Ultraviolet-A light increases mitochondrial anti-viral signaling protein in confluent human tracheal cells via cell-cell signaling.

Mitochondrial antiviral signaling (MAVS) protein mediates innate antiviral responses, including responses to certain coronaviruses such as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We have previously shown that ultraviolet-A (UVA) therapy can prevent virus-induced cell death in human ciliated tracheal epithelial cells (HTEpC) infected with coronavirus-229E (CoV-229E), and results in increased intracellular levels of MAVS. In this study, we explored the mechanisms by which UVA light can activate MAVS, and whether local UVA light application can activate MAVS at locations distant from the light source (e.g. via cell-to-cell communication). MAVS levels were compared in HTEpC exposed to 2 mW/cm2 narrow band (NB)-UVA for 20 min and in unexposed controls at 30-40% and at 100% confluency, and in unexposed HTEpC treated with supernatants or lysates from UVA-exposed cells or from unexposed controls. MAVS was also assessed in different sections of confluent monolayer plates where only one section was exposed to NB-UVA. Our results showed that UVA increases the expression of MAVS protein. Further, cells in a confluent monolayer exposed to UVA conferred an elevation in MAVS in cells adjacent to the exposed section, and also in cells in the most distant sections which were not exposed to UVA. In this study, human ciliated tracheal epithelial cells exposed to UVA demonstrate increased MAVS protein, and also appear to transmit this influence to confluent cells not exposed to UVA, likely via cell-cell signaling.

4-Methylumbelliferone administration enhances radiosensitivity of human fibrosarcoma by intercellular communication.

Hyaluronan synthesis inhibitor 4-methylumbelliferone (4-MU) is a candidate of radiosensitizers which enables both anti-tumour and anti-metastasis effects in X-ray therapy. The curative effects under such 4-MU administration have been investigated in vitro; however, the radiosensitizing mechanisms remain unclear. Here, we investigated the radiosensitizing effects under 4-MU treatment from cell experiments and model estimations. We generated experimental surviving fractions of human fibrosarcoma cells (HT1080) after 4-MU treatment combined with X-ray irradiation. Meanwhilst, we also modelled the pharmacological effects of 4-MU treatment and theoretically analyzed the synergetic effects between 4-MU treatment and X-ray irradiation. The results show that the enhancement of cell killing by 4-MU treatment is the greatest in the intermediate dose range of around 4 Gy, which can be reproduced by considering intercellular communication (so called non-targeted effects) through the model analysis. As supposed to be the involvement of intercellular communication in radiosensitization, the oxidative stress level associated with reactive oxygen species (ROS), which leads to DNA damage induction, is significantly higher by the combination of 4-MU treatment and irradiation than only by X-ray irradiation, and the radiosensitization by 4-MU can be suppressed by the ROS inhibitors. These findings suggest that the synergetic effects between 4-MU treatment and irradiation are predominantly attributed to intercellular communication and provide more efficient tumour control than conventional X-ray therapy.

Exosome-mediated radiosensitizing effect on neighboring cancer cells via increase in intracellular levels of reactive oxygen species.

The precise mechanism of intercellular communication between cancer cells following radiation exposure is unclear. Exosomes are membrane­enclosed small vesicles comprising lipid bilayers and are mediators of intercellular communication that transport a variety of intracellular components, including microRNAs (miRNAs or miRs). The present study aimed to identify novel roles of exosomes released from irradiated cells to neighboring cancer cells. In order to confirm the presence of exosomes in the human pancreatic cancer cell line MIAPaCa­2, ultracentrifugation was performed followed by transmission electron microscopy and nanoparticle tracking analysis (NanoSight) using the exosome­specific surface markers CD9 and CD63. Subsequent endocytosis of exosomes was confirmed by fluorescent microscopy. Cell survival following irradiation and the addition of exosomes was evaluated by colony forming assay. Expression levels of miRNAs in exosomes were then quantified by microarray analysis, while protein expression levels of Cu/Zn­ and Mn­superoxide dismutase (SOD1 and 2, respectively) enzymes in MIAPaCa­2 cells were evaluated by western blotting. Results showed that the uptake of irradiated exosomes was significantly higher than that of non­irradiated exosomes. Notably, irradiated exosomes induced higher intracellular levels of reactive oxygen species (ROS) and a higher frequency of DNA damage in MIAPaCa­2 cells, as determined by fluorescent microscopy and immunocytochemistry, respectively. Moreover, six up­ and five downregulated miRNAs were identified in 5 and 8 Gy­irradiated cells using miRNA microarray analyses. Further analysis using miRNA mimics and reverse transcription­quantitative PCR identified miR­6823­5p as a potential candidate to inhibit SOD1, leading to increased intracellular ROS levels and DNA damage. To the best of our knowledge, the present study is the first to demonstrate that irradiated exosomes enhance the radiation effect via increasing intracellular ROS levels in cancer cells. This contributes to improved understanding of the bystander effect of neighboring cancer cells.

Exosome-mediated cellular crosstalk within the tumor microenvironment upon irradiation.

Radiotherapy is one of the most effective treatment methods for various solid tumors. Bidirectional signal transduction between cancer cells and stromal cells within the irradiated microenvironment is important in cancer development and treatment responsiveness. Exosomes, initially considered as "garbage bins" for unwanted from cells, are now understood to perform a variety of functions in interactions within the tumor microenvironment. Exosome-mediated regulation processes are rebuilt under the irradiation stimuli, because the exosome production, uptake, and contents are markedly modified by irradiation. In turn, irradiation-modified exosomes may modulate the cell response to irradiation through feedback regulation. Here, we review current knowledge and discuss the roles of exosome-mediated interactions between cells under radiotherapy conditions.

Magnetic Stimulation Drives Macrophage Polarization in Cell to-Cell Communication with IL-1ß Primed Tendon Cells.

Inflammation is part of the natural healing response, but it has been simultaneously associated with tendon disorders, as persistent inflammatory events contribute to physiological changes that compromise tendon functions. The cellular interactions within a niche are extremely important for healing. While human tendon cells (hTDCs) are responsible for the maintenance of tendon matrix and turnover, macrophages regulate healing switching their functional phenotype to environmental stimuli. Thus, insights on the hTDCs and macrophages interactions can provide fundamental contributions on tendon repair mechanisms and on the inflammatory inputs in tendon disorders. We explored the crosstalk between macrophages and hTDCs using co-culture approaches in which hTDCs were previously stimulated with IL-1ß. The potential modulatory effect of the pulsed electromagnetic field (PEMF) in macrophage-hTDCs communication was also investigated using the magnetic parameters identified in a previous work. The PEMF influences a macrophage pro-regenerative phenotype and favors the synthesis of anti-inflammatory mediators. These outcomes observed in cell contact co-cultures may be mediated by FAK signaling. The impact of the PEMF overcomes the effect of IL-1ß-treated-hTDCs, supporting PEMF immunomodulatory actions on macrophages. This work highlights the relevance of intercellular communication in tendon healing and the beneficial role of the PEMF in guiding inflammatory responses toward regenerative strategies.

Radiation with STAT3 Blockade Triggers Dendritic Cell-T cell Interactions in the Glioma Microenvironment and Therapeutic Efficacy.

PURPOSE: Patients with central nervous system (CNS) tumors are typically treated with radiotherapy, but this is not curative and results in the upregulation of phosphorylated STAT3 (p-STAT3), which drives invasion, angiogenesis, and immune suppression. Therefore, we investigated the combined effect of an inhibitor of STAT3 and whole-brain radiotherapy (WBRT) in a murine model of glioma. EXPERIMENTAL DESIGN: C57BL/6 mice underwent intracerebral implantation of GL261 glioma cells, WBRT, and treatment with WP1066, a blood-brain barrier-penetrant inhibitor of the STAT3 pathway, or the two in combination. The role of the immune system was evaluated using tumor rechallenge strategies, immune-incompetent backgrounds, immunofluorescence, immune phenotyping of tumor-infiltrating immune cells (via flow cytometry), and NanoString gene expression analysis of 770 immune-related genes from immune cells, including those directly isolated from the tumor microenvironment. RESULTS: The combination of WP1066 and WBRT resulted in long-term survivors and enhanced median survival time relative to monotherapy in the GL261 glioma model (combination vs. control P < 0.0001). Immunologic memory appeared to be induced, because mice were protected during subsequent tumor rechallenge. The therapeutic effect of the combination was completely lost in immune-incompetent animals. NanoString analysis and immunofluorescence revealed immunologic reprograming in the CNS tumor microenvironment specifically affecting dendritic cell antigen presentation and T-cell effector functions. CONCLUSIONS: This study indicates that the combination of STAT3 inhibition and WBRT enhances the therapeutic effect against gliomas in the CNS by inducing dendritic cell and T-cell interactions in the CNS tumor.

Coupling of melanocyte signaling and mechanics by caveolae is required for human skin pigmentation.

Tissue homeostasis requires regulation of cell-cell communication, which relies on signaling molecules and cell contacts. In skin epidermis, keratinocytes secrete factors transduced by melanocytes into signaling cues promoting their pigmentation and dendrite outgrowth, while melanocytes transfer melanin pigments to keratinocytes to convey skin photoprotection. How epidermal cells integrate these functions remains poorly characterized. Here, we show that caveolae are asymmetrically distributed in melanocytes and particularly abundant at the melanocyte-keratinocyte interface in epidermis. Caveolae in melanocytes are modulated by ultraviolet radiations and keratinocytes-released factors, like miRNAs. Preventing caveolae formation in melanocytes increases melanin pigment synthesis through upregulation of cAMP signaling and decreases cell protrusions, cell-cell contacts, pigment transfer and epidermis pigmentation. Altogether, we identify that caveolae serve as molecular hubs that couple signaling outputs from keratinocytes to mechanical plasticity of pigment cells. The coordination of intercellular communication and contacts by caveolae is thus crucial to skin pigmentation and tissue homeostasis.

MicroRNA Profile of Exosomes and Parental Cells is Differently Affected by Ionizing Radiation.

Exosomes are key mediators of cell-to-cell communication involved in different aspects of the response to ionizing radiation. The functional role of exosomes depends on their molecular cargo, including protein and miRNA content. In this work, we compared the miRNA profile of cells exposed to a high-dose of radiation and the exosomes released by those cells. FaDu cells (derived from human head and neck cancer) were exposed to 2 and 8 Gy doses, exosomes were purified from culture media at 36 h postirradiation using a combination of differential centrifugation, ultrafiltration and precipitation, then microRNA was analyzed using the RNA-seq approach. There were 439 miRNA species quantified, and significant differences in their relative abundance were observed between the cells and exosomes; several low-abundance miRNAs were over-represented while high-abundance miRNA were under-represented in exosomes. There were a few miRNA species markedly affected in irradiated cells and in exosomes released by these cells. However, markedly different radiation-induced effects were observed in both miRNA sets, which could be exemplified by miR-3168 significantly downregulated in cells and upregulated in exosomes. On the other hand, both 2 and 8 Gy radiation doses induced similar effects. Radiation-affected miRNA species present in exosomes are linked to genes involved in the DNA damage and cytokine-mediated response, which may suggest their hypothetical role in the exosome-mediated radiation-induced bystander effect reported elsewhere.

Caveolin-1 regulates the ASMase/ceramide-mediated radiation response of endothelial cells in the context of tumor-stroma interactions.

The integral membrane protein caveolin-1 (CAV1) plays a central role in radioresistance-mediating tumor-stroma interactions of advanced prostate cancer (PCa). Among the tumor-stroma, endothelial cells (EC) evolved as critical determinants of the radiation response. CAV1 deficiency in angiogenic EC was already shown to account for increased apoptosis rates of irradiated EC. This study explores the potential impact of differential CAV1 levels in EC on the acid sphingomyelinase (ASMase)/ceramide pathway as a key player in the regulation of EC apoptosis upon irradiation and cancer cell radioresistance. Enhanced apoptosis sensitivity of CAV1-deficient EC was associated with increased ASMase activity, ceramide generation, formation of large lipid platforms, and finally an altered p38 mitogen-activated protein kinase (MAPK)/heat-shock protein 27 (HSP27)/AKT (protein kinase B, PKB) signaling. CAV1-deficient EC increased the growth delay of LNCaP and PC3 PCa cells upon radiation treatment in direct 3D spheroid co-cultures. Exogenous C6 and C16 ceramide treatment in parallel increased the growth delay of PCa spheroids and induced PCa cell apoptosis. Analysis of the respective ceramide species in PCa cells with increased CAV1 levels like those typically found in radio-resistant advanced prostate tumors further revealed an upregulation of unsaturated C24:1 ceramide that might scavenge the effects of EC-derived apoptosis-inducing C16 ceramide. Higher ASMase as well as ceramide levels could be confirmed by immunohistochemistry in human advanced prostate cancer specimen bearing characteristic CAV1 tumor-stroma alterations. Conclusively, CAV1 critically regulates the generation of ceramide-dependent (re-)organization of the plasma membrane that in turn affects the radiation response of EC and adjacent PCa cells. Understanding the CAV1-dependent crosstalk between tumor cells and the host-derived tumor microvasculature and its impact on radiosensitivity may allow to define a rational strategy for overcoming tumor radiation resistance improving clinical outcomes by targeting CAV1.

Therapeutic modulators of hepatic stellate cells for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common type of primary tumor in the liver and is a leading cause of cancer-related death worldwide. Activated hepatic stellate cells (HSCs) are key components of the HCC microenvironment and play an important role in the onset and progression of HCC through the secretion of growth factors and cytokines. Current treatment modalities that include chemotherapy, radiotherapy and ablation are able to activate HSCs and remodel the tumor microenvironment. Growing evidence has demonstrated that the complex interaction between activated HSCs and tumor cells can facilitate cancer chemoresistance and metastasis. Therefore, therapeutic targeting of activated HSCs has emerged as a promising strategy to improve treatment outcomes for HCC. This review summarizes the molecular mechanisms of HSC activation triggered by treatment modalities, the function of activated HSCs in HCC, as well as the crosstalk between tumor cells and activated HSCs. Pathways of activated HSC reduction are discussed, including inhibition, apoptosis, and reversion to the inactivated state. Finally, we outline the progress and challenges of therapeutic approaches targeting activated HSCs in the development of HCC treatment.

Tumor-derived extracellular vesicles: insights into bystander effects of exosomes after irradiation.

This review article aims to address the kinetic of TDEs in cancer cells pre- and post-radiotherapy. Radiotherapy is traditionally used for the treatment of multiple cancer types; however, there is growing evidence to show that radiotherapy exerts NTEs on cells near to the irradiated cells. In tumor mass, irradiated cells can affect non-irradiated cells in different ways. Of note, exosomes are nano-scaled cell particles releasing from tumor cells and play key roles in survival, metastasis, and immunosuppression of tumor cells. Recent evidence indicated that irradiation has the potential to affect the dynamic of different signaling pathways such as exosome biogenesis. Indeed, exosomes act as intercellular mediators in various cell communication through transmitting bio-molecules. Due to their critical roles in cancer biology, exosomes are at the center of attention. TDEs contain an exclusive molecular signature that they may serve as tumor biomarker in the diagnosis of different cancers. Interestingly, radiotherapy and IR could also contribute to altering the dynamic of exosome secretion. Most probably, the content of exosomes in irradiated cells is different compared to exosomes originated from the non-irradiated BCs. Irradiated cells release exosomes with exclusive content that mediate NTEs in BCs. Considering variation in cell type, IR doses, and radio-resistance or radio-sensitivity of different cancers, there is, however, contradictions in the feature and activity of irradiated exosomes on neighboring cells.

Interleukin-1 alpha derived from ultraviolet B-exposed keratinocytes is associated with a decrease of endocytic collagen receptor Endo180.

BACKGROUND: Endo180 contributes to the remodeling of the collagen fibers that comprise the dermal matrix due to the internalization of extracellular collagen fragments. In the sun-exposed elder skin, an accumulation of collagen fragments was observed in the dermal matrix which was associated with a reduction in Endo180 in the dermal fibroblasts. This suggests that the loss of Endo180 results in the accumulation of collagen fragments in the surrounding fibroblasts and causes interference with dermal matrix remodeling via collagen fibers. The purpose of the study was to identify a mechanism by which ultraviolet B (UVB) exposure induces a loss of Endo 180 with a specific focus on the crosstalk between keratinocytes and fibroblasts. METHODS: Endo180 from normal human dermal fibroblasts, which were cultured with a conditioned medium (CM) of UVB-exposed keratinocytes, was examined using mRNA expression, protein levels and collagen internalization by quantitative RT-PCR, ELISA, and flow cytometry, respectively. RESULTS: Although UVB irradiation to fibroblasts failed to reduce Endo180, the CM of UVB-exposed keratinocytes reduced Endo180 in the fibroblasts. Collagen internalization into the fibroblasts was decreased and was associated with a loss of Endo180. Among cytokines secreted from UVB-exposed keratinocytes, IL-1α solely reduced Endo180, and the reduction induced by the CM of UVB-exposed keratinocytes was abolished by the presence of IL-1RA. CONCLUSIONS: These results indicate that a substance secreted from UVB-exposed keratinocytes regulates Endo180 expression and that IL-1α may play an important role in the maintenance of Endo180.

Perspectives of cellular communication through tunneling nanotubes in cancer cells and the connection to radiation effects.

Direct cell-to-cell communication is crucial for the survival of cells in stressful situations such as during or after radiation exposure. This communication can lead to non-targeted effects, where non-treated or non-infected cells show effects induced by signal transduction from non-healthy cells or vice versa. In the last 15 years, tunneling nanotubes (TNTs) were identified as membrane connections between cells which facilitate the transfer of several cargoes and signals. TNTs were identified in various cell types and serve as promoter of treatment resistance e.g. in chemotherapy treatment of cancer. Here, we discuss our current understanding of how to differentiate tunneling nanotubes from other direct cellular connections and their role in the stress reaction of cellular networks. We also provide a perspective on how the capability of cells to form such networks is related to the ability to surpass stress and how this can be used to study radioresistance of cancer cells.

Effect of Ionizing Radiation on Human EA.hy926 Endothelial Cells under Inflammatory Conditions and Their Interactions with A549 Tumour Cells.

PURPOSE: Most tumours are characterized by an inflammatory microenvironment, and correlations between inflammation and cancer progression have been shown. Endothelial cells (ECs), as part of the tumour microenvironment, play a crucial role in inflammatory processes as well as in angiogenesis and could be critical targets of cancer therapy like irradiation. Therefore, in the present study we investigated the effect of ionizing radiation on endothelial cells under inflammatory conditions and their interactions with tumour cells. METHODS: Nonactivated and TNF-α treatment-activated human EC EA.hy926 were irradiated with doses between 0.1 Gy and 6 Gy with a linear accelerator. Using a multiplex assay, the accumulation of various chemokines (IL-8, MCP-1, E-selectin, and P-selectin) and soluble adhesion molecules (sICAM-1 and VCAM-1) as well as protein values of the vascular endothelial growth factor (VEGF) was measured in the supernatant at different time points. The adhesion capability of irradiated and nonirradiated A549 tumour cells to EA.hy926 cells was measured using flow cytometry, and the migration of tumour cells was investigated with a scratch motility assay. RESULTS: In contrast to unirradiated cells, IR of ECs resulted in a modified release of chemokines IL-8 and MCP-1 as well as the adhesion molecules sICAM-1 and VCAM-1 in the EC, whereas concentrations of E-selectin and P-selectin as well as VEGF were not influenced. IR always affected the adhesion capability of tumour cells to ECs with the effect dependent on the IR-treated cell type. TNF-α treatment generally increased adhesion ability of the tumour cells. Tumour cell migration was clearly inhibited after IR. This inhibitory effect was eliminated for radiation doses from 0.5 to 2 Gy when, additionally, an inflammatory environment was predominant. CONCLUSIONS: Our results support past findings suggesting that ECs, as part of the inflammatory microenvironment of tumours, are important regulators of the actual tumour response to radiation therapy.

Revisiting the Photon/Cell Interaction Mechanism in Low-Level Light Therapy.

Objective: Several reports claim that the enzyme cytochrome c oxidase (CCO) is the primary absorber for red-to-near-infrared (R-NIR) light in cells and causal for mitochondrial adenosine triphosphate (ATP) upregulation, and that pulsed R-NIR light has frequent therapeutic effects, which are superior to those of the continuous wave (CW) mode used in low-level light therapy (LLLT). Background data: Convincing evidence that the absorption of R-NIR photons by CCO is involved in mitochondrial ATP upregulations as well as a coherent explanation for the superiority of the pulsed irradiation mode is presently lacking in the literature. Methods: A comprehensive literature search and rigorous analysis of the data published on the idea that CCO is the primary absorber for R-NIR light, and of the claim that the effectivity of the pulsed irradiation mode can be derived from the absorption of R-NIR photons by CCO, reveal a number of severe inconsistencies. Results: A systematical analysis covering both the theory that CCO is the primary acceptor for R-NIR light and of its use to interpret differences between the biological effect of pulsed light and CW casts doubt on the general validity of the CCO-based hypothesis. Instead, we are offered a simple and conflict-free model accounting for both ATP upregulation and superiority of the pulsed mode in LLLT, which is in agreement with the results of recent laboratory experiments. Conclusions: CCO is not the primary acceptor for R-NIR light.

Near-Infrared Light-Activated DNA-Agonist Nanodevice for Nongenetically and Remotely Controlled Cellular Signaling and Behaviors in Live Animals.

Optogenetics provides promising tools for the precise control of receptor-mediated cell behaviors in a spatiotemporal manner. Yet, most photoreceptors require extensive genetic manipulation and respond only to ultraviolet or visible light, which are suboptimal for in vivo applications because they do not penetrate thick tissues. Here we report a novel near-infrared light-activated DNA agonist (NIR-DA) nanodevice for nongenetic manipulation of cell signaling and phenotype in deep tissues. This nanodevice is prepared by conjugating a preinactivated DNA agonist onto the gold nanorods (AuNRs). Upon NIR light treatment, the DNA agonist is released through the localized surface plasmon resonance (LSPR)-based photothermal effect of AuNRs and becomes active. The active DNA agonist dimerizes the DNA-modified chimeric or native receptor tyrosine kinase (RTK) on cell surfaces and activates downstream signal transduction in live cells. Such NIR-DA activation of RTK signaling enables the control of cytoskeletal remodeling, cell polarization, and directional migration. Furthermore, we demonstrate that the NIR-DA system can be used in vivo to mediate RTK signaling and skeletal muscle satellite cell migration and myogenesis, which are critical cellular behaviors in the process of skeletal muscle regeneration. Thus, the NIR-DA system offers a powerful and versatile platform for exogenous modulation of deep tissues for purposes such as regenerative medicine.

Collective radioresistance of T47D breast carcinoma cells is mediated by a Syncytin-1 homologous protein.

It is generally accepted that radiotherapy must target clonogenic cells, i.e., those cells in a tumour that have self-renewing potential. Focussing on isolated clonogenic cells, however, may lead to an underestimate or even to an outright neglect of the importance of biological mechanisms that regulate tumour cell sensitivity to radiation. We develop a new statistical and experimental approach to quantify the effects of radiation on cell populations as a whole. In our experiments, we change the proximity relationships of the cells by culturing them in wells with different shapes, and we find that the radiosensitivity of T47D human breast carcinoma cells in tight clusters is different from that of isolated cells. Molecular analyses show that T47D cells express a Syncytin-1 homologous protein (SyHP). We observe that SyHP translocates to the external surface of the plasma membrane of cells killed by radiation treatment. The data support the fundamental role of SyHP in the formation of intercellular cytoplasmic bridges and in the enhanced radioresistance of surviving cells. We conclude that complex and unexpected biological mechanisms of tumour radioresistance take place at the cell population level. These mechanisms may significantly bias our estimates of the radiosensitivity of breast carcinomas in vivo and thereby affect treatment plans, and they call for further investigations.

Decreased phototoxicity of photodynamic therapy by Cx32/Cx26-composed GJIC: A "Good Samaritan" effect.

BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT) has been widely used to treat malignant tumors. Our previous studies indicated that connexin (Cx) 32- and Cx26-composed gap junctional intercellular communication (GJIC) could improve the phototoxicity of PDT. However, the role of heterotypic Cx32/Cx26-formed GJIC in PDT phototoxicity is still unknown. Thus, the present study was aimed to investigate the effect of Cx32/Cx26-formed GJIC on PDT efficacy. METHODS: CCK8 assay was used to detect cell survival after PDT. Western blot assay was utilized to detect Cx32/Cx26 expression. "Parachute" dye-coupling assay was performed to measure the function of GJ channels. The intracellular Ca2+ concentrations were determined using flow cytometer. ELISA assay was performed to detect the intracellular levels of PGE2 and cAMP. RESULTS: The present study demonstrates there is a Cx32/Cx26-formed GJIC-dependent reduction of phototoxicity when cells were exposure to low concentration of Photofrin. Such a protective action is missing at low cell density due to the lack of GJ coupling. Under high-cell density condition, where there is opportunity for the cells to contact each other and form GJ, suppressing Cx32/Cx26-formed GJIC by either inhibiting the expression of Cx32/Cx26 or pretreating with GJ channel inhibitor augments PDT phototoxicity after cells were treated with at 2.5 µg/ml Photofrin. The above results suggest that at low Photofrin concentration, the presence of Cx32/Cx26-formed GJIC may decrease the phototoxicity of PDT, leading to the insensitivity of malignant cells to PDT treatment. The GJIC-mediated PDT insensitivity was associated with Ca2+ and prostaglandin E2 (PGE2 ) signaling pathways. CONCLUSION: The present study provides a cautionary note that for tumors expressing Cx32/Cx26, the presence of Cx32/Cx26-composed GJIC may cause the resistance of tumor cells to PDT. Oppositely, treatment strategies designed to downregulate the expression of Cx32/Cx26 or restrain the function of Cx32/Cx26-mediated GJIC may increase the sensitivity of malignant cell to PDT. Lasers Surg. Med. 51:301-308, 2019. © 2019 Wiley Periodicals, Inc.

Blue Light Switchable Cell-Cell Interactions Provide Reversible and Spatiotemporal Control Towards Bottom-Up Tissue Engineering.

Controlling cell-cell interactions is central for understanding key cellular processes and bottom-up tissue assembly from single cells. The challenge is to control cell-cell interactions dynamically and reversibly with high spatiotemporal precision noninvasively and sustainably. In this study, cell-cell interactions are controlled with visible light using an optogenetic approach by expressing the blue light switchable proteins CRY2 or CIBN on the surfaces of cells. CRY2 and CIBN expressing cells form specific heterophilic interactions under blue light providing precise control in space and time. Further, these interactions are reversible in the dark and can be repeatedly and dynamically switched on and off. Unlike previous approaches, these genetically encoded proteins allow for long-term expression of the interaction domains and respond to nontoxic low intensity blue light. In addition, these interactions are suitable to assemble cells into 3D multicellular architectures. Overall, this approach captures the dynamic and reversible nature of cell-cell interactions and controls them noninvasively and sustainably both in space and time. This provides a new way of studying cell-cell interactions and assembling cellular building blocks into tissues with unmatched flexibility.

BYSTANDER WI-38 CELLS MODULATE DNA DOUBLE-STRAND BREAK REPAIR IN MICROBEAM-TARGETED A549 CELLS THROUGH GAP JUNCTION INTERCELLULAR COMMUNICATION.

Bi-directional signaling involved in radiation-induced bystander effect (RIBE) between irradiated carcinoma cells and their surrounding non-irradiated normal cells is relevant to radiation cancer therapy. Using the SPICE-NIRS microbeam, we delivered 500 protons to A549-GFP lung carcinoma cells, stably expressing H2B-GFP, which were co-cultured with normal WI-38 cells. The level of γ-H2AX, a marker for DNA double-strand breaks (DSB), was subsequently measured up to 24-h post-irradiation in both targeted and bystander cells. As a result, inhibition of gap junction intercellular communication (GJIC) attenuated DSB repair in targeted A549-GFP cells, and suppressed RIBE in bystander WI-38 cells but not in distant A549-GFP cells. This suggests that GJIC plays a two-way role through propagating DNA damage effect between carcinoma to normal cells and reversing the bystander signaling, also called 'rescue effect' from bystander cells to irradiated cells, to enhance the DSB repair in targeted cells.