RESUMO
Thermal stability and iron saturation of lactoferrin (LF) are of great significance not only for the evaluation of the biological activities of LF but also for the optimization of the isolation and drying process parameters. Differential scanning calorimetry (DSC) is a well-established and efficient method for thermal stability and iron saturation detection in LF. However, multiple DSC measurements are typically performed sequentially, thus time-consuming and low throughput. Herein, we introduced the differential scanning fluorimetry (DSF) approach to overcome such limitations. The DSF can monitor LF thermal unfolding with a commonly available real-time PCR instrument and a fluorescent dye (SYPRO orange or Glomelt), and the measured melting temperature of LF is consistent with that determined by DSC. On the basis of that, a new quantification method was established for determination of iron saturation levels using the linear correlation of the degree of ion saturation of LF with DSF measurements. Such DSF method is simple, inexpensive, rapid (<15 min), and high throughput (>96 samples per experiment), and provides a valuable alternative tool for thermal stability detection of LF and other whey proteins.
Assuntos
Fluorometria , Ferro , Lactoferrina , Estabilidade Proteica , Lactoferrina/química , Lactoferrina/análise , Ferro/química , Fluorometria/métodos , Varredura Diferencial de Calorimetria/métodos , Temperatura , Ensaios de Triagem em Larga Escala/métodosRESUMO
BACKGROUND: Immune checkpoint inhibitors (ICIs) shed new light on triple-negative breast cancer (TNBC), but only a minority of patients demonstrate response. Therefore, adaptive immune resistance (AIR) needs to be further defined to guide the development of ICI regimens. METHODS: Databases, including The Cancer Genome Atlas, Gene Ontology Resource, University of California Santa Cruz Genome Browser, and Pubmed, were used to screen epigenetic modulators, regulators for CD8+ T cells, and transcriptional regulators of programmed cell death-ligand 1 (PD-L1). Human peripheral blood mononuclear cell (Hu-PBMC) reconstruction mice were adopted for xenograft transplantation. Tumor specimens from a TNBC cohort and the clinical trial CTR20191353 were retrospectively analyzed. RNA-sequencing, Western blotting, qPCR and immunohistochemistry were used to assess gene expression. Coculture assays were performed to evaluate the regulation of TNBC cells on T cells. Chromatin immunoprecipitation and transposase-accessible chromatin sequencing were used to determine chromatin-binding and accessibility. RESULTS: The epigenetic modulator AT-rich interaction domain 1A (ARID1A) gene demonstrated the highest expression association with AIR relative to other epigenetic modulators in TNBC patients. Low ARID1A expression in TNBC, causing an immunosuppressive microenvironment, promoted AIR and inhibited CD8+ T cell infiltration and activity through upregulating PD-L1. However, ARID1A did not directly regulate PD-L1 expression. We found that ARID1A directly bound the promoter of nucleophosmin 1 (NPM1) and that low ARID1A expression increased NPM1 chromatin accessibility as well as gene expression, further activating PD-L1 transcription. In Hu-PBMC mice, atezolizumab demonstrated the potential to reverse ARID1A deficiency-induced AIR in TNBC by reducing tumor malignancy and activating anti-tumor immunity. In CTR20191353, ARID1A-low patients derived more benefit from pucotenlimab compared to ARID1A-high patients. CONCLUSIONS: In AIR epigenetics, low ARID1A expression in TNBC contributed to AIR via the ARID1A/NPM1/PD-L1 axis, leading to poor outcome but sensitivity to ICI treatment.