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Oncolytic viruses, modified for tumor-restricted infection, are a promising cancer immunotherapeutic, yet much remains to be understood about factors driving their activity and outcome in the tumor microenvironment. Here, we report that oncolytic herpes simplex virus C134, previously found to exert T cell-dependent efficacy in mouse models of glioblastoma, exerts T cell-independent efficacy in mouse models of medulloblastoma, indicating this oncolytic virus uses different mechanisms in different tumors. We investigated C134's behavior in mouse medulloblastomas, using single cell RNA sequencing to map C134-induced gene expression changes across cell types, timepoints, and medulloblastoma subgroup models at whole-transcriptome resolution. Our work details substantial oncolytic virus-induced transcriptional remodeling of medulloblastoma-infiltrating immune cells, 10 subpopulations of monocytes and macrophages collectively demonstrating M1-like responses to C134, and suggests C134 be investigated as a potential new therapy for medulloblastoma.
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PURPOSE: We investigated why three patient-derived xenograft (PDX) childhood BRAFV600E-mutant brain tumor models are highly sensitive to trametinib. Mechanisms of acquired resistance selected in situ, and approaches to prevent resistance were also examined, which may translate to both low-grade glioma (LGG) molecular subtypes. EXPERIMENTAL DESIGN: Sensitivity to trametinib [MEK inhibitor (MEKi)] alone or in combination with rapamycin (TORC1 inhibitor), was evaluated in pediatric PDX models. The effect of combined treatment of trametinib with rapamycin on development of trametinib resistance in vivo was examined. PDX tissue and tumor cells from trametinib-resistant xenografts were characterized. RESULTS: In pediatric models TORC1 is activated through ERK-mediated inactivation of the tuberous sclerosis complex (TSC): consequently inhibition of MEK also suppressed TORC1 signaling. Trametinib-induced tumor regression correlated with dual inhibition of MAPK/TORC1 signaling, and decoupling TORC1 regulation from BRAF/MAPK control conferred trametinib resistance. In mice, acquired resistance to trametinib developed within three cycles of therapy in all three PDX models. Resistance to trametinib developed in situ is tumor-cell-intrinsic and the mechanism was tumor line specific. Rapamycin retarded or blocked development of resistance. CONCLUSIONS: In these three pediatric BRAF-mutant brain tumors, TORC1 signaling is controlled by the MAPK cascade. Trametinib suppressed both MAPK/TORC1 pathways leading to tumor regression. While low-dose intermittent rapamycin to enhance inhibition of TORC1 only modestly enhanced the antitumor activity of trametinib, it prevented or retarded development of trametinib resistance, suggesting future therapeutic approaches using rapamycin analogs in combination with MEKis that may be therapeutically beneficial in both KIAA1549::BRAF- and BRAFV600E-driven gliomas.
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Neoplasias Encefálicas , Glioma , Alvo Mecanístico do Complexo 1 de Rapamicina , Piridonas , Pirimidinonas , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Glioma/tratamento farmacológico , Glioma/genética , Glioma/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética , Piridonas/uso terapêutico , Pirimidinonas/uso terapêutico , SirolimoRESUMO
BACKGROUND: Glioblastoma (GBM) remains one of the least successfully treated cancers. It is essential to understand the basic biology of this lethal disease and investigate novel pharmacological targets to treat GBM. The aims of this study were to determine the biological consequences of elevated expression of ΔNp73, an N-terminal truncated isoform of TP73, and to evaluate targeting of its downstream mediators, the angiopoietin 1 (ANGPT1)/tunica interna endothelial cell kinase 2 (Tie2) axis, by using a highly potent, orally available small-molecule inhibitor (rebastinib) in GBM. METHODS: ΔNp73 expression was assessed in glioma sphere cultures, xenograft glioblastoma tumors, and glioblastoma patients by western blot, quantitative reverse transcription PCR, and immunohistochemistry. Immunoprecipitation, chromatin immunoprecipitation (ChiP) and sequential ChIP were performed to determine the interaction between ΔNp73 and E26 transformation-specific (ETS) proto-oncogene 2 (ETS2) proteins. The oncogenic consequences of ΔNp73 expression in glioblastomas were examined by in vitro and in vivo experiments, including orthotopic zebrafish and mouse intracranial-injection models. Effects of rebastinib on growth of established tumors and survival were examined in an intracranial-injection mouse model. RESULTS: ΔNp73 upregulates both ANGPT1 and Tie2 transcriptionally through ETS conserved binding sites on the promoters by interacting with ETS2. Elevated expression of ΔNp73 promotes tumor progression by mediating angiogenesis and survival. Therapeutic targeting of downstream ΔNp73 signaling pathways by rebastinib inhibits growth of established tumors and extends survival in preclinical models of glioblastoma. CONCLUSION: Aberrant expression of ΔNp73 in GBM promotes tumor progression through autocrine and paracrine signaling dependent on Tie2 activation by ANGPT1. Disruption of this signaling by rebastinib improves tumor response to treatment in glioblastoma.
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Antineoplásicos/administração & dosagem , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioblastoma/metabolismo , Glioblastoma/patologia , Proteína Proto-Oncogênica c-ets-2/metabolismo , Pirazóis/administração & dosagem , Piridinas/administração & dosagem , Quinolinas/administração & dosagem , Proteína Tumoral p73/metabolismo , Animais , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral/efeitos dos fármacos , Modelos Animais de Doenças , Glioblastoma/tratamento farmacológico , Humanos , Camundongos Transgênicos , Neovascularização Patológica/metabolismo , Proto-Oncogene Mas , Análise de Sobrevida , Peixe-ZebraRESUMO
Pediatric brain tumors including medulloblastoma and atypical teratoid/rhabdoid tumor are associated with significant mortality and treatment-associated morbidity. While medulloblastoma tumors within molecular subgroups 3 and 4 have a propensity to metastasize, atypical teratoid/rhabdoid tumors frequently afflict a very young patient population. Adjuvant treatment options for children suffering with these tumors are not only sub-optimal but also associated with many neurocognitive obstacles. A potentially novel treatment approach is oncolytic virotherapy, a developing therapeutic platform currently in early-phase clinical trials for pediatric brain tumors and recently US Food and Drug Administration (FDA)-approved to treat melanoma in adults. We evaluated the therapeutic potential of the clinically available oncolytic herpes simplex vector rRp450 in cell lines derived from medulloblastoma and atypical teratoid/rhabdoid tumor. Cells of both tumor types were supportive of virus replication and virus-mediated cytotoxicity. Orthotopic xenograft models of medulloblastoma and atypical teratoid/rhabdoid tumors displayed significantly prolonged survival following a single, stereotactic intratumoral injection of rRp450. Furthermore, addition of the chemotherapeutic prodrug cyclophosphamide (CPA) enhanced rRp450's in vivo efficacy. In conclusion, oncolytic herpes viruses with the ability to bioactivate the prodrug CPA within the tumor microenvironment warrant further investigation as a potential therapy for pediatric brain tumors.
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PURPOSE: Curative therapy for childhood glioma presents challenges when complete resection is not possible. Patients with recurrent low-grade tumors or anaplastic astrocytoma may receive radiation treatment; however, the long-term sequellae from radiation treatment can be severe. As many childhood gliomas are associated with activation of BRAF, we have explored the combination of ionizing radiation with MEK inhibition in a model of BRAF-mutant anaplastic astrocytoma. EXPERIMENTAL DESIGN: The regulation of TORC1 signaling by BRAF was examined in BT-40 (BRAF mutant) and BT-35 (BRAF wild type) xenografts, in a cell line derived from the BT-40 xenograft and two adult BRAF mutant glioblastoma cell lines. The effect of MEK inhibition (selumetinib), XRT (total dose 10 Gy as 2 Gy daily fractions), or the combination of selumetinib and XRT was evaluated in subcutaneous BT-40 xenografts. RESULTS: Inhibition of MEK signaling by selumetinib suppressed TORC1 signaling only in the context of the BRAF-mutant both in vitro and in vivo. Inhibition of MEK signaling in BT-40 cells or in xenografts lead to a complete suppression of FANCD2 and conferred hypersensitivity to XRT in BT-40 xenografts without increasing local skin toxicity. CONCLUSIONS: Selumetinib suppressed TORC1 signaling in the context of BRAF mutation. Selumetinib caused a rapid downregulation of FANCD2 and markedly potentiated the effect of XRT. These data suggest the possibility of potentiating the effect of XRT selectively in tumor cells by MEK inhibition in the context of mutant BRAF or maintaining tumor control at lower doses of XRT that would decrease long-term sequelae.
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Astrocitoma/genética , Astrocitoma/radioterapia , Benzimidazóis/efeitos adversos , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Radioterapia/efeitos adversos , Animais , Western Blotting , Linhagem Celular Tumoral , Feminino , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos SCID , Complexos Multiproteicos/metabolismo , Neoplasias Experimentais/genética , Neoplasias Experimentais/radioterapia , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Atypical teratoid rhabdoid tumor (AT/RT) is a rare, highly malignant pediatric tumor of the central nervous system that is usually refractory to available treatments. The aggressive growth, propensity to disseminate along the neuroaxis, and young age at diagnosis contribute to the poor prognosis. Previous studies have demonstrated the efficacy of using oncolytic measles virus (MV) against localized and disseminated models of medulloblastoma. The purpose of this study was to evaluate the oncolytic potential of MV in experimental models of AT/RT. METHODS: Following confirmation of susceptibility to MV infection and killing of AT/RT cells in vitro, nude mice were injected with BT-12 and BT-16 AT/RT cells stereotactically into the caudate nucleus (primary tumor model) or lateral ventricle (disseminated tumor model). Recombinant MV was administered either intratumorally or intravenously. Survival was determined for treated and control animals. Necropsy was performed on animals showing signs of progressive disease. RESULTS: All cell lines exhibited significant killing when infected with MV, all formed syncytia with infection, and all generated infectious virus after infection. Orthotopic xenografts displayed cells with rhabdoid-like cellular morphology, were negative for INI1 expression, and showed dissemination within the intracranial and spinal subarachnoid spaces. Intratumoral injection of live MV significantly prolonged the survival of animals with intracranial and metastatic tumors. CONCLUSION: These data demonstrate that AT/RT is susceptible to MV killing and suggest that the virus may have a role in treating this tumor in the clinical setting.
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Encéfalo/virologia , Vírus do Sarampo/fisiologia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/fisiologia , Tumor Rabdoide/virologia , Animais , Encéfalo/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Chlorocebus aethiops , Modelos Animais de Doenças , Feminino , Humanos , Estimativa de Kaplan-Meier , Camundongos , Camundongos Nus , Tumor Rabdoide/terapia , Células Vero , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Our intracranial implantation mouse model of ependymoma clearly demonstrates overexpression of the ephrin receptor EphB2 in Ink4a/Arf((-/-)) supratentorial embryonic neural stem cells (STeNSCs) to be essential for transformation and disease development; however the requirement for and consequence of receptor activation on transformation and neural stem cell function were not examined. We definitively illustrate the necessity for receptor activation in cellular transformation and the importance of implantation site and microenvironment in directing ependymoma development. In vitro assays of EphB2 overexpressing Ink4a/Arf((-/-)) STeNSCs showed no changes in their neural stem cell characteristics (stem cell marker expression and self-renewal) upon receptor activation, but EphB2 driven tumor cells were inhibited significantly in differentiation and exhibited increased tumorsphere formation and cellular proliferation in response to ephrin-B ligand mediated receptor activation. Additionally, we observed substantial differences in the phosphorylation state of several key proteins involved in Ras and p38 MAPK signaling when comparing EphB2 overexpressing Ink4a/Arf((-/-)) STeNSCs and tumor cells with relatively little change in total protein levels. We propose that EphB2 mediated ependymoma development is a multifactorial process requiring microenvironment directed receptor activation, resulting in changes in the phosphorylation status of key regulatory proteins, maintenance of a stem-like state and cellular proliferation.
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Neoplasias Encefálicas/genética , Transformação Celular Neoplásica/genética , Ependimoma/genética , Regulação Neoplásica da Expressão Gênica , Receptor EphB2/genética , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Diferenciação Celular , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Inibidor p16 de Quinase Dependente de Ciclina/genética , Modelos Animais de Doenças , Ependimoma/metabolismo , Ependimoma/patologia , Feminino , Humanos , Camundongos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Fosforilação , Receptor EphB2/metabolismo , Transdução de Sinais , Microambiente Tumoral , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
A successful oncolytic virus is one that selectively propagates and destroys cancerous tissue without causing excessive damage to the normal surrounding tissue. Oncolytic measles virus (MV) is one such virus that exhibits this characteristic and thus has rapidly emerged as a potentially useful anticancer modality. Derivatives of the Edmonston MV vaccine strain possess a remarkable safety record in humans. Promising results in preclinical animal models and evidence of biological activity in early phase trials contribute to the enthusiasm. Genetic modifications have enabled MV to evolve from a vaccine agent to a potential anticancer therapy. Specifically, alterations of the MV genome have led to improved tumor selectivity and delivery, therapeutic potency, and immune system modulation. In this article, we will review the advancements that have been made in the design and development of MV that have led to its use as a cancer therapy. In addition, we will discuss the evidence supporting its use, as well as the challenges associated with MV as a potential cancer therapeutic.
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BACKGROUND: Medulloblastoma is the most common type of pediatric brain tumor. Although numerous factors influence patient survival rates, more than 30% of all cases will ultimately be refractory to conventional therapies. Current standards of care are also associated with significant morbidities, giving impetus for the development of new treatments. We have previously shown that oncolytic measles virotherapy is effective against medulloblastoma, leading to significant prolongation of survival and even cures in mouse xenograft models of localized and metastatic disease. Because medulloblastomas are known to be highly vascularized tumors, we reasoned that the addition of angiogenesis inhibitors could further enhance the efficacy of oncolytic measles virotherapy. Toward this end, we have engineered an oncolytic measles virus that express a fusion protein of endostatin and angiostatin, two endogenous and potent inhibitors of angiogenesis. METHODS: Oncolytic measles viruses encoding human and mouse variants of a secretable endostatin/angiostatin fusion protein were designed and rescued according to established protocols. These viruses, known as MV-hE:A and MV-mE:A respectively, were then evaluated for their anti-angiogenic potential and efficacy against medulloblastoma cell lines and orthotopic mouse models of localized disease. RESULTS: Medulloblastoma cells infected by MV-E:A readily secrete endostatin and angiostatin prior to lysis. The inclusion of the endostatin/angiostatin gene did not negatively impact the measles virus' cytotoxicity against medulloblastoma cells or alter its growth kinetics. Conditioned media obtained from these infected cells was capable of inhibiting multiple angiogenic factors in vitro, significantly reducing endothelial cell tube formation, viability and migration compared to conditioned media derived from cells infected by a control measles virus. Mice that were given a single intratumoral injection of MV-E:A likewise showed reduced numbers of tumor-associated blood vessels and a trend for increased survival compared to mice treated with the control virus. CONCLUSIONS: These data suggest that oncolytic measles viruses encoding anti-angiogenic proteins may have therapeutic benefit against medulloblastoma and support ongoing efforts to target angiogenesis in medulloblastoma.
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Inibidores da Angiogênese/uso terapêutico , Angiostatinas/antagonistas & inibidores , Endostatinas/antagonistas & inibidores , Vírus do Sarampo/fisiologia , Meduloblastoma/terapia , Terapia Viral Oncolítica/efeitos adversos , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Vírus do Sarampo/genética , Meduloblastoma/patologia , Camundongos , Neoplasias Experimentais , Vírus Oncolíticos/genética , Células Vero , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Medulloblastoma is the most common malignant brain tumor of childhood. Although the clinical outcome for medulloblastoma patients has improved significantly, children afflicted with the disease frequently suffer from debilitating side effects related to the aggressive nature of currently available therapy. Alternative means for treating medulloblastoma are desperately needed. We have previously shown that oncolytic measles virus (MV) can selectively target and destroy medulloblastoma tumor cells in localized and disseminated models of the disease. MV-NIS, an oncolytic measles virus that encodes the human thyroidal sodium iodide symporter (NIS), has the potential to deliver targeted radiotherapy to the tumor site and promote a localized bystander effect above and beyond that achieved by MV alone. METHODS: We evaluated the efficacy of MV-NIS against medulloblastoma cells in vitro and examined their ability to incorporate radioiodine at various timepoints, finding peak uptake at 48 hours post infection. The effects of MV-NIS were also evaluated in mouse xenograft models of localized and disseminated medulloblastoma. Athymic nude mice were injected with D283med-Luc medulloblastoma cells in the caudate putamen (localized disease) or right lateral ventricle (disseminated disease) and subsequently treated with MV-NIS. Subsets of these mice were given a dose of 131I at 24, 48 or 72 hours later. RESULTS: MV-NIS treatment, both by itself and in combination with 131I, elicited tumor stabilization and regression in the treated mice and significantly extended their survival times. Mice given 131I were found to concentrate radioiodine at the site of their tumor implantations. In addition, mice with localized tumors that were given 131I either 24 or 48 hours after MV-NIS treatment exhibited a significant survival advantage over mice given MV-NIS alone. CONCLUSIONS: These data suggest MV-NIS plus radioiodine may be a potentially useful therapy for the treatment of medulloblastoma.
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Radioisótopos do Iodo/uso terapêutico , Vírus do Sarampo/fisiologia , Meduloblastoma/terapia , Vírus Oncolíticos/fisiologia , Simportadores/fisiologia , Animais , Transporte Biológico/fisiologia , Efeito Espectador/efeitos da radiação , Linhagem Celular Tumoral , Chlorocebus aethiops , Terapia Combinada , Células Gigantes/metabolismo , Células Gigantes/efeitos da radiação , Células Gigantes/virologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Radioisótopos do Iodo/farmacocinética , Medições Luminescentes/métodos , Vírus do Sarampo/genética , Meduloblastoma/patologia , Meduloblastoma/virologia , Camundongos , Camundongos Nus , Vírus Oncolíticos/genética , Análise de Sobrevida , Simportadores/genética , Glândula Tireoide/metabolismo , Resultado do Tratamento , Células Vero , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Medulloblastoma is the most common malignant brain tumor of childhood. Although the survival rate of afflicted children has improved considerably over the past several years, a subset of these patients will present with disseminated disease and face a much bleaker prognosis. In addition, patients may present with disseminated disease at recurrence. We previously demonstrated the efficacy of a recombinant oncolytic measles virus (MV) to treat localized medulloblastoma in a mouse xenograft model. In the present study, we sought to extend our findings to the treatment of disseminated disease. To this end, we developed and characterized a mouse xenograft model of disseminated medulloblastoma using serial bioluminescent imaging techniques in combination with histopathological examination. Mice injected with medulloblastoma cells into their right lateral ventricle showed tumor growth in their ventricles and in both intracranial and spinal subarachnoid spaces, closely recapitulating the human disease. Subsequent intraventricular administration of MV resulted in stabilization and shrinkage of the tumor, significantly prolonging the survival of the treated animals, compared with those treated with an inactivated virus. These data demonstrate that oncolytic MV may be of use in treating disseminated medulloblastoma. In addition, our protocol of intraventricular tumor cell injection, followed by bioluminescent imaging coupled with histopathological examination, provides a model for use in evaluating future recombinant oncolytic viruses and other preclinical therapeutic approaches for disseminated medulloblastoma.
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Neoplasias Cerebelares/terapia , Vírus do Sarampo , Meduloblastoma/terapia , Metástase Neoplásica/terapia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos , Animais , Líquido Cefalorraquidiano , Humanos , Camundongos , Camundongos Nus , Resultado do Tratamento , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although treatment of medulloblastoma has improved, at least 30% of patients with this tumor die of progressive disease. Unfortunately, many of the children who survive suffer long-term treatment-related morbidity. Previous studies have demonstrated the efficacy of using oncolytic viruses to eradicate brain tumors. The objective of this study was to test the efficacy of measles virus in treating medulloblastoma. To determine whether medulloblastoma cells are susceptible, 5 different human medulloblastoma cell lines were analyzed for the expression of the measles virus receptor CD46. Fluorescence-activated cell-sorting analysis confirmed expression of CD46 on all cell lines tested, with UW288-1 having the most prominent expression and D283med displaying the lowest expression. CD46 expression was also demonstrated, using immunohistochemistry, in 13 of 13 medulloblastoma tissue specimens. All 5 medulloblastoma cell lines were examined for their susceptibility to measles virus killing in vitro. A measles virus containing the green fluorescent protein (GFP) gene as a marker for infection (MV-GFP) was used. All cell lines exhibited significant killing when infected with MV-GFP, all formed syncytia with infection, all showed fluorescence, and all allowed viral replicaton after infection. In an intracerebral murine xenograft model, a statistically significant increase in survival was seen in animals treated with the active measles virus compared with those treated with inactivated virus. These data demonstrate that medulloblastoma is susceptible to measles virus killing and that the virus may have a role in treating this tumor in the clinical setting.
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Antineoplásicos/uso terapêutico , Neoplasias Cerebelares/terapia , Meduloblastoma/terapia , Terapia Viral Oncolítica/métodos , Animais , Linhagem Celular Tumoral , Separação Celular , Neoplasias Cerebelares/virologia , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Vírus do Sarampo , Meduloblastoma/virologia , Proteína Cofatora de Membrana/biossíntese , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Common sites of breast cancer metastasis include the lung, liver, and bone, and of these secondary metastatic sites, estrogen receptor alpha (ERalpha)-positive breast cancer often favors bone. Within secondary organs, cancer cells would predictably encounter tissue-specific fibroblasts or their soluble factors, yet our understanding of how tissue-specific fibroblasts directly affect cancer cell growth rates and survival remains largely unknown. Therefore, we tested the hypothesis that mesenchymal fibroblasts isolated from common sites of breast cancer metastasis provide a more favorable microenvironment with respect to tumor growth rates. We found a direct correlation between the ability of breast, lung, and bone fibroblasts to enhance ERalpha-positive breast cancer cell growth and the level of soluble interleukin-6 (IL-6) produced by each organ-specific fibroblast, and fibroblast-mediated growth enhancement was inhibited by the removal or inhibition of IL-6. Interestingly, mice coinjected with MCF-7 breast tumor cells and senescent skin fibroblasts, which secrete IL-6, developed tumors, whereas mice coinjected with presenescent skin fibroblasts that produce little to no IL-6 failed to form xenograft tumors. We subsequently determined that IL-6 promoted growth and invasion of breast cancer cells through signal transducer and activator of transcription 3-dependent up-regulation of Notch-3, Jagged-1, and carbonic anhydrase IX. These data suggest that tissue-specific fibroblasts and the factors they produce can promote breast cancer disease progression and may represent attractive targets for development of new therapeutics.
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Neoplasias da Mama/patologia , Divisão Celular/fisiologia , Fibroblastos/citologia , Interleucina-6/fisiologia , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Meios de Cultivo Condicionados , Humanos , Imunoprecipitação , Fosforilação , Interferência de RNA , Fator de Transcrição STAT3/metabolismoRESUMO
Bone is the primary anatomical site of breast cancer metastasis, and bone metastasis is associated with increased morbidity and mortality. Mesenchymal stem cells (MSC) are a predominant fibroblast cell population within the bone marrow, and metastatic breast cancer cells that seed within bone would predictably encounter MSC or their soluble factors. Therefore, we examined the impact of primary human MSC on a panel of estrogen receptor-alpha (ERalpha)-positive (MCF-7, T47D, BT474, and ZR-75-1) and ERalpha-negative (MDA-MB-231 and MDA-MB-468) human breast tumor cell lines. All ERalpha-positive breast tumor cell lines displayed low basal activation of signal transducer and activator of transcription 3 (STAT3) until exposed to MSC, which induced chronic phosphorylation of STAT3 on tyrosine-705. Paracrine IL-6 was found to be the principal mediator of STAT3 phosphorylation in coculture studies, and MSC induction of STAT3 phosphorylation was lost when IL-6 was depleted from MSC conditioned media or the IL-6 receptor was blocked on tumor cells. Enhanced tumor cell growth rates were observed in the ERalpha-positive mammary tumor cell line MCF-7 after paracrine and autocrine IL-6 exposure, where MCF-7 growth rates were enhanced by >2-fold when cocultured with MSC in vitro and even more pronounced in vivo with autocrine IL-6 production.
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Neoplasias da Mama/patologia , Receptor alfa de Estrogênio/metabolismo , Interleucina-6/fisiologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , HumanosRESUMO
Our understanding of the impact that fibroblasts have on cancer cell behavior in vivo has been limited by the complexities of in vivo tumor microenvironments, which contain many distinct cell populations that influence tumor growth and survival. Herein, we describe a novel, three-dimensional (3D), in vitro, fluorometric, Tumor Growth Assay (TGA) that allows for non-invasive measurements of cancer cell expansion in the presence of multiple tumor-associated cell types or soluble factors, while embedded in Cultrex or Matrigel Basement Membrane Extract (BME). Using this assay, we investigated the direct biological impact of primary human bone marrow stromal cells (hMSC) on the growth rates of a panel of metastatic breast cancer cell lines. Human MSC can be readily isolated from bone marrow, a principle site of breast cancer metastasis, and were found to significantly enhance the growth rate of MCF-7 (P-value<0.0001), an estrogen receptor-alpha (ERalpha) positive breast cancer cell line, in a soluble factor-dependent manner. MSC paracrine factors also enhanced the growth of other ERalpha positive breast cancer cell lines including T47D, BT474, and ZR-75-1 (P-value<0.05). In contrast, the ERalpha negative cell line MDA-MB-231 was unaffected by hMSC and the growth rate of another ERalpha negative cell line MDA-MB-468 was elevated in the presence of hMSC, albeit to a lesser extent than MCF-7 or the other ERalpha positive cell lines tested.