Molecular Imaging of Melanoma VEGF-expressing Tumors through [99mTc]Tc-HYNIC-Fab(Bevacizumab).

BACKGROUND: Angiogenesis is a process that many tumors depend on for growth, development, and metastasis. Vascular endothelial growth factor (VEGF) is one of the major players in tumor angiogenesis in several tumor types, including melanoma. VEGF inhibition is achieved by bevacizumab, a humanized monoclonal antibody that binds with high affinity to VEGF and prevents its function. In order to successfully enable in vivo VEGF expression imaging in a murine melanoma model, we previously labeled bevacizumab with [99mTc]Tc. We observed that this was feasible, but it had prolonged blood circulation and delayed tumor uptake. OBJECTIVE: The aim of this study was to develop a radiolabeled Fab bevacizumab fragment, [99mTc]Tc-HYNICFab( bevacizumab), for non-invasive in vivo VEGF expression molecular imaging. METHODS: Flow cytometry was used to examine VEGF presence in the murine melanoma cell line (B16-F10). Bevacizumab was digested with papain for six hours at 37°C to produce Fab(bevacizumab), which was then conjugated to NHS-HYNIC-Tfa for radiolabeling with [99mTc]Tc. Stability and binding affinity assays were also evaluated. Biodistribution and single photon emission computed tomography/computed tomography (SPECT/CT) were performed at 1, 3, and 6 h (n = 4) after injection of [99mTc]Tc-HYNIC-Fab(Bevacizumab) in normal and B16-F10 tumor-bearing C57Bl/6J mice. RESULTS: Using flow cytometry, it was shown that the B16-F10 murine melanoma cell line has intracellular VEGF expression. Papain incubation resulted in the complete digestion of bevacizumab with good purity and homogeneity. The radiolabeling yield of [99mTc]Tc-HYNIC-Fab(bevacizumab) was 85.00 ± 6.06%, with a specific activity of 291.87 ± 18.84 MBq/mg (n=3), showing in vitro stability. Binding assays demonstrated significant intracellular in vitro VEGF expression. Fast blood clearance and high kidney and tumor uptake were observed in biodistribution and SPECT/CT studies. CONCLUSIONS: We present the development and evaluation of [99mTc]Tc-HYNIC-Fab(bevacizumab), a novel molecular VEGF expression imaging agent that may be used for precision medicine in melanoma and potentially in other VEGF-expressing tumors.

Enhanced Tumor Targeting of Radiolabeled Mouse/Human Chimeric Anti-Tn Antibody in Losartan-Treated Mice Bearing Tn-Expressing Lung Tumors.

Aim: ChiTn, a mouse/human chimeric anti-Tn monoclonal antibody, was radiolabeled with iodine-131 (131I) and technetium-99m (99mTc) to assess its biodistribution and internalization in Tn-expressing (Tn+) and wild-type (Tn-) LL/2 lung cancer cells. Results: Selective accumulation and gradual internalization of ChiTn were observed in Tn+ cells. Biodistribution in mice with both Tn+ or Tn- lung tumors indicated that the uptake of radiolabeled ChiTn within tumors increased over time. Dual-labeling experiments with 99mTc and 131I showed different biodistribution patterns, with 99mTc exhibiting higher values in the liver, spleen, and kidneys, while 131I showed higher uptake in the thyroid and stomach. However, tumor uptake did not significantly differ between Tn+ and Tn- tumors. To improve tumor targeting, Losartan, an antihypertensive drug known to enhance tumor perfusion and drug delivery, was investigated. Biodistribution studies in Losartan-treated mice revealed significantly higher radiolabeled ChiTn uptake in Tn+ tumors. No significant changes were observed in the uptake of the control molecule IgG-HYNIC™99mTc. Conclusions: These findings demonstrate the enhanced tumor targeting of radiolabeled ChiTn in Losartan-treated mice with Tn-expressing lung tumors. They highlight the potential of ChiTn as a theranostic agent for cancer treatment and emphasize the importance of Losartan as an adjunctive treatment to improve tumor perfusion and drug delivery.

Mitofusin 1 silencing decreases the senescent associated secretory phenotype, promotes immune cell recruitment and delays melanoma tumor growth after chemotherapy.

Cellular senescence is a therapy endpoint in melanoma, and the senescence-associated secretory phenotype (SASP) can affect tumor growth and microenvironment, influencing treatment outcomes. Metabolic interventions can modulate the SASP, and mitochondrial energy metabolism supports resistance to therapy in melanoma. In a previous report we showed that senescence, induced by the DNA methylating agent temozolomide, increased the level of fusion proteins mitofusin 1 and 2 in melanoma, and silencing Mfn1 or Mfn2 expression reduced interleukin-6 secretion by senescent cells. Here we expanded these observations evaluating the secretome of senescent melanoma cells using shotgun proteomics, and explored the impact of silencing Mfn1 on the SASP. A significant increase in proteins reported to reduce the immune response towards the tumor was found in the media of senescent cells. The secretion of several of these immunomodulatory proteins was affected by Mfn1 silencing, among them was galectin-9. In agreement, tumors lacking mitofusin 1 responded better to treatment with the methylating agent dacarbazine, tumor size was reduced and a higher immune cell infiltration was detected in the tumor. Our results highlight mitochondrial dynamic proteins as potential pharmacological targets to modulate the SASP in the context of melanoma treatment.

99mTc-HYNIC/Cy7-Fab(Bevacizumab): su empleo como agente de imagen en mieloma múltiple / 99mTc-HYNIC/Cy7-Fab(Bevacizumab): its use as an imaging agent in Multiple Myeloma / 99mTc-HYNIC/Cy7-Fab (Bevacizumab): seu uso como agente de imagem em mieloma múltiplo

Introducción: el mieloma múltiple es un trastorno hematológico maligno y el segundo cáncer de la sangre más frecuente. El proceso de la angiogénesis tumoral es fundamental para el crecimiento y metástasis de muchos tipos de tumores, incluido en mieloma múltiple. Se sabe que la sobreexpresión del factor de crecimiento endothelial vascular se encuentra asociado a un mal pronóstico en esta patología, representando un blanco clave para la terapia anti-angiogénica en mieloma múltiple. El anticuerpo monoclonal Bevacizumab es capaz de unirse con gran afinidad al factor de crecimiento endothelial vascular bloqueando su acción. Objetivo: evaluar el Fab(Bevacizumab) marcado con 99mTc o Cy7 como potenciales agentes de imagen moleculares de la expresión de factor de crecimiento endothelial vascular en mieloma múltiple. Material y métodos: la expresión de factor de crecimiento endothelial vascular fue analizada mediante citometría de flujo en la línea celular huaman de mieloma múltiple, la MM1S. Fab(Bevacizumab) fue producido mediante digestión de Bevacizumab con papaína, conjugado a NHS-HYNIC-Tfa y radiomarcado con 99mTc. Se realizaron estudios de biodistribución y de tomografía computarizada por emisión del fotón simple. A su vez, Fab(Bevacizumab) fue marcado con Cy7 para obtener imágenes de fluorescencia in vivo hasta 96 horas. Resultados: el análisis por citometría de flujo en la línea celular MM1S reveló que la expresión de factor de crecimiento endothelial vascular es predominantemente intracelular. Los estudios de biodistribución y SPECT/CT del complejo 99mTc-HYNIC-Fab(Bevacizumab) mostraron una rápida eliminación sanguínea y una significativa captación a nivel renal y tumoral. Las imágenes por fluorescencia empleando Cy7-Fab(Bevacizumab) permitieron la visualización tumoral hasta 96 h p.i. Conclusiones: logramos visualizar la expresión de factor de crecimiento endothelial vascular in vivo en mieloma múltiple mediante el empleo del fragmento Fab del anticuerpo anti-VEGF (Bevacizumab) marcado con 99mTc y Cy7. Estos nuevos agentes de imagen molecular podrían ser empleados potencialmente en el ámbito clínico para la estadificación y el seguimiento de pacientes con mieloma múltiple, mediante la visualización radioactiva in vivo de la expresión de factor de crecimiento endothelial vascular en todo el cuerpo. La imagen óptica de estos trazadores mejoraría el muestreo tumoral y podría guiar la extirpación quirúrgica.

Introduction: Multiple myeloma is a hematologic malignancy and the second most common blood cancer. The process of tumor angiogenesis is central to the growth and metastasis of many types of tumors, including multiple myeloma. Overexpression of vascular endothelial growth factor is known to be associated with poor prognosis in this pathology, representing a key target for anti-angiogenic therapy in multiple myeloma. The monoclonal antibody Bevacizumab is able to bind with high affinity to vascular endothelial growth factor blocking its action. Objective: to evaluate 99mTc- or Cy7-labeled Fab(Bevacizumab) as potential molecular imaging agents of vascular endothelial growth factor expression in multiple myeloma. Methods: Vascular endothelial growth factor expression was analyzed by flow cytometry in the multiple myeloma huaman cell line, MM1S. Fab(Bevacizumab) was produced by digestion of Bevacizumab with papain, conjugated to NHS-HYNIC-Tfa and radiolabeled with 99mTc. Biodistribution and single photon emission computed tomography studies were performed. In turn, Fab(Bevacizumab) was labeled with Cy7 to obtain in vivo fluorescence images up to 96 hours. Results: Flow cytometry analysis in the MM1S cell line revealed that vascular endothelial growth factor expression is predominantly intracellular. Biodistribution and SPECT/CT studies of the 99mTc-HYNIC-Fab(Bevacizumab) complex showed rapid blood clearance and significant renal and tumor uptake. Fluorescence imaging using Cy7-Fab(Bevacizumab) allowed tumor visualization up to 96 h p.i. Conclusions: we were able to visualize vascular endothelial growth factor expression in vivo in multiple myeloma using the Fab fragment of the anti-VEGF antibody (Bevacizumab) labeled with 99mTc and Cy7. These new molecular imaging agents could potentially be employed in the clinical setting for staging and monitoring of patients with multiple myeloma by in vivo radioactive visualization of vascular endothelial growth factor expression throughout the body. Optical imaging of these tracers would improve tumor sampling and could guide surgical excision.

Introdução: O mieloma múltiplo é uma malignidade hematológica e o segundo câncer de sangue mais comum. O processo de angiogênese tumoral é fundamental para o crescimento e a metástase de muitos tipos de tumores, incluindo o mieloma múltiplo. Sabe-se que a superexpressão do fator de crescimento endotelial vascular está associada a um prognóstico ruim no mieloma múltiplo, representando um alvo importante para a terapia antiangiogênica no mieloma múltiplo. O anticorpo monoclonal Bevacizumab é capaz de se ligar com alta afinidade ao fator de crescimento endotelial vascular e bloquear sua ação. Objetivo: avaliar o Fab(Bevacizumab) marcado com 99mTc ou Cy7 como possíveis agentes de imagem molecular da expressão do fator de crescimento endotelial vascular no mieloma múltiplo. Métodos: A expressão do fator de crescimento endotelial vascular foi analisada por citometria de fluxo na linha celular de mieloma múltiplo MM1S. O Fab(Bevacizumab) foi produzido pela digestão do Bevacizumab com papaína, conjugado com NHS-HYNIC-Tfa e radiomarcado com 99mTc. Foram realizados estudos de biodistribuição e tomografia computadorizada por emissão de fóton único. Por sua vez, o Fab(Bevacizumab) foi marcado com Cy7 para geração de imagens de fluorescência in vivo por até 96 horas. Resultados: A análise de citometria de fluxo na linha celular MM1S revelou que a expressão do fator de crescimento endotelial vascular é predominantemente intracelular. Os estudos de biodistribuição e SPECT/CT do complexo 99mTc-HYNIC-Fab(Bevacizumab) mostraram uma rápida depuração sanguínea e uma captação renal e tumoral significativa. A imagem de fluorescência usando Cy7-Fab(Bevacizumab) permitiu a visualização do tumor até 96 horas p.i. Conclusões: Conseguimos visualizar a expressão do fator de crescimento endotelial vascular in vivo no mieloma múltiplo usando o fragmento Fab do anticorpo anti-VEGF (Bevacizumab) marcado com 99mTc e Cy7. Esses novos agentes de imagem molecular poderiam ser usados no cenário clínico para o estadiamento e o monitoramento de pacientes com mieloma múltiplo, visualizando radioativamente a expressão do fator de crescimento endotelial vascular in vivo em todo o corpo. A geração de imagens ópticas desses traçadores melhoraria a amostragem do tumor e poderia orientar a excisão cirúrgica.

99mTechnetium- or Cy7-Labeled Fab(Tocilizumab) as Potential Multiple Myeloma Imaging Agents.

BACKGROUND: Multiple Myeloma (MM) is a malignant hematologic disorder and the second most common blood cancer. Interleukin-6 (IL-6) has been identified as a crucial factor for the proliferation and survival of MM cells and the overexpression of IL-6 receptor is being studied as a molecular target for therapeutic and diagnostic use in myelomas and other comorbidities. Tocilizumab is a humanized monoclonal antibody that binds IL-6R. OBJECTIVE: We aim to label and evaluate Fab(Tocilizumab) with 99mTechnetium or Cy7 as potential MM imaging agents. METHODS: IL-6R distribution was analyzed by Laser Confocal Microscopy (LCM) in MM cell lines. Fab(Tocilizumab) was produced by the digestion of Tocilizumab with papain for 24h at 37°C, derivatized with NHS-HYNIC-Tfa and radiolabeled with 99mTc. Radiochemical stability and in vitro cell assays were evaluated. Biodistribution and SPECT/CT were performed. Also, Fab(Tocilizumab) was labeled with Cy7 for in vivo fluorescence imaging up to 72h. RESULTS: LCM analysis demonstrates IL-6R distribution on MM cell lines. Incubation with papain resulted in complete digestion of Tocilizumab and exhibited a good purity and homogeneity. Radiolabeling with 99mTc via NHS-HYNIC-Tfa was found to be fast, easy, reproducible and stable, revealing high radiochemical purity and without interfering with IL-6R recognition. Biodistribution and SPECT/CT studies showed a quick blood clearance and significant kidney and MM engrafted tumor uptake. Cy7-Fab(Tocilizumab) fluorescent imaging allowed MM1S tumor identification up to 72h p.i. CONCLUSION: These new molecular imaging agents could potentially be used in the clinical setting for staging and follow-up of MM through radioactive whole-body IL-6R expression visualization in vivo. The fluorescent version could be used for tissue sample evaluation and to guide surgical excision, if necessary.

Alterações ambientais independentes da resposta e sua interação com o relato verbal / Response-independent environmental changes and their interaction with verbal reports

Investigou-se os efeitos da exposição a eventos aversivos incontroláveis ou controláveis sobre o desempenho posterior a tal exposição e se estes efeitos seriam alterados pela solicitação de relatos do desempenho. Participaram 40 adultos distribuídos em 3 grupos experimentais: Controle, Fuga e Emparelhado. Na Fase 1 os participantes do Grupo Fuga e Emparelhado foram submetidos a 40 tentativas de apresentação de um som por até 5s: os participantes dos grupos Fuga podiam desligar os sons pressionando teclas no computador e os participantes do grupo Emparelhados foram submetidos à mesma distribuição e duração de sons de um participante dos grupo Fuga, mas não podiam desligar os sons. Os participantes também foram solicitados a fazer relatos sobre a tarefa e seu desempenho, variando-se o número de solicitações e as tentativas em que ocorreram. Todos os participantes passaram pela Fase 2 e todos podiam desligar o som clicando o mouse sobre ícones na tela do computador. Os resultados apontaram que em geral os participantes não tiveram desempenhos caracterizados como comportamento supersticioso ou desamparo aprendido e que padrões de respostas na Fase 1 estão relacionados com os desempenhos na Fase 2. Quanto ao relato, dizer que sabiam o que fazer não foi preditivo de sucesso na Fase 2, mas sucessivas solicitações de relato parecem ter promovido descrição, pelo participante, de seu comportamento e de auto-observação(AU)

The present study investigated the effects of exposure to both controllable and uncontrollable aversive events on the performances of adults. A second goal was to evaluate the effects of requests for verbal reports on participants’ performances. Forty adults were assigned to 3 groups: participants of the Escape Group were exposed to a Training Condition where an aversive sound could be turned off by their responses. Participants of the Yoked Group were exposed to the same Training Condition, but no responses were effective in turning off the sound. Participants of all Groups were exposed to a Test Condition where a different response turned off the sound. Participants of the Escape and Yoked Groups were assigned to 1 of 3 verbal report conditions where they were asked if they knew how to turn off the sound in given trials. Results did not suggest effects associated with helplessness or superstitious behavior, but specific patterns of responding to the Training Condition were closely related to participants’ performances in the Test Condition. Results showed that the verbal reports did not contribute to the emergence or prevention of helplessness(AU)

Alterações ambientais independentes da resposta e sua interação com o relato verbal / Response-independent environmental changes and their interaction with verbal reports

Investigou-se os efeitos da exposição a eventos aversivos incontroláveis ou controláveis sobre o desempenho posterior a tal exposição e se estes efeitos seriam alterados pela solicitação de relatos do desempenho. Participaram 40 adultos distribuídos em 3 grupos experimentais: Controle, Fuga e Emparelhado. Na Fase 1 os participantes do Grupo Fuga e Emparelhado foram submetidos a 40 tentativas de apresentação de um som por até 5s: os participantes dos grupos Fuga podiam desligar os sons pressionando teclas no computador e os participantes do grupo Emparelhados foram submetidos à mesma distribuição e duração de sons de um participante dos grupo Fuga, mas não podiam desligar os sons. Os participantes também foram solicitados a fazer relatos sobre a tarefa e seu desempenho, variando-se o número de solicitações e as tentativas em que ocorreram. Todos os participantes passaram pela Fase 2 e todos podiam desligar o som clicando o mouse sobre ícones na tela do computador. Os resultados apontaram que em geral os participantes não tiveram desempenhos caracterizados como comportamento supersticioso ou desamparo aprendido e que padrões de respostas na Fase 1 estão relacionados com os desempenhos na Fase 2. Quanto ao relato, dizer que sabiam o que fazer não foi preditivo de sucesso na Fase 2, mas sucessivas solicitações de relato parecem ter promovido descrição, pelo participante, de seu comportamento e de auto-observação.

The present study investigated the effects of exposure to both controllable and uncontrollable aversive events on the performances of adults. A second goal was to evaluate the effects of requests for verbal reports on participants’ performances. Forty adults were assigned to 3 groups: participants of the Escape Group were exposed to a Training Condition where an aversive sound could be turned off by their responses. Participants of the Yoked Group were exposed to the same Training Condition, but no responses were effective in turning off the sound. Participants of all Groups were exposed to a Test Condition where a different response turned off the sound. Participants of the Escape and Yoked Groups were assigned to 1 of 3 verbal report conditions where they were asked if they knew how to turn off the sound in given trials. Results did not suggest effects associated with helplessness or superstitious behavior, but specific patterns of responding to the Training Condition were closely related to participants’ performances in the Test Condition. Results showed that the verbal reports did not contribute to the emergence or prevention of helplessness.