RESUMEN
Bone metastasis is an incurable complication of breast cancer affecting 70-80 % of advanced patients. It is a multistep process that includes tumour cell mobilisation, intravasation, survival in the circulation, extravasation, migration and proliferation in the bone marrow/bone. Although novel findings demonstrate the bone marrow microenvironment significance in bone metastatic progression, a majority of studies have focused on end-stage disease and little is known about how the pre-metastatic niche arises in the bone marrow/bone tissues. We demonstrated a significant increase in patients' peripheral blood plasma ability to induce transendothelial migration of MCF-7 cells compared with healthy volunteers. Moreover, high RANKL, MIF and OPG levels in patients' peripheral blood could play a role in the intravasation, angiogenesis, survival and epithelial-mesenchymal transition of circulating tumour cells. Also, we observed a significant increase in patients' bone marrow plasma capacity to induce transendothelial migration of MDA-MB231 and MCF-7 cells compared with healthy volunteers. Furthermore, patients' bone marrow mesenchymal stem cells could control the recruitment of tumour cells, modifying the MCF-7 and MDA-MB231 cell migration. In addition, we found a significantly higher MDA-MB231 cell proliferation when we used patients' bone marrow plasma compared with healthy volunteers. Interestingly, PDGF-AB, ICAM-1 and VCAM-1 levels in patients' bone marrow were significantly higher than the values of healthy volunteers, suggesting that they could be involved in the cancer cell extravasation, bone resorption and cancer cell proliferation. We believe that these results can reveal new information about what alterations happen in the bone marrow of advanced breast cancer patients before bone colonisation, changes that create optimal soil for the metastatic cascade progression.
Asunto(s)
Médula Ósea/patología , Neoplasias Óseas/secundario , Neoplasias de la Mama/patología , Apoptosis , Neoplasias Óseas/sangre , Neoplasias de la Mama/sangre , Línea Celular Tumoral , Proliferación Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunohistoquímica , Metástasis de la NeoplasiaRESUMEN
Tumour cells can find in bone marrow (BM) a niche rich in growth factors and cytokines that promote their self-renewal, proliferation and survival. In turn, tumour cells affect the homeostasis of the BM and bone, as well as the balance among haematopoiesis, osteogenesis, osteoclastogenesis and bone-resorption. As a result, growth and survival factors normally sequestered in the bone matrix are released, favouring tumour development. Mesenchymal stem cells (MSCs) from BM can become tumour-associated fibroblasts, have immunosuppressive function, and facilitate metastasis by epithelial-to-mesenchymal transition. Moreover, MSCs generate osteoblasts and osteocytes and regulate osteoclastogenesis. Therefore, MSCs can play an important pro-tumorigenic role in the formation of a microenvironment that promotes BM and bone metastasis. In this study we showed that BM MSCs from untreated advanced breast and lung cancer patients, without bone metastasis, had low osteogenic and adipogenic differentiation capacity compared to that of healthy volunteers. In contrast, chondrogenic differentiation was increased. Moreover, MSCs from patients had lower expression of CD146. Finally, our data showed higher levels of Dkk-1 in peripheral blood plasma from patients compared with healthy volunteers. Because no patient had any bone disorder by the time of the study we propose that the primary tumour altered the plasticity of MSCs. As over 70 % of advanced breast cancer patients and 30-40 % of lung cancer patients will develop osteolytic bone metastasis for which there is no total cure, our findings could possibly be used as predictive tools indicating the first signs of future bone disease. In addition, as the MSCs present in the BM of these patients may not be able to regenerate bone after the tumour cells invasion into BM/bone, it is possible that they promote the cycle between tumour cell growth and bone destruction.
Asunto(s)
Médula Ósea/patología , Neoplasias de la Mama/patología , Neoplasias Pulmonares/patología , Células Madre Mesenquimatosas/patología , Neoplasias Óseas/secundario , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Osteólisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We have shown that bone marrow (BM) from untreated advanced lung and breast cancer patients (LCP and BCP) have a reduced number of colony-forming unit fibroblasts (CFU-Fs) or mesenchymal stem cells (MSCs). Factors that regulate the proliferation and differentiation of CFU-F are produced by the patients' BM microenvironment. We have now examined whether conditioned media (CM) from patients' CFU-F-derived stromal cells also inhibits the colony-forming efficiency (CFE) of CFU-F in primary cultures from healthy volunteers (HV)-BM. Thus the number and proliferation potential of HV-CFU-F were also found to be decreased and similar to colony numbers and colony size of patients' CFU-F. Stromal cells from both of these types of colonies appeared relatively larger and lacked the characteristic spindle morphology typically seen in healthy stromal cells. We developed an arbitrary mesenchymal stromal cell maturational index by taking three measures consisting of stromal cell surface area, longitudinal and horizontal axis. All stromal indices derived from HV-CFU-F grown in patients' CM were similar to those from stromal elements derived from patients' CFU-F. These indices were markedly higher than stromal indices typical of HV-CFU-F cultured in healthy CM or standard medium [alpha-medium plus 20% heat-inactivated fetal bovine serum (FBS)]. Patients' CM had increased concentrations of the CFU-F inhibitor, GM-CSF, and low levels of bFGF and Dkk-1, strong promoters of self-renewal of MSCs, compared to the levels quantified in CM from HV-CFU-F. Moreover, the majority of patients' MSCs were unresponsive in standard medium and healthy CM to give CFU-F, indicating that the majority of mesenchymal stromal cells from patients' CFU-F are locked in maturational arrest. These results show that alterations of GM-CSF, bFGF, and Dkk-1 are associated with deficient cloning and maturation arrest of CFU-F. Defective autocrine and paracrine mechanisms may be involved in the BM microenvironments of LCP and BCP.
Asunto(s)
Células de la Médula Ósea/metabolismo , Fibroblastos/metabolismo , Células Madre/metabolismo , Células del Estroma/metabolismo , Neoplasias de la Mama/sangre , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Medios de Cultivo Condicionados/metabolismo , Medios de Cultivo Condicionados/farmacología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neoplasias Pulmonares/sangre , Células Madre Mesenquimatosas/metabolismo , Factores de TiempoRESUMEN
Bone marrow (BM)-derived adult mesenchymal stem cells (MSCs) have the capacity to differentiate in vitro into different cell lines. This makes them a likely source for application in tissue repair therapies. Here, we report evidence indicating that, both in vivo and in vitro, IMT504, the prototype of the PyNTTTTGT class of immunostimulatory oligonucleotides, significantly increases the number of fibroblast colony-forming units (CFU-Fs) that originate MSCs. When rat BM cells were cultured with IMT504, the mean number of CFU-Fs increased about three times as compared with untreated controls (CFU-F: 19 +/- 6.3 vs. 6.8 +/- 2.0/2 x 10(6) seeded BM cells, p = .03). Furthermore, rats inoculated with IMT504 had a significantly higher number of CFU-Fs both in BM (CFU-F: 124 +/- 33 vs. 38 +/- 17/femur, p = .04) and in peripheral blood (animals with detectable CFU-Fs in circulation 8/12 vs. 2/12, p = .04) as compared with untreated animals. On the other hand, BM-derived adherent cells either treated in vitro with IMT504 or obtained from animals injected with IMT504 possess the capacity to differentiate to the osteogenic and adipogenic cell lineages as regular MSCs. Finally, we found that repair of a bone defect was accelerated in rats injected with IMT504 as compared with control animals (area with consolidated bone: 80% +/- 6.4% vs. 49% +/- 3.5%, p = .03, n = 10 rats per group). Importantly, when two human BM were cultured in the presence of IMT504, the mean number of fibroblastic adherent colonies also increased as compared with controls. These results suggest the possibility of clinical use of IMT504 in bone, and presumably other, tissue repair therapies.
Asunto(s)
Células de la Médula Ósea/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Oligodesoxirribonucleótidos/farmacología , Trasplante de Células Madre , Animales , Secuencia de Bases , Células de la Médula Ósea/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/fisiología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Previously, we reported a deficient cloning capacity of the bone marrow (BM) mesenchymal stem cells to give colony-forming unit fibroblast (CFU-F) and an inefficient confluence capacity of BM stromal cells in advanced untreated lung cancer patients (LCP) and breast cancer patients (BCP). Moreover, a decreased level of bFGF at day 7 in the conditioned media from BM CFU-F cultures was found in both cancer groups when compared to the normal range. The current study was specially undertaken, to evaluate the percentage of subconfluent fibroblasts expressing receptors (R) of interleukin-1 (IL-1), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor (TGF-beta), epidermal growth factor (EGF), and the proteins c-Fos and c-Myc in BM primary cultures from untreated LCP and BCP. An immunocytochemical study on subconfluent BM fibroblast cultures from 13 healthy patients, 16 LCP, and 8 BCP was performed, using as primary antibodies, anti-type I of IL-1 R (IL-1R-1), anti-alpha, beta chains of PDGF R (PDGFR-alpha, PDGFR-beta), anti-type I of FGF R (FGFR-I), anti-type I, II, and III of TGF-beta R (TGF-betaR-I, TGF- betaR-II, and TGF-betaR-III), anti-EGF R, anti-c-Fos, and anti-c-Myc. A diminished percentage of subconfluent fibroblasts expressing PDGFR-alpha, TGFbetaR-I, II, III, EGFR, and FGFR-I was found in LCP and BCP compared to healthy patients. A diminished percentage of subconfluent fibroblasts expressing c-Fos and c-Myc was found in patients when compared to healthy patients. The alterations we describe could help to explain the deficiency regarding the proliferative and confluence capacity of BM stroma cells in cancer patients.
Asunto(s)
Células de la Médula Ósea/metabolismo , Neoplasias de la Mama/metabolismo , Receptores ErbB/metabolismo , Neoplasias Pulmonares/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Receptores de Interleucina-1/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Células del Estroma/metabolismo , Animales , Células de la Médula Ósea/citología , Neoplasias de la Mama/patología , Células Cultivadas , Medios de Cultivo Condicionados , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Neoplasias Pulmonares/patología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Células del Estroma/citología , Ensayo de Tumor de Célula MadreRESUMEN
BACKGROUND: Bone marrow (BM) is an important tissue in the generation of immunocompetent and peripheral blood cells. The precursors of hematopoietic cells in BM undergo continuous proliferation and differentiation and are highly vulnerable to acute and chronic oxidative stress. Little is known about the oxidant and antioxidant status in the BM of untreated patients with nonhematologic tumors. In this study, oxidative stress was evaluated in peripheral blood plasma (PBP) and BM plasma (BMP) from lung carcinoma (LC) and breast carcinoma (BC) patients. METHODS: The sample included 13 consecutive untreated LC patients, 15 BC patients, and 11 healthy controls. Luminol-dependent chemiluminescence was used to evaluate oxygen radical generation by peripheral blood neutrophils. Lipid oxidation, assessed by 2-thiobarbituric acid-reactive substances (TBARS), and alpha-tocopherol, beta-carotene, and total ubiquinol-10 levels were determined in PBP and BMP. RESULTS: In LC and BC patients, neutrophil chemiluminescence was higher (128% and 264%, respectively) than in controls (P < 0.05). In cancer patients, TBARS levels were higher in both PBP (51% and 243% for LC and BC patients, respectively) and BMP (66% and 305% for LC and BC patients, respectively) than in plasma from controls (P < 0.01). alpha-Tocopherol and total ubiquinol-10 levels were significantly lower in BMP from BC patients compared with controls. In BC patients, alpha-tocopherol content in PBP was significantly lower than in controls. CONCLUSIONS: Untreated cancer patients presented an imbalance between oxidant generation and lipid-soluble antioxidant levels in favor of the former.