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1.
bioRxiv ; 2023 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-38077050

RESUMO

Decreased intra-tumor heterogeneity (ITH) correlates with increased patient survival and immunotherapy response. However, even highly homogenous tumors may display variability in their aggressiveness, and how immunologic-factors impinge on their aggressiveness remains understudied. Here we studied the mechanisms responsible for the immune-escape of murine tumors with low ITH. We compared the temporal growth of homogeneous, genetically-similar single-cell clones that are rejected vs. those that are not-rejected after transplantation in-vivo using single-cell RNA sequencing and immunophenotyping. Non-rejected clones showed high infiltration of tumor-associated-macrophages (TAMs), lower T-cell infiltration, and increased T-cell exhaustion compared to rejected clones. Comparative analysis of rejection-associated gene expression programs, combined with in-vivo CRISPR knockout screens of candidate mediators, identified Mif (macrophage migration inhibitory factor) as a regulator of immune rejection. Mif knockout led to smaller tumors and reversed non-rejection-associated immune composition, particularly, leading to the reduction of immunosuppressive macrophage infiltration. Finally, we validated these results in melanoma patient data.

2.
Cell Rep Med ; 4(8): 101142, 2023 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-37557179

RESUMO

EGFR-specific tyrosine kinase inhibitors (TKIs), especially osimertinib, have changed lung cancer therapy, but secondary mutations confer drug resistance. Because other EGFR mutations promote dimerization-independent active conformations but L858R strictly depends on receptor dimerization, we herein evaluate the therapeutic potential of dimerization-inhibitory monoclonal antibodies (mAbs), including cetuximab. This mAb reduces viability of cells expressing L858R-EGFR and blocks the FOXM1-aurora survival pathway, but other mutants show no responses. Unlike TKI-treated patient-derived xenografts, which relapse post osimertinib treatment, cetuximab completely prevents relapses of L858R+ tumors. We report that osimertinib's inferiority associates with induction of mutagenic reactive oxygen species, whereas cetuximab's superiority is due to downregulation of adaptive survival pathways (e.g., HER2) and avoidance of mutation-prone mechanisms that engage AXL, RAD18, and the proliferating cell nuclear antigen. These results identify L858R as a predictive biomarker, which may pave the way for relapse-free mAb monotherapy relevant to a large fraction of patients with lung cancer.


Assuntos
Receptores ErbB , Neoplasias Pulmonares , Humanos , Cetuximab/farmacologia , Cetuximab/uso terapêutico , Receptores ErbB/genética , Inibidores de Proteínas Quinases/farmacologia , Recidiva Local de Neoplasia/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Anticorpos Monoclonais/uso terapêutico , Biomarcadores , Proteínas de Ligação a DNA , Ubiquitina-Proteína Ligases
3.
Mater Today Bio ; 20: 100669, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37334185

RESUMO

Common methods to label cell surface proteins (CSPs) involve the use of fluorescently modified antibodies (Abs) or small-molecule-based ligands. However, optimizing the labeling efficiency of such systems, for example, by modifying them with additional fluorophores or recognition elements, is challenging. Herein we show that effective labeling of CSPs overexpressed in cancer cells and tissues can be obtained with fluorescent probes based on chemically modified bacteria. The bacterial probes (B-probes) are generated by non-covalently linking a bacterial membrane protein to DNA duplexes appended with fluorophores and small-molecule binders of CSPs overexpressed in cancer cells. We show that B-probes are exceptionally simple to prepare and modify because they are generated from self-assembled and easily synthesized components, such as self-replicating bacterial scaffolds and DNA constructs that can be readily appended, at well-defined positions, with various types of dyes and CSP binders. This structural programmability enabled us to create B-probes that can label different types of cancer cells with distinct colors, as well as generate very bright B-probes in which the multiple dyes are spatially separated along the DNA scaffold to avoid self-quenching. This enhancement in the emission signal enabled us to label the cancer cells with greater sensitivity and follow the internalization of the B-probes into these cells. The potential to apply the design principles underlying B-probes in therapy or inhibitor screening is also discussed here.

4.
Cancer Med ; 12(9): 10781-10790, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36934426

RESUMO

BACKGROUND: Proteasome inhibitors are in use in treating certain types of cancers. These drugs inhibit the catalytic activity of the 20S proteasome, shared by all the different proteasome complexes. Inhibitors of the 26S-associated deubiquitinating activity explicitly inhibit the 26S proteasomal degradation of ubiquitinylated substrates. We have previously reported an alternative strategy that is based on reducing the 26S/20S ratio by depleting PSMD1, 6, and 11, the subunits of the 19S proteasome regulatory complex. Given the addiction of the many cancer types to a high 26S/20S ratio, the depletion strategy is highly effective in killing many aggressive cancer cell lines but not mouse and human immortalized and normal cells. METHODS: We used two aggressive cell lines, MDA-MB-231, a triple-negative breast tumor cell line, and OVCAR8, a high-grade ovary adenocarcinoma. Cell culture, mouse MDA-MB-231, OVCAR8 xenografts, and patient-derived ovarian cancer xenograft (PDX) models were transduced with lentivectors expressing PSMD1 shRNA. Tumor size was measured to follow treatment efficacy. RESULTS: Using different experimental strategies of expressing shRNA, we found that PSMD1 depletion, either by expressing PSMD1 shRNA in an inducible manner or in a constitutive manner, robustly inhibited MDA-MB-231, and OVCAR8 xenograft tumor growth. Furthermore, the PSMD1 depletion strategy compromised the growth of the PDX of primary ovarian cancer. CONCLUSION: Our results suggest that reducing the 26S/20S ratio might be a valuable strategy for treating drug-resistant aggressive types of cancers.


Assuntos
Neoplasias Ovarianas , Complexo de Endopeptidases do Proteassoma , Feminino , Humanos , Linhagem Celular , Citoplasma/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Animais , Camundongos , Linhagem Celular Tumoral
5.
Cancer Discov ; 13(7): 1616-1635, 2023 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-36972357

RESUMO

Multiple studies have identified metabolic changes within the tumor and its microenvironment during carcinogenesis. Yet, the mechanisms by which tumors affect the host metabolism are unclear. We find that systemic inflammation induced by cancer leads to liver infiltration of myeloid cells at early extrahepatic carcinogenesis. The infiltrating immune cells via IL6-pSTAT3 immune-hepatocyte cross-talk cause the depletion of a master metabolic regulator, HNF4α, consequently leading to systemic metabolic changes that promote breast and pancreatic cancer proliferation and a worse outcome. Preserving HNF4α levels maintains liver metabolism and restricts carcinogenesis. Standard liver biochemical tests can identify early metabolic changes and predict patients' outcomes and weight loss. Thus, the tumor induces early metabolic changes in its macroenvironment with diagnostic and potentially therapeutic implications for the host. SIGNIFICANCE: Cancer growth requires a permanent nutrient supply starting from early disease stages. We find that the tumor extends its effect to the host's liver to obtain nutrients and rewires the systemic and tissue-specific metabolism early during carcinogenesis. Preserving liver metabolism restricts tumor growth and improves cancer outcomes. This article is highlighted in the In This Issue feature, p. 1501.


Assuntos
Fígado , Neoplasias Pancreáticas , Humanos , Fígado/metabolismo , Carcinogênese/patologia , Hepatócitos , Neoplasias Pancreáticas/patologia , Imunidade Inata , Microambiente Tumoral
6.
J Am Chem Soc ; 145(6): 3346-3360, 2023 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-36738297

RESUMO

Electrophiles for covalent inhibitors that are suitable for in vivo administration are rare. While acrylamides are prevalent in FDA-approved covalent drugs, chloroacetamides are considered too reactive for such purposes. We report sulfamate-based electrophiles that maintain chloroacetamide-like geometry with tunable reactivity. In the context of the BTK inhibitor ibrutinib, sulfamate analogues showed low reactivity with comparable potency in protein labeling, in vitro, and cellular kinase activity assays and were effective in a mouse model of CLL. In a second example, we converted a chloroacetamide Pin1 inhibitor to a potent and selective sulfamate acetamide with improved buffer stability. Finally, we show that sulfamate acetamides can be used for covalent ligand-directed release (CoLDR) chemistry, both for the generation of "turn-on" probes as well as for traceless ligand-directed site-specific labeling of proteins. Taken together, this chemistry represents a promising addition to the list of electrophiles suitable for in vivo covalent targeting.


Assuntos
Acetamidas , Inibidores de Proteínas Quinases , Camundongos , Animais , Ligantes , Inibidores de Proteínas Quinases/farmacologia
7.
Front Oncol ; 12: 959133, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36091130

RESUMO

We found that pediatric glioblastoma (PED-GBM) cell lines from diffuse intrinsic pontine glioma (DIPG) carrying the H3K27M mutation or from diffuse hemispheric glioma expressing the H3G34R mutation are sensitive to the combination of vorinostat (a histone deacetylase inhibitor) and PARP-1 inhibitors. The combined treatment increased the phosphorylation of eIF2α (P-eIF2α) relative to each drug alone and enhanced the decrease in cell survival. To explore the role played by increased P-eIF2α in modulating PED-GBM survival and response to treatments, we employed brain-penetrating inhibitors of P-eIF2α dephosphorylation: salubrinal and raphin-1. These drugs increased P-eIF2α, DNA damage, and cell death, similarly affecting the sensitivity of DIPG cells and derived neurospheres to PARP-1 inhibitors. Interestingly, these drugs also decreased the level of eIF2Bϵ (the catalytic subunit of eIF2B) and increased its phosphorylation, thereby enhancing the effect of increased P-eIF2α. Transient transfection with the S51D phosphomimetic eIF2α variant recapitulated the effect of salubrinal and raphin-1 on PED-GBM survival and sensitivity to PARP-1 inhibitors. Importantly, either salubrinal or raphin-1 dramatically increased the sensitivity of DIPG cells to radiation, the main treatment modality of PED-GBM. Finally, PED-GBM was more sensitive than normal human astrocytes to salubrinal, raphin-1, and the treatment combinations described herein. Our results indicate that combinations of histone deacetylase inhibitors and PARP-1 inhibitors should be evaluated for their toxicity and efficacy in PED-GBM patients and point to drugs that increase P-eIF2α or modulate its downstream effectors as a novel means of treating PED-GBM.

8.
Mol Cell ; 82(14): 2696-2713.e9, 2022 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-35716669

RESUMO

Cancer cells are highly heterogeneous at the transcriptional level and epigenetic state. Methods to study epigenetic heterogeneity are limited in throughput and information obtained per cell. Here, we adapted cytometry by time-of-flight (CyTOF) to analyze a wide panel of histone modifications in primary tumor-derived lines of diffused intrinsic pontine glioma (DIPG). DIPG is a lethal glioma, driven by a histone H3 lysine 27 mutation (H3-K27M). We identified two epigenetically distinct subpopulations in DIPG, reflecting inherent heterogeneity in expression of the mutant histone. These two subpopulations are robust across tumor lines derived from different patients and show differential proliferation capacity and expression of stem cell and differentiation markers. Moreover, we demonstrate the use of these high-dimensional data to elucidate potential interactions between histone modifications and epigenetic alterations during the cell cycle. Our work establishes new concepts for the analysis of epigenetic heterogeneity in cancer that could be applied to diverse biological systems.


Assuntos
Neoplasias do Tronco Encefálico , Glioma , Neoplasias do Tronco Encefálico/genética , Neoplasias do Tronco Encefálico/metabolismo , Neoplasias do Tronco Encefálico/patologia , Cromatina/genética , Epigênese Genética , Glioma/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Mutação
9.
Nat Chem Biol ; 17(9): 954-963, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33972797

RESUMO

The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Peptidilprolil Isomerase de Interação com NIMA/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas
10.
EMBO Mol Med ; 13(4): e13144, 2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33660397

RESUMO

Some antibacterial therapies entail sequential treatments with different antibiotics, but whether this approach is optimal for anti-cancer tyrosine kinase inhibitors (TKIs) remains open. EGFR mutations identify lung cancer patients who can derive benefit from TKIs, but most patients develop resistance to the first-, second-, and third-generation drugs. To explore alternatives to such whack-a-mole strategies, we simulated in patient-derived xenograft models the situation of patients receiving first-line TKIs. Monotherapies comprising approved first-line TKIs were compared to combinations with antibodies specific to EGFR and HER2. We observed uniform and strong superiority of all drug combinations over the respective monotherapies. Prolonged treatments, high TKI dose, and specificity were essential for drug-drug cooperation. Blocking pathways essential for mitosis (e.g., FOXM1), along with downregulation of resistance-conferring receptors (e.g., AXL), might underlie drug cooperation. Thus, upfront treatments using combinations of TKIs and antibodies can prevent emergence of resistance and hence might replace the widely applied sequential treatments utilizing next-generation TKIs.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Mutação , Compostos Orgânicos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico
11.
Cancers (Basel) ; 12(9)2020 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-32847130

RESUMO

Although two growth factor receptors, EGFR and HER2, are amongst the best targets for cancer treatment, no agents targeting HER3, their kinase-defective family member, have so far been approved. Because emergence of resistance of lung tumors to EGFR kinase inhibitors (EGFRi) associates with compensatory up-regulation of HER3 and several secreted forms, we anticipated that blocking HER3 would prevent resistance. As demonstrated herein, a neutralizing anti-HER3 antibody we generated can clear HER3 from the cell surface, as well as reduce HER3 cleavage by ADAM10, a surface metalloproteinase. When combined with a kinase inhibitor and an anti-EGFR antibody, the antibody completely blocked patient-derived xenograft models that acquired resistance to EGFRi. We found that the underlying mechanism involves posttranslational downregulation of HER3, suppression of MET and AXL upregulation, as well as concomitant inhibition of AKT signaling and upregulation of BIM, which mediates apoptosis. Thus, although HER3 is nearly devoid of kinase activity, it can still serve as an effective drug target in the context of acquired resistance. Because this study simulated in animals the situation of patients who develop resistance to EGFRi and remain with no obvious treatment options, the observations presented herein may warrant clinical testing.

12.
Nat Commun ; 11(1): 3296, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620799

RESUMO

Identifying robust, patient-specific, and predictive biomarkers presents a major obstacle in precision oncology. To optimize patient-specific therapeutic strategies, here we couple pathway knowledge with large-scale drug sensitivity, RNAi, and CRISPR-Cas9 screening data from 460 cell lines. Pathway activity levels are found to be strong predictive biomarkers for the essentiality of 15 proteins, including the essentiality of MAD2L1 in breast cancer patients with high BRCA-pathway activity. We also find strong predictive biomarkers for the sensitivity to 31 compounds, including BCL2 and microtubule inhibitors (MTIs). Lastly, we show that Bcl-xL inhibition can modulate the activity of a predictive biomarker pathway and re-sensitize lung cancer cells and tumors to MTI therapy. Overall, our results support the use of pathways in helping to achieve the goal of precision medicine by uncovering dozens of predictive biomarkers.


Assuntos
Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Transdução de Sinais/genética , Animais , Antineoplásicos/farmacologia , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Redes Reguladoras de Genes , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Medicina de Precisão/métodos , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
13.
Life Sci Alliance ; 3(4)2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32132179

RESUMO

Angiogenesis and lymphangiogenesis are key processes during embryogenesis as well as under physiological and pathological conditions. Vascular endothelial growth factor C (VEGFC), the ligand for both VEGFR2 and VEGFR3, is a central lymphangiogenic regulator that also drives angiogenesis. Here, we report that members of the highly conserved BACH (BTB and CNC homology) family of transcription factors regulate VEGFC expression, through direct binding to its promoter. Accordingly, down-regulation of bach2a hinders blood vessel formation and impairs lymphatic sprouting in a Vegfc-dependent manner during zebrafish embryonic development. In contrast, BACH1 overexpression enhances intratumoral blood vessel density and peritumoral lymphatic vessel diameter in ovarian and lung mouse tumor models. The effects on the vascular compartment correlate spatially and temporally with BACH1 transcriptional regulation of VEGFC expression. Altogether, our results uncover a novel role for the BACH/VEGFC signaling axis in lymphatic formation during embryogenesis and cancer, providing a novel potential target for therapeutic interventions.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Neovascularização Fisiológica/fisiologia , Fator C de Crescimento do Endotélio Vascular/genética , Proteínas de Peixe-Zebra/genética , Moduladores da Angiogênese/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linhagem Celular Tumoral , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Linfangiogênese/fisiologia , Vasos Linfáticos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Morfogênese , Transdução de Sinais , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismo
14.
Nat Commun ; 11(1): 896, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32060274

RESUMO

Predicting the outcome of immunotherapy treatment in melanoma patients is challenging. Alterations in genes involved in antigen presentation and the interferon gamma (IFNγ) pathway play an important role in the immune response to tumors. We describe here that the overexpression of PSMB8 and PSMB9, two major components of the immunoproteasome, is predictive of better survival and improved response to immune-checkpoint inhibitors of melanoma patients. We study the mechanism underlying this connection by analyzing the antigenic peptide repertoire of cells that overexpress these subunits using HLA peptidomics. We find a higher response of patient-matched tumor infiltrating lymphocytes against antigens diferentially presented after immunoproteasome overexpression. Importantly, we find that PSMB8 and PSMB9 expression levels are much stronger predictors of melanoma patients' immune response to checkpoint inhibitors than the tumors' mutational burden. These results suggest that PSMB8 and PSMB9 expression levels can serve as important biomarkers for stratifying melanoma patients for immune-checkpoint treatment.


Assuntos
Melanoma/imunologia , Melanoma/terapia , Complexo de Endopeptidases do Proteassoma/genética , Apresentação de Antígeno , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/imunologia , Humanos , Imunoterapia , Interferon gama/genética , Interferon gama/imunologia , Melanoma/diagnóstico , Melanoma/genética , Prognóstico , Complexo de Endopeptidases do Proteassoma/imunologia
15.
Nat Cancer ; 1(9): 894-908, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-35121952

RESUMO

Argininosuccinate synthase (ASS1) downregulation in different tumors has been shown to support cell proliferation and yet, in several common cancer subsets ASS1 expression associates with poor patient prognosis. Here we demonstrate that ASS1 expression under glucose deprivation is induced by c-MYC, providing survival benefit by increasing nitric oxide synthesis and activating the gluconeogenic enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase by S-nitrosylation. The resulting increased flux through gluconeogenesis enhances serine, glycine and subsequently purine synthesis. Notably, high ASS1-expressing breast cancer mice do not respond to immune checkpoint inhibitors and patients with breast cancer with high ASS1 have more metastases. We further find that inhibiting purine synthesis increases pyrimidine to purine ratio, elevates expression of the immunoproteasome and significantly enhances the response of autologous primary CD8+ T cells to anti-PD-1. These results suggest that treating patients with high-ASS1 cancers with purine synthesis inhibition is beneficial and may also sensitize them to immune checkpoint inhibition therapy.


Assuntos
Argininossuccinato Sintase , Neoplasias da Mama , Animais , Argininossuccinato Sintase/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Inibidores de Checkpoint Imunológico , Camundongos , Purinas
16.
Int J Cancer ; 139(8): 1788-98, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27242346

RESUMO

Metastatic ovarian cancer, the most lethal of gynecologic malignancies, is typically managed by debulking surgery, followed by chemotherapy. However, despite significant efforts, survival rate remains low. We have previously demonstrated, in mouse models, a specific systemic homing of labeled fibroblasts to solid ovarian tumors. Here, we demonstrate the feasibility of utilizing this specific homing of genetically modified fibroblasts for detection and targeted therapy of orthotopic metastatic ovarian carcinoma model in immune-deficient mice. Using an in vivo metastatic mouse model for ovarian cancer, we demonstrated that fibroblasts expressing fluorescent reporters injected intra-peritoneally, were specifically recruited to peritoneal tumor nodules (resulting in 93-100% co-localization). We further used fibroblasts over expressing the soluble receptor variant of VEGFR1 (s-Flt1). Mice bearing tumors were injected weekly with either control or s-Flt1 expressing fibroblasts. Injection of s-Flt1 expressing fibroblasts resulted in a significant reduction in the ascites volume, reduced vascularization of adherent metastases, and improved overall survival. Using fluorescently labeled fibroblasts for tumor detection with readily available intra-operative fluorescence imaging tools may be useful for tumor staging and directing biopsies or surgical efforts during exploratory or debulking surgery. Fibroblasts may serve as a beacon pointing to the otherwise invisible metastases in the peritoneal cavity of ovarian cancer patients. Utilizing the recruited fibroblasts also for targeted delivery of anti angiogenic or antitumor molecules may aid in controlling tumor progression. Thus, these results suggest a novel approach for targeting ovarian tumor metastases for both tumor detection and therapy.


Assuntos
Fibroblastos/patologia , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologia , Animais , Carcinoma Epitelial do Ovário , Linhagem Celular Transformada , Linhagem Celular Tumoral , Feminino , Fibroblastos/metabolismo , Fibroblastos/transplante , Corantes Fluorescentes/administração & dosagem , Corantes Fluorescentes/química , Haplorrinos , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Neoplasias Epiteliais e Glandulares/irrigação sanguínea , Neoplasias Epiteliais e Glandulares/diagnóstico por imagem , Neoplasias Epiteliais e Glandulares/terapia , Neoplasias Ovarianas/irrigação sanguínea , Neoplasias Ovarianas/diagnóstico por imagem , Neoplasias Ovarianas/terapia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese
17.
Sci Rep ; 6: 26550, 2016 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-27211820

RESUMO

Ferritin has gained significant attention as a potential reporter gene for in vivo imaging by magnetic resonance imaging (MRI). However, due to the ferritin ferrihydrite core, the relaxivity and sensitivity for detection of native ferritin is relatively low. We report here on a novel chimeric magneto-ferritin reporter gene - ferritin-M6A - in which the magnetite binding peptide from the magnetotactic bacteria magnetosome-associated Mms6 protein was fused to the C-terminal of murine h-ferritin. Biophysical experiments showed that purified ferritin-M6A assembled into a stable protein cage with the M6A protruding into the cage core, enabling magnetite biomineralisation. Ferritin-M6A-expressing C6-glioma cells showed enhanced (per iron) r2 relaxivity. MRI in vivo studies of ferritin-M6A-expressing tumour xenografts showed enhanced R2 relaxation rate in the central hypoxic region of the tumours. Such enhanced relaxivity would increase the sensitivity of ferritin as a reporter gene for non-invasive in vivo MRI-monitoring of cell delivery and differentiation in cellular or gene-based therapies.


Assuntos
Apoferritinas/metabolismo , Neoplasias Encefálicas/diagnóstico por imagem , Compostos Férricos/metabolismo , Óxido Ferroso-Férrico/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Apoferritinas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Genes Reporter , Engenharia Genética , Imageamento por Ressonância Magnética , Camundongos , Modelos Moleculares , Transplante de Neoplasias , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
18.
Pharmacol Ther ; 141(2): 192-208, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24134903

RESUMO

Cancer cells rely on extensive support from the stroma in order to survive, proliferate and invade. The tumor stroma is thus an important potential target for anti-cancer therapy. Typical changes in the stroma include a shift from the quiescence promoting-antiangiogenic extracellular matrix to a provisional matrix that promotes invasion and angiogenesis. These changes in the extracellular matrix are induced by changes in the secretion of extracellular matrix proteins and glucose amino glycans, extravasation of plasma proteins from hyperpermeable vessels and release of matrix modifying enzymes resulting in cleavage and cross-linking of matrix macromolecules. These in turn alter the rigidity of the matrix and the exposure and release of cytokines. Changes in matrix rigidity and vessel permeability affect drug delivery and mediate resistance to cytotoxic therapy. These stroma changes are brought about not only by the cancer cells, but also through the action of many cell types that are recruited by tumors including immune cells, fibroblasts and endothelial cells. Within the tumor, these normal host cells are activated resulting in loss of inhibitory and induction of cancer promoting activities. Key to the development of stroma-targeted therapies, selective biomarkers were developed for specific imaging of key aspects of the tumor stroma.


Assuntos
Neoplasias/diagnóstico , Animais , Biomarcadores Tumorais/metabolismo , Matriz Extracelular/metabolismo , Humanos , Neoplasias/metabolismo
19.
Cancer Immunol Immunother ; 58(1): 71-84, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18488222

RESUMO

Vascular-targeted photodynamic therapy (VTP) takes advantage of intravascular excitation of a photosensitizer (PS) to produce cytotoxic reactive oxygen species (ROS). These ROS are potent mediators of vascular damage inducing rapid local thrombus formation, vascular occlusion, and tissue hypoxia. This light-controlled process is used for the eradication of solid tumors with Pd-bacteriochlorophyll derivatives (Bchl) as PS. Unlike classical photodynamic therapy (PDT), cancer cells are not the primary target for VTP but instead are destroyed by treatment-induced oxygen deprivation. VTP initiates acute local inflammation inside the illuminated area accompanied by massive tumor tissue death. Consequently, in the present study, we addressed the possibility of immune response induction by the treatment that may be considered as an integral part of the mechanism of VTP-mediated tumor eradication. The effect of VTP on the host immune system was investigated using WST11, which is now in phase II clinical trials for age-related macular degeneration and intended to be evaluated for cancer therapy. We found that a functional immune system is essential for successful VTP. Long-lasting systemic antitumor immunity was induced by VTP involving both cellular and humoral components. The antitumor effect was cross-protective against mismatched tumors, suggesting VTP-mediated production of overlapping tumor antigens, possibly from endothelial origin. Based on our findings we suggest that local VTP might be utilized in combination with other anticancer therapies (e.g., immunotherapy) for the enhancement of host antitumor immunity in the treatment of both local and disseminated disease.


Assuntos
Formação de Anticorpos/efeitos dos fármacos , Bacterioclorofilas/farmacologia , Imunidade Celular/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Fotoquimioterapia , Animais , Vasos Sanguíneos/efeitos dos fármacos , Linhagem Celular Tumoral , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Feminino , Citometria de Fluxo , Imuno-Histoquímica , Interferon gama/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/imunologia , Fármacos Fotossensibilizantes/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
20.
Biochem Biophys Res Commun ; 344(4): 1271-7, 2006 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-16650824

RESUMO

We have previously shown that paraoxonase 1 action on macrophages produced lysophosphatidylcholine (LPC) and significantly decreased cell-mediated LDL oxidation. Thus, in the present study, we questioned whether LPC can directly inhibit macrophage-mediated oxidation of LDL. Addition of increasing LPC concentrations (0-5 microM) to J774A.1 macrophages, mouse peritoneal macrophages (MPM), or to human monocytes-derived macrophages (HMDM) resulted in up to 83%, 67%, and 75% inhibition in cell-mediated oxidation of LDL, respectively. The mechanism for this LPC effect involves up to 60% inhibition of superoxide anion release from MPM in response to phorbol ester (PMA), 26% inhibition of PMA-induced NADPH oxidase activation (p47phox translocation from the cytosol to the plasma membrane), and a 2-fold stimulation of the macrophage paraoxonase 2 (PON2) lactonase activity. We thus conclude that inhibition of macrophage-mediated oxidation of LDL by LPC can contribute to attenuation of macrophage foam cell formation and atherosclerotic lesion development.


Assuntos
Lipoproteínas LDL/metabolismo , Lisofosfatidilcolinas/farmacologia , Macrófagos/metabolismo , Animais , Arildialquilfosfatase/efeitos dos fármacos , Membrana Celular/enzimologia , Citoplasma/enzimologia , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Camundongos , NADPH Oxidases/efeitos dos fármacos , NADPH Oxidases/metabolismo , Oxirredução/efeitos dos fármacos , Ésteres de Forbol/farmacologia , Superóxidos/metabolismo
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