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1.
Cell ; 184(9): 2454-2470.e26, 2021 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-33857425

RESUMO

Glioblastoma multiforme (GBM) is an aggressive brain tumor for which current immunotherapy approaches have been unsuccessful. Here, we explore the mechanisms underlying immune evasion in GBM. By serially transplanting GBM stem cells (GSCs) into immunocompetent hosts, we uncover an acquired capability of GSCs to escape immune clearance by establishing an enhanced immunosuppressive tumor microenvironment. Mechanistically, this is not elicited via genetic selection of tumor subclones, but through an epigenetic immunoediting process wherein stable transcriptional and epigenetic changes in GSCs are enforced following immune attack. These changes launch a myeloid-affiliated transcriptional program, which leads to increased recruitment of tumor-associated macrophages. Furthermore, we identify similar epigenetic and transcriptional signatures in human mesenchymal subtype GSCs. We conclude that epigenetic immunoediting may drive an acquired immune evasion program in the most aggressive mesenchymal GBM subtype by reshaping the tumor immune microenvironment.


Assuntos
Neoplasias Encefálicas/imunologia , Epigênese Genética , Glioblastoma/imunologia , Evasão da Resposta Imune/imunologia , Células Mieloides/imunologia , Células-Tronco Neoplásicas/imunologia , Microambiente Tumoral/imunologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Proliferação de Células , Metilação de DNA , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Mieloides/metabolismo , Células Mieloides/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Proc Natl Acad Sci U S A ; 120(23): e2221707120, 2023 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-37253006

RESUMO

Steroid receptor coactivator 3 (SRC-3) is most strongly expressed in regulatory T cells (Tregs) and B cells, suggesting that it plays an important role in the regulation of Treg function. Using an aggressive E0771 mouse breast cell line syngeneic immune-intact murine model, we observed that breast tumors were "permanently eradicated" in a genetically engineered tamoxifen-inducible Treg-cell-specific SRC-3 knockout (KO) female mouse that does not possess a systemic autoimmune pathological phenotype. A similar eradication of tumor was noted in a syngeneic model of prostate cancer. A subsequent injection of additional E0771 cancer cells into these mice showed continued resistance to tumor development without the need for tamoxifen induction to produce additional SRC-3 KO Tregs. SRC-3 KO Tregs were highly proliferative and preferentially infiltrated into breast tumors by activating the chemokine (C-C motif) ligand (Ccl) 19/Ccl21/chemokine (C-C motif) receptor (Ccr)7 signaling axis, generating antitumor immunity by enhancing the interferon-γ/C-X-C motif chemokine ligand (Cxcl) 9 signaling axis to facilitate the entrance and function of effector T cells and natural killer cells. SRC-3 KO Tregs also show a dominant effect by blocking the immune suppressive function of WT Tregs. Importantly, a single adoptive transfer of SRC-3 KO Tregs into wild-type E0771 tumor-bearing mice can completely abolish preestablished breast tumors by generating potent antitumor immunity with a durable effect that prevents tumor reoccurrence. Therefore, treatment with SRC-3-deleted Tregs represents an approach to completely block tumor growth and recurrence without the autoimmune side effects that typically accompany immune checkpoint modulators.


Assuntos
Neoplasias da Mama , Neoplasias Mamárias Animais , Coativador 3 de Receptor Nuclear , Animais , Feminino , Masculino , Camundongos , Ligantes , Camundongos Knockout , Coativador 3 de Receptor Nuclear/genética , Linfócitos T Reguladores , Tamoxifeno/farmacologia
3.
Dev Neurosci ; 46(1): 22-43, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-37231843

RESUMO

In depth study of pediatric gliomas has been hampered due to difficulties in accessing patient tissue and a lack of clinically representative tumor models. Over the last decade, however, profiling of carefully curated cohorts of pediatric tumors has identified genetic drivers that molecularly segregate pediatric gliomas from adult gliomas. This information has inspired the development of a new set of powerful in vitro and in vivo tumor models that can aid in identifying pediatric-specific oncogenic mechanisms and tumor microenvironment interactions. Single-cell analyses of both human tumors and these newly developed models have revealed that pediatric gliomas arise from spatiotemporally discrete neural progenitor populations in which developmental programs have become dysregulated. Pediatric high-grade gliomas also harbor distinct sets of co-segregating genetic and epigenetic alterations, often accompanied by unique features within the tumor microenvironment. The development of these novel tools and data resources has led to insights into the biology and heterogeneity of these tumors, including identification of distinctive sets of driver mutations, developmentally restricted cells of origin, recognizable patterns of tumor progression, characteristic immune environments, and tumor hijacking of normal microenvironmental and neural programs. As concerted efforts have broadened our understanding of these tumors, new therapeutic vulnerabilities have been identified, and for the first time, promising new strategies are being evaluated in the preclinical and clinical settings. Even so, dedicated and sustained collaborative efforts are necessary to refine our knowledge and bring these new strategies into general clinical use. In this review, we will discuss the range of currently available glioma models, the way in which they have each contributed to recent developments in the field, their benefits and drawbacks for addressing specific research questions, and their future utility in advancing biological understanding and treatment of pediatric glioma.


Assuntos
Neoplasias Encefálicas , Glioma , Adulto , Humanos , Criança , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Glioma/genética , Glioma/patologia , Glioma/terapia , Microambiente Tumoral
4.
Cytometry A ; 105(9): 659-665, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39107997

RESUMO

This panel was designed to characterize the immune cell landscape in the mouse tumor microenvironment as well as mouse lymphoid tissues (e.g., spleen). As an example, using the MC-38 mouse syngeneic tumor model, we demonstrated that we could measure the frequency and characterize the functional status of CD4 T cells, CD8 T cells, regulatory T cells, NK cells, B cells, macrophages, granulocytes, monocytes, and dendritic cells. This panel is especially useful for understanding the immune landscape in "cold" preclinical tumor models with very low immune cell infiltration and for investigating how therapeutic treatments may modulate the immune landscape.


Assuntos
Neoplasias do Colo , Citometria de Fluxo , Baço , Microambiente Tumoral , Animais , Baço/imunologia , Baço/patologia , Camundongos , Citometria de Fluxo/métodos , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Microambiente Tumoral/imunologia , Linhagem Celular Tumoral , Linfócitos T CD8-Positivos/imunologia , Células Matadoras Naturais/imunologia , Células Dendríticas/imunologia , Modelos Animais de Doenças , Linfócitos T CD4-Positivos/imunologia , Macrófagos/imunologia , Macrófagos/patologia , Linfócitos T Reguladores/imunologia , Linfócitos B/imunologia
5.
Mol Ther ; 31(3): 676-685, 2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36518079

RESUMO

A chromosome 14 inversion was found in a patient who developed bone marrow aplasia following treatment with allogeneic chimeric antigen receptor (CAR) Tcells containing gene edits made with transcription activator-like effector nucleases (TALEN). TALEN editing sites were not involved at either breakpoint. Recombination signal sequences (RSSs) were found suggesting recombination-activating gene (RAG)-mediated activity. The inversion represented a dominant clone detected in the context of decreasing absolute CAR Tcell and overall lymphocyte counts. The inversion was not associated with clinical consequences and wasnot detected in the drug product administered to this patient or in any drug product used in this or other trials using the same manufacturing processes. Neither was the inversion detected in this patient at earlier time points or in any other patient enrolled in this or other trials treated with this or other product lots. This case illustrates that spontaneous, possibly RAG-mediated, recombination events unrelated to gene editing can occur in adoptive cell therapy studies, emphasizes the need for ruling out off-target gene editing sites, and illustrates that other processes, such as spontaneous V(D)J recombination, can lead to chromosomal alterations in infused cells independent of gene editing.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Receptores de Antígenos Quiméricos , Humanos , Edição de Genes , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Linfócitos T , Receptores de Antígenos Quiméricos/genética , Imunoterapia Adotiva/efeitos adversos
6.
Biol Res ; 57(1): 59, 2024 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-39223638

RESUMO

BACKGROUND: Tumour dormancy, a resistance mechanism employed by cancer cells, is a significant challenge in cancer treatment, contributing to minimal residual disease (MRD) and potential relapse. Despite its clinical importance, the mechanisms underlying tumour dormancy and MRD remain unclear. In this study, we employed two syngeneic murine models of myeloid leukemia and melanoma to investigate the genetic, epigenetic, transcriptomic and protein signatures associated with tumour dormancy. We used a multiomics approach to elucidate the molecular mechanisms driving MRD and identify potential therapeutic targets. RESULTS: We conducted an in-depth omics analysis encompassing whole-exome sequencing (WES), copy number variation (CNV) analysis, chromatin immunoprecipitation followed by sequencing (ChIP-seq), transcriptome and proteome investigations. WES analysis revealed a modest overlap of gene mutations between melanoma and leukemia dormancy models, with a significant number of mutated genes found exclusively in dormant cells. These exclusive genetic signatures suggest selective pressure during MRD, potentially conferring resistance to the microenvironment or therapies. CNV, histone marks and transcriptomic gene expression signatures combined with Gene Ontology (GO) enrichment analysis highlighted the potential functional roles of the mutated genes, providing insights into the pathways associated with MRD. In addition, we compared "murine MRD genes" profiles to the corresponding human disease through public datasets and highlighted common features according to disease progression. Proteomic analysis combined with multi-omics genetic investigations, revealed a dysregulated proteins signature in dormant cells with minimal genetic mechanism involvement. Pathway enrichment analysis revealed the metabolic, differentiation and cytoskeletal remodeling processes involved in MRD. Finally, we identified 11 common proteins differentially expressed in dormant cells from both pathologies. CONCLUSIONS: Our study underscores the complexity of tumour dormancy, implicating both genetic and nongenetic factors. By comparing genomic, transcriptomic, proteomic, and epigenomic datasets, our study provides a comprehensive understanding of the molecular landscape of minimal residual disease. These results provide a robust foundation for forthcoming investigations and offer potential avenues for the advancement of targeted MRD therapies in leukemia and melanoma patients, emphasizing the importance of considering both genetic and nongenetic factors in treatment strategies.


Assuntos
Modelos Animais de Doenças , Melanoma , Neoplasia Residual , Animais , Melanoma/genética , Melanoma/patologia , Camundongos , Leucemia/genética , Leucemia/patologia , Variações do Número de Cópias de DNA , Sequenciamento do Exoma , Camundongos Endogâmicos C57BL , Proteômica , Transcriptoma , Perfilação da Expressão Gênica , Multiômica
7.
Pediatr Surg Int ; 40(1): 195, 2024 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-39017743

RESUMO

BACKGROUND: We previously showed that total tumor resection enhances metastatic growth in a syngeneic metastatic mouse model of neuroblastoma. In this study, we further investigated which surgical factors contributed most to metastatic growth. METHODS: Tumor cells derived from MYCN transgenic mice were subcutaneously injected into wild-type mice. Mice were randomly assigned to receive partial resection (PR group), subcutaneous implantation of a sponge (Sp group), or observation (Obs group). The lymph node metastasis volume and the frequency of lung metastasis were compared 14 days after assignment by measuring C-reactive protein (CRP) and interleukin-6 (IL-6) levels. RESULTS: The lymph node metastasis volume in the Sp group was larger than in the Obs group (148.4 [standard deviation {SD}: 209.5] vs. 10.2 [SD 12.8] mm3). The frequency of lung metastasis was greater in the Sp group than in the PR group (11.9 [SD 12.2] vs. 6.6 [SD 4.0] counts/slide). The CRP level in the Sp group was higher than in the PR group (2.3 [SD 0.5] vs. 1.5 [SD 0.4] µg/mL), and the IL-6 level in the Sp group was higher than in the PR or Obs groups (28.4 [SD 34.5] vs. 12.4 [SD 19.0] vs. 5.4 [SD 8.1] pg/mL). CONCLUSION: Metastatic growth may be enhanced by systemic inflammation.


Assuntos
Proteína C-Reativa , Modelos Animais de Doenças , Inflamação , Neoplasias Pulmonares , Neuroblastoma , Animais , Neuroblastoma/patologia , Camundongos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Proteína C-Reativa/metabolismo , Inflamação/patologia , Interleucina-6 , Metástase Linfática , Camundongos Transgênicos
8.
Int J Cancer ; 152(9): 1916-1932, 2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-36637144

RESUMO

Basal-like breast cancer (BLBC) is the most aggressive and heterogeneous breast cancer (BC) subtype. Conventional chemotherapies represent next to surgery the most frequently employed treatment options. Unfortunately, resistant tumor phenotypes often develop, resulting in therapeutic failure. To identify the early events occurring upon the first drug application and initiating chemotherapy resistance in BLBC, we leveraged the WAP-T syngeneic mammary carcinoma mouse model and we developed a strategy combining magnetic-activated cell sorting (MACS)-based tumor cell enrichment with high-throughput transcriptome analyses. We discovered that chemotherapy induced a massive gene expression reprogramming toward stemness acquisition to tolerate and survive the cytotoxic treatment in vitro and in vivo. Retransplantation experiments revealed that one single cycle of cytotoxic drug combination therapy (Cyclophosphamide, Adriamycin and 5-Fluorouracil) suffices to induce resistant tumor cell phenotypes in vivo. We identified Axl and its ligand Pros1 as highly induced genes driving cancer stem cell (CSC) properties upon chemotherapy in vivo and in vitro. Furthermore, from our analysis of BLBC patient datasets, we found that AXL expression is also strongly correlated with CSC-gene signatures, a poor response to conventional therapies and worse survival outcomes in those patients. Finally, we demonstrate that AXL inhibition sensitized BLBC-cells to cytotoxic treatment in vitro. Together, our data support AXL as a promising therapeutic target to optimize the efficiency of conventional cytotoxic therapies in BLBC.


Assuntos
Antineoplásicos , Carcinoma , Camundongos , Animais , Antineoplásicos/farmacologia , Transdução de Sinais , Ciclofosfamida/farmacologia , Células-Tronco Neoplásicas/metabolismo , Carcinoma/metabolismo , Linhagem Celular Tumoral
9.
EMBO J ; 38(1)2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30396996

RESUMO

Targeting immune checkpoints, such as PD-L1 and its receptor PD-1, has opened a new avenue for treating cancers. Understanding the regulatory mechanism of PD-L1 and PD-1 will improve the clinical response rate and efficacy of PD-1/PD-L1 blockade in cancer patients and the development of combinatorial strategies. VGLL4 inhibits YAP-induced cell proliferation and tumorigenesis through competition with YAP for binding to TEADs. However, whether VGLL4 has a role in anti-tumor immunity is largely unknown. Here, we found that disruption of Vgll4 results in potent T cell-mediated tumor regression in murine syngeneic models. VGLL4 deficiency reduces PD-L1 expression in tumor cells. VGLL4 interacts with IRF2BP2 and promotes its protein stability through inhibiting proteasome-mediated protein degradation. Loss of IRF2BP2 results in persistent binding of IRF2, a transcriptional repressor, to PD-L1 promoter. In addition, YAP inhibits IFNγ-inducible PD-L1 expression partially through suppressing the expression of VGLL4 and IRF1 by YAP target gene miR-130a. Our study identifies VGLL4 as an important regulator of PD-L1 expression and highlights a central role of VGLL4 and YAP in the regulation of tumor immunity.


Assuntos
Antígeno B7-H1/genética , Fatores de Transcrição/genética , Evasão Tumoral/genética , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Células Cultivadas , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Oncogenes/genética , Interferência de RNA , Fatores de Transcrição/fisiologia , Proteínas de Sinalização YAP
10.
J Transl Med ; 21(1): 376, 2023 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-37296466

RESUMO

BACKGROUND: Infection with high-risk human papillomavirus (HPV) strains is one of the risk factors for the development of oral squamous cell carcinoma (OSCC). Some patients with HPV-positive OSCC have a better prognosis and respond better to various treatment modalities, including radiotherapy or immunotherapy. However, since HPV can only infect human cells, there are only a few immunocompetent mouse models available that enable immunological studies. Therefore, the aim of our study was to develop a transplantable immunocompetent mouse model of HPV-positive OSCC and characterize it in vitro and in vivo. METHODS: Two monoclonal HPV-positive OSCC mouse cell lines were established by inducing the expression of HPV-16 oncogenes E6 and E7 in the MOC1 OSCC cell line using retroviral transduction. After confirming stable expression of HPV-16 E6 and E7 with quantitative real-time PCR and immunofluorescence staining, the cell lines were further characterized in vitro using proliferation assay, wound healing assay, clonogenic assay and RNA sequencing. In addition, tumor models were characterized in vivo in C57Bl/6NCrl mice in terms of their histological properties, tumor growth kinetics, and radiosensitivity. Furthermore, immunofluorescence staining of blood vessels, hypoxic areas, proliferating cells and immune cells was performed to characterize the tumor microenvironment of all three tumor models. RESULTS: Characterization of the resulting MOC1-HPV cell lines and tumor models confirmed stable expression of HPV-16 oncogenes and differences in cell morphology, in vitro migration capacity, and tumor microenvironment characteristics. Although the cell lines did not differ in their intrinsic radiosensitivity, one of the HPV-positive tumor models, MOC1-HPV K1, showed a significantly longer growth delay after irradiation with a single dose of 15 Gy compared to parental MOC1 tumors. Consistent with this, MOC1-HPV K1 tumors had a lower percentage of hypoxic tumor area and a higher percentage of proliferating cells. Characteristics of the newly developed HPV-positive OSCC tumor models correlate with the transcriptomic profile of MOC1-HPV cell lines. CONCLUSIONS: In conclusion, we developed and characterized a novel immunocompetent mouse model of HPV-positive OSCC that exhibits increased radiosensitivity and enables studies of immune-based treatment approaches in HPV-positive OSCC.


Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Infecções por Papillomavirus , Humanos , Animais , Camundongos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço , Infecções por Papillomavirus/complicações , Microambiente Tumoral
11.
J Transl Med ; 21(1): 843, 2023 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-37996891

RESUMO

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease. This is due to its aggressive course, late diagnosis and its intrinsic drugs resistance. The complexity of the tumor, in terms of cell components and heterogeneity, has led to the approval of few therapies with limited efficacy. The study of the early stages of carcinogenesis provides the opportunity for the identification of actionable pathways that underpin therapeutic resistance. METHODS: We analyzed 43 Intraductal papillary mucinous neoplasms (IPMN) (12 Low-grade and 31 High-grade) by Spatial Transcriptomics. Mouse and human pancreatic cancer organoids and T cells interaction platforms were established to test the role of mucins expression on T cells activity. Syngeneic mouse model of PDAC was used to explore the impact of mucins downregulation on standard therapy efficacy. RESULTS: Spatial transcriptomics showed that mucin O-glycosylation pathway is increased in the progression from low-grade to high-grade IPMN. We identified GCNT3, a master regulator of mucins expression, as an actionable target of this pathway by talniflumate. We showed that talniflumate impaired mucins expression increasing T cell activation and recognition using both mouse and human organoid interaction platforms. In vivo experiments showed that talniflumate was able to increase the efficacy of the chemotherapy by boosting immune infiltration. CONCLUSIONS: Finally, we demonstrated that combination of talniflumate, an anti-inflammatory drug, with chemotherapy effectively improves anti-tumor effect in PDAC.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Intraductais Pancreáticas , Neoplasias Pancreáticas , Humanos , Animais , Camundongos , Mucinas , Gencitabina , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/patologia
12.
Cell Tissue Res ; 391(2): 323-337, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36447073

RESUMO

Systemic sclerosis associated with lung interstitial lung disease (SSc-ILD) is the most common cause of death among patients with SSc. Mesenchymal stem cell (MSCs) transplantations had been treated by SSc patients that showed in the previous case report. The therapeutic mechanisms and effects of MSCs on SSc-ILD are still obscure. In this study, we investigated the therapeutic effects and mechanisms of treatment of BM-MSC derived from C57BL/6 on the topoisomerase I (TOPO I) induced SSc-ILD-like mice model. The mice were immunized with a mixture of recombinant human TOPO I in PBS solution (500 U/mL) and completed Freund's adjuvant [CFA; 1:1 (volume/volume)] twice per week for 9 weeks. On week 10, the mice were sacrificed to analyze the related pathological parameters. Lung and skin pathologies were analyzed using histochemical staining. CD4 T-helper (TH) cell differentiation in lung and skin-draining lymph nodes was detected using flow cytometry. Our results revealed that allogeneic and syngeneic MSCs exhibited similar repressive effects on TOPO I-induced IgG1 and IgG2a in the SSc group. After intravascular (IV) treatment with syngeneic or allogeneic MSCs, the dermal thickness and fibrosis dramatically condensed and significantly reduced airway hyperresponsiveness. These findings showed that both allogeneic and syngeneic MSCs have therapeutic potential for SSc-ILD.


Assuntos
Doenças Pulmonares Intersticiais , Células-Tronco Mesenquimais , Pneumonia , Escleroderma Sistêmico , Humanos , Animais , Camundongos , DNA Topoisomerases Tipo I , Camundongos Endogâmicos C57BL , Fibrose , Escleroderma Sistêmico/complicações , Escleroderma Sistêmico/terapia , Doenças Pulmonares Intersticiais/etiologia , Doenças Pulmonares Intersticiais/patologia , Pulmão/patologia , Pneumonia/patologia
13.
Mol Pharm ; 20(5): 2415-2425, 2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-37014648

RESUMO

Tumor-associated macrophages (TAMs) are large phagocytic cells that play numerous roles in cancer biology and are an important component of the relationship between immune system response and tumor progression. The peptide, RP832c, targets the Mannose Receptor (CD206) expressed on M2-like macrophages and is cross-reactive to both human and murine CD206. Additionally, it exhibits therapeutic properties through its ability to shift the population of TAMs from an M2-like (protumor) toward an M1-like phenotype (antitumor) and has demonstrated promise in inhibiting tumor resistance in PD-L1 unresponsive melanoma murine models. In addition, it has shown inhibition in bleomycin-induced pulmonary fibrosis through interactions with CD206 macrophages.1,2 Our work aims to develop a novel CD206 positron emission tomography (PET) imaging probe based on RP832c (Kd = 5.64 µM) as a direct, noninvasive method for the assessment of TAMs in mouse models of cancer. We adapted RP832c to incorporate the chelator DOTA to allow for radiolabeling with the PET isotope 68Ga (t1/2 = 68 min; ß+ = 89%). In vitro stability studies were conducted in mouse serum up to 3 h. The in vitro binding characteristics of [68Ga]RP832c to CD206 were determined by a protein plate binding assay and Surface Plasmon Resonance (SPR). PET imaging and biodistribution studies were conducted in syngeneic tumor models. Stability studies in mouse serum demonstrated that 68Ga remained complexed up to 3 h (less than 1% free 68Ga). Binding affinity studies demonstrated high binding of [68Ga]RP832c to mouse CD206 protein and that the binding of the tracer was able to be blocked significantly when incubated with a blocking solution of native RP832c. PET imaging and biodistribution studies in syngeneic tumor models demonstrated uptake in tumor and CD206 expressing organs of [68Ga]RP832c. A significant correlation was found between the percentage of CD206 present in each tumor imaged with [68Ga]RP832c and PET imaging mean standardized uptake values in a CT26 mouse model of cancer. The data shows that [68Ga]RP832c represents a promising candidate for macrophage imaging in cancer and other diseases.


Assuntos
Radioisótopos de Gálio , Neoplasias , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Radioisótopos de Gálio/química , Macrófagos/metabolismo , Neoplasias/metabolismo , Peptídeos/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Distribuição Tecidual , Receptor de Manose/metabolismo
14.
Mol Pharm ; 20(8): 4319-4330, 2023 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-37485886

RESUMO

Patients with pancreatic ductal adenocarcinoma (PDAC) have a dismal 5 year survival of 9%. One important limiting factor for treatment efficacy is the dense tumor-supporting stroma. The cancer-associated fibroblasts in this stroma deposit excessive amounts of extracellular matrix components and anti-inflammatory mediators, which hampers the efficacy of chemo- and immunotherapies. Systemic depletion of all activated fibroblasts is, however, not feasible nor desirable and therefore a local approach should be pursued. Here, we provide a proof-of-principle of using fibroblast activation protein (FAP)-targeted photodynamic therapy (tPDT) to treat PDAC. FAP-targeting antibody 28H1 and irrelevant control antibody DP47GS were conjugated to the photosensitizer IRDye700DX (700DX) and the chelator diethylenetriaminepentaacetic acid. In vitro binding and cytotoxicity were evaluated using the fibroblast cell-line NIH-3T3 stably transfected with FAP. Biodistribution of 111In-labeled antibody-700DX constructs was determined in mice carrying syngeneic tumors of the murine PDAC cell line PDAC299, and in a genetically engineered PDAC mouse model (CKP). Then, tPDT was performed by exposing the subcutaneous or the spontaneous PDAC tumors to 690 nm light. Induction of apoptosis after treatment was assessed using automated analyses of immunohistochemistry for cleaved caspase-3. 28H1-700DX effectively bound to 3T3-FAP cells and induced cytotoxicity upon exposure to 690 nm light, whereas no binding or cytotoxic effects were observed for DP47GS-700DX. Although both 28H1-700DX and DP47GS-700DX accumulated in subcutaneous PDAC299 tumors, autoradiography demonstrated that only 28H1-700DX reached the tumor core. On the contrary, control antibody DP47GS-700DX was only present at the tumor rim. In CKP mice, both antibodies accumulated in the tumor, but tumor-to-blood ratios of 28H1-700DX were higher than that of the control. Notably, in vivo FAP-tPDT caused upregulation of cleaved caspase-3 staining in both subcutaneous and in spontaneous tumors. In conclusion, we have shown that tPDT is a feasible approach for local depletion of FAP-expressing stromal cells in murine models for PDAC.


Assuntos
Fibroblastos Associados a Câncer , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Fotoquimioterapia , Camundongos , Animais , Serina Endopeptidases/metabolismo , Caspase 3/metabolismo , Distribuição Tecidual , Modelos Animais de Doenças , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/patologia , Fibroblastos/metabolismo , Anticorpos/metabolismo , Linhagem Celular Tumoral , Neoplasias Pancreáticas
15.
Immunol Invest ; 52(6): 749-766, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37403798

RESUMO

BACKGROUND: Tumor innervation has been shown to be utilized by some solid cancers to support tumor initiation, growth, progression, and metastasis, as well as confer resistance to immune checkpoint blockade through suppression of antitumor immunologic responses. Since botulinum neurotoxin type A1 (BoNT/A1) blocks neuronal cholinergic signaling, its potential use as an anticancer drug in combination with anti-PD-1 therapy was investigated in four different syngeneic mouse tumor models. METHODS: Mice implanted with breast (4T1), lung (LLC1), colon (MC38), and melanoma (B16-F10) tumors were administered a single intratumoral injection of 15 U/kg BoNT/A1, repeated intraperitoneal injections of 5 mg/kg anti-PD-1 (RMP1-14), or both. RESULTS: Compared to the single-agent treatments, anti-PD-1 and BoNT/A1 combination treatment elicited significant reduction in tumor growth among B16-F10 and MC38 tumor-bearing mice. The combination treatment also lowered serum exosome levels in these mice compared to the placebo control group. In the B16-F10 syngeneic mouse tumor model, anti-PD-1 + BoNT/A1 combination treatment lowered the proportion of MDSCs, negated the increased proportion of Treg cells, and elicited a higher number of tumor-infiltrating CD4+ and CD8+ T lymphocytes into the tumor microenvironment compared to anti-PD-1 treatment alone. CONCLUSION: Our findings demonstrate the synergistic antitumor effects of BoNT/A1 and PD-1 checkpoint blockade in mouse tumor models of melanoma and colon carcinoma. These findings provide some evidence on the potential application of BoNT/A1 as an anticancer drug in combination with immune checkpoint blockade and should be further explored.


Assuntos
Antineoplásicos , Toxinas Botulínicas , Melanoma , Animais , Camundongos , Receptor de Morte Celular Programada 1 , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Toxinas Botulínicas/farmacologia , Colo , Microambiente Tumoral , Linfócitos T CD8-Positivos
16.
Int J Mol Sci ; 24(20)2023 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-37894963

RESUMO

There is a clear need to expand the toolkit of adequate mouse models and cell lines available for preclinical studies of high-grade neuroendocrine lung carcinoma (small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC)). SCLC and LCNEC are two highly aggressive tumor types with dismal prognoses and few therapeutic options. Currently, there is an extreme paucity of material, particularly in the case of LCNEC. Given the lack of murine cell lines and transplant models of LCNEC, the need is imperative. In this study, we generated and examined new models of LCNEC and SCLC transplantable cell lines derived from our previously developed primary mouse LCNEC and SCLC tumors. RNA-seq analysis demonstrated that our cell lines and syngeneic tumors maintained the transcriptome program from the original transgenic primary tumor and displayed strong similarities to human SCLC or LCNEC. Importantly, the SCLC transplanted cell lines showed the ability to metastasize and mimic this characteristic of the human condition. In summary, we generated mouse cell line tools that allow further basic and translational research as well as preclinical testing of new treatment strategies for SCLC and LCNEC. These tools retain important features of their human counterparts and address the lack of LCNEC disease models.


Assuntos
Carcinoma de Células Grandes , Carcinoma Neuroendócrino , Carcinoma de Células Pequenas , Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Humanos , Animais , Camundongos , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Células Pequenas/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Carcinoma Neuroendócrino/genética , Carcinoma Neuroendócrino/patologia , Carcinoma de Células Grandes/genética , Carcinoma de Células Grandes/patologia , Pulmão/patologia
17.
Int J Mol Sci ; 24(6)2023 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-36982737

RESUMO

Estrogen receptor-positive breast cancers (ER+ BCas) are the most common form of BCa and are increasing in incidence, largely due to changes in reproductive practices in recent decades. Tamoxifen is prescribed as a component of standard-of-care endocrine therapy for the treatment and prevention of ER+ BCa. However, it is poorly tolerated, leading to low uptake of the drug in the preventative setting. Alternative therapies and preventatives for ER+ BCa are needed but development is hampered due to a paucity of syngeneic ER+ preclinical mouse models that allow pre-clinical experimentation in immunocompetent mice. Two ER-positive models, J110 and SSM3, have been reported in addition to other tumour models occasionally shown to express ER (for example 4T1.2, 67NR, EO771, D2.0R and D2A1). Here, we have assessed ER expression and protein levels in seven mouse mammary tumour cell lines and their corresponding tumours, in addition to their cellular composition, tamoxifen sensitivity and molecular phenotype. By immunohistochemical assessment, SSM3 and, to a lesser extent, 67NR cells are ER+. Using flow cytometry and transcript expression we show that SSM3 cells are luminal in nature, whilst D2.0R and J110 cells are stromal/basal. The remainder are also stromal/basal in nature; displaying a stromal or basal Epcam/CD49f FACS phenotype and stromal and basal gene expression signatures are overrepresented in their transcript profile. Consistent with a luminal identity for SSM3 cells, they also show sensitivity to tamoxifen in vitro and in vivo. In conclusion, the data indicate that the SSM3 syngeneic cell line is the only definitively ER+ mouse mammary tumour cell line widely available for pre-clinical research.


Assuntos
Neoplasias da Mama , Receptores de Estrogênio , Tamoxifeno , Humanos , Linhagem Celular Tumoral , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Animais , Camundongos , Modelos Animais de Doenças , Receptores de Estrogênio/genética , Tamoxifeno/farmacologia , Fenótipo , Imuno-Histoquímica , Citometria de Fluxo , Transcriptoma , Camundongos da Linhagem 129 , RNA-Seq , Células Epiteliais , Glândulas Mamárias Animais/citologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/genética
18.
Int J Mol Sci ; 24(22)2023 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-38003463

RESUMO

Stimulator of interferon genes (STING) agonists have shown potent anti-tumor efficacy in various mouse tumor models and have the potential to overcome resistance to immune checkpoint inhibitors (ICI) by linking the innate and acquired immune systems. First-generation STING agonists are administered intratumorally; however, a systemic delivery route would greatly expand the clinical use of STING agonists. Biochemical and cell-based experiments, as well as syngeneic mouse efficacy models, were used to demonstrate the anti-tumoral activity of ALG-031048, a novel STING agonist. In vitro, ALG-031048 is highly stable in plasma and liver microsomes and is resistant to degradation via phosphodiesterases. The high stability in biological matrices translated to good cellular potency in a HEK 293 STING R232 reporter assay, efficient activation and maturation of primary human dendritic cells and monocytes, as well as long-lasting, antigen-specific anti-tumor activity in up to 90% of animals in the CT26 mouse colon carcinoma model. Significant reductions in tumor growth were observed in two syngeneic mouse tumor models following subcutaneous administration. Combinations of ALG-031048 and ICIs further enhanced the in vivo anti-tumor activity. This initial demonstration of anti-tumor activity after systemic administration of ALG-031048 warrants further investigation, while the combination of systemically administered ALG-031048 with ICIs offers an attractive approach to overcome key limitations of ICIs in the clinic.


Assuntos
Neoplasias do Colo , Neoplasias , Camundongos , Animais , Humanos , Células HEK293 , Neoplasias/patologia , Neoplasias do Colo/tratamento farmacológico , Modelos Animais de Doenças , Imunoterapia , Microambiente Tumoral
19.
Int J Mol Sci ; 24(9)2023 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-37175837

RESUMO

Erythroid cells are emerging players in immunological regulation that have recently been shown to play a crucial role in fetomaternal tolerance in mice. In this work, we set ourselves the goal of discovering additional information about the molecular mechanisms of this process. We used flow cytometry to study placental erythroid cells' composition and BioPlex for the secretome profiling of 23 cytokines at E12.5 and E19.5 in both allogeneic and syngeneic pregnancies. We found that (1) placental erythroid cells are mainly represented by CD45+ erythroid cells; (2) the secretomes of CD71+ placental erythroid cells differ from the ones in syngeneic pregnancy; (3) CCL2, CCL3, CCL4 and CXCL1 chemokines were secreted on each day of embryonic development and in both types of pregnancy studied. We believe that these chemokines lure placental immune cells towards erythroid cells so that erythroid cells can induce anergy in those immune cells via cell-bound ligands such as PD-L1, enzymes such as ARG1, and secreted factors such as TGFß-1.


Assuntos
Células Eritroides , Placenta , Animais , Feminino , Camundongos , Gravidez , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocinas , Citometria de Fluxo , Imunossupressores
20.
Molecules ; 28(10)2023 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-37241912

RESUMO

Cold atmospheric plasma (CAP) may have applications in treating various types of malignant tumors. This study assessed the anticancer effects of CAP using melanoma and colon cancer cell lines. CAP treatment significantly reduced the in vitro viability of melanoma and colon cancer cell lines and had a negligible effect on the viability of normal human melanocytes. Additionally, CAP and epidermal growth factor receptor (EGFR) inhibitor had an additive anticancer effect in a CAP-resistant melanoma cell line. Reactive oxygen and nitrogen species known to be generated by CAP enhanced the anticancer effects of CAP and EGFR inhibitors. The in vivo anticancer activities of CAP were evaluated by testing its effects against syngeneic tumors induced in mice by melanoma and colon cancer cells. CAP treatment reduced tumor volume and weight in both cancer models, with the extent of tumor reduction dependent on the duration and number of CAP treatments. Histologic examination also revealed the tumoricidal effects of CAP in both tumor models. In conclusion, CAP inhibits the growth of mouse melanoma and colon cancer cell lines in vitro and shows tumoricidal effects against mouse models of melanoma and colon cancer in vivo.


Assuntos
Neoplasias do Colo , Melanoma , Gases em Plasma , Humanos , Animais , Camundongos , Gases em Plasma/farmacologia , Gases em Plasma/uso terapêutico , Linhagem Celular Tumoral , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Neoplasias do Colo/tratamento farmacológico , Receptores ErbB
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