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Photodynamic therapy (PDT) is a light-based anticancer therapy that can induce tumor necrosis and/or apoptosis. Two important factors contributing to the efficacy of PDT are the concentration of the photosensitizer in the tumor tissue and its preferential accumulation in the tumor tissue compared to that in normal tissues. In this study, we investigated the use of optical imaging for monitoring whole-body bio-distribution of the fluorescent (660 nm) photosensitizer Bremachlorin in vivo, in a murine pancreatic ductal adenocarcinoma (PDAC) model. Moreover, we non-invasively, examined the induction of tumor necrosis after PDT treatment using near-infrared fluorescent imaging of the necrosis avid cyanine dye IRDye®-800CW Carboxylate. Using whole-body fluorescence imaging, we observed that Bremachlorin preferentially accumulated in pancreatic tumors. Furthermore, in a longitudinal study we showed that 3 hours after Bremachlorin administration, the fluorescent tumor signal reached its maximum. In addition, the tumor-to-background ratio at all-time points was approximately 1.4. Ex vivo, at 6 hours after Bremachlorin administration, the tumor-to-muscle or -normal pancreas ratio exhibited a greater difference than it did at 24 hours, suggesting that, in terms of efficacy, 6 hours after Bremachlorin administration was an effective time point for PDT treatment of PDAC. In vivo administration of the near infrared fluorescence agent IRDye®-800CW Carboxylate showed that PDT, 6 hours after administration of Bremachlorin, selectively induced necrosis in the tumor tissues, which was subsequently confirmed histologically. In conclusion, by using in vivo fluorescence imaging, we could non-invasively and longitudinally monitor, the whole-body distribution of Bremachlorin. Furthermore, we successfully used IRDye®-800CW Carboxylate, a near-infrared fluorescent necrosis avid agent, to image PDT-induced necrotic cell death as a measure of therapeutic efficacy. This study showed how fluorescence can be applied for optimizing, and assessing the efficacy of, PDT.
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Carcinoma Ductal Pancreático , Indóis , Necrose , Imagem Óptica , Neoplasias Pancreáticas , Fotoquimioterapia , Fármacos Fotossensibilizantes , Animais , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/farmacocinética , Camundongos , Carcinoma Ductal Pancreático/diagnóstico por imagem , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/diagnóstico por imagem , Indóis/química , Distribuição Tecidual , Modelos Animais de Doenças , Linhagem Celular Tumoral , Imagem Corporal Total/métodos , Feminino , Combinação de Medicamentos , PorfirinasRESUMO
[This corrects the article DOI: 10.7150/thno.37949.].
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PURPOSE: Multifocal disease in PTC is associated with an increased recurrence rate. Multifocal disease (MD) is underdiagnosed with the current gold standard of pre-operative ultrasound staging. Here, we evaluate the use of EMI-137 targeted molecular fluorescence-guided imaging (MFGI) and spectroscopy as a tool for the intra-operative detection of uni- and multifocal papillary thyroid cancer (PTC) aiming to improve disease staging and treatment selection. METHODS: A phase-1 study (NCT03470259) with EMI-137 was conducted to evaluate the possibility of detecting PTC using MFGI and quantitative fiber-optic spectroscopy. RESULTS: Fourteen patients underwent hemi- or total thyroidectomy (TTX) after administration of 0.09 mg/kg (n = 1), 0.13 mg/kg (n = 8), or 0.18 mg/kg (n = 5) EMI-137. Both MFGI and spectroscopy could differentiate PTC from healthy thyroid tissue after administration of EMI-137, which binds selectively to MET in PTC. 0.13 mg/kg was the lowest dosage EMI-137 that allowed for differentiation between PTC and healthy thyroid tissue. The smallest PTC focus detected by MFGI was 1.4 mm. MFGI restaged 80% of patients from unifocal to multifocal PTC compared to ultrasound. CONCLUSION: EMI-137-guided MFGI and spectroscopy can be used to detect multifocal PTC. This may improve disease staging and treatment selection between hemi- and total thyroidectomy by better differentiation between unifocal and multifocal disease. TRIAL REGISTRATION: NCT03470259.
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AIMS: The aim of our study was to determine the effect of histone deacetylase (HDAC) inhibitors (HDACis) on somatostatin type-2 receptor (SSTR2) expression and [111In]In-/[177Lu]Lu-DOTA-TATE uptake in vitro and in vivo. MATERIALS AND METHODS: The human cell lines NCI-H69 (small-cell lung carcinoma) and BON-1 (pancreatic neuroendocrine tumor) were treated with HDACis (i.e. entinostat, mocetinostat (MOC), LMK-235, CI-994 or panobinostat (PAN)), and SSTR2 mRNA expression levels and [111In]In-DOTA-TATE uptake were measured. Furthermore, vehicle- and HDACi-treated NCI-H69 and BON-1 tumor-bearing mice were injected with radiolabeled DOTA-TATE followed by biodistribution studies. Additionally, SSTR2 and HDAC mRNA expression of xenografts, and of NCI-H69, BON-1, NCI-H727 (human pulmonary carcinoid) and GOT1 (human midgut neuroendocrine tumor) cells were determined. KEY FINDINGS: HDACi treatment resulted in the desired effects in vitro. However, no significant increase in tumoral DOTA-TATE uptake was observed after HDACi treatment in NCI-H69 tumor-bearing animals, whereas tumoral SSTR2 mRNA and/or protein expression levels were significantly upregulated after treatment with MOC, CI-994 and PAN, i.e. a maximum of 2.1- and 1.3-fold, respectively. Analysis of PAN-treated BON-1 xenografts solely demonstrated increased SSTR2 mRNA expression levels. Comparison of HDACs and SSTR2 expression in BON-1 and NCI-H69 xenografts showed a significantly higher expression of 6/11 HDACs in BON-1 xenografts. Of these HDACs, a significant inverse correlation was found between HDAC3 and SSTR2 expression (Pearson r = -0.92) in the studied cell lines. SIGNIFICANCE: To conclude, tumoral uptake levels of radiolabeled DOTA-TATE were not enhanced after HDACi treatment in vivo, but, depending on the applied inhibitor, increased SSTR2 expression levels were observed.
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Receptores de Somatostatina , Somatostatina , Humanos , Camundongos , Animais , Receptores de Somatostatina/genética , Receptores de Somatostatina/metabolismo , Distribuição Tecidual , Somatostatina/metabolismo , Linhagem Celular Tumoral , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Introduction: The location of T-cells during tumor progression and treatment provides crucial information in predicting the response in vivo. Methods: Here, we investigated, using our bioluminescent, dual color, T-cell reporter mouse, termed TbiLuc, T-cell location and function during murine PDAC tumor growth and checkpoint blockade treatment with anti-PD-1 and anti-CTLA-4. Using this model, we could visualize T-cell location and function in the tumor and the surrounding tumor microenvironment longitudinally. We used murine PDAC clones that formed in vivo tumors with either high T-cell infiltration (immunologically 'hot') or low T-cell infiltration (immunologically 'cold'). Results: Differences in total T-cell bioluminescence could be seen between the 'hot' and 'cold' tumors in the TbiLuc mice. During checkpoint blockade treatment we could see in the tumor-draining lymph nodes an increase in bioluminescence on day 7 after treatment. Conclusions: In the current work, we showed that the TbiLuc mice can be used to monitor T-cell location and function during tumor growth and treatment.
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Neoplasias , Camundongos , Animais , Linfócitos T CD8-Positivos , Testes Imunológicos , Microambiente TumoralRESUMO
The development of new drugs for idiopathic pulmonary fibrosis strongly relies on preclinical experimentation, which requires the continuous improvement of animal models and integration with in vivo imaging data. Here, we investigated the lung distribution of bleomycin (BLM) associated with the indocyanine green (ICG) dye by fluorescence imaging. A long-lasting lung retention (up to 21 days) was observed upon oropharyngeal aspiration (OA) of either ICG or BLM + ICG, with significantly more severe pulmonary fibrosis, accompanied by the progressive appearance of emphysema-like features, uniquely associated with the latter combination. More severe and persistent lung fibrosis, together with a progressive air space enlargement uniquely associated with the BLM + ICG group, was confirmed by longitudinal micro-computed tomography (CT) and histological analyses. Multiple inflammation and fibrosis biomarkers were found to be increased in the bronchoalveolar lavage fluid of BLM- and BLM + ICG-treated animals, but with a clear trend toward a much stronger increase in the latter group. Similarly, in vitro assays performed on macrophage and epithelial cell lines revealed a significantly more marked cytotoxicity in the case of BLM + ICG-treated mice. Also unique to this group was the synergistic upregulation of apoptotic markers both in lung sections and cell lines. Although the exact mechanism underlying the more intense lung fibrosis phenotype with emphysema-like features induced by BLM + ICG remains to be elucidated, we believe that this combination treatment, whose overall effects more closely resemble the human disease, represents a valuable alternative model for studying fibrosis development and for the identification of new antifibrotic compounds.
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Enfisema , Fibrose Pulmonar Idiopática , Enfisema Pulmonar , Humanos , Camundongos , Animais , Bleomicina , Microtomografia por Raio-X , Pulmão/diagnóstico por imagem , Pulmão/patologia , Enfisema Pulmonar/induzido quimicamente , Enfisema Pulmonar/diagnóstico por imagem , Enfisema Pulmonar/patologia , Líquido da Lavagem Broncoalveolar , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/diagnóstico por imagem , Fibrose Pulmonar Idiopática/patologia , Enfisema/patologia , Modelos Animais de Doenças , Camundongos Endogâmicos C57BLRESUMO
Photodynamic therapy (PDT) has a great therapeutic potential because it induces local cellular cytotoxicity upon application of a laser light that excites a photosensitizer, leading to toxic reactive oxygen species. Nevertheless, PDT still is underutilized in the clinic, mostly because of damage induced to normal surrounding tissues. Efforts have been made to improve the specificity. Nanobody-targeted PDT is one of such approaches, in which the variable domain of heavy-chain antibodies, i.e., nanobodies, are used to target photosensitizers selectively to cancer cells. In vitro studies are certainly very valuable to evaluate the therapeutic potential of PDT approaches, but many aspects such as bio-distribution of the photosensitizers, penetration through tissues, and clearance are not taken into account. In vivo studies are therefore essential to assess the influence of such factors, in order to gain more insights into the therapeutic potential of a treatment under development. This chapter describes the development of an orthotopic model of head and neck cancer, to which nanobody-targeted PDT is applied, and the therapeutic potential is assessed by immunohistochemistry one day after PDT.
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Neoplasias de Cabeça e Pescoço , Fotoquimioterapia , Anticorpos de Domínio Único , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Humanos , Luz , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Anticorpos de Domínio Único/farmacologia , Anticorpos de Domínio Único/uso terapêuticoRESUMO
PURPOSE: Patients undergoing prophylactic central compartment dissection (PCLND) for papillary thyroid cancer (PTC) are often overtreated. This study aimed to determine if molecular fluorescence-guided imaging (MFGI) and spectroscopy can be useful for detecting PTC nodal metastases (NM) and to identify negative central compartments intraoperatively. METHODS: We used a data-driven prioritization strategy based on transcriptomic profiles of 97 primary PTCs and 80 normal thyroid tissues (NTT) to identify tumor-specific antigens for a clinically available near-infrared fluorescent tracer. Protein expression of the top prioritized antigen was immunohistochemically validated with a tissue microarray containing primary PTC (n = 741) and NTT (n = 108). Staining intensity was correlated with 10-year locoregional recurrence-free survival (LRFS). A phase 1 study (NCT03470259) with EMI-137, targeting MET, was conducted to evaluate safety, optimal dosage for detecting PTC NM with MFGI, feasibility of NM detection with quantitative fiber-optic spectroscopy, and selective binding of EMI-137 for MET. RESULTS: MET was selected as the most promising antigen. A worse LRFS was observed in patients with positive versus negative MET staining (81.9% versus 93.2%; p = 0.02). In 19 patients, no adverse events related to EMI-137 occurred. 0.13 mg/kg EMI-137 was selected as optimal dosage for differentiating NM from normal lymph nodes using MFGI (p < 0.0001) and spectroscopy (p < 0.0001). MFGI identified 5/19 levels (26.3%) without NM. EMI-137 binds selectively to MET. CONCLUSION: MET is overexpressed in PTC and associated with increased locoregional recurrence rates. Perioperative administration of EMI-137 is safe and facilitates NM detection using MFGI and spectroscopy, potentially reducing the number of negative PCLNDs with more than 25%. CLINICAL TRIAL REGISTRATION: NCT03470259.
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Carcinoma Papilar , Carcinoma , Neoplasias da Glândula Tireoide , Carcinoma/patologia , Carcinoma Papilar/diagnóstico por imagem , Humanos , Linfonodos/patologia , Recidiva Local de Neoplasia/patologia , Análise Espectral , Câncer Papilífero da Tireoide/diagnóstico por imagem , Neoplasias da Glândula Tireoide/patologia , TireoidectomiaRESUMO
PURPOSE: To assess our improved NACA for the detection of tumor necrosis. METHODS: We increased the blood circulation time of our NACA by adding an albumin-binding domain to the molecular structure. We tested the necrosis avidity on dead or alive cultured cells and performed SPECT and fluorescence imaging of both spontaneous and treatment-induced necrosis in murine breast cancer models. We simultaneously recorded [18F]FDG-PET and bioluminescence images for complementary detection of tumor viability. RESULTS: We generated two albumin-binding IRDye800CW derivatives which were labeled with indium-111 with high radiochemical purity. Surprisingly, both albumin-binding NACAs had >10x higher in vitro binding towards dead cells. We selected [111In]3 for in vivo experiments which showed higher dead cell binding in vitro and in vivo stability. The doxorubicin-treated tumors showed increased [111In]3-uptake (1.74 ± 0.08%ID/g after saline treatment, 2.25 ± 0.16%ID/g after doxorubicin treatment, p = 0.044) and decreased [18F]FDG-uptake (3.02 ± 0.51%ID/g after saline treatment, 1.79 ± 0.11%ID/g after doxorubicin treatment, p = 0.040), indicating therapy efficacy. Moreover, we detected increased [111In]3-uptake and tumor necrosis in more rapidly growing EMT6 tumors. CONCLUSIONS: Our albumin-binding NACA based on IRDye800CW facilitates tumor-necrosis imaging for assessment of therapy efficacy and aggressiveness in solid tumors using both fluorescence and SPECT imaging.
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BACKGROUND: There is a growing body of nuclear contrast agents that are repurposed for fluorescence-guided surgery. New contrast agents are obtained by substituting the radioactive tag with, or adding a fluorescent cyanine to the molecular structure of antibodies or peptides. This enables intra-operative fluorescent detection of cancerous tissue, leading to more complete tumor resection. However, these fluorescent cyanines can have a remarkable influence on pharmacokinetics and tumor uptake, especially when labeled to smaller targeting vectors such as peptides. Here we demonstrate the effect of cyanine-mediated dead cell-binding of Ac-Lys0(IRDye800CW)-Tyr3-octreotate (800CW-TATE) and how this can be used as an advantage for fluorescence-guided surgery. RESULTS: Binding of 800CW-TATE could be blocked with DOTA0-Tyr3-octreotate (DOTA-TATE) on cultured SSTR2-positive U2OS cells and was absent in SSTR2 negative U2OS cells. However, strong binding was observed to dead cells, which could not be blocked with DOTA-TATE and was also present in dead SSTR2 negative cells. No SSTR2-mediated binding was observed in frozen tumor sections, possibly due to disruption of the cells in the process of sectioning the tissue before exposure to the contrast agent. DOTA-TATE blocking resulted in an incomplete reduction of 61.5 ± 5.8% fluorescence uptake by NCI-H69-tumors in mice. Near-infrared imaging and dead cell staining on paraffin sections from resected tumors revealed that fluorescence uptake persisted in necrotic regions upon blocking with DOTA-TATE. CONCLUSION: This study shows that labeling peptides with cyanines can result in dead cell binding. This does not hamper the ultimate purpose of fluorescence-guided surgery, as necrotic tissue appears in most solid tumors. Hence, the necrosis binding can increase the overall tumor uptake. Moreover, necrotic tissue should be removed as much as possible: it cannot be salvaged, causes inflammation, and is tumorigenic. However, when performing binding experiments to cells with disrupted membrane integrity, which is routinely done with nuclear probes, this dead cell-binding can resemble non-specific binding. This study will benefit the development of fluorescent contrast agents.
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Fluorescence-guided surgery is an intraoperative optical imaging method that provides surgeons with real-time guidance for the delineation of tumours. Currently, in phase 1 and 2 clinical trials, evaluation of fluorescence-guided surgery is primarily focused on its diagnostic performance, although the corresponding outcome variables do not inform about the added clinical benefit of fluorescence-guided surgery and are challenging to assess objectively. Nonetheless, the effect of fluorescence-guided surgery on intraoperative decision making is the most objective outcome measurement to assess the clinical value of this imaging method. In this Review, we explore the study designs of existing trials of fluorescence-guided surgery that allow us to extract information on potential changes in intraoperative decision making, such as additional or more conservative resections. On the basis of this analysis, we offer recommendations on how to report changes in intraoperative decision making that result from fluorescence imaging, which is of utmost importance for the widespread clinical implementation of fluorescence-guided surgery.
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Tomada de Decisões , Neoplasias/cirurgia , Imagem Óptica/métodos , Cirurgia Assistida por Computador/métodos , Ensaios Clínicos como Assunto , Fluorescência , Humanos , Período Intraoperatório , Projetos de PesquisaRESUMO
Necrosis only occurs in pathological situations and is directly related to disease severity and, therefore, is an important biomarker. Tumor necrosis occurs in most solid tumors due to improperly functioning blood vessels that cannot keep up with the rapid growth, especially in aggressively growing tumors. The amount of necrosis per tumor volume is often correlated to rapid tumor proliferation and can be used as a diagnostic tool. Furthermore, efficient therapy against solid tumors will directly or indirectly lead to necrotic tumor cells, and detection of increased tumor necrosis can be an early marker for therapy efficacy. We propose the application of necrosis avid contrast agents to detect therapy-induced tumor necrosis. Herein, we advance gallium-68-labeled IRDye800CW, a near-infrared fluorescent dye that exhibits excellent necrosis avidity, as a potential PET tracer for in vivo imaging of tumor necrosis. We developed a reliable labeling procedure to prepare [68Ga]Ga-DOTA-PEG4-IRDye800CW ([68Ga]Ga-1) with a radiochemical purity of >96% (radio-HPLC). The prominent dead cell binding of fluorescence and radioactivity from [68Ga]Ga-1 was confirmed with dead and alive cultured 4T1-Luc2 cells. [68Ga]Ga-1 was injected in 4T1-Luc2 tumor-bearing mice, and specific fluorescence and PET signal were observed in the spontaneously developing tumor necrosis. The ip injection of D-luciferin enabled simultaneous bioluminescence imaging of the viable tumor regions. Tumor necrosis binding was confirmed ex vivo by colocalization of fluorescence uptake with TUNEL dead cell staining and radioactivity uptake in dichotomized tumors and frozen tumor sections. Our presented study shows that [68Ga]Ga-1 is a promising PET tracer for the detection of tumor necrosis.
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Meios de Contraste , Radioisótopos de Gálio , Animais , Linhagem Celular Tumoral , Camundongos , Necrose/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Compostos RadiofarmacêuticosRESUMO
Tissue acidosis plays a pivotal role in tumor progression: in particular, interstitial acidosis promotes tumor cell invasion, and is a major contributor to the dysregulation of tumor immunity and tumor stromal cells. The cell membrane and integral membrane proteins commonly act as important sensors and transducers of altered pH. Cell adhesion molecules and cation channels are prominent membrane proteins, the majority of which is regulated by protons. The pathophysiological consequences of proton-sensitive ion channel function in cancer, however, are scarcely considered in the literature. Thus, the main focus of this review is to highlight possible events in tumor progression and tumor immunity where the pH sensitivity of cation channels could be of great importance.
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PURPOSE: Current clinical measurements for tumor treatment efficiency rely often on changes in tumor volume measured as shrinkage by CT or MRI, which become apparent after multiple lines of treatment and pose a physical and psychological burden on the patient. Detection of therapy-induced cell death in the tumor can be a fast measure for treatment efficiency. However, there are no reliable clinical tools for detection of tumor necrosis. Previously, we studied the necrosis avidity of cyanine-based fluorescent dyes, which suffered long circulation times before tumor necrosis could be imaged due to low hydrophilicity. We now present the application of radiolabeled 800CW, a commercially available cyanine with high hydrophilicity, to image tumor necrosis in a mouse model. PROCEDURES: We conjugated 800CW to DOTA via a PEG linker, for labeling with single-photon emission-computed tomography isotope indium-111, yielding [111In]In-DOTA-PEG4-800CW. We then investigated specific [111In]In-DOTA-PEG4-800CW uptake by dead cells in vitro, using both fluorescence and radioactivity as detection modalities. Finally, we investigated [111In]In-DOTA-PEG4-800CW uptake into necrotic tumor regions of a 4T1 breast tumor model in mice. RESULTS: We successfully prepared a precursor and developed a reliable procedure for labeling 800CW with indium-111. We detected specific [111In]In-DOTA-PEG4-800CW uptake by dead cells, using both fluorescence and radioactivity. Albeit with a tumor uptake of only 0.37%ID/g at 6 h post injection, we were able to image tumor necrosis with a tumor to background ratio of 7:4. Fluorescence and radioactivity in cryosections from the dissected tumors were colocalized with tumor necrosis, confirmed by TUNEL staining. CONCLUSIONS: [111In]In-DOTA-PEG4-800CW can be used to image tumor necrosis in vitro and in vivo. Further research will elucidate the application of [111In]In-DOTA-PEG4-800CW or other radiolabeled hydrophilic cyanines for the detection of necrosis caused by chemotherapy or other anti-cancer therapies. This can provide valuable prognostic information in treatment of solid tumors.
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Meios de Contraste/química , Radioisótopos de Índio/química , Indóis/química , Coloração e Rotulagem , Animais , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Compostos Heterocíclicos com 1 Anel/síntese química , Compostos Heterocíclicos com 1 Anel/química , Indóis/síntese química , Camundongos Endogâmicos BALB C , Camundongos Nus , Necrose , Polietilenoglicóis/síntese química , Polietilenoglicóis/química , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/química , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
RATIONALE: A substantial number of breast cancer patients with an overexpression of the human epidermal growth factor receptor 2 (HER2) have residual disease after neoadjuvant therapy or become resistant to trastuzumab. Photodynamic therapy (PDT) using nanobodies targeted to HER2 is a promising treatment option for these patients. Here we investigate the in vitro and in vivo antitumor efficacy of HER2-targeted nanobody-photosensitizer (PS) conjugate PDT. METHODS: Nanobodies targeting HER2 were obtained from phage display selections. Monovalent nanobodies were engineered into a biparatopic construct. The specificity of selected nanobodies was tested in immunofluorescence assays and their affinity was evaluated in binding studies, both performed in a panel of breast cancer cells varying in HER2 expression levels. The selected HER2-targeted nanobodies 1D5 and 1D5-18A12 were conjugated to the photosensitizer IRDye700DX and tested in in vitro PDT assays. Mice bearing orthotopic HCC1954 trastuzumab-resistant tumors with high HER2 expression or MCF-7 tumors with low HER2 expression were intravenously injected with nanobody-PS conjugates. Quantitative fluorescence spectroscopy was performed for the determination of the local pharmacokinetics of the fluorescence conjugates. After nanobody-PS administration, tumors were illuminated to a fluence of 100 Jâcm-2, with a fluence rate of 50 mWâcm-2, and thereafter tumor growth was measured with a follow-up until 30 days. RESULTS: The selected nanobodies remained functional after conjugation to the PS, binding specifically and with high affinity to HER2-positive cells. Both nanobody-PS conjugates potently and selectively induced cell death of HER2 overexpressing cells, either sensitive or resistant to trastuzumab, with low nanomolar LD50 values. In vivo, quantitative fluorescence spectroscopy showed specific accumulation of nanobody-PS conjugates in HCC1954 tumors and indicated 2 h post injection as the most suitable time point to apply light. Nanobody-targeted PDT with 1D5-PS and 1D5-18A12-PS induced significant tumor regression of trastuzumab-resistant high HER2 expressing tumors, whereas in low HER2 expressing tumors only a slight growth delay was observed. CONCLUSION: Nanobody-PS conjugates accumulated selectively in vivo and their fluorescence could be detected through optical imaging. Upon illumination, they selectively induced significant tumor regression of HER2 overexpressing tumors with a single treatment session. Nanobody-targeted PDT is therefore suggested as a new additional treatment for HER2-positive breast cancer, particularly of interest for trastuzumab-resistant HER2-positive breast cancer. Further studies are now needed to assess the value of this approach in clinical practice.
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Neoplasias da Mama , Fotoquimioterapia , Anticorpos de Domínio Único , Animais , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Fármacos Fotossensibilizantes/uso terapêutico , Receptor ErbB-2 , Trastuzumab , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Targeted photodynamic therapy (PDT) has the potential to selectively damage tumor tissue and to increase tumor vessel permeability. Here we characterize the tissue biodistribution of two EGFR-targeted nanobody-photosensitizer conjugates (NB-PS), the monovalent 7D12-PS and the biparatopic 7D12-9G8-PS. In addition, we report on the local and acute phototoxic effects triggered by illumination of these NB-PS which have previously shown to lead to extensive tumor damage. Methods: Intravital microscopy and the skin-fold chamber model, containing OSC-19-luc2-cGFP tumors, were used to investigate: a) the fluorescence kinetics and distribution, b) the vascular response and c) the induction of necrosis after illumination at 1 or 24 h post administration of 7D12-PS and 7D12-9G8-PS. In addition, dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) of a solid tumor model was used to investigate the microvascular status 2 h after 7D12-PS mediated PDT. Results: Image analysis showed significant tumor colocalization for both NB-PS which was higher for 7D12-9G8-PS. Intravital imaging showed clear tumor cell membrane localization 1 and 2 h after administration of 7D12-9G8-PS, and fluorescence in or close to endothelial cells in normal tissue for both NB-PS. PDT lead to vasoconstriction and leakage of tumor and normal tissue vessels in the skin-fold chamber model. DCE-MRI confirmed the reduction of tumor perfusion after 7D12-PS mediated PDT. PDT induced extensive tumor necrosis and moderate normal tissue damage, which was similar for both NB-PS conjugates. This was significantly reduced when illumination was performed at 24 h compared to 1 h after administration. Discussion: Although differences were observed in distribution of the two NB-PS conjugates, both led to similar necrosis. Clearly, the response to PDT using NB-PS conjugates is the result of a complex mixture of tumor cell responses and vascular effects, which is likely to be necessary for a maximally effective treatment.
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Receptores ErbB/metabolismo , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Nanopartículas/química , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Células Endoteliais/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Microscopia Intravital/métodos , Imageamento por Ressonância Magnética/métodos , Camundongos , Imagem Óptica/métodos , Fármacos Fotossensibilizantes/química , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Distribuição Tecidual/efeitos dos fármacosRESUMO
Stem-cell-based therapy may be used to replace damaged or lost neurons in the cochlear nerve of patients suffering from severe-to-profound sensorineural hearing loss. In order to achieve functional recovery in future clinical trials, knowledge about survival of grafted cells and their differentiation into functional neurons is a prerequisite. This calls for non-invasive in vivo visualization of cells and long-term monitoring of their survival and fate after cochlear transplantation. We have investigated if molecular optical imaging enables visualization of exogenous cells in the intact cochlea of guinea pig cadaver heads. Transduced (stem) cells, stably co-expressing fluorescent (copGFP) and bioluminescent (Luc2) reporter molecules, were injected into the internal auditory meatus or directly into the cochlea through the round window. After injection of the cells into the internal auditory meatus, a bright bioluminescent signal was observed in the cavum conchae of the auricle, indicating that light generated by Luc2 is passing through the tympanic membrane and the external auditory meatus. Similar results were obtained after injection of the cells through the round window membrane, either directly into the scala tympani or in Rosenthal's canal within the modiolus of the basal cochlear turn. Imaging of the auditory bulla demonstrated that the bioluminescent signal passes through the tympanic membrane and crevices in the bony wall of the bulla. After opening the auditory bulla, the bioluminescent signal was emanating from the round window. This is the first study demonstrating that bioluminescence imaging enables visualization of luciferase-expressing cells injected into the intact guinea pig cochlea. Anat Rec, 303:427-440, 2020. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.
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Cóclea/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular/fisiologia , Cobaias , Medições Luminescentes , Transplante de Células-TroncoAssuntos
Transplante de Fígado/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Preservação de Órgãos/métodos , Perfusão/métodos , Aloenxertos/metabolismo , Animais , Feminino , Humanos , Interleucina-6/análise , Interleucina-6/metabolismo , Interleucina-8/análise , Interleucina-8/metabolismo , Fígado/metabolismo , Células-Tronco Mesenquimais/metabolismo , Modelos Animais , Comunicação Parácrina , Sus scrofa , Transplante Heterólogo/métodosRESUMO
Chemoimmunotherapy is an emerging combinatorial modality for the treatment of cancers resistant to common first-line therapies, such as chemotherapy and checkpoint blockade immunotherapy. We used biodegradable nanoparticles as delivery vehicles for local, slow and sustained release of doxorubicin, two immune adjuvants and one chemokine for the treatment of resistant solid tumors. Methods: Bio-compatible poly(lactic-co-glycolic acid)-PEG nanoparticles were synthesized in an oil/water emulsion, using a solvent evaporation-extraction method. The nanoparticles were loaded with a NIR-dye for theranostic purposes, doxorubicin cytostatic agent, poly (I:C) and R848 immune adjuvants and CCL20 chemokine. After physicochemical and in vitro characterization the nanoparticles therapeutic efficacy were carried-out on established, highly aggressive and treatment resistant TC-1 lung carcinoma and MC-38 colon adenocarcinoma models in vivo. Results: The yielded nanoparticles average size was 180 nm and -14 mV surface charge. The combined treatment with all compounds was significantly superior than separate compounds and the compounds nanoparticle encapsulation was required for effective tumor control in vivo. The mechanistic studies confirmed strong induction of circulating cancer specific T cells upon combined treatment in blood. Analysis of the tumor microenvironment revealed a significant increase of infiltrating leukocytes upon treatment. Conclusion: The multi-drug loaded nanoparticles mediated delivery of chemoimmunotherapy exhibited excellent therapeutic efficacy gain on two treatment resistant cancer models and is a potent candidate strategy to improve cancer therapy of solid tumors resistant to first-line therapies.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Imunoterapia/métodos , Nanopartículas/administração & dosagem , Adenocarcinoma/tratamento farmacológico , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/farmacocinética , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linhagem Celular Tumoral , Quimiocina CCL20/administração & dosagem , Quimiotaxia/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Doxorrubicina/farmacocinética , Feminino , Imidazóis/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Camundongos Endogâmicos C57BL , Nanopartículas/química , Tamanho da Partícula , Polietilenoglicóis/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Microambiente Tumoral/efeitos dos fármacosRESUMO
Cell death is a natural process for the turnover of aged cells, but it can also arise as a result of pathological conditions. Cell death is recognized as a key feature in both acute and chronic hepatobiliary diseases caused by drug, alcohol, and fat uptake; by viral infection; or after surgical intervention. In the case of chronic disease, cell death can lead to (chronic) secondary inflammation, cirrhosis, and the progression to liver cancer. In liver transplantation, graft preservation and ischemia/reperfusion injury are associated with acute cell death. In both cases, so-called programmed cell death modalities are involved. Several distinct types of programmed cell death have been described of which apoptosis and necroptosis are the most well known. Parenchymal liver cells, including hepatocytes and cholangiocytes, are susceptible to both apoptosis and necroptosis, which are triggered by distinct signal transduction pathways. Apoptosis is dependent on a proteolytic cascade of caspase enzymes, whereas necroptosis induction is caspase-independent. Moreover, different from the "silent" apoptotic cell death, necroptosis can cause a secondary inflammatory cascade, so-called necroinflammation, triggered by the release of various damage-associated molecular patterns (DAMPs). These DAMPs activate the innate immune system, leading to both local and systemic inflammatory responses, which can even cause remote organ failure. Therapeutic targeting of necroptosis by pharmacological inhibitors, such as necrostatin-1, shows variable effects in different disease models.