicles and Murine Marrow Cells Is Influenced By Circadian Rhythm

Introduction: Extracellular vesicles (EVs) are membrane-bound, subcellular fragments that contain DNA, RNA, protein and lipids, and play an important role in intercellular communication. They have been implicated in myriad physiologic and pathologic processes within the hematopoietic system including thrombosis and leukemia, and as such, have tremendous therapeutic potential if manipulated appropriately. In order to fully harness their clinical potential, it will be important to more fully characterize the factors that modulate their biogenesis and function. Numerous factors are thought to influence EVs and their ability to communicate with target marrow cells, but little is known about how circadian oscillations alter EV function. Using a well-established model of lung-derived EV modulation of marrow phenotype, we report here our studies exploring the effects of circadian rhythm on the ability of lung-derived EVs to alter the transcriptome of bone marrow target cells.Methods: C57BL/6 mice were housed on a 12-hour light/dark schedule. For the first set of experiments, lungs, blood and bone marrow were harvested (6-16 mice/time) at three different circadian time-points (8,12 and 24 hours after light onset (HALOs). EVs were isolated from each tissue at each time-point and quantified by nanoparticle tracking analysis. Second, to determine if circadian rhythm influences the ability of lung-derived EVs to modulate the transcriptome of target whole bone marrow cells (WBM), lungs were harvested (3 mice/time-point x 2 experiments) at 4,12,16, and 24 hours after light onset and co-cultured, across a cell-impermeable membrane, with murine WBM. The WBM was harvested after 24hrs in co-culture and analyzed for the induction of pulmonary specific mRNA expression by RT-PCR. Finally, to determine if altering the circadian timepoint of the target whole bone marrow influenced the ability of EVs to modulate marrow phenotype, lungs harvested from mice sacrificed at HALO 2 were cultured for 48hrs. EVs were isolated from the lung-conditioned media by ultracentrifugation. WBM was then harvested at different circadian time-points (HALO 4, 8, 12, 16, 20, and 24; 3 mice/time-point x 2 experiments), cultured with or without the lung-derived EVs for 24hrs, and then analyzed for pulmonary-specific mRNAs by RT-PCR.Results: There were changes in EV quantity related to circadian timepoint. There were significantly increased numbers of EVs generated from lungs harvested at HALO 24 and HALO 8 when compared to HALO 16. Similarly, marrow harvested at HALO 8 had significantly increased number of EVs as compared to WBM harvested at HALO 16 and 24. In addition to circadian-related differences in EV quantity, there were clear oscillations in the expression patterns of pulmonary specific mRNAs when both the circadian timepoint of the lung donor and the circadian timepoint of the recipient marrow cells were altered. If lung was harvested at HALO 12 or HALO 16, the co-cultured WBM showed significantly increased expression of pulmonary-specific mRNAs when compared to WBM co-cultured with lung harvested at HALO 4 or HALO 24. Likewise, when WBM harvested at different circadian time-points was incubated with lung-derived EVs, there was significantly increased expression of surfactant A, surfactant B, and aquaporin 5 when WBM was harvested at HALO 16 when compared to HALO 12 or HALO 24.Conclusions: Based on these data, we conclude that circadian rhythm is likely an important modulator of EV-mediated intercellular communication with whole bone marrow cells and warrants more detailed study. DisclosuresNo relevant conflicts of interest to declare.

Abstract B10: Soluble lung-derived factors mediate breast cancer cell migration and growth via CD44 receptor-ligand interactions

Abstract Background: The majority of breast cancer deaths occur due to metastatic disease. Clinical studies have identified a specific pattern for the metastatic spread of breast cancer, where preferential secondary sites include the lymph node, lung, liver, bone, and brain. However, it is not clear whether properties of the cancer cells (the “seeds”); properties of the organ microenvironment (the “soil”), or a combination of both are responsible for this observed organ tropism of breast cancer. It has been proposed that small subsets of stem-like tumor cells are responsible for tumor initiation and progression. In breast cancer, these cells are identified by their high aldehyde dehydrogenase (ALDH) activity and/or CD44+CD24- phenotype. We have previously shown that ALDHhiCD44+ breast cancer cells exhibit key metastatic behaviors in vitro and in vivo. However, the mechanisms underlying the organ-specific metastatic capacity of these cells requires further elucidation. Hypothesis: Breast cancer cells exhibit differential growth and migration patterns in specific organ microenvironments; and ALDHhiCD44+ breast cancer cells exhibit increased organ-specific migration and growth (1) preferentially in the lung microenvironment and (2) compared to their ALDHlowCD44- counterparts. Methods: A novel ex vivo model system comprised of organ-conditioned media generated from athymic nude mice was used to test organotropic migration/growth of whole populations of MDA-MB-231, MDA-MB-468, SUM149, and SUM159 human breast cancer cell lines, and ALDHhiCD44+ and ALDHlowCD44- populations isolated from the MDA-MB-231 cell line. Protein arrays were used to interrogate lung-conditioned media and identify soluble factors of interest. Bead-based immunodepletion of target proteins was performed on lung-conditioned media. The migration/growth response of MDA-MB-231 breast cancer cells was then assessed following exposure to depleted vs. non-depleted lung-conditioned media. Results: Whole populations of all 4 human breast cancer cell lines examined responded via cell line specific patterns of migration towards organ-conditioned media. Notably, all lines demonstrated increased migration towards lung-conditioned media (p&amp;lt;0.05). These observations closely mirror previously observed patterns of in vivo metastatic behavior, providing validation for our ex vivo model system. ALDHhiCD44+ cells responded in a chemotactic manner towards organ-conditioned media in a similar pattern to whole population results. Notably, ALDHhiCD44+ cells showed preferential migration towards lung-conditioned media relative to all other organ-conditioned media tested (p&amp;lt;0.05). Several CD44 ligands including osteopontin (OPN) and E-, L-, and P-selectin were identified by protein array analysis of lung-conditioned media. Immunodepletion of L-selectin or OPN from lung-conditioned media resulted in decreased migration of MDA-MB-231 cells, however only depletion of OPN also resulted in decreased growth relative to non-depleted media. These effects could be abrogated by pre-treatment with a functional CD44 receptor blocking antibody (p&amp;lt;0.05). Conclusions: Our data suggest an important role for CD44 receptor-ligand interactions in mediating lung-specific breast cancer cell migration and growth. To the best of our knowledge, this is the first study to undertake ex vivo investigation of the role of the microenvironment in metastatic organ tropism in the context of the cancer stem cell hypothesis. Citation Format: Ying Xia, Jenny E. Chu, Benjamin Chin-Yee, David Goodale, Alysha K. Croker, Alison L. Allan. Soluble lung-derived factors mediate breast cancer cell migration and growth via CD44 receptor-ligand interactions. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B10.

Progenitor/Stem Cell Fate Determination: Interactive Dynamics of Cell Cycle and Microvesicles

We have shown that hematopoietic stem/progenitor cell phenotype and differentiative potential change throughout cell cycle. Lung-derived microvesicles (LDMVs) also change marrow cell phenotype by inducing them to express pulmonary epithelial cell-specific mRNA and protein. These changes are accentuated when microvesicles isolated from injured lung. We wish to determine if microvesicle-treated stem/progenitor cell phenotype is linked to cell cycle and to the injury status of the lung providing microvesicles. Lineage depleted, Sca-1+ (Lin-/Sca-1+) marrow isolated from mice were cultured with interleukin 3 (IL-3), IL-6, IL-11, and stem cell factor (cytokine-cultured cells), removed at hours zero (cell cycle phase G0/G1), 24 (late G1/early S), and 48 (late S/early G2/M), and cocultured with lung tissue, lung conditioned media (LCM), or LDMV from irradiated or nonirradiated mice. Alternatively, Lin-/Sca-1+ cells not exposed to exogenous cytokines were separated into G0/G1 and S/G2/M cell cycle phase populations by fluorescence-activated cell sorting (FACS) and used in coculture. Separately, LDMV from irradiated and nonirradiated mice were analyzed for the presence of adhesion proteins. Peak pulmonary epithelial cell-specific mRNA expression was seen in G0/G1 cytokine-cultured cells cocultured with irradiated lung and in late G1/early S cells cocultured with nonirradiated lung. The same pattern was seen in cytokine-cultured Lin-/Sca-1 cells cocultured with LCM and LDMV and when FACS-separated Lin-/Sca-1 cells unexposed to exogenous cytokines were used in coculture. Cells and LDMV expressed adhesion proteins whose levels differed based on cycle status (cells) or radiation injury (LDMV), suggesting a mechanism for microvesicle entry. These data demonstrate that microvesicle modification of progenitor/stem cells is influenced by cell cycle and the treatment of the originator lung tissue.

Alteration of Marrow Cell Gene Expression, Protein Production, and Engraftment into Lung by Lung-Derived Microvesicles: A Novel Mechanism for Phenotype Modulation

Numerous animal studies have demonstrated that adult marrow-derived cells can contribute to the cellular component of the lung. Lung injury is a major variable in this process; however, the mechanism remains unknown. We hypothesize that injured lung is capable of inducing epigenetic modifications of marrow cells, influencing them to assume phenotypic characteristics of lung cells. We report that under certain conditions, radiation-injured lung induced expression of pulmonary epithelial cell-specific genes and prosurfactant B protein in cocultured whole bone marrow cells separated by a cell-impermeable membrane. Lung-conditioned media had a similar effect on cocultured whole bone marrow cells and was found to contain pulmonary epithelial cell-specific RNA-filled microvesicles that entered whole bone marrow cells in culture. Also, whole bone marrow cells cocultured with lung had a greater propensity to produce type II pneumocytes after transplantation into irradiated mice. These findings demonstrate alterations of marrow cell phenotype by lung-derived microvesicles and suggest a novel mechanism for marrow cell-directed repair of injured tissue.

The role of PTEN in prostate cancer cell tropism to the bone micro-environment

Little is known about the role of the tumor suppressor gene phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in prostate cancer bone metastasis. To explore this, we used a pTetOn PTEN cell line in which PTEN expression was reconstituted in a PTEN-null bone metastatic human prostate cancer cell line, LnCaP-C4-2. We found that C4-2 cells selectively migrated toward conditioned medium from primary mouse calvaria cells compared with that derived from lung fibroblasts. Further evaluation with conditioned medium from an established mouse calvaria osteoblast cell line and control non-osteoblast cell line indicates that osteoblastic characteristics convey this specific migration to C4-2 cells. We evaluated promiscuously metastatic PC-3 prostate as well as T24T and UMUC-3 bladder cells and found they did not have a specific migratory response to calvaria-conditioned medium as did C4-2. Induction of PTEN expression inhibited the motility of C4-2 cells toward calvaria-conditioned medium but had no effect on migration toward lung-conditioned medium and this inhibitory effect was dependent on the PTEN lipid phosphatase activity. Calvaria- but not lung-conditioned medium induced activation of the small GTPase Rac1. Constitutively active Rac1 but not focal adhesion kinase or Cdc42 could rescue cells from the inhibitory effect of PTEN on cell migration and PTEN induction was observed to inhibit Rac1 activation in response to calvaria-conditioned medium. Our results support the notion that loss of PTEN function in human prostate cancer may specifically facilitate bone rather than other organ metastasis and suggest that Rac1, as a PTEN effector, may contribute to this metastatic tropism.

Irradiation induces increased production of haemopoietic and proinflammatory cytokines in the mouse lung

Purpose : To investigate cytokine expression following irradiation of mice, predominantly in lung tissue but also in selected other tissues. Materials and methods : Mice of strain ICR were whole-body (unilaterally) exposed to 3-20 Gy of 60 Co γ-rays. Colony-stimulating activity (CSA) of lung-conditioned media (LCM), and also other non-haemopoietic and haemopoietic organs, and blood serum of mice was assayed using a GM-CFC bioassay. The production of GM-CSF, IL-6 and TNF- α protein in LCM and sera was determined by an ELISA method. Results : Greatest CSA was detected in conditioned media from the lungs and was induced in a dose- and time-dependent fashion, peaking at 3-9 days after irradiation with a lethal dose of 9 Gy. Conditioned medium prepared from lungs that had been irradiated with a dose of 9 Gy in vitro did not exhibit an increase in CSA. However, whereas the lung-conditioned medium from irradiated mice was found to produce CSA, sera from normal or irradiated mice did not lead to this effect. A significant increase in CSA in sera was observed in the presence of a suboptimal concentration of IL-3, implying that they comprise the co-stimulatory activity (CoSA). The results showed that radiation exposure increased GM-CSF and TNF- α protein levels but did not affect IL-6 production in LCM. In contrast, IL-6 and TNF- α protein levels in serum were increased after irradiation but no GM-CSF production could be detected. Conclusion : Whole-body irradiation enhances CSA in lungs as well as in other haemopoietic and non-haemopoietic organs. The increase of CSA correlates with increased levels of haemopoietic and proinflammatory cytokines in lung.

Apoptotic cell death in mammary adenocarcinoma cells is prevented by soluble factors present in the target organ of metastasis.

Target organ of metastasis determines the fate of metastasis. The soluble factors released from one or more cell types in the new stroma may influence growth and survival of metastatic cells. In the present study, we used conditioned media from the kidney, liver and lung, the latter being the target organ of metastasis of murine mammary adenocarcinoma cell lines LM3, LMM3 and F3II, to assess whether the soluble factors released from these organs could modulate in vitro survival of these cell lines after apoptosis-inducing treatments and to investigate the mechanisms involved in this effect. We demonstrate that conditioned medium from lung, but not from liver or kidney, promotes survival of these cells after doxorubicin, cisplatin, agonistic anti-Fas antibody and serum withdrawal treatments. Furthermore, LMM3 cells treated with lung conditioned medium after doxorubicin exposure maintained their tumorigenic capacity and metastatic potential. Neither IGF nor EGF could promote survival but, surprisingly, TGF-beta could reduce sensitivity of LMM3 cells to doxorubicin in vitro. Doxorubicin treatment induced Bax expression and down-regulated Bcl-2 expression. In contrast, lung conditioned medium increased Bcl-2 expression and inhibited doxorubicin-mediated Bcl-2 down-regulation. Neither of those treatments alone modified Bcl-X(L) expression, although co-treatment induced a 3- to 5-fold increase of its expression. These results suggest that the lung microenvironment could promote metastasis of these adenocarcinoma cell lines by increasing survival of metastatic cells, possibly by modulation of Bcl-2 protein family expression.

Evaluation Of Myelotoxicity In Cotton Rats (Sigmodon hispidus) Exposed To Environmental Contaminants. I. In Vitro Bone-Marrow Progenitor Culture

Bone marrow is extremely sensitive to toxicants, and in vitro culture of bone-marrow progenitor cells has been shown to be a sensitive indicator of bone-marrow injury in laboratory rodents. The ability of a bone-marrow progenitor cell assay to detect myelotoxicity in a wild rodent model (cotton rat, Sigmodon hispidus) that inhabits many contaminated ecosystems in the southern United States was examined. Responsiveness of progenitor cells to recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) and cotton rat lung-conditioned medium (LCM) was determined to optimize culture conditions for cotton rats. Myelotoxicity was induced in cotton rats by treating animals with either cyclophosphamide (8 or 80 mg/kg) or dexamethasone (500 µg/kg) over a 5-d period. Administration of a high dose of cyclophosphamide caused nearly total suppression of colony formation of granulocyte-macrophage progenitor cells (CFU-GM). Marked histological changes in both the bone marrow and spleen were also observed in cotton rats treated with a high dose of cyclophosphamide. Although histological lesions were not apparent, the number of CFU-GM in the bone marrow of low-dose cyclophosphamide- and dexamethasone-treated cotton rats was significantly suppressed compared to controls. The number of CFU-GM was consistently higher using LCM than recombinant murine GM-CSF. This reproducible, quantitative, in vitro bone-marrow progenitor cell culture system was a sensitive indicator of myelotoxicity in wild cotton rats and should be useful for monitoring chronic exposures to low levels of environmental toxicants in wild rodent populations.

Kappa-Opioid receptors in marrow stroma mediate the hematopoietic effects of melatonin-induced opioid cytokines.

Melatonin exerts colony-stimulating activity and rescues myeloid progenitors from apoptosis, induced either in vivo or in vitro by cancer chemotherapy compounds in tumor-bearing mice. These effects are mediated mainly by T-helper cell-derived opioid cytokines with an apparent molecular mass of 15 kDa and 67 kDa that are recognized both by anti-interleukin-4 and anti-dynorphin B antibodies. These putative new cytokines were named melatonin-induced-opioid (MIO). The most active and naltrexone-sensitive MIO was the smaller molecule, which was called MIO15 and found to act on an opioid-binding site present in adherent bone marrow cells. However, the hematopoietic action of MIO15 was dependent on the presence of colony-stimulating factors (CSF). To investigate this point, we studied the ability of melatonin to rescue granulocyte/macrophage colony-forming units (GM-CFU) in the bone marrow of tumor-free animals treated with cancer chemotherapeutic compounds. We found that melatonin not only is unable to protect bone marrow GM-CFU unless the mice are transplanted with Lewis lung carcinoma (LLC), but also that melatonin seems to increase the myelotoxicity of cyclophosphamide in tumor-free mice. In both tumor-bearing or healthy mice, the effect of melatonin is negated by naltrexone, indicating the involvement of MIO15. Competition studies classified the target opioid-binding site as a kappa-opioid receptor with low affinity in tumor-free mice and high affinity in LLC-implanted mice. LLC is known to release CSF. Consistently, addition of CSF in the form of lung-conditioned medium (LCM) to adherent bone marrow cells increased the affinity of the kappa-opioid receptor. Addition of antigranulocyte/macrophage colony-stimulating factor (GM-CSF) mAbs neutralized the effect of LCM. In conclusion, the affinity state of the kappa-opioid receptors in stromal bone marrow cells seems to modulate the hematopoietic effect of melatonin and/or MIO15.

Modification of PDGFalpha receptor expression or function alters the metastatic phenotype of 3LL cells.

Functional PDGFalpha receptors are selectively expressed on highly lung-metastasizing clones of the 3LL Lewis lung carcinoma, but not on low-mestastatic clones. The highly metastatic clones are also growth induced in vitro by PDGF and lung conditioned medium. To investigate whether modification of PDGFalpha receptor expression or function can affect metastatic capability, we transfected cells of a low-metastatic 3LL clone with a full length PDGFalpha receptor gene and cells of a highly-metastatic clone with a truncated kinase domain PDGFalpha receptor gene. Introduction of the full length PDGFalpha receptor conferred upon low-metastatic cells the ability to grow in vitro in the presence of PDGF-AA and to colonize the lung in experimental and spontaneous metastases assays. Conversely, introduction of a truncated version of the PDGFalpha receptor into highly metastatic cells reduced their metastatic load to control levels. Accordingly, their responses to PDGF-AA, including growth stimulation and receptor autophosphorylation, were reduced. These results demonstrate that PDGFalpha receptor expression and function can control the capacity of tumor cells to generate metastases in the lung. The response of this receptor to lung-derived PDGF-like factors may define a paracrine mode of metastatic cell growth in the target organ.

Mesenchymal regulation of differentiation of intestinal epithelial cells.

Intestinal epithelial differentiation requires the presence of mesenchyme, but the exact nature of this epithelial-mesenchymal interaction is unknown. The role of humoral and extracellular matrix factors in determining intestinal epithelial differentiation was examined by exposing a nontransformed, immature, rat small-intestinal cell line (IEC-6) to intestinal fibroblast-conditioned media or by growing the cells on matrix deposited by intestinal fibroblasts. Differentiation was assessed morphologically and by induction of the disaccharidase sucrase, which is a marker of intestinal epithelial maturation. When exposed to fetal intestinal fibroblast-conditioned medium or grown on extracellular matrix prepared from fetal intestinal fibroblasts, IEC-6 cells showed a significant increase in sucrase activity. The addition of cortisol to IEC-6 cells cultured on intestinal matrix also led to increased sucrase expression, as measured by enzyme activity and protein and messenger RNA levels. Ultrastructurally, cells cultured on intestinal fibroblast matrix in the presence of cortisol displayed a more differentiated phenotype. In contrast, there was no induction of sucrase activity in cells exposed to lung-conditioned media or grown on lung fibroblast matrix. These studies suggest that both humoral and matrix factors from intestinal mesenchyme are involved in intestinal epithelial differentiation and that these factors appear to be organ specific.

The development and magnitude of thermotolerance during chronic hyperthermia in murine granulocyte-macrophage progenitors: II.

We have previously reported that murine granulocyte-macrophage progenitors (CFU-GM) are capable of developing thermotolerance during chronic hyperthermia at temperatures of 40 to 42 degrees C. However, a differential profile of intrinsic thermal response and, in particular, the capability of developing thermotolerance during chronic heating was identified between CFU-GM and macrophage colony-forming units (CFU-M) stimulated respectively, by lung conditioned medium (LCM) and L929 cell conditioned medium (CCM). Nucleated marrow cells treated in vitro were cultured in McCoy's 5A medium plus 15% fetal bovine serum (FBS) in semisolid agar with 10% of CCM. Two different treatment protocols were used in this study to determine the kinetics of thermotolerance in CFU-M: (1) nucleated marrow from mouse tibia and femur were chronically heated in vitro at temperatures of 40, 41 and 42 degrees C (up to 480 min) or (2) nucleated marrow cells were heated over a period of 90 min stepwise from 37 to 42 degrees C, at a heating rate of 0.056 degrees C/min, before exposure to 42 degrees C. The amount of thermotolerance developed was analysed at various times after chronic incubation at 40-42 degrees C by a challenge with 15 min at 44 degrees C. In contrast to CFU-GM, the surviving fraction of CFU-M heated with 15 min at 44 degrees C did not increase during chronic hyperthermia at 40 degrees C for up to 480 min indicating failure to develop thermotolerance. However, CFU-M were able to develop thermotolerance during prolonged incubation at 41 and 42 degrees C, although to a much less extent than observed in CFU-GM. In other words, there was much less development of thermotolerance in murine CFU-M compared to that in CFU-GM. Furthermore, a slow temperature transit from 37 to 42 degrees C over 90 min before exposure to 42 degrees C induced CFU-M to develop thermotolerance. The thermotolerance ratio (TTR, the ratio of the surviving fraction at maximum tolerance versus normotolerance) increased from a maximum of 3.5 after 180 min at 42 degrees C (no warm-up) to a maximum of 4.1 after 60 min at 42 degrees C when the cells received a slow warm-up to 42 degrees C. This implies that in the murine bone marrow granulocyte/macrophage lineage, CFU-M does not normally develop thermotolerance during hyperthermia and that the colony forming unit-granulocyte (CFU-G) and CFU-GM play a more critical role than CFU-M in the initiation and promotion of thermotolerance during chronic hyperthermia. However, in a situation that simulates the slow heat-up used clinically in wholebody hyperthermia, e.g., the 90 min slow warm-up from 37 to 42 degrees C, stimulated CFU-M to develop greater thermotolerance more rapidly than during rapid heating.

The development and magnitude of thermotolerance during chronic hyperthermia in murine bone marrow granulocyte-macrophage progenitors: I.

Murine bone marrow granulocyte-macrophage progenitors (CFU-GM) are capable of developing thermotolerance during exposure to temperatures < 42.5 degrees C. Bone marrow from the tibia and femora was heated to 40-42 degrees C (i.e. chronic hyperthermia), and challenged immediately with 15 min at 44 degrees C at regular intervals during treatment (step-up heating). CFU-GM were heated and cultured in McCoy's 5A medium + 15% FBS (fetal bovine serum) and lung-conditioned medium (source of colony stimulating factor) in semisolid agar. The kinetics of thermotolerance development and decay, and the magnitude of the thermotolerance during chronic heating with temperatures of 40-41.5 degrees C were similar. Survival increased rapidly to a maxima by approximately 120 min of hyperthermia (temperatures of 40-41.5 degrees C) and thereafter decreased with a slope similar to the controls. Normalization for cell killing by chronic hyperthermia that occurred during "step-up' heating permitted analysis of thermotolerance in the surviving cells. The surviving fraction after 15 min at 44 degrees C, during incubation at 40, 41 and 41.5 degrees C increased from 0.13 to maxima of 0.56 +/- 0.04, 0.71 +/- 0.03 and 0.82 +/- 0.03 respectively, by 150 min and did not decrease for up to 480 min during chronic hyperthermia. The surviving fraction after 15 min at 44 degrees C during incubation at 42 degrees C increased more slowly than during incubations at 40-41.5 degrees C. The survival of thermotolerant cells after exposure to 15 min at 44 degrees C during 42 degrees C chronic hyperthermia was maximal at 0.87 +/- 0.08 by 120 min and then decreased after approximately 150 min of exposure to 42 degrees C. The thermotolerance ratios (TTR's) were 4.0, 5.4, 6.7 and 6.9 for temperatures of 40, 41, 41.5 and 42 degrees C respectively. The results suggest that chronic hyperthermia temperatures (i.e. 40-42 degrees C) induce rapid thermotolerance development in CFU-GM during the thermal exposure and protect this normal marrow progenitor during whole body hyperthermia or ex vivo purging of leukaemic cells.

Low doses of radiation induce systemic production of cytokines: possible contribution to leukemogenesis.

Experiments were conducted to explore the possible effect of low-dose irradiation on cytokine production in mice. SJL/J and C57BL/6J mice were exposed to 3 Gy and 4 Gy respectively. At various time intervals thereafter, lung-conditioned media, bone-marrow-conditioned media and sera were collected. Interleukin-6 and macrophage-colony-stimulating-factor activities were tested. Interleukin-6 in lung-conditioned media from both strains was found to be significantly induced by irradiation. Macrophage-colony-stimulating-factor activity was greatly enhanced in sera of irradiated SJL/J mice as well as in bone-marrow-conditioned media of both strains. The kinetics of the radiation-induced interleukin 6 and macrophage-colony-stimulating-factor activity differed in the 2 strains. SJL/J and C57BL/6J mice have been previously shown to be susceptible to low-dose-radiation-induced leukemia. The coleukemogenic effects of both cytokines in 3 different models of experimental leukemogenesis is demonstrated.

Inhibition of the immunosuppressive activity of resident pulmonary alveolar macrophages by granulocyte/macrophage colony-stimulating factor.

Resident pulmonary alveolar macrophages (PAM) play an important role in the maintenance of immunological homeostasis in the lung via downmodulation of local T cell responses in the steady state. The present study demonstrates that this pathway for T cell suppression is reversible via granulocyte/macrophage colony-stimulating factor (GM-CSF). Thus, freshly isolated PAM strongly inhibit mitogen-induced T cell proliferation, and pretreatment of the PAM with cytokine-rich lung-conditioned medium (LCM) generated by exposure of lung to bacterial lipopolysaccharide (LPS) abrogated this suppressive activity. LCM from lungs of normal and athymic nude mice exhibited identical activity. Moreover, the PAM-modulating activity of LCM was inhibited by blocking antibody specific for GM-CSF, and the activity of LCM could be reproduced by recombinant GM-CSF. This suggests that secretion of GM-CSF by mesenchymal cells and/or macrophages under stimulation from agents such as LPS provides a potential mechanism for upregulation of local T cell responsiveness during acute inflammation. In addition, experiments with a range of cytokines indicated that interleukin 4, transforming growth factor beta 1 and tumor necrosis factor alpha (TNF-alpha) exhibited weaker (but significant) modulatory effects on PAM, and (in the case of TNF-alpha) amplified the effects of GM-CSF.