Prakash Gangadaran

Fascin overexpression promotes neoplastic progression in oral squamous cell carcinoma

BackgroundFascin is a globular actin cross-linking protein, which plays a major role in forming parallel actin bundles in cell protrusions and is found to be associated with tumor cell invasion and metastasis in various type of cancers including oral squamous cell carcinoma (OSCC). Previously, we have demonstrated that fascin regulates actin polymerization and thereby promotes cell motility in K8-depleted OSCC cells. In the present study we have investigated the role of fascin in tumor progression of OSCC.MethodsTo understand the role of fascin in OSCC development and/or progression, fascin was overexpressed along with vector control in OSCC derived cells AW13516. The phenotype was studied using wound healing, Boyden chamber, cell adhesion, Hanging drop, soft agar and tumorigenicity assays. Further, fascin expression was examined in human OSCC samples (N = 131) using immunohistochemistry and level of its expression was correlated with clinico-pathological parameters of the patients.ResultsFascin overexpression in OSCC derived cells led to significant increase in cell migration, cell invasion and MMP-2 activity. In addition these cells demonstrated increased levels of phosphorylated AKT, ERK1/2 and JNK1/2. Our in vitro results were consistent with correlative studies of fascin expression with the clinico-pathological parameters of the OSCC patients. Fascin expression in OSCC showed statistically significant correlation with increased tumor stage (P = 0.041), increased lymph node metastasis (P = 0.001), less differentiation (P = 0.005), increased recurrence (P = 0.038) and shorter survival (P = 0.004) of the patients.ConclusionIn conclusion, our results indicate that fascin promotes tumor progression and activates AKT and MAPK pathways in OSCC-derived cells. Further, our correlative studies of fascin expression in OSCC with clinico-pathological parameters of the patients indicate that fascin may prove to be useful in prognostication and treatment of OSCC.

Loss of keratin 8 phosphorylation leads to increased tumor progression and correlates with clinico-pathological parameters of OSCC patients

Background Keratins are cytoplasmic intermediate filament proteins expressed in tissue specific and differentiation dependent manner. Keratins 8 and 18 (K8 and K18) are predominantly expressed in simple epithelial tissues and perform both mechanical and regulatory functions. Aberrant expression of K8 and K18 is associated with neoplastic progression, invasion and poor prognosis in human oral squamous cell carcinomas (OSCCs). K8 and K18 undergo several post-translational modifications including phosphorylation, which are known to regulate their functions in various cellular processes. Although, K8 and K18 phosphorylation is known to regulate cell cycle, cell growth and apoptosis, its significance in cell migration and/or neoplastic progression is largely unknown. In the present study we have investigated the role of K8 phosphorylation in cell migration and/or neoplastic progression in OSCC. Methodology and Principal Findings To understand the role of K8 phosphorylation in neoplastic progression of OSCC, shRNA-resistant K8 phospho-mutants of Ser73 and Ser431 were overexpressed in K8-knockdown human AW13516 cells (derived from SCC of tongue; generated previously). Wound healing assays and tumor growth in NOD-SCID mice were performed to analyze the cell motility and tumorigenicity respectively in overexpressed clones. The overexpressed K8 phospho-mutants clones showed significant increase in cell migration and tumorigenicity as compared with K8 wild type clones. Furthermore, loss of K8 Ser73 and Ser431 phosphorylation was also observed in human OSCC tissues analyzed by immunohistochemistry, where their dephosphorylation significantly correlated with size, lymph node metastasis and stage of the tumor. Conclusion and Significance Our results provide first evidence of a potential role of K8 phosphorylation in cell migration and/or tumorigenicity in OSCC. Moreover, correlation studies of K8 dephosphorylation with clinico-pathological parameters of OSCC patients also suggest its possible use in prognostication of human OSCC.

In Vivo therapeutic potential of mesenchymal stem cell-derived extracellular vesicles with optical imaging reporter in tumor mice model

Mesenchymal stem cells (MSCs) can be used as a therapeutic armor for cancer. Extracellular vesicles (EVs) from MSCs have been evaluated for anticancer effects. In vivo targeting of EVs to the tumor is an essential requirement for successful therapy. Therefore, non-invasive methods of monitoring EVs in animal models are crucial for developing EV-based cancer therapies. The present study to develop bioluminescent EVs using Renilla luciferase (Rluc)-expressing MSCs. The EVs from MSC/Rluc cells (EV-MSC/Rluc) were visualized in a murine lung cancer model. The anticancer effects of EVs on Lewis lung carcinoma (LLC) and other cancer cells were assessed. EV-MSC/Rluc were visualized in vivo in the LLC-efffuc tumor model using optical imaging. The induction of apoptosis was confirmed with Annexin-V and propidium iodide staining. EV-MSC/Rluc and EV-MSCs showed a significant cytotoxic effect against LLC-effluc cells and 4T1; however, no significant effect on CT26, B16F10, TC1 cells. Moreover, EV-MSC/Rluc inhibited LLC tumor growth in vivo. EV-MSC/Rluc-mediated LLC tumor inhibitory mechanism revealed the decreased pERK and increased cleaved caspase 3 and cleaved PARP. We successfully developed luminescent EV-MSC/Rluc that have a therapeutic effect on LLC cells in both in vitro and in vivo. This bioluminescent EV system can be used to optimize EV-based therapy.

Exosomes Derived From Natural Killer Cells Exert Therapeutic Effect in Melanoma

Objective: Exosomes are nanovesicles that are released from normal and tumor cells and are detectable in cell culture supernatant and human biological fluids. Although previous studies have explored exosomes released from cancer cells, little is understood regarding the functions of exosomes released by normal cells. Natural killer (NK) cells display rapid immunity to metastatic or hematological malignancies, and efforts have been undertaken to clinically exploit the antitumor properties of NK cells. However, the characteristics and functions of exosomes derived from NK cells remain unknown. In this study, we explored NK cell-derived exosome-mediated antitumor effects against aggressive melanoma in vitro and in vivo. Methods: B16F10 cells were transfected with enhanced firefly luciferase (effluc) and thy1.1 genes, and thy1.1-positive cells were immunoselected using microbeads. The resulting B16F10/effluc cells were characterized using reverse transcriptase polymerase chain reaction (RT-PCR), western blotting, and luciferase activity assays. Exosomes derived from NK-92MI cells (NK-92 Exo) were isolated by ultracentrifugation and density gradient ultracentrifugation. NK-92 Exo were characterized by transmission electron microscopy and western blotting. We also performed an enzyme-linked immunosorbent assay to measure cytokines retained in NK-92 Exo cells. The in vitro cytotoxicity of NK-92 Exo against the cancer cells was determined using a bioluminescence imaging system (BLI) and CCK-8 assays. To investigate the possible side effects of NK-92 Exo on healthy cells, we also performed the BLI and CCK-8 assays using the human kidney Phoenix™-Ampho cell line. Flow cytometry and western blotting confirmed that NK-92 Exo induced apoptosis in the B16F10/effluc cells. In vivo, we used a B16F10/effluc cell xenograft model to detect the immunotherapeutic effect of NK-92 Exo. We injected NK-92 Exo into tumors, and tumor growth progression was monitored using the IVIS Lumina imaging system and ultrasound imaging. Tumor mass was monitored after in vivo experiments. Results: RT-PCR and western blotting confirmed effluc gene expression and protein levels in B16F10/effluc cells. B16F10/effluc activity was found to increase with increasing cell numbers, using BLI assay. For NK-92 Exo characterization, western blotting was performed on both ultracentrifuged and density gradient-isolated exosomes. The results confirmed that NK cell-derived exosomes express two typical exosome proteins, namely CD63 and ALIX. We demonstrated by western blot analysis that NK-92 Exo presented two functional NK proteins, namely perforin and FasL. Moreover, we confirmed the membrane expression of FasL. The enzyme-linked immunosorbent assay results indicated that NK-92 Exo can secrete tumor necrosis factor (TNF)-α, which affected the cell proliferation signaling pathway. The antitumor effect of NK-92 Exo against B16F10/effluc cells in vitro was confirmed by BLI (p < 0.001) and CCK-8 assays (p < 0.001). Furthermore, in normal healthy cells, even after 24 h of co-culture, NK-92 Exo did not exhibit significant side effects. In the in vivo experiments, tumors in the vehicle control group were significantly increased, compared with those in the NK-92 Exo-treated group (p < 0.05). Conclusion: The results of the current study suggest that exosomes derived from NK cells exert cytotoxic effects on melanoma cells and thus warrant further development as a potential immunotherapeutic strategy for cancer.

Current Perspectives on In Vivo Noninvasive Tracking of Extracellular Vesicles with Molecular Imaging

Clinical and preclinical in vivo tracking of extracellular vesicles (EVs) are a crucial tool for the development and optimization of EV-based diagnosis and treatment. EVs have gained interest due to their unique properties that make them excellent candidates for biological applications. Noninvasive in vivo EV tracking has allowed marked progress towards elucidating the mechanisms and functions of EVs in real time in preclinical and clinical studies. In this review, we summarize several molecular imaging methods that deal with EVs derived from different cells, which have allowed investigations of EV biodistribution, as well as their tracking, delivery, and tumor targeting, to determine their physiological functions and to exploit imaging-derived information for EV-based theranostics.

Advances in Molecular Imaging Strategies for In Vivo Tracking of Immune Cells

Tracking of immune cells in vivo is a crucial tool for development and optimization of cell-based therapy. Techniques for tracking immune cells have been applied widely for understanding the intrinsic behavior of immune cells and include non-radiation-based techniques such as optical imaging and magnetic resonance imaging (MRI), radiation-based techniques such as computerized tomography (CT), and nuclear imaging including single photon emission computerized tomography (SPECT) and positron emission tomography (PET). Each modality has its own strengths and limitations. To overcome the limitations of each modality, multimodal imaging techniques involving two or more imaging modalities are actively applied. Multimodal techniques allow integration of the strengths of individual modalities. In this review, we discuss the strengths and limitations of currently available preclinical in vivo immune cell tracking techniques and summarize the value of immune cell tracking in the development and optimization of immune cell therapy for various diseases.

Natural Killer Cell (NK-92MI)-Based Therapy for Pulmonary Metastasis of Anaplastic Thyroid Cancer in a Nude Mouse Model

Objective Natural killer (NK) cells represent the third largest population of lymphocytes, and they play an important role in immune surveillance against tumors. The lungs are a common metastatic site for anaplastic thyroid cancer (ATC), and metastasis is one of the most frequent causes of mortality in this type of cancer. In the current study, we evaluated the effects of NK cell-based immunotherapy for pulmonary metastasis of ATC and determined how it affects the effector molecules of NK cells. Methods Human NK cells (NK-92MI) were retrovirally transduced to express the effluc gene. Human ATC cells (CAL-62) were transduced with the effluc and Rluc genes. The cytotoxicity of NK cells against CAL-62 cells was assessed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay system. Pulmonary metastases of ATC were developed by i.v. injection of CAL-62, and metastasis growth was monitored using bioluminescence imaging (BLI). To treat the metastases, five million NK-92MI cells were injected twice into the caudal vein of nude mice. To assess the targetability of NK cells to ATC tumors, NK-92MI cells expressing the effluc gene (NK/F) were administered through the tail vein of nude mice with a pulmonary metastasis or tumor xenograft. BLI was subsequently performed at 1, 3, 24, and 48 h. Results NK/F and CAL-62 cells expressing the effluc or Rluc gene (CAL-62/F, CAL-62/R) were successfully established. Expression of the effluc and Rluc genes in NK/F, CAL-62/F, and CAL-62/R cells was verified by RT-polymerase chain reaction, western blotting, and luciferase assay. After coculture of NK-92MI and CAL-62/F cells for 24 h, the BLI signal intensity of CAL-62/F cells proportionally decreased with the number of cocultured NK cells. An ATC pulmonary metastasis mouse model was successfully generated, and NK cells significantly inhibited the growth of the metastasis (p < 0.01). The NK/F cells exhibited targetability to the pulmonary metastasis and tumor xenograft in the mouse model. Conclusion The results of present study suggest that NK cells are able to target ATC tumors and that NK cell-based immunotherapy may serve as an effective therapeutic approach for pulmonary metastases of ATC.

Molecular Imaging: A Useful Tool for the Development of Natural Killer Cell-Based Immunotherapies

Molecular imaging is a relatively new discipline that allows visualization, characterization, and measurement of the biological processes in living subjects, including humans, at a cellular and molecular level. The interaction between cancer cells and natural killer (NK) cells is complex and incompletely understood. Despite our limited knowledge, progress in the search for immune cell therapies against cancer could be significantly improved by dynamic and non-invasive visualization and tracking of immune cells and by visualization of the response of cancer cells to therapies in preclinical and clinical studies. Molecular imaging is an essential tool for these studies, and a multimodal molecular imaging approach can be applied to monitor immune cells in vivo, for instance, to visualize therapeutic effects. In this review, we discuss the usefulness of NK cells in cancer therapies and the preclinical and clinical usefulness of molecular imaging in NK cell-based therapies. Furthermore, we discuss different molecular imaging modalities for use with NK cell-based therapies, and their preclinical and clinical applications in animal and human subjects. Molecular imaging has contributed to the development of NK cell-based therapies against cancers in animal models and to the refinement of current cell-based cancer immunotherapies. Developing sensitive and reproducible non-invasive molecular imaging technologies for in vivo NK cell monitoring and for real-time assessment of therapeutic effects will accelerate the development of NK cell therapies.

Extracellular vesicles from mesenchymal stem cells activates VEGF receptors and accelerates recovery of hindlimb ischemia

&NA; Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are potential therapies for various diseases, but their angiogenic mechanisms of therapeutic efficacy remain unclear. Here, we describe how MSC‐EVs, activates VEGF receptors and downstream angiogenesis pathways. Mouse MSC‐EVs were isolated from cell culture medium and characterized using transmission electron microscopy, nanoparticle analysis, and western blotting. In vitro migration, proliferation, and tube formation assays using endothelial cells were used to assess the angiogenic potential of MSC‐EVs, and revealed higher levels of cellular migration, proliferation, and tube formation after treatment. qRT‐PCR and western blotting (WB) revealed higher protein and mRNA expression of the angiogenic genes VEGFR1 and VEGFR2 in mouse SVEC‐4 endothelial cells after MSC‐EVs treatment. Additionally, other vital pro‐angiogenic pathways (SRC, AKT, and ERK) were activated by in vitro MSC‐EV treatment. WB and qRT‐PCR revealed enriched presence of VEGF protein and miR‐210‐3p in MSC‐EV. The hindlimb ischemia mouse model was established and MSC‐EVs with or without Matrigel (EV‐MSC + Gel) were injected into the ischemic area and blood reperfusion was monitored using molecular imaging techniques. The in vivo administration of MSC‐EVs increased both blood reperfusion and the formation of new blood vessels in the ischemic limb, with the addition of matrigel enhancing this effect further by releasing EVs slowly. MSC‐EVs enhance angiogenesis in ischemic limbs, most likely via the overexpression of VEGFR1 and VEGFR2 in endothelial cells. These findings reveal a novel mechanism of activating receptors by MSC‐EVs influence the angiogenesis. Graphical abstract Figure. No caption available.

Role of pulmonary macrophages in initiation of lung metastasis in anaplastic thyroid cancer.

Several clinical studies have demonstrated that increased macrophage infiltration into tumors confers metastatic potential and poor prognosis in cancer. Preclinical studies are needed to develop new strategies for countering metastasis. Our study was designed to investigate the impact of pulmonary macrophages on lung metastasis of anaplastic thyroid cancer (ATC). ATC (CAL‐62) and macrophage (Raw264.7) were transfected with the effluc (CAL‐62/effluc, Raw264.7/effluc). Coculture and migration assays were used to assess the effect of Raw264.7 or THP1 (human macrophage) (or conditioned medium) on the proliferation and/or migration of CAL‐62/effluc cells in vitro. The effect of clodro‐lipo or PBS‐lipo on macrophage depletion was confirmed in vitro and in vivo. CAL‐62/effluc cells (1 × 106) were intravenously injected into nude mice 24 h after clodro‐lipo or PBS‐lipo administration. Effect of clodro‐lipo on the lung metastasis of CAL‐62/effluc was assessed by bioluminescence imaging (BLI). Micro computed tomography (micro‐CT) and histology. BLI signals of CAL‐62/effluc and Raw264.7/effluc increased to cell number. Raw264.7 cells and THP1 cells promoted CAL‐62/effluc proliferation, and conditioned medium of Raw264.7 cells promoted CAL‐62/effluc migration. Clodro‐lipo significantly depleted pulmonary macrophages in vitro and in vivo. Intensity of BLI signals in ATC lung metastasis was weaker in the clodro‐lipo group than PBS‐lipo control. Micro‐CT imaging and hematoxylin/eosin staining revealed smaller tumor masses in the clodro‐lipo group than PBS‐lipo control. Our findings indicate that pulmonary macrophages have an important role in initiation of lung metastasis of ATC. New therapeutic strategies that preclude initiation of pulmonary metastasis could potentially be developed by targeting pulmonary macrophages.