Macrophage-based drug delivery systems, such as macrophage-liposome conjugates (Mϕ-Lip), leverage the natural tumor-homing ability of macrophages and offer a potential solution for overcoming biological barriers and delivering chemotherapy drugs to challenging tumor regions. However, reliable platforms to assess the tumor-targeting efficiency, penetration capabilities, and therapeutic effectiveness of drug-laden macrophages remain largely unavailable. In this study, we developed a three-dimensional (3D) cell culture platform that mimics the structural and biological complexity of in vivo tumors, enabling real-time observation and analysis of Mϕ-Lip as they migrate, penetrate, and exert anti-tumor effects. Beyond evaluating the delivery process, this work focuses on the rational design and optimization of dosage regimens for co-delivering cisplatin (CDDP) and paclitaxel (Taxol) using Mϕ-Lip. Experimental results demonstrated that the drugs encapsulated within the liposomes influenced the invasive behavior of Mϕ-Lip, which in turn impacted their tumor-killing efficiency. Using this 3D cell culture platform, we identified optimal dosage regimens for co-delivering combination chemotherapy drugs through the Mϕ-Lip. This newly developed approach provides a reliable and versatile tool not only for evaluating but also for fine-tuning cell-based drug delivery strategies. It holds significant promise for advancing targeted chemotherapy strategies and improving therapeutic outcomes for solid tumors.

Both red-light (RL) illumination and direct-current electric fields (dcEFs) have independently been shown to promote neurite outgrowth in two-dimensional (2D) neural cell cultures. However, their combined effects in a three-dimensional (3D) culture environment remain unexplored. In this study, we examined the combined effects of RL and dcEFs on neurite extension in human neuroblastoma (SH-SY5Y) and mouse neuroblastoma (N2a) cells embedded within a 3D collagen gel matrix. Our results demonstrated that optimized dcEF stimulation and RL exposure significantly enhanced neurite elongation and directional alignment. RNA sequencing further identified Neuropeptide Y (NPY) and its receptors as key mediators of these effects. Moreover, electrophysiological assessments revealed that neurons subjected to the combined stimulation exhibited enhanced functional maturation. These findings provide compelling evidence supporting the potential applications of RL and dcEFs in neural regeneration and repair.

BackgroundDengue virus (DENV) causes the most significant mosquito-borne viral disease with a wide spectrum of clinical manifestation, including neurological symptoms associated with lethal dengue diseases. Dopamine receptors are expressed in central nervous system, and dopamine antagonists have been reported to exhibit antiviral activity against DENV infection in vivo and in vitro. Although identification of host-cell receptor is critical to understand dengue neuropathogenesis and neurotropism, the involvement of dopamine receptors in DENV infection remains unclear.ResultsWe exploited the sensitivity and precision of force spectroscopy to address whether dopamine type-2 receptors (D2R) directly interact with DENV particles at the first step of infection. Using optical tweezers, we quantified and characterized DENV binding to D2R expressed on Chinese hamster ovary (CHO) cells. Our finding suggested that the binding was D2R- and DENV-dependent, and that the binding force was in the range of 50-60 pN. We showed that dopamine antagonists prochlorperazine (PCZ) and trifluoperazine (TFP), previously reported to inhibit dengue infection, interrupt the DENV-D2R specific binding.ConclusionsThis study demonstrates that D2R could specifically recognize DENV particles and function as an attachment factor on cell surfaces for DENV. We propose D2R as a host receptor for DENV and as a potential therapeutic target for anti-DENV drugs.

We used pulsed electrical signals of 70 V, 1 kHz, 50 sec width for 2 hours, followed by 10 mW/cm(2)red light for 48 hours to improve neurite length in N2a cells in 3D culture.

Fibrosis and fibroblast activation usually occur in the tissues surrounding a malignant tumor; therefore, anti-fibrotic drugs are used in addition to chemotherapy. A reliable technique for evaluating the combined effects of anti-fibrotic drugs and anticancer drugs would be beneficial for the development of an appropriate treatment strategy. In this study, we manufactured a three-dimensional (3D) co-culture system of fibroblasts and lung cancer cell spheroids in Matrigel supplemented with fibrin (fibrin/Matrigel) that simulated the tissue microenvironment around a solid tumor. We compared the efficacy of an anticancer drug (cisplatin) with or without pretreatments of two anti-fibrotic drugs, nintedanib and pirfenidone, on the growth and invasion of cancer cells co-cultured with fibroblasts. The results showed that the addition of nintedanib improved cisplatin's effects on suppressing the growth of cancer cell spheroids and the invasion of cancer cells. In contrast, pirfenidone did not enhance the anticancer activity of cisplatin. Nintedanib also showed higher efficacy than pirfenidone in reducing the expression of four genes in fibroblasts associated with cell adhesion, invasion, and extracellular matrix degradation. This study demonstrated that the 3D co-cultures in fibrin/Matrigel would be useful for assessing the effects of drug combinations on tumor growth and invasion.

The applications of red-light photobiomodulation (PBM) to enhance neurite growth have been proposed for many years. However, the detailed mechanisms require further studies. In the present work we used a focused red-light spot to illuminate the junction of the longest neurite and the soma of a neuroblastoma cell (N2a), and demonstrated enhanced neurite growth at 620 nm and 760 nm with adequate illumination energy fluences. In contrast, 680 nm light showed no effect on neurite growth. The neurite growth was accompanied with the in-crease of intracellular reactive oxygen species (ROS). Using Trolox to reduce the ROS level, this red light-induced neurite growth was hindered. Suppressing the activities of cytochrome c oxidase (CCO) by using either a small-molecule inhibitor or siRNA abrogated the red light-induced neurite growth. These results suggest that red light-induced ROS production through the activation of CCO could be beneficial for neurite growth.

Background Tunneling nanotubes (TNTs) are special membrane structures for intercellular communications. Vital cargoes (such as mitochondria) could be delivered from healthy cells to rescue damaged ones through TNTs. The TNTs could be utilized for the purpose of systematic delivery of therapeutic agents between cells. However, there are insufficient studies on the controlled enhancement of TNT formations. The purpose of this study is to understand how macrophages influence the TNT formation in cancer cells. Results Here we compared the capabilities of inducing TNTs in human pancreatic cancer cells (PANC-1) of the media conditioned by M0, M1 and M2 macrophages derived from THP-1 cells. The M0 and M1 macrophage conditioned media promoted TNT formation. Using a focused ion beam to cut through a TNT, we observed tunnel-like structures inside dense cytoskeletons with scanning electron microscopy. The TNT formation correlated with raised motility, invasion, and epithelial-mesenchymal transition in the PANC-1 cells. Mitochondria and lysosomes were also found to be transported in the TNTs. Conclusions These results suggest that TNT formation could be one of the responses to the immune stress in pancreatic cancer cells caused by M0 and M1 macrophages. This finding is valuable for the development of macrophage-targeting cancer therapy.

Fibroblast migration is closely regulated by the mechanical characteristics in surrounding microenvironment. While increased interstitial hydrostatic pressure (HP) is a hallmark in many pathological and physiological conditions, little is known about how the HP affects fibroblast motility. Using cell-culture chips with elevated HP conditions, we showed that 20 cmH2O HP significantly accelerated fibroblast migration. The HP-induced migration acceleration was dependent on the augmentation of transforming growth factor-β1, and correlated with the activation of filamin A via the phosphorylation of p38 mitogen-activated protein kinase. Our results suggest that interstitial HP elevation associated with various pathological states could significantly regulate fibroblast migration.

We investigated the effects of cigarette smoke extract (CSE) on lung fibroblasts and found that the invasiveness of lung cancer cells was facilitated by the conditioned medium from CSE-treated fibroblasts. CSE induced autophagy in fibroblasts and increased the expression of autophagy-related proteins, including optineurin and Ras-related protein Rab1B. Afterward, the fibroblasts produced high levels of interleukin-8 (IL-8), which promoted cancer cell invasion. The inhibition of either optineurin or Rab1B abrogated a rise in microtubule-associated protein 1 light chain 3 β and a decrease in p62 protein, as well as the production of IL-8, in CSE-treated fibroblasts. A three-dimensional invasion assay using cancer cell spheroids revealed that the invasion of cancer cells alone and the fibroblast-led cancer cell invasion were both enhanced by the conditioned media from CSE-treated fibroblasts. These results suggest that cigarette smoke may induce autophagy and IL-8 secretion in lung fibroblasts and modify the microenvironment to favor invasion of lung cancer cells.

The interaction of light with biological tissues has been considered for various therapeutic applications. Light-induced neurite growth has the potential to be a clinically useful technique for neuron repair. However, most previous studies used either a large illumination area to accelerate overall neurite growth or employed a light spot to guide a growing neurite. It is not clear if optical stimulation can induce the regrowth of a retracted neurite. In the present work, we used blue light (wavelength: 473 nm) to cause neurite retraction, and we proved that using a red-light (wavelength: 650 nm) spot to illuminate the soma near the junction of the retracted neurite could induce neurite regrowth. As a comparison, we found that green light (wavelength 550 nm) had a 62% probability of inducing neurite regrowth, while red light had a 75% probability of inducing neurite regrowth at the same power level. Furthermore, the neurite regrowth length induced by red light was increased by the pre-treatment with inhibitors of myosin functions. We also observed actin propagation from the soma to the tip of the re-growing neurite following red-light stimulation of the soma. The red light-induced extension and regrowth were abrogated in the calcium-free medium. These results suggest that illumination with a red-light spot on the soma may trigger the regrowth of a neurite after the retraction caused by blue-light illumination.

We demonstrated that 473 nm blue light caused the neurite retraction in mouse neuroblastoma cell N2a, while 650 nm red light induced neurite regrowth. The myosin II inhibitor blebbistatin enhanced this red light-induced neurite regeneration. We also observed the actin wave propagating from the soma to the tip of a re-growing neurite. This red light-induced neurite regrowth did not occur in the calcium-free culture medium.

Recent studies indicate that changing the physical properties of lipid bilayers may profoundly change the function of membrane proteins. Here, the effects of dissolved nitrogen and oxygen molecules on the mechanical properties and stability of lipid bilayers are investigated using differential confocal microscopy, atomic force microscopy, and molecular dynamics simulations. All experiments evidence the presence of dissolved air gas in lipid bilayers prepared without gas control. The lipid bilayers in degassed solutions are softer and less stable than those in ambient solutions. High concentrations of nitrogen increase the bending moduli and stability of the lipid bilayers and impede phase separation in ternary lipid bilayers. The effect of oxygen is less prominent. Molecular dynamics simulations indicate that higher nitrogen affinity accounts for increased rigidity. These findings have fundamental and wide implications for phenomena related to lipid bilayers and cell membranes, including the origin of life.

We demonstrated that a 473 nm light spot caused neurite retraction of a neuroblastoma cell; while a 650 nm light spot on soma plus a myosin II inhibitor stimulated the neurite re-growth effectively.

Abstract Membrane topography of living cells has been considered as an effective parameter that reflects cellular statuses. With the improvements in spatial and temporal resolutions of various measurement techniques, the changes of membrane topography in response to various external stimulations in the culture environments can be accurately recorded. Membrane roughness is a useful parameter to evaluate the changes in membrane topography. At present, atomic force microscopy (AFM) is the most common technique to measure the membrane topography and roughness of living cells. On the other hand, with the non-contact profiling capability, many optical techniques are also employed in this field of research. This review briefly introduces the evolution and applications of AFM on measuring membrane roughness. Meanwhile, quantifying the cell membrane topography and roughness with a non-scanning, non-contact, and label-free optical technique, non-interferometric wide-field optical profilometry (NIWOP), is also presented.

Cell membrane roughness has been proposed as a sensitive feature to reflect cellular physiological conditions. In order to know whether membrane roughness is associated with the substrate properties, we employed the non-interferometric wide-field optical profilometry (NIWOP) technique to measure the membrane roughness of living mouse embryonic fibroblasts with different conditions of the culture substrate. By controlling the surface density of fibronectin (FN) coated on the substrate, we found that cells exhibited higher membrane roughness as the FN density increased in company with larger focal adhesion (FA) sizes. The examination of membrane roughness was also confirmed with atomic force microscopy. Using reagents altering actin or microtubule cytoskeletons, we provided evidence that the dynamics of actin filaments rather than that of microtubules plays a crucial role for the regulation of membrane roughness. By changing the substrate rigidity, we further demonstrated that the cells seeded on compliant gels exhibited significantly lower membrane roughness and smaller FAs than the cells on rigid substrate. Taken together, our data suggest that the magnitude of membrane roughness is modulated by way of actin dynamics in cells responding to substrate properties.

We proposed to use cellular spheroids of co-cultured lung cancer cells and fibroblasts as a platform to evaluate the efficacy of anti-cancer drug combinations. We labelled the cancer cells and fibroblast with dyes of different colors, and employed selective plane illumination microscopy (SPIM) [1, 2] to provide a three-dimensional (3D) perspective of relative positions and amounts of the co-cultured cells. Therefore we were able to evaluate the drug effects on individual types of cells in the 3D co-culture environment. The size of the spheroids strongly influences the evaluation of the drug effects. In order to unify the spheroid size, we used a microfluidic culture device that contained cubic chambers for confining the cellular spheroids [3]. Figure 1 shows the scheme and photo of the device used in the present work. The side wall of these culture chambers was flat such that the illuminating light sheet could propagate through without distortion. In the present work, the spheroids were kept in cubic chambers with a side length of 250 µm. We found that the co-culture of CL1-0 lung cancer cells and MRC-5 fibroblasts could form a spheroid (diameter ~ 200 µm) much easier than the cancer cell alone. The fibroblasts were enclosed by the cancer cells in a spheroid, regardless of the seeding sequences. In contrast, while the cancer cells were co-cultured with bronchial epithelial cells BEAS-2B, the latter did not invade into the cancer cell aggregation (Fig. 2). This result implied that fibroblasts could play an essential role in the early stage of tumor formation. Next, we used the co-culture spheroids to test the efficacy of a common anti-cancer drug cisplatin (CDDP) in combination with chloroquine (CQ), an inhibitor of cellular autophagy. We first used the total intensity of the fluorescent dye-labelled cells as a parameter to judge the drug efficacy. The addition of CQ enhanced the CDDP efficacy at 0.2 and 2.0 µM. With the SPIM images, we further realized that the survival rate of cancer cells in the co-culture spheroids was reduced by the addition of CQ, in comparison with the cases with CDDP only. In other words, CQ might selectively enhance the injury to the cancer cells in the co-culture spheroids. This result could not be revealed with the conventional cell viability test on the whole spheroid. We will report the results of other drug combinations in the conference. The co-culture spheroids consist of cancer cells and stromal cells combined with 3D SPIM imaging could serve as a useful platform to investigate the tumor formation process and to test the drug combination efficacy. For the evaluation of therapeutic strategies, the results from experiments in 3D microenvironments could be more relevant than those from 2D experiments. REFERENCES: Huisken et al., "Optical sectioning deep inside live embryos by selective plane illumination microscopy," Science 2004, 305, 1007-1009. J. Verveer et al., "High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy," Nat. Methods 2007, 4, 311-313. Patra et al., "Migration and vascular lumen formation of endothelial cells in cancer cell spheroids of various sizes," Biomicrofluidics 2014, 8, 052109.

T lymphocytes are important mediators of adoptive immunity but the mechanism of T cell receptor (TCR) triggering remains uncertain. The interspatial distance between engaged T cells and antigen-presenting cells (APCs) is believed to be important for topological rearrangement of membrane tyrosine phosphatases and initiation of TCR signaling. We investigated the relationship between ligand topology and affinity by generating a series of artificial APCs that express membrane-tethered anti-CD3 scFv with different affinities (OKT3, BC3, and 2C11) in addition to recombinant class I and II pMHC molecules. The dimensions of membrane-tethered anti-CD3 and pMHC molecules were progressively increased by insertion of different extracellular domains. In agreement with previous studies, elongation of pMHC molecules or low-affinity anti-CD3 scFv caused progressive loss of T cell activation. However, elongation of high-affinity ligands (BC3 and OKT3 scFv) did not abolish TCR phosphorylation and T cell activation. Mutation of key amino acids in OKT3 to reduce binding affinity to CD3 resulted in restoration of topological dependence on T cell activation. Our results show that high-affinity TCR ligands can effectively induce TCR triggering even at large interspatial distances between T cells and APCs.

Membrane topography of living cells has been considered as an effective parameter that reflects cellular statuses. With the improvements in spatial and temporal resolutions of various measurement techniques, the changes of membrane topography in response to various external stimulations in the culture environments can be accurately recorded. Membrane roughness is a useful parameter to evaluate the changes in membrane topography. At present, atomic force microscopy (AFM) is the most common technique to measure the membrane topography and roughness of living cells. On the other hand, with the non-contact profiling capability, many optical techniques are also employed in this field of research. This review briefly introduces the evolution and applications of AFM on measuring membrane roughness. Meanwhile, quantifying the cell membrane topography and roughness with a non-scanning, non-contact, and label-free optical technique, non-interferometric wide-field optical profilometry (NIWOP), is also presented.

Conventional two-dimensional (2D) monolayer cell cultures lack the three-dimensional (3D) architectures as those of real tissues in vivo. In cancer research, comparing 2D cell cultures with 3D cellular spheroids, the latter have features closer to those of a real tumor, such as cell-cell interaction, molecule responses [1]. Cigarette smoke is a main risk factor for lung cancers because it may participate in lung tumor invasion and metastasis [2]. However, the effect of cigarette smoke in 3D microenvironment of a lung tumor is still unclear. In the present study, we treated 3D cellular spheroids of co-cultured lung cancer cells and fibroblasts with cigarette smoke extract (CSE) to observe the variations of cell viability and invasion ability with light-sheet fluorescence microscopy (LSFM). LSFM is suitable for the observations on cellular spheroids because of its low phototoxicity and 3D imaging capability [3]. Figure 1 shows the LSFM systems used in this work. We employed a microfluidic culture device to form co-culture cellular spheroids of lung fibroblast MRC-5 and lung cancer cell CL1-0 [4]. The cancer cells and fibroblasts were labelled with different dyes, such that we could employ LSFM to measure the intensity variation of individual types of cells. Figure 2 is the LSFM images of the co-culture spheroids without and with the treatments of CSE. Our experimental data show that CSE reduced the signal of fibroblasts, while that of the cancer cells sustained. It seems that the cancer cells have a higher resistance to the toxicity of CSE. We also verified the invasion ability of co-culture spheroid under the CSE treatment. Figure 3 shows that CSE could enhance the invasion ability of both fibroblasts and lung cancer cells as the spheroid was placed in Matrigel®. Previous studies indicated CSE could improve the growth of tumor by reverse Warburg effect through the enhancement of autophagy in the surrounding fibroblasts [5]. We are now analyzing the expressions of specific proteins related to autophagy in the fibroblasts under the treatment of CSE. The preliminary results showed that the CSE treatment increased the expression of LC3B and decreased p62 in fibroblast. These changes in protein expressions indicated the promotion of autophagy. Therefore, we hypothesized that the autophagy of fibroblast was involved in the viability and invasion ability of cancer cells under the CSE treatment. More data will be presented in the Conference. REFERENCES: Pampaloni, F., E.G. Reynaud, and E.H.K. Stelzer, The third dimension bridges the gap between cell culture and live tissue. Nat. Rev. Mol. Cell Biol., 2007. 8(10): p. 839-845. Wang, Q., et al., Activation of uPAR is required for cigarette smoke extract-induced epithelial-mesenchymal transition in lung epithelial cells. Oncol. Res., 2013. 21(6): p. 295-305. Pampaloni, F., B.J. Chang, and E.H. Stelzer, Light sheet-based fluorescence microscopy (LSFM) for the quantitative imaging of cells and tissues. Cell Tissue Res, 2015. 360(1): p. 129-41. Patra, B., et al., Migration and vascular lumen formation of endothelial cells in cancer cell spheroids of various sizes. Biomicrofluidics, 2014. 8: p. 052109. Salem, A.F., et al., Cigarette smoke metabolically promotes cancer, via autophagy and premature aging in the host stromal microenvironment. Cell Cycle, 2013. 12(5): p. 818-825.