FOXF1 promotes tumor vessel normalization and prevents lung cancer progression through FZD4.

Cancer cells re-program normal lung endothelial cells (EC) into tumor-associated endothelial cells (TEC) that form leaky vessels supporting carcinogenesis. Transcriptional regulators that control the reprogramming of EC into TEC are poorly understood. We identified Forkhead box F1 (FOXF1) as a critical regulator of EC-to-TEC transition. FOXF1 was highly expressed in normal lung vasculature but was decreased in TEC within non-small cell lung cancers (NSCLC). Low FOXF1 correlated with poor overall survival of NSCLC patients. In mice, endothelial-specific deletion of FOXF1 decreased pericyte coverage, increased vessel permeability and hypoxia, and promoted lung tumor growth and metastasis. Endothelial-specific overexpression of FOXF1 normalized tumor vessels and inhibited the progression of lung cancer. FOXF1 deficiency decreased Wnt/ß-catenin signaling in TECs through direct transcriptional activation of Fzd4. Restoring FZD4 expression in FOXF1-deficient TECs through endothelial-specific nanoparticle delivery of Fzd4 cDNA rescued Wnt/ß-catenin signaling in TECs, normalized tumor vessels and inhibited the progression of lung cancer. Altogether, FOXF1 increases tumor vessel stability, and inhibits lung cancer progression by stimulating FZD4/Wnt/ß-catenin signaling in TECs. Nanoparticle delivery of FZD4 cDNA has promise for future therapies in NSCLC.

Disseminated cysticercosis with tongue involvement: a rare case report from Nepal.

Introduction and importance: Cysticercosis is a condition in which humans are infected by the larval form of the pork tapeworm Tenia solium. Cysticercosis in humans is common in the cerebral tissue but rare in the tongue. Case presentation: Here, the authors report a rare case of a 38-year-old male with neurocysticercosis and cysticercosis of the tongue. The patient presented with a complaint of loss of consciousness for 4-5 min. Local examination of his oral cavity revealed a swelling of ~2×2 cm on the tongue. An MRI of the brain showed various stages of neurocysticercosis involving the neuroparenchyma and tongue. For this, he was started on low-dose prednisolone of 50 mg tapered over 6 weeks and levetiracetam of 500 mg BD continued for his seizure episodes. He is responding well with the medications and is planned to start antiparasitic agent only after the perilesional edema decreases. Clinical discussion: Cysticercosis may involve the central nervous system, muscle, heart, lungs, peritoneum, eye, and subcutaneous tissue. Oral cavity and perioral involvement by cysticercous larva is rare in humans. Radiologic imaging, serology, and tissue biopsy can be used to confirm a diagnosis of cysticercosis. The most common locations for oral cysticercosis are the tongue, buccal mucosa, lower lip, and upper lip.Only 102 cases of oral cysticercosis have been reported based on a PubMed English-language literature search. Conclusion: Oral cysticercosis is a rare event, and it represents a difficulty in clinical diagnosis. But a patient with a mass in the tongue should be considered as a possible case of cysticercosis especially in endemic regions like Nepal.

Ultra-high dose-rate proton FLASH improves tumor control.

BACKGROUND AND PURPOSE: Proton radiotherapy (PRT) offers potential benefits over other radiation modalities, including photon and electron radiotherapy. Increasing the rate at which proton radiation is delivered may provide a therapeutic advantage. Here, we compared the efficacy of conventional proton therapy (CONVpr) to ultrahigh dose-rate proton therapy, FLASHpr, in a mouse model of non-small cell lung cancers (NSCLC). MATERIALS AND METHODS: Mice bearing orthotopic lung tumors received thoracic radiation therapy using CONVpr (<0.05 Gy/s) and FLASHpr (>60 Gy/s) dose rates. RESULTS: Compared to CONVpr, FLASHpr was more effective in reducing tumor burden and decreasing tumor cell proliferation. Furthermore, FLASHpr was more efficient in increasing the infiltration of cytotoxic CD8+ T-lymphocytes inside the tumor while simultaneously reducing the percentage of immunosuppressive regulatory T-cells (Tregs) among T-lymphocytes. Also, compared to CONVpr, FLASHpr was more effective in decreasing pro-tumorigenic M2-like macrophages in lung tumors, while increasing infiltration of anti-tumor M1-like macrophages. Finally, FLASHpr treatment reduced expression of checkpoint inhibitors in lung tumors, indicating reduced immune tolerance. CONCLUSIONS: Our results suggest that FLASH dose-rate proton delivery modulates the immune system to improve tumor control and might thus be a promising new alternative to conventional dose rates for NSCLC treatment.

Lung endothelial cells regulate pulmonary fibrosis through FOXF1/R-Ras signaling.

Pulmonary fibrosis results from dysregulated lung repair and involves multiple cell types. The role of endothelial cells (EC) in lung fibrosis is poorly understood. Using single cell RNA-sequencing we identified endothelial transcription factors involved in lung fibrogenesis, including FOXF1, SMAD6, ETV6 and LEF1. Focusing on FOXF1, we found that FOXF1 is decreased in EC within human idiopathic pulmonary fibrosis (IPF) and mouse bleomycin-injured lungs. Endothelial-specific Foxf1 inhibition in mice increased collagen depositions, promoted lung inflammation, and impaired R-Ras signaling. In vitro, FOXF1-deficient EC increased proliferation, invasion and activation of human lung fibroblasts, and stimulated macrophage migration by secreting IL-6, TNFα, CCL2 and CXCL1. FOXF1 inhibited TNFα and CCL2 through direct transcriptional activation of Rras gene promoter. Transgenic overexpression or endothelial-specific nanoparticle delivery of Foxf1 cDNA decreased pulmonary fibrosis in bleomycin-injured mice. Nanoparticle delivery of FOXF1 cDNA can be considered for future therapies in IPF.

Demonstration of Safety in Wild Type Mice of npFOXF1, a Novel Nanoparticle-Based Gene Therapy for Alveolar Capillary Dysplasia with Misaligned Pulmonary Veins.

Introduction: Alveolar Capillary Dysplasia with Misaligned Pulmonary Veins (ACDMPV) is a fatal congenital disease resulting from a pulmonary vascular endothelial deficiency of FOXF1, producing abnormal morphogenesis of alveolar capillaries, malpositioned pulmonary veins and disordered development of lung lobes. Affected neonates suffer from cyanosis, severe breathing insufficiency, pulmonary hypertension, and death typically within days to weeks after birth. Currently, no treatment exists for ACDMPV, although recent murine research in the Kalinichenko lab demonstrates nanoparticle delivery improves survival and reconstitutes normal alveolar-capillary architecture. The aim of the present study is to investigate the safety of intravenous administration of FOXF1-expressing PEI-PEG nanoparticles (npFOXF1), our pioneering treatment for ACDMPV. Methods: npFOXF1 was constructed, validated, and subsequently administered in a single dose to postnatal day 14 (P14) mice via retro-orbital injection. Biochemical, serologic, and histologic safety were monitored at postnatal day 16 (P16) and postnatal day 21 (P21). Results: With treatment we observed no lethality, and the general condition of mice revealed no obvious abnormalities. Serum chemistry, whole blood, and histologic toxicity was assayed on P16 and P21 and revealed no abnormality. Discussion: In conclusion, npFOXF1 has a very good safety profile and combined with preceding studies showing therapeutic efficacy, npFOXF1 can be considered as a good candidate therapy for ACDMPV in human neonates.

Hypoxia represses FOXF1 in lung endothelial cells through HIF-1α.

Introduction: Forkhead Box F1 (FOXF1) transcription factor plays a critical role in lung angiogenesis during embryonic development and lung repair after injury. FOXF1 expression is decreased in endothelial cells after lung injury; however, molecular mechanisms responsible for the FOXF1 transcript changes in injured lung endothelium remain unknown. Methods: We used immunostaining of injured mouse lung tissues, FACS-sorted lung endothelial cells from hypoxia-treated mice, and data from patients diagnosed with hypoxemic respiratory failure to demonstrate that hypoxia is associated with decreased FOXF1 expression. Endothelial cell cultures were used to induce hypoxia in vitro and identify the upstream molecular mechanism through which hypoxia inhibits FOXF1 gene expression. Results: Bleomycin-induced lung injury induced hypoxia in the mouse lung tissue which was associated with decreased Foxf1 expression. Human FOXF1 mRNA was decreased in the lungs of patients diagnosed with hypoxemic respiratory failure. Mice exposed to hypoxia exhibited reduced Foxf1 expression in the lung tissue and FACS-sorted lung endothelial cells. In vitro, hypoxia (1% of O2) or treatment with cobalt (II) chloride increased HIF-1α protein levels but inhibited FOXF1 expression in three endothelial cell lines. Overexpression of HIF-1α in cultured endothelial cells was sufficient to inhibit Foxf1 expression. siRNA-mediated depletion of HIF-1α prevented the downregulation of Foxf1 gene expression after hypoxia or cobalt (II) chloride treatment. Conclusion: Hypoxia inhibits FOXF1 expression in endothelial cells in a HIF-1α dependent manner. Our data suggest that endothelial cell-specific inhibition of HIF-1α via gene therapy can be considered to restore FOXF1 and improve lung repair in patients with severe lung injury.

Probing endogenous collagen by laser-induced autofluorescence in burn wound biopsies: A pilot study.

The focus of the current study was to interrogate the predictive potential of laser-induced autofluorescence (LIAF) by objectively assessing collagen synthesis in burn wound granulation tissues ex vivo. Prior grafting, granulation tissues (20 samples) following burn injury were collected from 17 subjects of age range 18 to 60 years with patient/donor consent and the corresponding autofluorescence spectra were recorded at 325 nm He-Cd laser (≈2 mW) excitations. The resulting endogenous collagen intensity from the above tissue samples was computed by normalizing the nicotinamide adenine dinucleotide levels. In addition, the hydroxyproline content was also estimated biochemically from the same granulation tissues. A comparative assessment of both LIAF and biochemical estimations for endogenous collagen by hydroxyproline resulted in strong positive correlation among them. The above relevant observations suggest that LIAF is equally informative as that of biochemical estimations, in evaluating endogenous collagen content in wound granulation tissues. Thus, it can be concluded that LIAF has the predictive potential, as a noninvasive objective tool to measure the endogenous collagen levels in wound biopsy tissues and provide complementary data conducive for making clinical decisions.