Histological pattern of tumor inflammation and stromal density correlate with patient demographics and immuno-oncologic transcriptional profile in oral squamous cell carcinoma.

Introduction: Oral squamous cell carcinoma (OSCC) is the most prevalent oral malignancy, with emerging interest in the characterization of its tumor microenvironment. Herein, we present a comprehensive histological analysis of OSCC stromal density and inflammation and their relationship with patient demographics, clinicopathologic features and immuno-oncologic signatures. Materials-methods: Eighty-seven completely excised OSCC tissues were prospectively collected and scored for histopathologic inflammatory subtypes [HIS]-inflamed (INF), immune-excluded (IE) and immune-desert (ID), peritumoral stromal inflammation (PTSI), and peritumoral stromal fibrosis (PTSF). Scoring of inflammation was complemented by Semaphorin 4D immunohistochemistry. NanoString differential gene expression (DGE) analysis was conducted for eight OSCC cases representative of the inflammatory and stromal subtypes and the demographic groups. Results: PTSF correlated with male gender (p = 0.0043), smoking (p = 0.0455), alcohol consumption (p = 0.0044), increased tumor size (p = 0.0054), and advanced stage (p = 0.002). On the contrary, PTSI occurred predominantly in females (p = 0.0105), non-drinkers (p = 0.0329), and small tumors (p = 0.0044). Transcriptionally, decreased cytokine signaling, and oncogenic pathway activation were observed in HIS-IE. Smokers and males displayed decreased global immune-cell levels and myeloid-cell predominance. Conclusion: Our work describes OSCC stromal and inflammatory phenotypes in correlation with distinct patient groups and DGE, highlighting the translational potential of characterizing the tumor microenvironment for optimal patient stratification.

Immuno-oncologic signature of malignant transformation in oral squamous cell carcinoma.

OBJECTIVE: The purpose of this study is to identify the immuno-oncologic (IO) signature at the surgical tumor margin (TM) of oral squamous cell carcinoma (OSCC) that is involved in the process of malignant transformation. STUDY DESIGN: Under institutional review board approval, TM of 73 OSCC were investigated using immunohistochemistry for the immune biomarker, programmed death ligand-1 (PD-L1). NanoString 770 IO-focused gene set was analyzed in 5 pairs of TM and invasive tumor (T). PD-L1 regulation in response to interferon-gamma (IFN-γ) was investigated in an oral potentially malignant cell line (OPMC). RESULTS: Programmed death ligand-1 expression in the epithelial margin directly correlated with its expression in the underlying immune cells (P = .0082). Differential gene expression showed downregulation of PD-L1 and IFN-γ 6 gene signature in the TM relative to T pair.CD8 and macrophages were higher in TM. CNTFR, LYZ, C7, RORC, and FGF13 downregulation in T relative to TM. TDO2, ADAM12, MMP1, LAMC2, MB21D1, TYMP, OASL, COL5A1, exhausted_CD8, Tregs,and NK_CD56dim were upregulated in T relative to TM. Finally, IFN-γ induced upregulation of PD-L1 in the OPMC. CONCLUSIONS: Our work suggests a role for IFN-γ in PD-L1 upregulation in OPMC and presents novel IO transcriptional signatures for frankly invasive OSCC relative to TM.

Biocompatible PCL-nanofibers scaffold with immobilized fibronectin and laminin for neuronal tissue regeneration.

Recent advances in regenerative medicine have given hope in overcoming and rehabilitating complex medical conditions. In this regard, the biopolymer poly-ε-caprolactone (PCL) may be a promising candidate for tissue regeneration, despite lacking the essential bioactivity. The present study used PCL nanofibers (NFs) scaffold decorated with the extracellular matrix proteins fibronectin and laminin combined for neuronal regeneration. The potential for the dual proteins to support neuronal cells and promote axonal growth was investigated. Two NFs scaffolds were produced with PLC concentrations of 12% or 15%. Under scanning electron microscopy, both scaffolds evidenced uniform diameter distribution in the range of 358 nm and 887 nm, respectively, with >80% porosity. The Brunauer-Emmett-Teller (BET) test confirmed that the fabricated NFs mats had a high surface area, especially for the 12% NFs with 652 m2/g compared to 254 m2/g for the 15% NFs. The proteins of interest were successfully conjugated to the 12% PCL scaffold through chemical carbodiimide reaction as confirmed by Fourier-transform infrared spectroscopy. The addition of fibronectin and laminin together was shown to be the most favorable for cellular attachment and elongation of neuroblastoma SH-SY5Y cells compared to other formulations. Light microscopy revealed longer neurite outgrowth, higher cellular projected area, and lower shape index for the cells cultured on the combined proteins conjugated fibers, indicating enhanced cellular spread on the scaffold. This preliminary study suggests that PCL nanoscaffolding conjugated with matrix proteins can support neuronal cell viability and neurite growth.

Soluble Sema4D in Plasma of Head and Neck Squamous Cell Carcinoma Patients Is Associated With Underlying Non-Inflamed Tumor Profile.

Semaphorin 4D (Sema4D) is a glycoprotein that is expressed by several tumors and immune cells. It can function as a membrane bound protein or as a cleaved soluble protein (sSema4D). We sought to investigate the translational potential of plasma sSema4D as an immune marker in plasma of patients with head and neck squamous cell carcinoma (HNSCC). Paired peripheral blood and tumor tissue samples of 104 patients with HNSCC were collected at the same time point to allow for real time analysis. Scoring of the histological inflammatory subtype (HIS) was carried out using Sema4D immunohistochemistry on the tumor tissue. sSema4D was detected in plasma using direct ELISA assay. Defining elevated sSema4D as values above the 95th percentile in healthy controls, our data showed that sSema4D levels in plasma were elevated in 25.0% (95% CI, 16.7-34.9%) of the patients with HNSCC and showed significant association with HIS immune excluded (HIS-IE) (p = 0.007), Sema4D+ve tumor cells (TCs) (p = 0.018) and PD-L1+ve immune cells (ICs) (p = 0.038). A multi-variable logistic regression analysis showed that HIS was significantly (P = 0.004) associated with elevated sSema4D, an association not explained by available patient-level factors. Using the IO-360 nanoString platform, differential gene expression (DGE) analysis of 10 HNSCC tumor tissues showed that patients with high sSema4D in plasma (HsS4D) clustered as IFN-γ negative tumor immune signature and were mostly HIS-IE. The IC type in the HsS4D paired tumor tissue was predominantly myeloid, while the lymphoid compartment was higher in the low sSema4D (LsS4D). The Wnt signaling pathway was upregulated in the HsS4D group. Further analysis using the IO-360, 770 gene set, showed significant non-inflamed profile of the HsS4D tumors compared to the LsS4D. In conclusion, our data reveals an association between sSema4D and the histological inflammatory subtype.

Lactoferrin, a multi-functional glycoprotein: Active therapeutic, drug nanocarrier & targeting ligand.

Recent progress in protein-based nanomedicine, inspired by the success of Abraxane® albumin-paclitaxel nanoparticles, have resulted in novel therapeutics used for treatment of challenging diseases like cancer and viral infections. However, absence of specific drug targeting, poor pharmacokinetics, premature drug release, and off-target toxicity are still formidable challenges in the clinic. Therefore, alternative protein-based nanomedicines were developed to overcome those challenges. In this regard, lactoferrin (Lf), a glycoprotein of transferrin family, offers a promising biodegradable well tolerated material that could be exploited both as an active therapeutic and drug nanocarrier. This review highlights the major pharmacological actions of Lf including anti-cancer, antiviral, and immunomodulatory actions. Delivery technologies of Lf to improve its pries and enhance its efficacy were also reviewed. Moreover, different nano-engineering strategies used for fabrication of drug-loaded Lf nanocarriers were discussed. In addition, the use of Lf for functionalization of drug nanocarriers with emphasis on tumor-targeted drug delivery was illustrated. Besides its wide application in oncology nano-therapeutics, we discussed the recent advances of Lf-based nanocarriers as efficient platforms for delivery of anti-parkinsonian, anti-Alzheimer, anti-viral drugs, immunomodulatory and bone engineering applications.

Dual-Targeted Lactoferrin Shell-Oily Core Nanocapsules for Synergistic Targeted/Herbal Therapy of Hepatocellular Carcinoma.

Herein, both strategies of synergistic drug combination together with dual active tumor targeting were combined for effective therapy of hepatocellular carcinoma (HCC). Therefore, based on the tumor sensitizing action, the herbal quercetin (QRC) was co-delivered with the targeted therapeutic drug sorafenib (SFB), preformulated as phospholipid complex, via protein shell-oily core nanocapsules (NCs). Inspired by the targeting action of lactoferrin (LF) via binding to LF receptors overexpressed by HCC cells, LF shell was electrostatically deposited onto the drug-loaded oily core to elaborate LF shell-oily core NCs. For dual tumor targeting, lactobionic acid (LA) or glycyrrhetinic acid (GA) was individually coupled to LF shell for binding to asialoglycoprotein and GA receptors on liver cancer cells, respectively. Compared to LF and GA/LF NCs, the dual-targeted LA/LF-NCs showed higher internalization into HepG2 cells with 2-fold reduction in half-maximal inhibitory concentration compared to free combination therapy after 48 h. Moreover, dual-targeted LF-NCs showed powerful in vivo antitumor efficacy. It was revealed as significant downregulation of the mRNA expression levels of nuclear factor-kappa B and tumor necrosis factor α as well as suppression of Ki-67 protein expression level in diethylnitrosamine (DEN)-induced HCC mice (P < 0.05). Furthermore, dual-targeted LF-NCs attenuated the liver toxicity induced by DEN in animal models. Overall, this study proposes dual-targeted LF-NCs for combined delivery of SFB and QRC as a potential therapeutic HCC strategy.