Tumour Microenvironment as a Potential Immune Therapeutic Target for Tongue Cancer Management.

Immunotherapy is a promising approach in the management of human cancers and has been proven to provide a durable response in many cancers. It is helpful as an adjuvant therapy for cancers and at present is considered as a fourth pillar supporting surgery, chemotherapy and radiotherapy. In the treatment of oral cancer, immunotherapy is approved in late-stage diseases where surgical resection cannot be carried out or fails, leading to recurrences and metastasis. Evidences suggest that when given as a first-line treatment, it can elicit an immune response that shrinks tumours, which could provide long-term benefit for patients. But unlike the traditional approach which follows the uniform protocol for all oral cancer patients, effective immunotherapy requires a more site-specific personalized approach. The aim of this paper is to review the various immune evasive mechanisms adopted by tumour cells and their relevance as potential targets for immunotherapy in oral tongue squamous cell carcinoma.

5-FU mediated depletion of myeloid suppressor cells enhances T-cell infiltration and anti-tumor response in immunotherapy-resistant lung tumor.

Tumor microenvironment (TME) is a heterogeneous system consisting of both cellular and acellular components. The growth and progression of tumors rely greatly on the nature of TME, marking it as an important target in cancer immunotherapy. Lewis Lung Carcinoma (LLC) is an established murine lung cancer model representing immunologically 'cold' tumors characterized by very few infiltrated cytotoxic T-cells, high levels of Myeloid-Derived Suppressor Cells (MDSCs) and Tumor-Associated Macrophages (TAMs). Here, we report various strategies we applied to reverse the non-immunogenic character of this cold tumor by imparting: a) immunogenic cell death using Hypericin nanoparticle-based photodynamic therapy (PDT), b) repolarising TAM using a TLR7/8 agonist, resiquimod, c) immune checkpoint inhibition using anti-PD-L1 and d) depleting MDSCs using low-dose 5-fluorouracil (5-FU) chemotherapy. Interestingly, the nano-PDT, resiquimod or anti-PD-L1 treatment had no major impact on tumor growth, whereas low-dose 5-FU-mediated depletion of MDSCs showed significant anti-tumor effect, primarily caused by the increased infiltration of CD8+ cytotoxic T-cells (∼96%). Though we have tested combining PDT with resiquimod or 5-FU for any synergistic effect, low-dose 5-FU alone showed better response than combinations. In effect, we show that depletion of MDSCs using low-dose 5-FU was one of the best methods to augment infiltration of CD8+ cytotoxic T-cells into a cold tumor, which is resistant to conventional therapies including immune checkpoint inhibitors.

Intracranial nanomedicine-gel with deep brain-penetration for glioblastoma therapy.

Glioblastoma Multiforme (GBM) is one of the challenging tumors to treat as it recurs, almost 100%, even after surgery, radiation, and chemotherapy. In many cases, recurrence happens within 2-3cm depth of the resected tumor margin, indicating the inefficacy of current anti-glioma drugs to penetrate deep into the brain tissue. Here, we report an injectable nanoparticle-gel system, capable of providing deep brain penetration of drug up to 4 cm, releasing in a sustained manner up to >15 days. The system consists of ∼222 nm sized PLGA nanoparticles (NP-222) loaded with an anti-glioma drug, Carmustine (BCNU), and coated with a thick layer of polyethylene glycol (PEG). Upon release of the drug from PLGA core, it will interact with the outer PEG-layer leading to the formation of PEG-BCNU nanocomplexes of size ∼33 nm (BCNU-NC-33), which could penetrate >4 cm deep into the brain tissue compared to the free drug (< 5 mm). In vitro drug release showed sustained release of drug for 15 days by BCNU-NP gel, and enhanced cytotoxicity by BCNU-NC-33 drug-nanocomplexes in glioma cell lines. Ex vivo goat-brain phantom studies showed drug diffusion up to 4 cm in tissue and in vivo brain-diffusion studies showed almost complete coverage within the rat brain (∼1.2 cm), with ∼55% drug retained in the tissue by day-15, compared to only ∼5% for free BCNU. Rat orthotopic glioma studies showed excellent anti-tumor efficacy by BCNU-NP gel compared to free drug, indicating the potential of the gel-system for anti-glioma therapy. In effect, we demonstrate a unique method of sustained release of drug in the brain using larger PLGA nanoparticles acting as a reservoir while deep-penetration of the released drug was achieved by in situ formation of drug-nanocomplexes of size <50 nm which is less than the native pore size of brain tissue (> 100 nm). This method will have a major impact on a challenging field of brain drug delivery.

Enhanced oral bioavailability and antitumor therapeutic efficacy of sorafenib administered in core-shell protein nanoparticle.

Orally delivered molecularly targeted small-molecule drugs play a significant role in managing cancer as a chronic disease. However, due to the poor oral bioavailability of some of these molecules, high-dose administration is required leading to dose-limiting toxicity especially when delivered daily for a long duration. Here, we report an oral nanoformulation for small-molecule multi-kinase inhibitor, sorafenib tosylate, showing nearly two fold enhancement in the oral bioavailability and enhanced therapeutic efficacy with a better safety profile compared to the current clinical formulation. Using a scalable process involving high-pressure homogenization, sorafenib was loaded into an albumin nanocarrier at ~ 50 w/w%. Repeated preparation of gram-scale batches (n = 7) showed an average particle size of 180 ± 9 nm, encapsulation efficiency of 95 [Formula: see text] 2%, and drug-loading efficiency of 48 [Formula: see text] 0.7%. Further, surface engineering with a mucoadhesive layer on nanoparticles (referred to as ABSORF) resulted in the final size of 299 ± 38 nm and surface charge of -54 ± 8 mV. Single-dose and multidose pharmacokinetic studies showed two fold enhancement in the plasma concentration of sorafenib compared to current clinically used tablets. Antitumor efficacy studies in the orthotopic rat liver tumor model showed significant tumor regression (p value = 0.0037) even at half dose (eqv. to 200 mg of human equivalent dose) of ABSORF compared to clinical control (eqv. to 400 mg). The biodistribution of sorafenib from ABSORF was higher in the liver; however, liver and kidney function test parameters were comparable with that of the 2 × dose of clinical control. No abnormalities and signs of toxicity were seen in the histopathological analysis for ABSORF-treated animals. In summary, we demonstrate a scalable preparation of small-molecule drug-loaded nanoformulation with approximately two fold enhancement in oral bioavailability, improved antitumor efficacy, and acceptable toxicity profile.

Discovery of Anticancer Hybrid Molecules by Supervised Machine Learning Models and in Vitro Validation in Drug Resistant Chronic Myeloid Leukemia Cells.

The concept of hybrid drugs for targeting multiple aberrant pathways of cancer, by combining the key pharmacophores of clinically approved single-targeted drugs, has emerged as a promising approach for overcoming drug-resistance. Here, we report the design of unique hybrid molecules by combining the two pharmacophores of clinically approved BCR-ABL inhibitor (ponatinib) and HDAC inhibitor (vorinostat) and results of in vitro studies in drug-resistant CML cells. Robust 2D-QSAR and 3D-pharmacophore machine learning supervised models were developed for virtual screening of the hybrid molecules based on their predicted BCR-ABL and HDAC inhibitory activity. The developed 2D-QSAR model showed five information rich molecular descriptors while the 3D-pharmacophore model of BCR-ABL showed five different chemical features (hydrogen bond acceptor, donor, hydrophobic group, positive ion group, and aromatic rings) and the HDAC model showed four different chemical features (hydrogen bond acceptor, donor, positive ion group, and aromatic rings) for potent BCR-ABL and HDAC inhibition. Virtual screening of the 16 designed hybrid molecules identified FP7 and FP10 with better potential of inhibitory activity. FP7 was the most effective molecule with predicted IC50 using the BCR-ABL based 2D-QSAR model of 0.005 µM and that of the HDAC model of 0.153 µM, and that using the BCR-ABL based 3D-pharmacophore model was 0.02 µM and that with HDAC model was 0.014 µM. In vitro study (dose-response relationship) of FP7 in wild type and imatinib-resistant CML cell lines harboring Thr315Ile or Tyr253His mutations showed growth inhibitory IC50 values of 0.000 16, 0.0039, and 0.01 µM, respectively. This molecule also showed better biocompatibility when tested in whole blood and in PBMCs as compared to ponatinib or vorinostat.

Exploiting the preferential phagocytic uptake of nanoparticle-antigen conjugates for the effective treatment of autoimmunity.

Tolerance induction is central to the suppression of autoimmunity. Here, we engineered the preferential uptake of nano-conjugated autoantigens by spleen-resident macrophages to re-introduce self-tolerance and suppress autoimmunity. The brain autoantigen, myelin oligodendrocyte glycoprotein (MOG), was conjugated to 200 or 500 nm silica nanoparticles (SNP) and delivered to the spleen and liver-resident macrophages of experimental autoimmune encephalomyelitis (EAE) mice, used as a model of multiple sclerosis. MOG-SNP conjugates significantly reduced signs of EAE at a very low dose (50 µg) compared to the higher dose (>800 µg) of free-MOG. This was associated with reduced proliferation of splenocytes and pro-inflammatory cytokines secretion, decreased spinal cord inflammation, demyelination and axonal damage. Notably, biodegradable porous SNP showed an enhanced disease suppression assisted by elevated levels of regulatory T cells and programmed-death ligands (PD-L1/2) in splenic and lymph node cells. Our results demonstrate that targeting nano-conjugated autoantigens to tissue-resident macrophages in lymphoid organs can effectively suppress autoimmunity.

Intracranially injectable multi-siRNA nanomedicine for the inhibition of glioma stem cells.

BACKGROUND: Nanoparticle siRNA-conjugates are promising clinical therapeutics as indicated by recent US-FDA approval. In glioma stem cells (GSC), multiple stemness associated genes were found aberrant. We report intracranially injectable, multi-gene-targeted siRNA nanoparticle gel (NPG) for the combinatorial silencing of 3 aberrant genes, thus inhibiting the tumorogenic potential of GSCs. METHODS: NPG loaded with siRNAs targeted against FAK, NOTCH-1, and SOX-2 were prepared by the self-assembly of siRNAs with protamine-hyaluronic acid combination. Electron microscopy, DLS, and agarose gel electrophoresis were used for the physicochemical characterization. Cell transfection and gene-silencing efficiency were studied using human mesenchymal stem cells and rat C6 glioma-derived GSCs. Neurosphere inhibition was tested in vitro using GSCs derived from C6 cell line and glioma patient samples. Patient-derived xenograft model and orthotopic rat glioma model were used to test the effect of NPG on in vivo tumorigenicity. RESULTS: The siRNA nanoparticles with an average size ~ 250 nm and ~ 95% loading efficiency showed cellular uptake in ~95.5% GSCs. Simultaneous gene silencing of FAK, NOTCH-1, and SOX-2 led to the inhibition of neurosphere formation by GSCs, whereas normal stem cells remained unaffected and retained neuronal differentiation capability. GBM PDX models manifested significant impairment in the tumorigenic potential of NPG treated GSCs. Intracranial injection of NPG inhibited tumor growth in orthotopic rat brain tumor model. CONCLUSION: Intracranially injectable n-siRNA NPG targeted to multiple stem-cell signaling impairs glioma initiation capabilities of GSCs and inhibited tumor growth in vivo.

Low PDL1 Expression in Tumour Infiltrating Lymphocytes Predicts Local Recurrence in Oral Squamous Cell Carcinoma.

In oral squamous cell carcinoma (OSCC), expression of PDL1 is controversial with expressions showing a positive and negative correlation with survival in previous studies. Additionally, it is unclear whether expression on the tumour or tumour infiltrating lymphocytes (TIL) is a better predictor of survival. We performed this study on a cohort of Indian patients with OSCC to determine impact of PDL1 expression on survival. Retrospective analysis of 64 patients of OSCC treated with curative intent surgery with or without adjuvant therapy was performed. Stored tissue blocks were extracted and quantitative immunohistochemistry was performed for PDL1 expression separately on the tumour and the TIL using commercially available Dako kits. Correlation of clinical and pathological variables with PDL1 expression was performed using chi-square test. Survival analysis was performed using Kaplan-Meier method and Cox proportional hazards ratio. In our cohort, PDL1 expression was low, both in tumour (92% had <1% expression) and TIL (56% had <1% expression). Tumour low PDL1 expression (<1%) was associated with a higher risk of lymphovascular invasion (p = 0.044) and bone invasion (p = 0.01) but did not impact survival. Low TIL PDL1 expression (<1%) was more common in younger patients (<45 years) (p = 0.023) significantly predicting local recurrence (p = 0.02). PDL1 expression in OSCC was low. Low TIL PDL1 was common in younger patients and predicted local recurrence. Further study is required to better understand the relationship between age, tumour microenvironment and local recurrence.

Cancer nanomedicine developed from total human serum: a novel approach for making personalized nanomedicine.

Aim: To develop a method for making total serum nanoparticles (TSN) loaded with cytotoxic chemodrugs for cancer therapy. Materials & methods: TSN loaded with paclitaxel (PTX) or piperlongumine (PL) were prepared using high-pressure homogenization and tested for immunogenicity in healthy animals and antitumor properties in pancreatic cancer xenograft models. Results: TSN-PL nanoparticles of average size 104 nm and encapsulation efficiency approximately 50% showed enhanced dose-dependent cytotoxicity compared with TSN-PTX or clinically used combination of gemcitabine and nano-PTX in two pancreatic cell lines. Significant antitumor efficacy was also established in the pancreatic xenograft model. Conclusion: We developed a unique method of converting total blood serum into chemo drug-loaded nanoparticles and demonstrated its efficacy in vitro and in vivo.

Sex-Dependent Bioaccumulation of Nano Zinc Oxide and Its Adverse Effects on Sexual Behavior and Reproduction in Japanese Medaka.

This study investigated the adverse effects of 200 nm zinc oxide particles (nZnO) on sexual behavior and reproduction in Japanese medaka in comparison with ZnSO4 and correlated the consequences with the bioaccumulation pattern of the particles in associated organs. nZnO exposure impaired sexual and territorial behaviors and affected fertility by altering sperm viability and motility in males through reactive oxygen species (ROS) induction. Conversely, none of these effects other than behavior loss was seen in males exposed to ZnSO4. nZnO exposure to females induced ROS in ovaries, causing follicular growth arrest, atresia, and subfertility. Further, sex-steroid levels were altered by both nZnO and ZnSO4 in males and by nZnO but not ZnSO4 in females. Biodistribution studies revealed the deposition of nZnO as particulate matter in the brain, gills, gut, kidney, and ovary. Particle accumulation in the brain was sex specific, as the particles were found in the brain of males but not that of females. A similar trend was seen for zinc levels in males and females exposed to ZnSO4. Importantly, the female sex hormone, 17ß-estradiol was found to prevent nZnO accumulation in the female brain, emphasizing the need for biodistribution profiling of nanoparticle-based drug delivery vehicles separately in males and females before they are commercialized. This study has demonstrated that the toxic effects of nZnO on the reproductive system were mainly caused by ROS induction, while zinc ions were predominantly responsible for the adverse impact of ZnSO4.

nCP:Fe Nanocontrast Agent for Magnetic Resonance Imaging-Based Early Detection of Liver Cirrhosis and Hepatocellular Carcinoma.

Early detection of liver tumors and cirrhotic lesions by magnetic resonance imaging (MRI) remains a great challenge. Here, we report a biomineral nanocontrast agent based on iron-doped nanocalcium phosphate (nCP:Fe-CA) for magnetic resonance imaging of early-stage liver cirrhotic and hepatocellular carcinoma nodules using rat models. We have optimized an intravenously injectable, aqueous suspension of nCP:Fe-CA having an average size of 137.6 nm, a spherical shape, magnetic relaxivity of 63 mM-1S-1, and colloidal stability for 48 h, post-resuspension in an aqueous phase. Compared to superparamagnetic iron oxide nanoparticles (SPIONs), the optimized nCP:Fe-CA could detect liver tumor lesions as small as ∼0.25 cm, whereas the current clinical detection limit is ∼1 cm. In addition, multiple cirrhotic nodules of size <0.2 cm could be detected by nCP:Fe-CA-assisted MRI. The number of nodules observed after injecting nCP:Fe-CA was ∼3 times higher than that without CA (5-10 nodules). A biocompatibility study on healthy rats injected with nCP:Fe-CA showed unaltered liver transaminases, blood urea nitrogen, serum creatinine, and insignificant hemolysis. Furthermore, hepatobiliary clearance of nCP:Fe-CA was observed in 72 h compared to prolonged retention of SPIONs for 30 days when tested under identical conditions. Overall, the nCP:Fe-CA nanoparticles showed promising results as a biocompatible, MR contrast (T2) agent for the early-stage imaging of liver cirrhosis and hepatocellular carcinoma.

A Novel Surface Enhanced Raman Catheter for Rapid Detection, Classification, and Grading of Oral Cancer.

Fabrication and testing of a novel nanostructured surface-enhanced Raman catheter device is reported for rapid detection, classification, and grading of normal, premalignant, and malignant tissues with high sensitivity and accuracy. The sensor part of catheter is formed by a surface-enhanced Raman scattering (SERS) substrate made up of leaf-like TiO2 nanostructures decorated with 30 nm sized Ag nanoparticles. The device is tested using a total of 37 patient samples wherein SERS signatures of oral tissues consisting of malignant oral squamous cell carcinoma (OSCC), verrucous carcinoma, premalignant leukoplakia, and disease-free conditions are detected and classified with an accuracy of 97.24% within a short detection-cum-processing time of nearly 25-30 min per patient. Neoplastic grade changes detected using this device correlate strongly with conventional pathological data, enabling correct classification of tumors into three grades with an accuracy of 97.84% in OSCC. Thus, the potential of a SERS catheter device as a point-of-care pathological tool is shown for the rapid and accurate detection, classification, and grading of solid tumors.

Magnetic 3D scaffold: A theranostic tool for tissue regeneration and non-invasive imaging in vivo.

We report an osteoconducting magnetic 3D scaffold using Fe2+ doped nano-hydroxyapatite-Alginate-Gelatin (AGHFe1) for Magnetic Resonance Imaging based non-invasive monitoring of bone tissue regeneration. In rat cranial defect model, the scaffold facilitated non-invasive monitoring of cell migration, inflammatory response and matrix deposition by unique changes in transverse relaxation time (T2). Cell infiltration resulted in a considerable increase in T2 from ~37 to ~62 ms, which gradually returned to that of native bone (~23 ms) by 90 days. We used this method to compare in vivo performance of scaffold with bone-morphogenic protein-2 (AGHFe2) or faster degrading (AGHFe3). MRI and histological analysis over 90 days showed non-uniform bone formation in AGHFe1 with ∆T2 (T2Native bone - T2 Regenerated bone) ~13 ms, whereas, AGHFe2 and AGHFe3 showed ∆T2 ~ 09 and 05 ms respectively, suggesting better bone formation in AGHFe3. Thus, we show that MR-contrast enabled scaffold can help better assessment of bone-regeneration non-invasively.

nCP:Fe-A Biomineral Magnetic Nanocontrast Agent for Tracking Implanted Stem Cells in Brain Using MRI.

In vivo tracking of transplanted stem cells to monitor their migration, biodistribution, and engraftment in the host tissue is important for assessing the efficacy of stem cell therapeutics. Here, we report a biomineral nanocontrast agent, iron doped calcium phosphate nanoparticles (nCP:Fe), for the in vivo tracking of stem cells in brain using magnetic resonance imaging (MRI). We have synthesized ∼100 nm sized nCP nanoparticles doped with 9.81 wt % Fe3+. In vitro studies using mesenchymal stem cells (MSCs) showed excellent biocompatibility for nCP:Fe with ∼87% labeling efficiency under optimized conditions (100 µg/mL, 6 h). Most importantly, the labeling was not found to affect the neurogenic differentiation potential of MSCs. MRI of labeled cells (∼22.34 pg Fe/cell) showed significant reduction in T2 relaxation time from 195 to 89 ms, rendering dark contrast. In vivo transplantation of labeled cells (1 × 106 cells) in external capsule of healthy rat brain showed a clearly distinguishable hypointense (dark) region in T2 weighted MR images, which remained visible up to 30 days. Subsequently, MRI tracking of labeled MSCs transplanted intracerebrally, 3 mm near to the LPS induced inflammatory site in brain, showed successful migration of labeled MSCs toward the site of inflammation. The cell migration was confirmed ex vivo by Prussian-blue (Fe3+) and Alizarin-red (Ca2+) staining of tissue sections, where individual cells were found migrated to the site of inflammation over a period of 30 days. In summary, our results clearly show that, as a biocompatible mineral composition, nCP:Fe is a promising magnetic nanocontrast agent for MRI based cell tracking in vivo.

Brain-Tumor-Regenerating 3D Scaffold-Based Primary Xenograft Models for Glioma Stem Cell Targeted Drug Screening.

Glioma stem cells (GSC) present a critical therapeutic challenge for glioblastoma multiforme (GBM). Drug screening against GSC demands development of novel in vitro and in vivo platforms that can mimic brain microenvironment and support GSC maintenance and tumorigenesis. Here, we report, a 3-dimensionel (3D) biomimetic macro-porous scaffold developed by incorporating hyaluronic acid, porcine brain extra cellular matrix (ECM) and growth factors that facilitates regeneration of GBM from primary GSCs, ex vivo and in vivo. After characterizing with human and rat GBM cell lines and neurospheres, human GSCs expressing Notch1, Sox-2, Nestin, and CD133 biomarkers were isolated from GBM patients, cultured in the 3D scaffold, and implanted subcutaneously in nude mice to develop patient derived xenograft (PDX) models. Aggressive growth pattern of PDX with formation of intratumoral vascularization was monitored by magnetic resonance imaging (MRI). Histopathological and phenotypial features of the original tumors were retained in the PDX models. We used this regenerated GBM platform to screen novel siRNA nanotherapeutics targeting Notch, Sox-2, FAK signaling for its ability to inhibit the tumorigenic potential of GSCs. Current clinical drug, Temozolomide and an anticancer phytochemical, nanocurcumin, were used as controls. The siRNA nanoparticles showed excellent efficacy in inhibiting tumorigenesis by GSCs in vivo. Our study suggests that the brain-ECM mimicking scaffold can regenerate primary gliomas from GSCs in vitro and in vivo, and the same can be used as an effective platform for screening drugs against glioma stem cells.