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1.
J Proteome Res ; 21(10): 2331-2340, 2022 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-36049057

RESUMO

The fibronectin (FN) isoform including the extradomain B (EDB) segment (EDB + FN) is a promising tumor target and is highly expressed in some tumor types, such as breast, head, and neck cancer. To date, mostly immunohistochemistry (IHC) and Western blot have been used for the analysis of EDB + FN. However, complete quantitative measurements of EDB + FN expression in a tumor and circulation are important for the development of anti-EDB therapeutics. To this end, a method using protein enrichment followed by online antipeptide antibody enrichment coupled with a nanoflow LC-MS/MS was developed to quantify EDB + FN in human and cynomolgus plasma, patient-derived xenograft (PDX) tumors, and PDX formalin-fixed paraffin-embedded (FFPE) samples. Mouse plasma EDB + FN was analyzed using a protein immunoaffinity method followed by nanoflow LC-MS/MS. EDB + FN concentrations were 63.1 pmol/g in PDX breast cancer tumor and 49.6 pmol/g in PDX head and neck tumor. Mean plasma concentration was 1.1 nM (pmol/mL, 47.4 ng/mL) in normal healthy humans and 0.35 nM (15.1 ng/mL) in naive cynomolgus. The assay sensitivity was 0.018 nM based on calibration with recombinant human EDB + FN (rhEDB + FN).


Assuntos
Neoplasias da Mama , Fibronectinas , Animais , Neoplasias da Mama/patologia , Cromatografia Líquida , Modelos Animais de Doenças , Feminino , Fibronectinas/análise , Formaldeído , Xenoenxertos , Humanos , Camundongos , Inclusão em Parafina , Isoformas de Proteínas/metabolismo , Espectrometria de Massas em Tandem
2.
Proc Natl Acad Sci U S A ; 115(50): E11671-E11680, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30487219

RESUMO

Tumorigenesis depends on intricate interactions between genetically altered tumor cells and their surrounding microenvironment. While oncogenic drivers in lung squamous carcinoma (LUSC) have been described, the role of stroma in modulating tissue architecture, particularly cell polarity, remains unclear. Here, we report the establishment of a 3D coculture system of LUSC epithelial cells with cancer-associated fibroblasts (CAFs) and extracellular matrix that together capture key components of the tumor microenvironment (TME). Single LUSC epithelial cells develop into acinar-like structures with 0.02% efficiency, and addition of CAFs provides proper tumor-stromal interactions within an appropriate 3D architectural context. Using this model, we recapitulate key pathological changes during tumorigenesis, from hyperplasia to dysplasia and eventually invasion, in malignant LUSC spheroids that undergo phenotypic switching in response to cell intrinsic and extrinsic changes. Overexpression of SOX2 is sufficient to mediate the transition from hyperplasia to dysplasia in LUSC spheroids, while the presence of CAFs makes them invasive. Unexpectedly, CAFs suppress the activity of high SOX2 levels, restore hyperplasia, and enhance the formation of acinar-like structures. Taken together, these observations suggest that stromal factors can override cell intrinsic oncogenic changes in determining the disease phenotype, thus providing fundamental evidence for the existence of dynamic reciprocity between the nucleus and the TME of LUSC.


Assuntos
Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Fatores de Transcrição SOXB1/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/genética , Polaridade Celular , Técnicas de Cocultura , Regulação Neoplásica da Expressão Gênica , Humanos , Hiperplasia , Neoplasias Pulmonares/genética , Modelos Biológicos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fatores de Transcrição SOXB1/genética , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologia , Microambiente Tumoral/genética , Regulação para Cima
3.
EMBO J ; 34(13): 1773-85, 2015 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-25979827

RESUMO

Lat1 (SLC7A5) is an amino acid transporter often required for tumor cell import of essential amino acids (AA) including Methionine (Met). Met is the obligate precursor of S-adenosylmethionine (SAM), the methyl donor utilized by all methyltransferases including the polycomb repressor complex (PRC2)-specific EZH2. Cell populations sorted for surface Lat1 exhibit activated EZH2, enrichment for Met-cycle intermediates, and aggressive tumor growth in mice. In agreement, EZH2 and Lat1 expression are co-regulated in models of cancer cell differentiation and co-expression is observed at the invasive front of human lung tumors. EZH2 knockdown or small-molecule inhibition leads to de-repression of RXRα resulting in reduced Lat1 expression. Our results describe a Lat1-EZH2 positive feedback loop illustrated by AA depletion or Lat1 knockdown resulting in SAM reduction and concomitant reduction in EZH2 activity. shRNA-mediated knockdown of Lat1 results in tumor growth inhibition and points to Lat1 as a potential therapeutic target.


Assuntos
Aminoácidos/metabolismo , Epigênese Genética/fisiologia , Transportador 1 de Aminoácidos Neutros Grandes/fisiologia , Complexo Repressor Polycomb 2/fisiologia , Animais , Transporte Biológico/genética , Proliferação de Células/genética , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Tumorais Cultivadas
4.
Mol Cancer Ther ; 21(9): 1462-1472, 2022 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-35793468

RESUMO

Extra domain B splice variant of fibronectin (EDB+FN) is an extracellular matrix protein (ECM) deposited by tumor-associated fibroblasts, and is associated with tumor growth, angiogenesis, and invasion. We hypothesized that EDB+FN is a safe and abundant target for therapeutic intervention with an antibody-drug conjugate (ADC). We describe the generation, pharmacology, mechanism of action, and safety profile of an ADC specific for EDB+FN (EDB-ADC). EDB+FN is broadly expressed in the stroma of pancreatic, non-small cell lung (NSCLC), breast, ovarian, head and neck cancers, whereas restricted in normal tissues. In patient-derived xenograft (PDX), cell-line xenograft (CLX), and mouse syngeneic tumor models, EDB-ADC, conjugated to auristatin Aur0101 through site-specific technology, demonstrated potent antitumor growth inhibition. Increased phospho-histone H3, a pharmacodynamic biomarker of response, was observed in tumor cells distal to the target site of tumor ECM after EDB-ADC treatment. EDB-ADC potentiated infiltration of immune cells, including CD3+ T lymphocytes into the tumor, providing rationale for the combination of EDB-ADC with immune checkpoint therapy. EDB-ADC and anti-PD-L1 combination in a syngeneic breast tumor model led to enhanced antitumor activity with sustained tumor regressions. In nonclinical safety studies in nonhuman primates, EDB-ADC had a well-tolerated safety profile without signs of either on-target toxicity or the off-target effects typically observed with ADCs that are conjugated through conventional conjugation methods. These data highlight the potential for EDB-ADC to specifically target the tumor microenvironment, provide robust therapeutic benefits against multiple tumor types, and enhance activity antitumor in combination with checkpoint blockade.


Assuntos
Neoplasias da Mama , Imunoconjugados , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Fibronectinas/metabolismo , Humanos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Camundongos , Neovascularização Patológica/metabolismo , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Cell Rep Med ; 2(5): 100279, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34095881

RESUMO

Aberrant NOTCH3 signaling and overexpression is oncogenic, associated with cancer stem cells and drug resistance, yet therapeutic targeting remains elusive. Here, we develop NOTCH3-targeted antibody drug conjugates (NOTCH3-ADCs) by bioconjugation of an auristatin microtubule inhibitor through a protease cleavable linker to two antibodies with differential abilities to inhibit signaling. The signaling inhibitory antibody rapidly induces ligand-independent receptor clustering and internalization through both caveolin and clathrin-mediated pathways. The non-inhibitory antibody also efficiently endocytoses via clathrin without inducing receptor clustering but with slower lysosomal co-localization kinetics. In addition, DLL4 ligand binding to the NOTCH3 receptor mediates transendocytosis of NOTCH3-ADCs into ligand-expressing cells. NOTCH3-ADCs internalize into receptor and ligand cells independent of signaling and induce cell death in both cell types representing an atypical mechanism of ADC cytotoxicity. Treatment of xenografts with NOTCH3-ADCs leads to sustained tumor regressions, outperforms standard-of-care chemotherapy, and allows targeting of tumors that overexpress NOTCH3 independent of signaling inhibition.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Imunoconjugados/farmacologia , Receptor Notch3/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Humanos , Imunoconjugados/metabolismo , Oncogenes/efeitos dos fármacos , Receptor Notch3/imunologia , Receptores Notch/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto
6.
J Vis Exp ; (157)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32250351

RESUMO

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.


Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias Pulmonares/patologia , Transformação Celular Neoplásica/metabolismo , Técnicas de Cocultura , Progressão da Doença , Matriz Extracelular/patologia , Humanos , Organoides/metabolismo , Cultura Primária de Células , Microambiente Tumoral
7.
Mol Cancer Ther ; 19(10): 2068-2078, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32747418

RESUMO

The approval of ado-trastuzumab emtansine (T-DM1) in HER2+ metastatic breast cancer validated HER2 as a target for HER2-specific antibody-drug conjugates (ADC). Despite its demonstrated clinical efficacy, certain inherent properties within T-DM1 hamper this compound from achieving the full potential of targeting HER2-expressing solid tumors with ADCs. Here, we detail the discovery of PF-06804103, an anti-HER2 ADC designed to have a widened therapeutic window compared with T-DM1. We utilized an empirical conjugation site screening campaign to identify the engineered ĸkK183C and K290C residues as those that maximized in vivo ADC stability, efficacy, and safety for a four drug-antibody ratio (DAR) ADC with this linker-payload combination. PF-06804103 incorporates the following novel design elements: (i) a new auristatin payload with optimized pharmacodynamic properties, (ii) a cleavable linker for optimized payload release and enhanced antitumor efficacy, and (iii) an engineered cysteine site-specific conjugation approach that overcomes the traditional safety liabilities of conventional conjugates and generates a homogenous drug product with a DAR of 4. PF-06804103 shows (i) an enhanced efficacy against low HER2-expressing breast, gastric, and lung tumor models, (ii) overcomes in vitro- and in vivo-acquired T-DM1 resistance, and (iii) an improved safety profile by enhancing ADC stability, pharmacokinetic parameters, and reducing off-target toxicities. Herein, we showcase our platform approach in optimizing ADC design, resulting in the generation of the anti-HER2 ADC, PF-06804103. The design elements of identifying novel sites of conjugation employed in this study serve as a platform for developing optimized ADCs against other tumor-specific targets.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Imunoconjugados/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Gástricas/tratamento farmacológico , Animais , Neoplasias da Mama/patologia , Feminino , Humanos , Imunoconjugados/farmacologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Neoplasias Gástricas/patologia
8.
Oncotarget ; 6(5): 2928-38, 2015 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-25671303

RESUMO

In addition to genetic alterations, cancer cells are characterized by myriad epigenetic changes. EZH2 is a histone methyltransferase that is over-expressed and mutated in cancer. The EZH2 gain-of-function (GOF) mutations first identified in lymphomas have recently been reported in melanoma (~2%) but remain uncharacterized. We expressed multiple EZH2 GOF mutations in the A375 metastatic skin melanoma cell line and observed both increased H3K27me3 and dramatic changes in 3D culture morphology. In these cells, prominent morphological changes were accompanied by a decrease in cell contractility and an increase in collective cell migration. At the molecular level, we observed significant alteration of the axonal guidance pathway, a pathway intricately involved in the regulation of cell shape and motility. Furthermore, the aggressive 3D morphology of EZH2 GOF-expressing melanoma cells (both endogenous and ectopic) was attenuated by EZH2 catalytic inhibition. Finally, A375 cells expressing exogenous EZH2 GOF mutants formed larger tumors than control cells in mouse xenograft studies. This study not only demonstrates the first functional characterization of EZH2 GOF mutants in non-hematopoietic cells, but also provides a rationale for EZH2 catalytic inhibition in melanoma.


Assuntos
Movimento Celular , Proliferação de Células , Forma Celular , Epigênese Genética , Melanoma/genética , Mutação , Complexo Repressor Polycomb 2/genética , Neoplasias Cutâneas/genética , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Metilação de DNA , Proteína Potenciadora do Homólogo 2 de Zeste , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Melanoma/tratamento farmacológico , Melanoma/enzimologia , Melanoma/patologia , Camundongos Nus , Terapia de Alvo Molecular , Invasividade Neoplásica , Complexo Repressor Polycomb 2/metabolismo , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/patologia , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Mol Cancer Ther ; 12(1): 38-47, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23223830

RESUMO

Antibody-drug conjugates (ADC) represent a promising therapeutic modality for the clinical management of cancer. We sought to develop a novel ADC that targets 5T4, an oncofetal antigen expressed on tumor-initiating cells (TIC), which comprise the most aggressive cell population in the tumor. We optimized an anti-5T4 ADC (A1mcMMAF) by sulfydryl-based conjugation of the humanized A1 antibody to the tubulin inhibitor monomethylauristatin F (MMAF) via a maleimidocaproyl linker. A1mcMMAF exhibited potent in vivo antitumor activity in a variety of tumor models and induced long-term regressions for up to 100 days after the last dose. Strikingly, animals showed pathologic complete response in each model with doses as low as 3 mg antibody/kg dosed every 4 days. In a non-small cell lung cancer patient-derived xenograft model, in which 5T4 is preferentially expressed on the less differentiated tumor cells, A1mcMMAF treatment resulted in sustained tumor regressions and reduced TIC frequency. These results highlight the potential of ADCs that target the most aggressive cell populations within tumors, such as TICs. In exploratory safety studies, A1mcMMAF exhibited no overt toxicities when administered to cynomolgus monkeys at doses up to 10 mg antibody/kg/cycle × 2 and displayed a half-life of 5 days. The preclinical efficacy and safety data established a promising therapeutic index that supports clinical testing of A1mcMMAF.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Glicoproteínas de Membrana/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Anticorpos Monoclonais Humanizados/metabolismo , Anticorpos Monoclonais Humanizados/toxicidade , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Macaca fascicularis , Masculino , Dose Máxima Tolerável , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/efeitos dos fármacos , Indução de Remissão , Distribuição Tecidual , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Cancer Res ; 71(12): 4236-46, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21540235

RESUMO

Poorly differentiated tumors in non-small cell lung cancer (NSCLC) have been associated with shorter patient survival and shorter time to recurrence following treatment. Here, we integrate multiple experimental models with clinicopathologic analysis of patient tumors to delineate a cellular hierarchy in NSCLC. We show that the oncofetal protein 5T4 is expressed on tumor-initiating cells and associated with worse clinical outcome in NSCLC. Coexpression of 5T4 and factors involved in the epithelial-to-mesenchymal transition were observed in undifferentiated but not in differentiated tumor cells. Despite heterogeneous expression of 5T4 in NSCLC patient-derived xenografts, treatment with an anti-5T4 antibody-drug conjugate resulted in complete and sustained tumor regression. Thus, the aggressive growth of heterogeneous solid tumors can be blocked by therapeutic agents that target a subpopulation of cells near the top of the cellular hierarchy.


Assuntos
Antígenos de Neoplasias/análise , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Imunotoxinas/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Glicoproteínas de Membrana/análise , Células-Tronco Neoplásicas/imunologia , Animais , Antígeno CD24/análise , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Humanos , Receptores de Hialuronatos/análise , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Glicoproteínas de Membrana/fisiologia , Camundongos
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