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1.
Int J Radiat Biol ; : 1-9, 2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31914348

RESUMO

Purpose: Pulmonary inflammation is an adverse consequence of radiation therapy in breast cancer. The aim of this study was to elucidate biological pathways leading to this pathology.Materials and methods: Lung endothelial cells were isolated 24 h after thorax-irradiation (sham or 10 Gy X-ray) from female C57Bl/6 mice and cultivated for 6 days.Results: Quantitative proteomic analysis of lung endothelial cells was done using data independent acquisition (DIA) mass spectrometry. The data were analyzed using Ingenuity Pathway Analysis and STRINGdb. In total, 4220 proteins were identified using DIA of which 60 were dysregulated in the irradiated samples (fold change ≥2.00 or ≤0.50; q-value <0.05). Several (12/40) upregulated proteins formed a cluster of inflammatory proteins with STAT1 and IRF3 as predicted upstream regulators. The several-fold increased expression of STAT1 and STAT-associated ISG15 was confirmed by immunoblotting. The expression of antioxidant proteins SOD1 and PRXD5 was downregulated suggesting radiation-induced oxidative stress. Similarly, the phosphorylated (active) forms of STING and IRF3, both members of the cGAS/STING pathway, were downregulated.Conclusions: These data suggest the involvement of JAK/STAT and cGas/STING pathways in the genesis of radiation-induced lung inflammation. These pathways may be used as novel targets for the prevention of radiation-induced lung damage.

2.
Bone ; 133: 115251, 2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31978616

RESUMO

Angiogenesis is critical in bone development and growth. Dense, large-scale, and multi-layered vascular networks formed by thin-walled sinusoidal vessels perfuse the plate bones and play an important role in bone repair. Yet, the intricate functional morphology of skull microvasculature remains poorly understood as it is difficult to visualize using existing intravital microscopy techniques. Here we introduced an intravital, fully-transcranial imaging approach based on hybrid optoacoustic and ultrasound bio-microscopy for large-scale observations and quantitative analysis of the vascular morphology, angiogenesis, vessel remodeling, and subsurface roughness in murine skulls. Our approach revealed radiation-inhibited angiogenesis in the skull bone. We also observed previously undocumented sinusoidal vascular networks spanning the entire skullcap, thus opening new vistas for studying the complex interactions between calvarial, pial, and cortical vascular systems.

3.
Int J Hyperthermia ; 37(1): 55-65, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31918587

RESUMO

Introduction: An abscopal effect is a clinical observation whereby a local treatment is associated with regression of metastatic cancer at a site distant from the primary location of treatment. Here, we describe the clinical systemic effect induced by regional hyperthermia combined with low-dose chemotherapy and provide immunologic correlates.Case presentation: A 15-year-old patient had been diagnosed with alveolar rhabdomyosarcoma (ARMS). All previous treatment options failed in the patient including haploidentical stem cell transplantation and donor lymphocyte infusion. The patient presented with local and metastatic disease, and upon admission, underwent regional hyperthermia combined with low-dose chemotherapy. Immediately following therapy severe skin reactions were observed. Skin biopsies revealed an intraepithelial lymphocytic infiltration dominated by CD3+/CD8+ T cells with a regular network of dendritic cells. Clinical images compared before and during sequential treatment cycles showed complete metabolic response of the local tumor for more than 10 months of therapy. In addition, metastases completely regressed although they were not direct targets of regional hyperthermia. The systemic effect was associated with enhanced frequency of NK cells and T cells expressing the lectin-like natural-killer group 2 D activating receptor (NKG2D), an increase of the CD56bright subset of NK cells, as well as an increase of effector/memory and effector CD8+ and CD4+ T cells in the blood while the percentage of CD25+FOXP3+ regulatory T cells declined.Conclusions: Regional hyperthermia combined with low-dose chemotherapy had the potential to create a systemic effect which was associated with activation of NK cells and T cells.

4.
Adv Exp Med Biol ; 1232: 131-143, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31893404

RESUMO

Hypoxia, one of the hallmarks of cancer, is caused by an insufficient oxygen supply, mostly due to a chaotic, deficient tumor microcirculation. Apart from a hypoxia-mediated resistance to standard therapies, modulated gene and protein expression, genetic instability and malignant progression, hypoxia also plays a pivotal role in anti-cancer immune responses by (a) reducing survival, cytolytic and migratory activity of effector cells such as CD4+ cells, CD8+ cytotoxic T cells, natural killer-like T cells and natural killer cells, (b) reducing the production and release of effector cytokines, (c) supporting immunosuppressive cells such as regulatory T cells, myeloid-derived suppressor cells and M2 macrophages, (d) increasing the production and release of immunosuppressive cytokines, and (e) inducing the expression of immune checkpoint inhibitors. In this minireview, immunosuppressive effects of hypoxia- and HIF-1a-driven traits in cancers are described.


Assuntos
Hipóxia , Células Supressoras Mieloides , Neoplasias , Humanos , Hipóxia/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias/imunologia , Linfócitos T Reguladores/imunologia , Microambiente Tumoral/imunologia
5.
Adv Exp Med Biol ; 1232: 169-176, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31893407

RESUMO

Inhospitable conditions within the tumor microenvironment (TME) are a characteristic feature ('hallmark') of most solid malignancies. Regional tumor hypoxia is a primary deficiency since it plays a key role in malignant progression. Severe hypoxia is often associated with other detrimental conditions in the TME as a consequence of hypoxia-/HIF-1α-induced (with/without oncogene-direction and/or reciprocal interaction of cancer cells with TME cells) metabolic re-programming, exorbitant extracellular adenosine (ADO) generation and VEGF overexpression/VEGF-R activation. Re-programming of the tumor metabolism inter alia includes a 'selfish' upregulation of aerobic glycolysis/glycolytic flux ('Warburg effect'), a strongly enhanced glutaminolysis in tumor cells, ketogenesis in cancer-associated fibroblasts, and an acceleration of the tryptophan uptake/intensified catabolism yielding kynurenine, which can support the malignant phenotype. Aerobic glycolysis and glutaminolysis result in lactate accumulation (up to 40 mM), and together with the enhanced ketogenesis and CO2/carbonic acid production lead to extracellular acidosis (pHe < 6.8). These traits of the TME individually or collectively operate towards cancer progression via e.g. promotion of genetic instability and mutation, resistance to apoptosis, clonal selection, limitless cell survival and sustained proliferation, continuous angiogenesis and tumor growth, local invasion and distant metastasis, anti-tumor immunosuppression and resistance to therapy.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia , Neoplasias , Hipóxia Tumoral , Microambiente Tumoral , Linhagem Celular Tumoral , Progressão da Doença , Glicólise , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias/fisiopatologia , Fenótipo
6.
Expert Opin Biol Ther ; 20(1): 23-33, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31714156

RESUMO

Introduction: Lung cancer is a devastating disease with poor overall survival. Despite significant advances in the treatment of lung cancers using radiochemotherapy, targeted therapies and/or immune therapies prognosis remains poor. The capacity of natural killer (NK) cells to provide a first line of defense that can bridge and orchestrate innate and 'downstream' adaptive immune responses renders them to be an ideal platform on which to base new cancer therapeutics.Areas covered: We provide an overview of the mechanisms controlling the effector functions of NK cells, tumor-directed immune escape, the impact and influence of NK cells on the development of effective, protective anti-tumor immunity and the therapeutic potential of combined cytokine-, complement-dependent- and antibody-dependent cellular cytotoxicity (CDC/ADCC), NK-92-, KIR mismatch- and CAR-NK cell-based therapies.Expert opinion: Despite promising results of immuno-oncological approaches, a relevant proportion of patients do not profit from these therapies, partly due to an ineffective NK cell activation, a lack of tumor-specific NK cells, an upregulated expression of checkpoint pathways, and a low mutational burden, which hinders the development of long-term adaptive immunity. Strategies that re-activate NK cells in combination with other therapies are therefore likely to be beneficial for the clinical outcome of patients with lung cancer.Abbreviations: ADCC: antibody-dependent cell-mediated cytotoxicity; ALK: anaplastic lymphoma kinase; CAR: chimeric antigen receptor; CDC: complement-dependent cytotoxicity; CEACAM-1: carcinoembryonic antigen-related cell adhesion molecule 1; DC: dendritic cell; DNAM: activating, maturation receptor; EGFR, epidermal growth factor receptor; EMT: epithelial-to-mesenchymal transition; EpCAM: epithelial cell adhesion molecule; GM-CSF: granulocyte monocyte colony stimulating factor; HIF: hypoxia inducible factor; IDO, indoleamine 2,3-dioxygenase; IFN: interferon; IL: interleukin; ITIM/ITAM: immune tyrosine-based inhibitory/activatory motif; KIR: killer cell immunoglobulin-like receptor; LAG-3: lymphocyte activation gene 3; MDSC: myeloid derived suppressor cells; MICA/B: MHC class I-related proteins A/B; MHC: major histocompatibility complex; mTOR: mechanistic target of rapamycin; NCAM: neuronal adhesion molecule; NCR: natural cytotoxicity receptor; NK: natural killer; NSCLC: non-small cell lung cancer; PD-1: programmed cell death 1; PS: phosphatidylserine; SCLC: small cell lung cancer; STAT: signal transducer and activator of transcription; TAM: tumor-associated M2 macrophages; TCR: T cell receptor; TIGIT: T cell immunoglobulin and ITIM domain; Tim-3: T cell immunoglobulin- and mucin domain-containing 3; TNF: tumor necrosis factor; ULBP: UL16-binding protein.

7.
Int J Mol Sci ; 20(21)2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31652993

RESUMO

Most molecular chaperones belonging to heat shock protein (HSP) families are known to protect cancer cells from pathologic, environmental and pharmacological stress factors and thereby can hamper anti-cancer therapies. In this review, we present data on inhibitors of the heat shock response (particularly mediated by the chaperones HSP90, HSP70, and HSP27) either as a single treatment or in combination with currently available anti-cancer therapeutic approaches. An overview of the current literature reveals that the co-administration of chaperone inhibitors and targeting drugs results in proteotoxic stress and violates the tumor cell physiology. An optimal drug combination should simultaneously target cytoprotective mechanisms and trigger the imbalance of the tumor cell physiology.

8.
Cells ; 8(10)2019 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-31569342

RESUMO

The inhibition of heat shock protein 90 (Hsp90) a molecular chaperone for multiple oncogenic client proteins is considered as a promising approach to overcome radioresistance. Since most Hsp90 inhibitors activate HSF-1 that induces the transcription of cytoprotective and tumor-promoting stress proteins such as Hsp70 and Hsp27, a combined approach consisting of HSF-1 knockdown (k.d.) and Hsp90 inhibition was investigated. A specific HSF-1 k.d. was achieved in H1339 lung cancer cells using RNAi-Ready pSIRENRetroQ vectors with puromycin resistance. The Hsp90 inhibitor NVP-AUY922 was evaluated at low concentrations-ranging from 1-10 nM-in control and HSF-1 k.d. cells. Protein expression (i.e., Hsp27/Hsp70, HSF-1, pHSF-1, Akt, ß-actin) and transcriptional activity was assessed by western blot analysis and luciferase assays and radiosensitivity was measured by proliferation, apoptosis (Annexin V, active caspase 3), clonogenic cell survival, alkaline comet, γH2AX, 53BP1, and Rad51 foci assays. The k.d. of HSF-1 resulted in a significant reduction of basal and NVP-AUY922-induced Hsp70/Hsp27 expression levels. A combined approach consisting of HSF-1 k.d. and low concentrations of the Hsp90 inhibitor NVP-AUY922 reduces the Hsp90 client protein Akt and potentiates radiosensitization, which involves an impaired homologous recombination mediated by Rad51. Our findings are key for clinical applications of Hsp90 inhibitors with respect to adverse hepatotoxic effects.

9.
Cell Stress Chaperones ; 24(5): 969-978, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31375996

RESUMO

The concentration of circulating heat shock protein 70 (Hsp70) was measured in liquid biopsies of canine tumor patients as a potential biomarker. Compared with rodent tumor models, spontaneously occurring tumors in pet dogs reflect the clinical situation of human patients better, as dogs cohabitate with their owners in the same environment, reach a much older age than rodents, can provide blood samples much more frequently, and receive up-to-date medical care and, similar to humans, their tumors show a high genetic heterogeneity. Due to the species-specific sequence homology of human and canine Hsp70, two human enzyme-linked immunosorbent assay (ELISA) systems (R&D and lipHsp70) were used to measure canine Hsp70 concentrations in serum and plasma. In general, higher Hsp70 concentrations were found in serum compared with plasma samples of dogs, and the lipHsp70 ELISA detected higher peak concentrations of Hsp70 in a broader range than the R&D ELISA. Compared with a tumor-free control group, serum Hsp70 concentrations were higher in tumor-bearing dogs, irrespective of breed, age, body weight, and gender. A sub-classification of the different tumors according to their cytological characteristics revealed significantly elevated Hsp70 serum concentrations in dogs with round cell tumors (p < 0.01), a heterogeneous group of malignancies with hematopoietic origin such as mast cells, plasma cells, lymphocytes, histiocytes, and melanomas. Future studies with larger patient cohorts and well-defined tumor sizes are necessary to elucidate the role of serum Hsp70 as a biomarker for tumor detection and monitoring of outcome in pet animals.

10.
Clin Cancer Res ; 25(19): 5997-6008, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31196853

RESUMO

PURPOSE: The innate tumor homing potential of mesenchymal stem cells (MSCs) has been used for a targeted delivery of the theranostic sodium iodide symporter (NIS) transgene into solid tumors. We have previously shown that external beam radiotherapy (EBRT) results in the enhanced recruitment of NIS-expressing MSCs into human hepatocellular carcinoma (HuH7). In parallel, the tumor-associated cytokine TGFB1 becomes strongly upregulated in HuH7 tumors in response to EBRT. EXPERIMENTAL DESIGN: We therefore evaluated the effects of combining focused EBRT (5 Gy) with MSC-mediated systemic delivery of the theranostic NIS transgene under control of a synthetic TGFB1-inducible SMAD-responsive promoter (SMAD-NIS-MSCs) using 123I-scintigraphy followed by 131I therapy in CD1 nu/nu mice harboring subcutaneous human hepatocellular carcinoma (HuH7). RESULTS: Following tumor irradiation and SMAD-NIS-MSC application, tumoral iodide uptake monitored in vivo by 123I-scintigraphy was enhanced as compared with nonirradiated tumors. Combination of EBRT and SMAD-NIS-MSC-mediated 131I therapy resulted in a significantly improved delay in tumor growth and prolonged survival in therapy mice as compared with the combined therapy using CMV-NIS-MSCs or to control groups receiving EBRT or saline only, or EBRT together with SMAD-NIS-MSCs and saline applications. CONCLUSIONS: MSC-based NIS-mediated 131I therapy after EBRT treatment dramatically enhanced therapeutic efficacy when a TGFB1-inducible SMAD-responsive promoter was used to drive NIS expression in adoptively applied MSCs. The remarkable therapeutic effect seen is thought to be linked in large part to the enhanced TGFB1 produced in this context, which leads to a highly selective and focused amplification of MSC-based NIS expression within the tumor milieu.

11.
Radiat Oncol ; 14(1): 78, 2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-31077235

RESUMO

BACKGROUND: Breast cancer is the most common invasive tumor in women worldwide and the second cause of cancer-related deaths. After breast conserving surgery the tumor bed gets irradiated. Radiation-induced tumor cell death has been found to be associated with the release of damage-associated molecular patterns (DAMPs) including free Hsp70 that can stimulate inflammatory immune responses. Therefore, Hsp70 serum levels as well as the composition of lymphocyte subpopulations have been measured in breast cancer patients during therapy and in the follow-up period as potential predictors for clinical outcome. METHODS: The serum of 40 breast cancer patients, who received a breast-conserving surgery and adjuvant radiotherapy (RT) was examined for soluble, free Hsp70 using the R&D Human HSP70 DuoSet and lipHsp70 ELISA. Lymphocyte subpopulations and total lymphocyte counts were analysed by multiparameter flow cytometry in the peripheral blood. Blood samples were collected before (t1), after 30 Gy (t2) and 60 Gy (t3), 6 weeks (t4), 6 months (t5) and 1 year (t6) after RT. Clinical responses were assessed regularly up to 5 years after RT. RESULTS: Patients who developed a contralateral recurrence or metastases within the first 2 years after RT had significantly higher serum Hsp70 values at the end of RT (t3; p = 0.03) up to 6 weeks after RT (t4; p = 0.007) compared to patients who either remained disease-free or developed a secondary endometrial carcinoma. Clinicopathological parameters such as age, tumor size, grading and TNM-stage of the resected tumors, adjuvant chemotherapy and irradiation dose did not affect serum Hsp70 levels. Elevated free Hsp70 levels might be indicative for a chronic inflammatory response which could support tumor recurrence. Lymphocyte subpopulation analysis revealed lower NK cell counts after RT in recurrence/metastases patients as compared to disease-free patients. In contrast, no significant changes were observed in the proportion of T and B cells. CONCLUSION: Longitudinal elevated serum levels of free Hsp70 up to 6 weeks after RT and dropping NK cell counts might be predictive for an unfavourable prognosis in patients with breast cancer.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/radioterapia , Proteínas de Choque Térmico HSP70/sangue , Células Matadoras Naturais/patologia , Recidiva Local de Neoplasia/diagnóstico , Radioterapia Adjuvante/efeitos adversos , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Feminino , Humanos , Células Matadoras Naturais/imunologia , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/sangue , Recidiva Local de Neoplasia/etiologia , Prognóstico
12.
Front Immunol ; 10: 454, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30967859

RESUMO

Heat shock protein 70 (Hsp70) which is expressed on the plasma membrane of highly aggressive tumors including non-small cell lung carcinoma and glioblastoma multiforme serves as a target for Hsp70-targeting NK cells. Herein, we aimed to investigate the antitumor effects of a combined therapy consisting of ex vivo Hsp70-peptide TKD/IL-2-activated NK cells in combination with mouse/human anti-PD-1 antibody in a syngeneic glioblastoma and a xenograft lung cancer mouse model. Mice with membrane Hsp70 positive syngeneic GL261 glioblastoma or human xenograft A549 lung tumors were sham-treated with PBS or injected with ex vivo TKD/IL-2-activated mouse/human NK cells and mouse/human PD-1 antibody either as a single regimen or in combination. Tumor volume was assessed by MR scanning and tumor-infiltrating CD8+ T, NK, and PD-1+ cells were quantified by immunohistochemistry (IHC). We could show that the adoptive transfer of ex vivo TKD/IL-2-activated mouse NK cells or the inhibition of PD-1 resulted in tumor growth delay and an improved overall survival (OS) in a syngeneic glioblastoma mouse model. A combination of both therapies was well-tolerated and significantly more effective with respect to both outcome parameters than either of the single regimens. A combined treatment in a xenograft lung cancer model showed identical effects in immunodeficient mice bearing human lung cancer after adoptive transfer of TKD/IL-2-activated human effector cells and a human PD-1 antibody. Tumor control was associated with a massive infiltration with CD8+ T and NK cells in both tumor models and a decreased in PD-1 expression on immune effector cells. In summary, a combined approach consisting of activated NK cells and anti-PD-1 therapy is safe and results in a long-term tumor control which is accompanied by a massive tumor immune cell infiltration in 2 preclinical tumor models.

13.
Front Oncol ; 9: 156, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30941308

RESUMO

Radiotherapy (RT) has been applied for decades as a treatment modality in the management of various types of cancer. Ionizing radiation induces tumor cell death, which in turn can either elicit protective anti-tumor immune responses or immunosuppression in the tumor micromilieu that contributes to local tumor recurrence. Immunosuppression is frequently accompanied by the attraction of immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs), M2 tumor-associated macrophages (TAMs), T regulatory cells (Tregs), N2 neutrophils, and by the release of immunosuppressive cytokines (TGF-ß, IL-10) and chemokines. Immune checkpoint pathways, particularly of the PD-1/PD-L1 axis, have been determined as key regulators of cancer immune escape. While IFN-dependent upregulation of PD-L1 has been extensively investigated, up-to-date studies indicated the importance of DNA damage signaling in the regulation of PD-L1 expression following RT. DNA damage dependent PD-L1 expression is upregulated by ATM/ATR/Chk1 kinase activities and cGAS/STING-dependent pathway, proving the role of DNA damage signaling in PD-L1 induced expression. Checkpoint blockade immunotherapies (i.e., application of anti-PD-1 and anti-PD-L1 antibodies) combined with RT were shown to significantly improve the objective response rates in therapy of various primary and metastatic malignancies. Further improvements in the therapeutic potential of RT are based on combinations of RT with other immunotherapeutic approaches including vaccines, cytokines and cytokine inducers, and an adoptive immune cell transfer (DCs, NK cells, T cells). In the current review we provide immunological rationale for a combination of RT with various immunotherapies as well as analysis of the emerging preclinical evidences for these therapies.

14.
Small ; 15(13): e1900205, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30828968

RESUMO

Functionalized superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as potential clinical tools for cancer theranostics. Membrane-bound 70 kDa heat shock protein (mHsp70) is ubiquitously expressed on the cell membrane of various tumor types but not normal cells and therefore provides a tumor-specific target. The serine protease granzyme B (GrB) that is produced as an effector molecule by activated T and NK cells has been shown to specifically target mHsp70 on tumor cells. Following binding to Hsp70, GrB is rapidly internalized into tumor cells. Herein, it is demonstrated that GrB functionalized SPIONs act as a contrast enhancement agent for magnetic resonance imaging and induce specific tumor cell apoptosis. Combinatorial regimens employing stereotactic radiotherapy and/or magnetic targeting are found to further enhance the therapeutic efficacy of GrB-SPIONs in different tumor mouse models.

15.
Strahlenther Onkol ; 195(4): 352-361, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30747241

RESUMO

BACKGROUND: Membrane heat shock protein 70 (mHsp70) is indicative of high-risk tumors and serves as a tumor-specific target for natural killer (NK) cells stimulated with Hsp70 peptide (TKD) and Interleukin(IL)-2. Radiochemotherapy (RCT), mHsp70-targeting NK cells, and programmed death(PD)-1 inhibition were combined to improve the efficacy of tumor-specific immune cells in a non-small cell lung carcinoma (NSCLC) patient. PATIENT: Following simultaneous RCT (64.8 Gy), a patient with inoperable NSCLC (cT4, cN3, cM0, stage IIIb) was treated with 4 cycles of autologous ex vivo TKD/IL-2-activated NK cells and the PD-1 antibody nivolumab as a second-line therapy. Blood samples were taken for immunophenotyping during the course of therapy. RESULTS: Adoptive transfer of ex vivo TKD/IL-2-activated NK cells after RCT combined with PD-1 blockade is well tolerated and results in superior overall survival (OS). No viable tumor cells but a massive immune cell infiltration in fibrotic tissue was detected after therapy. Neither tumor progression nor distant metastases were detectable by CT scanning 33 months after diagnosis. Therapy response was associated with significantly increased CD3-/NKG2D+/CD94+ NK cell counts, elevated CD8+ to CD4+ T cell and CD3-/CD56bright to CD3-/CD56dim NK cell ratios, and significantly reduced regulatory T cells (Tregs) in the peripheral blood. CONCLUSION: A combined therapy consisting of RCT, mHsp70-targeting NK cells, and PD-1 antibody inhibition is well tolerated, induces anti-tumor immunity, and results in long-term tumor control in one patient with advanced NSCLC. Further, randomized studies are necessary to confirm the efficacy of this combination therapy.


Assuntos
Transferência Adotiva , Carcinoma Pulmonar de Células não Pequenas/terapia , Quimiorradioterapia/métodos , Células Matadoras Naturais/transplante , Neoplasias Pulmonares/terapia , Nivolumabe/uso terapêutico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/patologia , Terapia Combinada , Proteínas de Choque Térmico HSP70/sangue , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Planejamento da Radioterapia Assistida por Computador , Tomografia Computadorizada por Raios X
16.
Prog Lipid Res ; 74: 18-30, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30710597

RESUMO

Beyond guarding the cellular proteome the major stress inducible heat shock protein Hsp70 has been shown to interact with lipids. Non-cytosolic Hsp70 stabilizes membranes during stress challenges and, in pathophysiological states, facilitates endocytosis, counteracts apoptotic mechanisms, sustains survival pathways or represents a signal that can be recognized by the immune system. Disease-coupled lipid-associated functions of Hsp70 may be targeted via distinct subcellular localizations of Hsp70 itself or its specific interacting lipids. With a special focus on interacting lipids, here we discuss localization-dependent roles of the membrane-bound Hsp70 in the context of its therapeutic potential, particularly in cancer and neurodegenerative diseases.


Assuntos
Membrana Celular/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Lipídeos de Membrana/metabolismo , Neoplasias/metabolismo , Doenças Neurodegenerativas/metabolismo , Humanos , Neoplasias/patologia , Doenças Neurodegenerativas/patologia
17.
Cell Stress Chaperones ; 24(1): 213-221, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30632067

RESUMO

Heat shock protein Hsp70 (Hsp70) is found on the cell surface of a large variety of human and mouse tumor cell types including U87, GL261 glioblastoma, and 4T1 mammary carcinoma cells. We studied the role of membrane-bound Hsp70 (mHsp70) in the formation of cell-to-cell connections via tunneling nanotubes (TNTs) using live-cell STED nanoscopy. This technique allows the visualization of microstructures in the 100-nm range in the living cells. We could show that the presence of tumor-derived mHsp70 in TNTs with a diameter ranging from 120 to 140 nm predominantly originates from cholesterol-rich-microdomains containing the lipid compound globoyltriaosylceramide (Gb3). Under non-stress conditions, Hsp70 and Gb3 are structurally clustered in the membrane of TNTs of tumor cells that showed tumor type specific variations in the amount of cell-to-cell connection networks. Furthermore depletion of cholesterol and ionizing radiation as a stress factor results in a complete loss of Hsp70-containing TNTs.


Assuntos
Comunicação Celular , Membrana Celular/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Nanotubos/química , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Microscopia , Modelos Biológicos , Triexosilceramidas/metabolismo
18.
Front Oncol ; 8: 497, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30443493

RESUMO

The presence of circulating tumor cells (CTCs) in the peripheral blood is a pre-requisite for progression, invasion, and metastatic spread of cancer. Consequently, the enumeration and molecular characterization of CTCs from the peripheral blood of patients with solid tumors before, during and after treatment serves as a valuable tool for categorizing disease, evaluating prognosis and for predicting and monitoring therapeutic responsiveness. Many of the techniques for isolating CTCs are based on the expression of epithelial cell surface adhesion molecule (EpCAM, CD326) on tumor cells. However, the transition of adherent epithelial cells to migratory mesenchymal cells (epithelial-to-mesenchymal transition, EMT)-an essential element of the metastatic process-is frequently associated with a loss of expression of epithelial cell markers, including EpCAM. A highly relevant proportion of mesenchymal CTCs cannot therefore be isolated using techniques that are based on the "capture" of cells expressing EpCAM. Herein, we provide evidence that a monoclonal antibody (mAb) directed against a membrane-bound form of Hsp70 (mHsp70)-cmHsp70.1-can be used for the isolation of viable CTCs from peripheral blood of tumor patients of different entities in a more quantitative manner. In contrast to EpCAM, the expression of mHsp70 remains stably upregulated on migratory, mesenchymal CTCs, metastases and cells that have been triggered to undergo EMT. Therefore, we propose that approaches for isolating CTCs based on the capture of cells that express mHsp70 using the cmHsp70.1 mAb are superior to those based on EpCAM expression.

19.
Cancer Res ; 78(21): 6268-6281, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30228173

RESUMO

High precision in vivo PET/CT imaging of solid tumors improves diagnostic credibility and clinical outcome of patients. An epitope of the oligomerization domain of Hsp70 is exclusively exposed on the membrane of a large variety of tumor types, but not on normal cells, and thus provides a universal tumor-specific target. Here we developed a novel PET tracer TPP-PEG24-DFO[89Zr] based on the tumor cell-penetrating peptide probe TPP, which specifically recognizes membrane Hsp70 (mHsp70) on tumor cells. The implemented PEG24 moiety supported tracer stability and improved biodistribution characteristics in vivo The K d of the tracer ranged in the low nanomolar range (18.9 ± 11.3 nmol/L). Fluorescein isothiocyanate (FITC)-labeled derivatives TPP-[FITC] and TPP-PEG24-[FITC] revealed comparable and specific binding to mHsp70-positive 4T1, 4T1+, a derivative of the 4T1 cell line sorted for high Hsp70 expression, and CT26 tumor cells, but not to mHsp70-negative normal fibroblasts. The rapid internalization kinetics of mHsp70 into the cytosol and the favorable biodistribution of the peptide-based tracer TPP-PEG24-DFO[89Zr] in vivo enabled a tumor-specific accumulation with a high tumor-to-background contrast and renal body clearance. The tumor-specific enrichment of the tracer in 4T1+ (6.2 ± 1.1%ID/g), 4T1 (4.3 ± 0.7%ID/g), and CT26 (2.6 ± 0.6%ID/g) mouse tumors with very high, high, and intermediate mHsp70 densities, respectively, reflected mHsp70 expression profiles of the different tumor types, whereas benign mHsp70-negative fibroblastic hyperplasia showed no tracer accumulation (0.2 ± 0.03%ID/g). The ability of our chemically optimized peptide-based tracer TPP-PEG24-DFO[89Zr] to detect mHsp70 in vivo suggests its broad applicability in targeting and imaging with high specificity for any tumor type that exhibits surface expression of Hsp70.Significance: A novel peptide-based PET tracer against the oligomerization domain of Hsp70 has potential for universal tumor-specific imaging in vivo across many tumor type. Cancer Res; 78(21); 6268-81. ©2018 AACR.


Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Proteínas de Choque Térmico HSP70/metabolismo , Peptídeos/química , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons/métodos , Radioisótopos/química , Animais , Anticorpos Monoclonais/metabolismo , Ligação Competitiva , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/metabolismo , Epitopos/química , Fibroblastos/metabolismo , Fluoresceína-5-Isotiocianato/química , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoconjugados/química , Concentração Inibidora 50 , Cinética , Camundongos , Microscopia de Fluorescência , Distribuição Tecidual , Zircônio/química
20.
Adv Exp Med Biol ; 1072: 171-175, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30178341

RESUMO

The metabolic tumor microenvironment (TME) is characterized inter alia by critical oxygen depletion (hypoxia/anoxia), extracellular acidosis (pH ≤ 6.8), high lactate levels (up to 40 mM in heterogeneously distributed areas), strongly elevated adenosine concentrations (10-100 µM) and declining nutrient resources. These TME features are major drivers, e.g., for genetic instability, intratumor heterogeneity, malignant progression and development of resistance to conventional anticancer therapies. In this context, hypoxia-dependent (and non-hypoxic) HIF-1α activation plays a key role in orchestrating a multifaceted (local) suppression of innate and adaptive antitumor immune responses (and of immune-based tumor treatment). Besides the characteristic traits mentioned, the immune-suppressive actions can additionally be triggered by an (over-)expression of VEGF and activation of VEGFR, and externalisation of phosphatidylserine from the inner to the outer membrane leaflet of cells and exosomes. Altogether, and even individually, these features provide strong immune-suppressive signals. The downstream effects of an enhanced HIF-1α expression include (a) an activation of immune-suppressive effects (recruitment and stimulation of immune-suppressor cells [e.g., Treg, MDSC], secretion of immune-suppressive TH2-type cytokines), and (b) inhibition of antitumor immune responses (inhibition of immune cell actions [e.g., NK, NKT, CD4+, CD8+], inhibition of antigen-presenting cells [e.g., DC], reduced production of immune-stimulatory TH1-type cytokines).


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias/patologia , Evasão Tumoral/fisiologia , Hipóxia Tumoral/fisiologia , Microambiente Tumoral/fisiologia , Animais , Progressão da Doença , Humanos , Neoplasias/metabolismo
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