Previously, we found that dysfunctional natural killer (NK) cells with low interferon gamma (IFN-γ) were restored in acute myeloid leukemia (AML) by the FLT4 antagonist MAZ51. Here, we developed 12 peptides targeting FLT4 for clinical application and examined whether they restored the frequency of lymphocytes, especially T cells and NK cells, and high IFN-γ expression, as MAZ51 treatment did in our previous study. Although clinical data from using peptides are currently available, peptides targeting FLT4 to modulate immune cells have not been fully elucidated. In this study, we focus on novel peptide 4 (P4) from the intracellular domain of FLT4 because it had dominant negative activity. Similar to MAZ51, high IFN-γ levels were expressed in AML-mononuclear cells exposed to P4. Additionally, T and NK cell levels were restored, as were high IFN-γ levels, in a leukemic environment when P4 was treated. Interestingly, the regulatory T cells were significantly decreased by P4, implying the role of peptide in tumor niche. Overall, we demonstrated the therapeutic value of functionally modulating lymphocytes using a peptide targeting FLT4 and proposed the development of advanced therapeutic approaches against AML by using immune cells.
© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.

Fms-related tyrosine kinase-4 (FLT4) is involved in tumor progression. Previously, we found that dysfunctional natural killer (NK) cells with low interferon-gamma (IFN-γ) were restored in acute myeloid leukemia (AML) by the FLT4 antagonist MAZ51. In this work, we developed 12 peptides targeting FLT4 for clinical application and ultimately selected 4 of them to examine whether they restored the frequency of lymphocytes, especially T cells and NK cells, and high IFN-γ expression, as MAZ51 treatment did in our previous study. Although clinical data from using intracellular kinase domain–targeting peptides are currently available, peptides targeting FLT4 to modulate immune cells have not been fully elucidated. In this study, we focus on novel peptide 4 (P4) from the intracellular domain of FLT4 because it had dominant negative activity. Similar to MAZ51, high IFN-γ levels were expressed in AML-mononuclear cells (MNCs) exposed to P4. In addition, T and NK cell levels were restored, as were high IFN-γ levels, in a leukemic environment when P4 was co-cultured with cytosine β-D-arabinofuranoside. Interestingly, the frequency of regulatory T cells was significantly decreased by P4, implying that the peptide plays a role in modulating the tumor niche. Overall, we demonstrated the therapeutic value of functionally modulating lymphocytes using a peptide targeting FLT4 and propose the development of advanced therapeutic approaches against AML by using immune cells.

The increased expression for the Fms-related tyrosine kinase-4 (FLT-4, known as VEGFR-3) is relevant to dysfunctional natural killer (NK) cells in acute myeloid leukemia (AML). MAZ51 (M), a VEGFR-3 inhibiting chemical, was effectively restored the function of NK cells via the high expression of interferon- gamma (IFN-γ) in NK cells, as shown in our previous study. Although tremendous amount of clinical data using peptides are currently available in real clinic, peptides targeting FLT-4 in modulating immune cells such as NK cells are not fully elucidated.
In present study, we developed peptides targeting FLT-4 (P), which is inhibiting an affinity for AML-NK expressing FLT-4 in vitro and in vivo. Bone marrow (BM) and peripheral blood (PB) mononuclear cells (MNCs) from AML patients were treated with combinational cocktails of the three agents including P, M, ara-C (A) and FLT-4 expression and IFN-γ release were examined. In an AML mouse model, IFN-γ expression were examined in T and NK cells from mouse BM, spleen, and liver to address relevance between peptides and immune cell activation. We found that AML-NK cells both in human and mouse samples showed a gradual increase the IFN-γ levels compared to the controls. There was a trend toward a reduction in leukemic blasts in the BM, spleen, and liver from the AML mice, when we compared the effects of combinational treatments.
Our results suggest that the function of AML-NK cells was synergistically activated by P in combination with M or A.

Liquid biopsy is promising for early detection, monitoring of response, and recurrence of cancer. The blood-brain barrier (BBB) limits the shedding of biomarker, such as cell-free DNA (cfDNA), into the blood from brain tumors, and their detection by conventional assays. Transcranial MR-guided focused ultrasound (MRgFUS) can safely and transiently open the BBB, providing an opportunity for less-invasive access to brain pathology. We hypothesized that MRgFUS can enrich the signal of circulating brain-derived biomarkers to aid in liquid biopsy.
Nine patients were treated in a prospective single-arm, open-label trial to investigate serial MRgFUS and adjuvant temozolomide combination in patients with glioblastoma (NCT03616860). Blood samples were collected as an exploratory measure within the hours before and after sonication, with control samples from non-brain tumor patients undergoing BBB opening (BBBO) alone (NCT03739905).
Brain regions averaging 7.8 ± 6.0 cm3 (range 0.8-23.1 cm3) were successfully treated within 111 ± 39 minutes without any serious adverse events. We found MRgFUS acutely enhanced plasma cfDNA (2.6 ± 1.2-fold, P < .01, Wilcoxon signed-rank test), neuron-derived extracellular vesicles (3.2 ± 1.9-fold, P < .01), and brain-specific protein S100b (1.4 ± 0.2-fold, P < .01). Further comparison of the cfDNA methylation profiles suggests a signature that is disease- and post-BBBO-specific, in keeping with our hypothesis. We also found cfDNA-mutant copies of isocitrate dehydrogenase 1 (IDH1) increased, although this was in only one patient known to harbor the tumor mutation.
This first-in-human proof-of-concept study shows MRgFUS enriches the signal of circulating brain-derived biomarkers, demonstrating the potential of the technology to support liquid biopsy for the brain.
© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.

Pulmonary mesenchymal cells are critical players in both the mouse and human during lung development and disease states. They are increasingly recognized as highly heterogeneous, but there is no consensus on subpopulations or discriminative markers for each subtype. We completed scRNA-seq analysis of mesenchymal cells from the embryonic, postnatal, adult and aged fibrotic lungs of mice and humans. We consistently identified and delineated the transcriptome of lipofibroblasts, myofibroblasts, smooth muscle cells, pericytes, mesothelial cells, and a novel population characterized by Ebf1 expression. Subtype selective transcription factors and putative divergence of the clusters during development were described. Comparative analysis revealed orthologous subpopulations with conserved transcriptomic signatures in murine and human lung mesenchymal cells. All mesenchymal subpopulations contributed to matrix gene expression in fibrosis. This analysis would enhance our understanding of mesenchymal cell heterogeneity in lung development, homeostasis and fibrotic disease conditions.
© 2021 The Authors.