RESUMEN
The immune response to SARS-CoV-2 antigen after infection or vaccination is defined by the durable production of antibodies and T cells. Population-based monitoring typically focuses on antibody titer, but there is a need for improved characterization and quantification of T cell responses. Here, we used multimodal sequencing technologies to perform a longitudinal analysis of circulating human leukocytes collected before and after immunization with the mRNA vaccine BNT162b2. Our data indicated distinct subpopulations of CD8+ T cells, which reliably appeared 28 days after prime vaccination. Using a suite of cross-modality integration tools, we defined their transcriptome, accessible chromatin landscape and immunophenotype, and we identified unique biomarkers within each modality. We further showed that this vaccine-induced population was SARS-CoV-2 antigen-specific and capable of rapid clonal expansion. Moreover, we identified these CD8+ T cell populations in scRNA-seq datasets from COVID-19 patients and found that their relative frequency and differentiation outcomes were predictive of subsequent clinical outcomes.
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Linfocitos T CD8-positivos , COVID-19 , Humanos , Vacunas contra la COVID-19 , SARS-CoV-2 , Vacuna BNT162 , COVID-19/prevención & control , Vacunación , Anticuerpos AntiviralesRESUMEN
Given mounting evidence of the importance of gut-microbiota/immune-cell interactions in immune homeostasis and responsiveness, surprisingly little is known about leukocyte movements to, and especially from, the gut. We address this topic in a minimally perturbant manner using Kaede transgenic mice, which universally express a photoconvertible fluorescent reporter. Transcutaneous exposure of the cervical lymph nodes to violet light permitted punctual tagging of immune cells specifically therein, and subsequent monitoring of their immigration to the intestine; endoscopic flashing of the descending colon allowed specific labeling of intestinal leukocytes and tracking of their emigration. Our data reveal an unexpectedly broad movement of leukocyte subsets to and from the gut at steady state, encompassing all lymphoid and myeloid populations examined. Nonetheless, different subsets showed different trafficking proclivities (e.g., regulatory T cells were more restrained than conventional T cells in their exodus from the cervical lymph nodes). The novel endoscopic approach enabled us to evidence gut-derived Th17 cells in the spleens of K/BxN mice at the onset of their genetically determined arthritis, thereby furnishing a critical mechanistic link between the intestinal microbiota, namely segmented filamentous bacteria, and an extraintestinal autoinflammatory disease.
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Sistema Digestivo/inmunología , Sistema Digestivo/microbiología , Leucocitos/inmunología , Leucocitos/fisiología , Microbiota/inmunología , Inmunidad Adaptativa , Animales , Artritis Experimental/inmunología , Artritis Experimental/microbiología , Artritis Experimental/patología , Movimiento Celular/inmunología , Sistema Digestivo/citología , Genes Reporteros , Inmunidad Innata , Antígenos Comunes de Leucocito/metabolismo , Proteínas Luminiscentes/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Transgénicos , Procesos FotoquímicosRESUMEN
The hallmark of type 1 diabetes is autoimmune destruction of the insulin-producing ß-cells of the pancreatic islets. Autoimmune diabetes has been difficult to study or treat because it is not usually diagnosed until substantial ß-cell loss has already occurred. Imaging agents that permit noninvasive visualization of changes in ß-cell mass remain a high-priority goal. We report on the development and testing of a near-infrared fluorescent ß-cell imaging agent. Based on the amino acid sequence of exendin-4, we created a neopeptide via introduction of an unnatural amino acid at the K(12) position, which could subsequently be conjugated to fluorophores via bioorthogonal copper-catalyzed click-chemistry. Cell assays confirmed that the resulting fluorescent probe (E4(×12)-VT750) had a high binding affinity (~3 nM). Its in vivo properties were evaluated using high-resolution intravital imaging, histology, whole-pancreas visualization, and endoscopic imaging. According to intravital microscopy, the probe rapidly bound to ß-cells and, as demonstrated by confocal microscopy, it was internalized. Histology of the whole pancreas showed a close correspondence between fluorescence and insulin staining, and there was an excellent correlation between imaging signals and ß-cell mass in mice treated with streptozotocin, a ß-cell toxin. Individual islets could also be visualized by endoscopic imaging. In short, E4(×12)-VT750 showed strong and selective binding to glucose-like peptide-1 receptors and permitted accurate measurement of ß-cell mass in both diabetic and nondiabetic mice. This near-infrared imaging probe, as well as future radioisotope-labeled versions of it, should prove to be important tools for monitoring diabetes, progression, and treatment in both experimental and clinical contexts.
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Células Secretoras de Insulina/metabolismo , Lisina/metabolismo , Péptidos/metabolismo , Ponzoñas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Línea Celular Tumoral , Diabetes Mellitus Tipo 1/inducido químicamente , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Exenatida , Colorantes Fluorescentes/química , Receptor del Péptido 1 Similar al Glucagón , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/patología , Laparoscopía/instrumentación , Laparoscopía/métodos , Lisina/química , Lisina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Microscopía Fluorescente , Datos de Secuencia Molecular , Células 3T3 NIH , Páncreas/metabolismo , Páncreas/patología , Péptidos/química , Péptidos/genética , Receptores de Glucagón/genética , Receptores de Glucagón/metabolismo , Estreptozocina/toxicidad , Ponzoñas/química , Ponzoñas/genéticaRESUMEN
The gastrointestinal tract is continuously exposed to foreign antigens in food and commensal microbes with potential to induce adaptive immune responses. Peripherally induced T regulatory (pTreg) cells are essential for mitigating inflammatory responses to these agents1-4. While RORγt+ antigen-presenting cells (RORγt-APCs) were shown to program gut microbiota-specific pTregs5-7, understanding of their characteristics remains incomplete, and the APC subset responsible for food tolerance has remained elusive. Here, we demonstrate that RORγt-APCs are similarly required for differentiation of food antigen-specific pTregs and establishment of oral tolerance. The ability of these cells to direct both food and microbiota-specific pTreg cell differentiation is contingent on expression of RORγt and on a unique cis-regulatory element within the Rorc gene locus (Rorc(t) +7kb). Absent this +7kb element, there was a notable increase in food antigen-specific T helper 2 (Th2) cells in lieu of pTregs, leading to compromised tolerance in a mouse asthma model. By employing single-cell analyses across these models, as well as freshly resected mesenteric lymph nodes from a human organ donor, we identified a rare subset of evolutionarily conserved APCs that are dependent on RORγt, uniquely express the Prdm16 transcription factor, and are endowed with essential mediators for inducing pTreg cell differentiation. Our findings suggest that a better understanding of how RORγt-APCs develop and how they regulate T cell responses to food and microbial antigens could offer new insights into developing therapeutic strategies for autoimmune and allergic diseases as well as organ transplant tolerance.
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Food antigens elicit immune tolerance through the action of regulatory T cells (Tregs) in the intestine. Although antigens that trigger common food allergies are known, the epitopes that mediate tolerance to most foods have not been described. Here, we identified murine T cell receptors specific for maize, wheat, and soy, and used expression cloning to de-orphan their cognate epitopes. All of the epitopes derive from seed storage proteins that are resistant to degradation and abundant in the edible portion of the plant. Multiple unrelated T cell clones were specific for an epitope at the C-terminus of 19 kDa alpha-zein, a protein from maize kernel. An MHC tetramer loaded with this antigen revealed that zein-specific T cells are predominantly Tregs localized to the intestine. These cells, which develop concurrently with weaning, constitute up to 2% of the peripheral Treg pool. Bulk and single-cell RNA sequencing revealed that these cells express higher levels of immunosuppressive markers and chemokines compared to other Tregs. These data suggest that immune tolerance to plant-derived foods is focused on a specific class of antigens with common features, and they reveal the functional properties of naturally occurring food-specific Tregs.
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The human immune response to SARS-CoV-2 antigen after infection or vaccination is defined by the durable production of antibodies and T cells. Population-based monitoring typically focuses on antibody titer, but there is a need for improved characterization and quantification of T cell responses. Here, we utilize multimodal sequencing technologies to perform a longitudinal analysis of circulating human leukocytes collected before and after BNT162b2 immunization. Our data reveal distinct subpopulations of CD8 + T cells which reliably appear 28 days after prime vaccination (7 days post boost). Using a suite of cross-modality integration tools, we define their transcriptome, accessible chromatin landscape, and immunophenotype, and identify unique biomarkers within each modality. By leveraging DNA-oligo-tagged peptide-MHC multimers and T cell receptor sequencing, we demonstrate that this vaccine-induced population is SARS-CoV-2 antigen-specific and capable of rapid clonal expansion. Moreover, we also identify these CD8 + populations in scRNA-seq datasets from COVID-19 patients and find that their relative frequency and differentiation outcomes are predictive of subsequent clinical outcomes. Our work contributes to our understanding of T cell immunity, and highlights the potential for integrative and multimodal analysis to characterize rare cell populations.
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BACKGROUND & AIMS: Bone marrow stromal cells (MSCs) are being evaluated as a cellular therapeutic for immune-mediated diseases. We investigated the effects of MSCs in mice with chemically induced colitis and determined the effects of CD11b(+) cells based on the hypothesis that MSCs increase numbers of regulatory T cells. METHODS: Colitis was induced in mice using trinitrobenzene sulfonic acid; symptoms were monitored as a function of MSC delivery. An immunomodulatory response was determined by measuring numbers of regulatory T cells in mesenteric lymph nodes. In vitro cocultures were used to assess the interaction of MSCs with regulatory T cells and CD11b(+) cells; findings were supported using near-infrared tracking of MSCs in vivo. We chemically and surgically depleted splenic CD11b(+) cells before colitis was induced with trinitrobenzene sulfonic acid to monitor the effects of MSCs. We adoptively transferred CD11b(+) cells that were cocultured with MSCs into mice with colitis. RESULTS: Intravenous grafts of MSCs prevented colitis and increased survival times of mice. Numbers of Foxp3(+) regulatory T cells increased in mesenteric lymph nodes in mice given MSCs. MSCs increased the numbers of Foxp3(+) splenocytes in a CD11b(+) cell-dependent manner. Transplanted MSCs colocalized near splenic CD11b(+) cells in vivo. Loss of CD11b(+) cells eliminated the therapeutic effect of MSCs. MSCs increased the anticolitis effects of CD11b(+) cells in mice. CONCLUSIONS: MSC transplants, delivered by specific parameters, reduce colitis in mice. Interactions between MSC and CD11b(+) regulatory T cells might be used to develop potency assays for MSCs, to identify nonresponders to MSC therapy, and to create new cell grafts that are composed of CD11b(+) cells preconditioned by MSCs.
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Trasplante de Médula Ósea , Antígeno CD11b/metabolismo , Colitis/prevención & control , Colon/inmunología , Ganglios Linfáticos/inmunología , Bazo/inmunología , Células del Estroma/trasplante , Linfocitos T Reguladores/inmunología , Traslado Adoptivo , Animales , Comunicación Celular , Rastreo Celular , Técnicas de Cocultivo , Colitis/inducido químicamente , Colitis/inmunología , Colitis/patología , Colon/patología , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/metabolismo , Inmunofenotipificación , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Fenotipo , Factores de Tiempo , Ácido TrinitrobencenosulfónicoRESUMEN
An arthritogenic strain of Subdoligranulum in the gut elicits a local immune response, a precursor to systemic autoimmunity (Chriswell et al.).
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Autoinmunidad , Microbioma GastrointestinalRESUMEN
Importance: There is clinical equipoise for COVID-19 convalescent plasma (CCP) use in patients hospitalized with COVID-19. Objective: To determine the safety and efficacy of CCP compared with placebo in hospitalized patients with COVID-19 receiving noninvasive supplemental oxygen. Design, Setting, and Participants: CONTAIN COVID-19, a randomized, double-blind, placebo-controlled trial of CCP in hospitalized adults with COVID-19, was conducted at 21 US hospitals from April 17, 2020, to March 15, 2021. The trial enrolled 941 participants who were hospitalized for 3 or less days or presented 7 or less days after symptom onset and required noninvasive oxygen supplementation. Interventions: A unit of approximately 250 mL of CCP or equivalent volume of placebo (normal saline). Main Outcomes and Measures: The primary outcome was participant scores on the 11-point World Health Organization (WHO) Ordinal Scale for Clinical Improvement on day 14 after randomization; the secondary outcome was WHO scores determined on day 28. Subgroups were analyzed with respect to age, baseline WHO score, concomitant medications, symptom duration, CCP SARS-CoV-2 titer, baseline SARS-CoV-2 serostatus, and enrollment quarter. Outcomes were analyzed using a bayesian proportional cumulative odds model. Efficacy of CCP was defined as a cumulative adjusted odds ratio (cOR) less than 1 and a clinically meaningful effect as cOR less than 0.8. Results: Of 941 participants randomized (473 to placebo and 468 to CCP), 556 were men (59.1%); median age was 63 years (IQR, 52-73); 373 (39.6%) were Hispanic and 132 (14.0%) were non-Hispanic Black. The cOR for the primary outcome adjusted for site, baseline risk, WHO score, age, sex, and symptom duration was 0.94 (95% credible interval [CrI], 0.75-1.18) with posterior probability (P[cOR<1] = 72%); the cOR for the secondary adjusted outcome was 0.92 (95% CrI, 0.74-1.16; P[cOR<1] = 76%). Exploratory subgroup analyses suggested heterogeneity of treatment effect: at day 28, cORs were 0.72 (95% CrI, 0.46-1.13; P[cOR<1] = 93%) for participants enrolled in April-June 2020 and 0.65 (95% CrI, 0.41 to 1.02; P[cOR<1] = 97%) for those not receiving remdesivir and not receiving corticosteroids at randomization. Median CCP SARS-CoV-2 neutralizing titer used in April to June 2020 was 1:175 (IQR, 76-379). Any adverse events (excluding transfusion reactions) were reported for 39 (8.2%) placebo recipients and 44 (9.4%) CCP recipients (P = .57). Transfusion reactions occurred in 2 (0.4) placebo recipients and 8 (1.7) CCP recipients (P = .06). Conclusions and Relevance: In this trial, CCP did not meet the prespecified primary and secondary outcomes for CCP efficacy. However, high-titer CCP may have benefited participants early in the pandemic when remdesivir and corticosteroids were not in use. Trial Registration: ClinicalTrials.gov Identifier: NCT04364737.
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Transfusión de Componentes Sanguíneos , COVID-19/terapia , Enfermedad Crítica/terapia , Adulto , Anciano , Método Doble Ciego , Femenino , Hospitalización/estadística & datos numéricos , Humanos , Inmunización Pasiva , Masculino , Persona de Mediana Edad , Respiración Artificial/estadística & datos numéricos , Resultado del Tratamiento , Estados Unidos , Sueroterapia para COVID-19RESUMEN
Eosinophils are multifunctional leukocytes that degrade and remodel tissue extracellular matrix through production of proteolytic enzymes, release of proinflammatory factors to initiate and propagate inflammatory responses, and direct activation of mucus secretion and smooth muscle cell constriction. Thus, eosinophils are central effector cells during allergic airway inflammation and an important clinical therapeutic target. Here we describe the use of an injectable MMP-targeted optical sensor that specifically and quantitatively resolves eosinophil activity in the lungs of mice with experimental allergic airway inflammation. Through the use of real-time molecular imaging methods, we report the visualization of eosinophil responses in vivo and at different scales. Eosinophil responses were seen at single-cell resolution in conducting airways using near-infrared fluorescence fiberoptic bronchoscopy, in lung parenchyma using intravital microscopy, and in the whole body using fluorescence-mediated molecular tomography. Using these real-time imaging methods, we confirmed the immunosuppressive effects of the glucocorticoid drug dexamethasone in the mouse model of allergic airway inflammation and identified a viridin-derived prodrug that potently inhibited the accumulation and enzyme activity of eosinophils in the lungs. The combination of sensitive enzyme-targeted sensors with noninvasive molecular imaging approaches permitted evaluation of airway inflammation severity and was used as a model to rapidly screen for new drug effects. Both fluorescence-mediated tomography and fiberoptic bronchoscopy techniques have the potential to be translated into the clinic.
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Androstenos/farmacología , Antiinflamatorios/farmacología , Bacteriocinas/farmacología , Broncoscopía/métodos , Dexametasona/farmacología , Profármacos/farmacología , Hipersensibilidad Respiratoria/tratamiento farmacológico , Hipersensibilidad Respiratoria/patología , Tomografía Óptica/métodos , Androstenos/uso terapéutico , Animales , Antiinflamatorios/uso terapéutico , Bacteriocinas/uso terapéutico , Dexametasona/uso terapéutico , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos/métodos , Eosinófilos/enzimología , Eosinófilos/patología , Inflamación/tratamiento farmacológico , Inflamación/enzimología , Inflamación/patología , Inflamación/fisiopatología , Pulmón/enzimología , Pulmón/patología , Pulmón/fisiopatología , Ratones , Ratones Endogámicos BALB C , Ratones Mutantes , Contracción Muscular/efectos de los fármacos , Músculo Liso/enzimología , Músculo Liso/patología , Profármacos/uso terapéutico , Hipersensibilidad Respiratoria/enzimología , Hipersensibilidad Respiratoria/fisiopatologíaRESUMEN
Glowing tags: a series of activatable ("turn-on") tetrazine-conjugated fluorescent probes was developed, which react rapidly in an inverse-electron-demand [4+2] cycloaddition with strained dienophiles such as trans-cyclooctene, thereby strongly increasing the fluorescence intensity. The novel turn-on probes were applied for intracellular live-cell imaging of a microtubuli-binding trans-cyclooctene modified taxol.
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Colorantes Fluorescentes/química , Imagen Molecular/métodos , Animales , Microscopía Confocal , Procesos FotoquímicosRESUMEN
OBJECTIVES: Cytoreductive surgery is a cornerstone of therapy in metastatic ovarian cancer. While conventional white light (WL) inspection detects many obvious tumor foci, careful histologic comparison has shown considerable miss rates for smaller foci. The goal of this study was to compare tumor detection using WL versus near infrared (NIR) imaging with a protease activatable probe, as well as to evaluate the ability to quantify NIR fluorescence using a novel quantitative optical imaging system. METHODS: A murine model for peritoneal carcinomatosis was generated and metastatic foci were imaged using WL and NIR imaging following the i.v. administration of the protease activatable probe ProSense750. The presence of tumor was confirmed by histology. Additionally, the ability to account for variations in fluorescence signal intensity due to changes in distance between the catheter and target lesion during laparoscopic procedures was evaluated. RESULTS: NIR imaging with a ProSense750 significantly improved upon the target-to-background ratios (TBRs) of tumor foci in comparison to WL imaging (minimum improvement was approximately 3.5 fold). Based on 52 histologically validated samples, the sensitivity for WL imaging was 69%, while the sensitivity for NIR imaging was 100%. The effects of intraoperative distance changes upon fluorescence intensity were corrected in realtime, resulting in a decrease from 89% to 5% in signal variance during fluorescence laparoscopy. CONCLUSIONS: With its molecular specificity, low background autofluorescence, high TBRs, and quantitative signal, optical imaging with NIR protease activatable probes greatly improves upon the intraoperative detection of ovarian cancer metastases.
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Neoplasias Ováricas/diagnóstico , Neoplasias Ováricas/patología , Neoplasias Peritoneales/diagnóstico , Neoplasias Peritoneales/secundario , Espectroscopía Infrarroja Corta/métodos , Algoritmos , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Colorantes Fluorescentes/farmacocinética , Humanos , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/cirugía , Péptido Hidrolasas/metabolismo , Neoplasias Peritoneales/enzimologíaRESUMEN
The genetic basis of pancreatic ductal adenocarcinoma, which constitutes the most common type of pancreatic malignancy, involves the sequential activation of oncogenes and inactivation of tumor suppressor genes. Among the pivotal genetic alterations are Ki-RAS oncogene activation and p53 tumor suppressor gene inactivation. We explain that the combination of these genetic events facilitates pancreatic carcinogenesis as revealed in novel three-dimensional cell (spheroid cyst) culture and in vivo subcutaneous and orthotopic xenotransplantation models. N-cadherin, a member of the classic cadherins important in the regulation of cell-cell adhesion, is induced in the presence of Ki-RAS mutation but subsequently downregulated with the acquisition of p53 mutation as revealed by gene microarrays and corroborated by reverse transcription-PCR and Western blotting. N-cadherin modulates the capacity of pancreatic ductal cells to migrate and invade, in part via complex formation with keratinocyte growth factor receptor and neural cell adhesion molecule and in part via interaction with p120-catenin. However, modulation of these complexes by Ki-RAS and p53 leads to enhanced cell migration and invasion. This preferentially induces the downstream effector AKT over mitogen-activated protein kinase to execute changes in cellular behavior. Thus, we are able to define molecules that in part are directly affected by Ki-RAS and p53 during pancreatic ductal carcinogenesis, and this provides a platform for potential new molecularly based therapeutic interventions.
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Cadherinas/metabolismo , Movimiento Celular , Proteína Oncogénica p21(ras)/metabolismo , Conductos Pancreáticos/citología , Conductos Pancreáticos/patología , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Uniones Adherentes/patología , Animales , Carcinoma Ductal Pancreático/patología , Técnicas de Cultivo de Célula , Transformación Celular Neoplásica , Quistes/patología , Activación Enzimática , Regulación Neoplásica de la Expresión Génica , Ratones , Mutación/genética , Invasividad Neoplásica/patología , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Proteína Oncogénica p21(ras)/genética , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
PURPOSE: Current therapy for lung cancer involves multimodality therapies. However, many patients are either refractory to therapy or develop drug resistance. KRAS and epidermal growth factor receptor (EGFR) mutations represent some of the most common mutations in lung cancer, and many studies have shown the importance of these mutations in both carcinogenesis and chemoresistance. Genetically engineered murine models of mutant EGFR and KRAS have been developed that more accurately recapitulate human lung cancer. Recently, using cell-based experiments, we showed that platinum-based drugs and the antidiabetic drug rosiglitazone (PPARgamma ligand) interact synergistically to reduce cancer cell and tumor growth. Here, we directly determined the efficacy of the PPARgamma/carboplatin combination in these more relevant models of drug resistant non-small cell lung cancer. EXPERIMENTAL DESIGN: Tumorigenesis was induced by activation of either mutant KRAS or EGFR. Mice then received either rosiglitazone or carboplatin monotherapy, or a combination of both drugs. Change in tumor burden, pathology, and evidence of apoptosis and cell growth were assessed. RESULTS: Tumor burden remained unchanged or increased in the mice after monotherapy with either rosiglitazone or carboplatin. In striking contrast, we observed significant tumor shrinkage in mice treated with these drugs in combination. Immunohistochemical analyses showed that this synergy was mediated via both increased apoptosis and decreased proliferation. Importantly, this synergy between carboplatin and rosiglitazone did not increase systemic toxicity. CONCLUSIONS: These data show that the PPARgamma ligand/carboplatin combination is a new therapy worthy of clinical investigation in lung cancers, including those cancers that show primary resistance to platinum therapy or acquired resistance to targeted therapy.
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Antineoplásicos/uso terapéutico , Carboplatino/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Hipoglucemiantes/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Tiazolidinedionas/uso terapéutico , Animales , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quimioterapia Combinada , Receptores ErbB/fisiología , Genes ras/fisiología , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Imagen por Resonancia Magnética , Ratones , Ratones Transgénicos , PPAR gamma/agonistas , Inhibidores de Proteínas Quinasas/efectos adversos , Rosiglitazona , Uteroglobina/fisiologíaRESUMEN
PURPOSE: To use near-infrared (NIR) optical imaging to assess the therapeutic susceptibility and drug dosing of orthotopic human breast cancers implanted in mice treated with molecularly targeted therapy. MATERIALS AND METHODS: This study was approved by the institutional animal care and use committee. Imaging probes were synthesized by conjugating the human epidermal growth factor receptor type 2 (HER2)-specific antibody trastuzumab with fluorescent dyes. In vitro probe binding was assessed with flow cytometry. HER2-normal and HER2-overexpressing human breast cancer cells were orthotopically implanted in nude mice. Intravital laser scanning fluorescence microscopy was used to evaluate the in vivo association of the probe with the tumor cells. Mice bearing 3-5-mm-diameter tumors were intravenously injected with 0.4 nmol of HER2 probe before or after treatment. A total of 123 mice were used for all in vivo tumor growth and imaging experiments. Tumor fluorescence intensity was assessed, and standard fluorescence values were determined. Statistical significance was determined by performing standard analysis of variance across the imaging cohorts. RESULTS: HER2 probe enabled differentiation between HER2-normal and HER2-overexpressing human breast cancer cells in vitro and in vivo, with binding levels correlating with tumor trastuzumab susceptibility. Serial imaging before and during trastuzumab therapy revealed a significant reduction (P < .05) in probe binding with treatment and thus provided early evidence of successful HER2 inhibition days before the overall reduction in tumor growth was apparent. CONCLUSION: NIR imaging with HER2-specific imaging probes enables evaluation of the therapeutic susceptibility of human mammary tumors and of drug dosing during HER2-targeted therapy with trastuzumab. This approach, combined with tomographic imaging techniques, has potential in the clinical setting for determining patient eligibility for and adequate drug dosing in molecularly targeted cancer therapies.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Sistemas de Liberación de Medicamentos/métodos , Microscopía Fluorescente/métodos , Receptor ErbB-2/metabolismo , Animales , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Ratones , Ratones Desnudos , Técnicas de Sonda Molecular , PronósticoRESUMEN
New cancer therapies are increasingly molecular-pathway specific. The evaluation of these novel therapies would be greatly facilitated by the development of noninvasive methods to assess multiple tumor cellular and molecular parameters. Using fluorescent probes specific for HER2/neu (AF750-trastuzumab) and apoptosis (Cy5.5-Annexin), we demonstrate a multichannel near infrared molecular imaging approach that yields accurate and early assessment of treatment susceptibility, drug target inhibition and tumor response during HER2-targeted therapy of orthotopic human mammary carcinomas in mice with trastuzumab (Herceptin). This combined approach detects both partial treatment response (tumor growth inhibition without regression) as well as therapeutic resistance before alterations in tumor growth are apparent. Partially responsive tumors exhibit increased Annexin signal when trastuzumab is combined with a cytotoxic agent (paclitaxel), which predicts subsequent tumor regression and suggests that imaging can guide therapy optimization. This multiparametric imaging approach has great potential in the clinical setting for determining patient eligibility, adequate drug dosing and early biological response of molecularly-targeted cancer therapies.
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Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Neoplasias/diagnóstico , Neoplasias/tratamiento farmacológico , Paclitaxel/farmacología , Receptor ErbB-2/análisis , Espectrometría de Fluorescencia , Espectroscopía Infrarroja Corta , Animales , Anexinas , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales Humanizados , Antineoplásicos/administración & dosificación , Antineoplásicos Fitogénicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Diagnóstico Precoz , Femenino , Regulación Neoplásica de la Expresión Génica , Glioma/diagnóstico , Glioma/tratamiento farmacológico , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Microscopía Confocal , Microscopía Fluorescente , Neoplasias/química , Paclitaxel/administración & dosificación , Valor Predictivo de las Pruebas , Receptor ErbB-2/efectos de los fármacos , Trastuzumab , Regulación hacia ArribaRESUMEN
A number of exendin derivatives have been developed to target glucagon-like peptide 1 (GLP-1) receptors on beta cells in vivo. Modifications of exendin analogues have been shown to have significant effects on pharmacokinetics and, as such, have been used to develop a variety of therapeutic compounds. Here, we show that an exendin-4, modified at position 12 with a cysteine conjugated to a tetrazine, can be labeled with 18F-trans-cyclooctene and converted into a PET imaging agent at high yields and with good selectivity. The agent accumulates in beta cells in vivo and has sufficiently high accumulation in mouse models of insulinomas to enable in vivo imaging.
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Cancer cells interact with surrounding stromal fibroblasts during tumorigenesis, but the complex molecular rules that govern these interactions remain poorly understood thus hindering the development of therapeutic strategies to target cancer stroma. We have taken a mathematical approach to begin defining these rules by performing the first large-scale quantitative analysis of fibroblast effects on cancer cell proliferation across more than four hundred heterotypic cell line pairings. Systems-level modeling of this complex dataset using singular value decomposition revealed that normal tissue fibroblasts variably express at least two functionally distinct activities, one which reflects transcriptional programs associated with activated mesenchymal cells, that act either coordinately or at cross-purposes to modulate cancer cell proliferation. These findings suggest that quantitative approaches may prove useful for identifying organizational principles that govern complex heterotypic cell-cell interactions in cancer and other contexts.