Tapered fibertrodes for optoelectrical neural interfacing in small brain volumes with reduced artefacts.

Deciphering the neural patterns underlying brain functions is essential to understanding how neurons are organized into networks. This deciphering has been greatly facilitated by optogenetics and its combination with optoelectronic devices to control neural activity with millisecond temporal resolution and cell type specificity. However, targeting small brain volumes causes photoelectric artefacts, in particular when light emission and recording sites are close to each other. We take advantage of the photonic properties of tapered fibres to develop integrated 'fibertrodes' able to optically activate small brain volumes with abated photoelectric noise. Electrodes are positioned very close to light emitting points by non-planar microfabrication, with angled light emission allowing the simultaneous optogenetic manipulation and electrical read-out of one to three neurons, with no photoelectric artefacts, in vivo. The unconventional implementation of two-photon polymerization on the curved taper edge enables the fabrication of recoding sites all around the implant, making fibertrodes a promising complement to planar microimplants.

Depth-resolved fiber photometry with a single tapered optical fiber implant.

Fiber photometry is increasingly utilized to monitor fluorescent sensors of neural activity in the brain. However, most implementations are based on flat-cleaved optical fibers that can only interface with shallow tissue volumes adjacent to the fiber. We exploit modal properties of tapered optical fibers (TFs) to enable light collection over an extent of up to 2 mm of tissue and multisite photometry along the taper. Using a single TF, we simultaneously observed distinct dopamine transients in dorsal and ventral striatum in freely moving mice performing a simple, operant conditioning task. Collection volumes from TFs can also be engineered in both shape and size by microstructuring the nonplanar surface of the taper, to optically target multiple sites not only in the deep brain but, in general, in any biological system or organ in which light collection is beneficial but challenging because of light scattering and absorption.

Ray tracing models for estimating light collection properties of microstructured tapered optical fibers for optical neural interfaces.

Tapered optical fibers (TFs) were recently employed for depth-resolved monitoring of functional fluorescence in subcortical brain structures, enabling light collection from groups of a few cells through small optical windows located on the taper edge [Pisano et al., Nat. Methods16, 1185 (2019)1548-709110.1038/s41592-019-0581-x]. Here we present a numerical model to estimate light collection properties of microstructured TFs implanted in scattering brain tissue. Ray tracing coupled with the Henyey-Greenstein scattering model enables the estimation of both light collection and fluorescence excitation fields in three dimensions, whose combination is employed to retrieve the volume of tissue probed by the device.

Coenzyme Q10 + alpha lipoic acid for chronic COVID syndrome.

Chronic COVID syndrome is characterized by chronic fatigue, myalgia, depression and sleep disturbances, similar to chronic fatigue syndrome (CFS) and fibromyalgia syndrome. Implementations of mitochondrial nutrients (MNs) with diet are important for the clinical effects antioxidant. We examined if use of an association of coenzyme Q10 and alpha lipoic acid (Requpero®) could reduce chronic covid symptoms. The Requpero study is a prospective observational study in which 174 patients, who had developed chronic-covid syndrome, were divided in two groups: The first one (116 patients) received coenzyme Q10 + alpha lipoic acid, and the second one (58 patients) did not receive any treatment. Primary outcome was reduction in Fatigue Severity Scale (FSS) in treatment group compared with control group. complete FSS response was reached most frequently in treatment group than in control group. A FSS complete response was reached in 62 (53.5%) patients in treatment group and in two (3.5%) patients in control group. A reduction in FSS core < 20% from baseline at T1 (non-response) was observed in 11 patients in the treatment group (9.5%) and in 15 patients in the control group (25.9%) (p < 0.0001). To date, this is the first study that tests the efficacy of coenzyme Q10 and alpha lipoic acid in chronic Covid syndrome. Primary and secondary outcomes were met. These results have to be confirmed through a double blind placebo controlled trial of longer duration.

Toward Plasmonic Neural Probes: SERS Detection of Neurotransmitters through Gold-Nanoislands-Decorated Tapered Optical Fibers with Sub-10 nm Gaps.

Integration of plasmonic nanostructures with fiber-optics-based neural probes enables label-free detection of molecular fingerprints via surface-enhanced Raman spectroscopy (SERS), and it represents a fascinating technological horizon to investigate brain function. However, developing neuroplasmonic probes that can interface with deep brain regions with minimal invasiveness while providing the sensitivity to detect biomolecular signatures in a physiological environment is challenging, in particular because the same waveguide must be employed for both delivering excitation light and collecting the resulting scattered photons. Here, a SERS-active neural probe based on a tapered optical fiber (TF) decorated with gold nanoislands (NIs) that can detect neurotransmitters down to the micromolar range is presented. To do this, a novel, nonplanar repeated dewetting technique to fabricate gold NIs with sub-10 nm gaps, uniformly distributed on the wide (square millimeter scale in surface area), highly curved surface of TF is developed. It is experimentally and numerically shown that the amplified broadband near-field enhancement of the high-density NIs layer allows for achieving a limit of detection in aqueous solution of 10-7  m for rhodamine 6G and 10-5  m for serotonin and dopamine through SERS at near-infrared wavelengths. The NIs-TF technology is envisioned as a first step toward the unexplored frontier of in vivo label-free plasmonic neural interfaces.

Orthogonalization of far-field detection in tapered optical fibers for depth-selective fiber photometry in brain tissue.

The field of implantable optical neural interfaces has recently enabled the interrogation of neural circuitry with both cell-type specificity and spatial resolution in sub-cortical structures of the mouse brain. This generated the need to integrate multiple optical channels within the same implantable device, motivating the requirement of multiplexing and demultiplexing techniques. In this article, we present an orthogonalization method of the far-field space to introduce mode-division demultiplexing for collecting fluorescence from the implantable tapered optical fibers. This is achieved by exploiting the correlation between the transversal wavevector k t of the guided light and the position of the fluorescent sources along the implant, an intrinsic property of the taper waveguide. On these bases, we define a basis of orthogonal vectors in the Fourier space, each of which is associated with a depth along the taper, to simultaneously detect and demultiplex the collected signal when the probe is implanted in fixed mouse brain tissue. Our approach complements the existing multiplexing techniques used in silicon-based photonics probes with the advantage of a significant simplification of the probe itself.

Comparative study of autofluorescence in flat and tapered optical fibers towards application in depth-resolved fluorescence lifetime photometry in brain tissue.

As the scientific community seeks efficient optical neural interfaces with sub-cortical structures of the mouse brain, a wide set of technologies and methods is being developed to monitor cellular events through fluorescence signals generated by genetically encoded molecules. Among these technologies, tapered optical fibers (TFs) take advantage of the modal properties of narrowing waveguides to enable both depth-resolved and wide-volume light collection from scattering tissue, with minimized invasiveness with respect to standard flat fiber stubs (FFs). However, light guided in patch cords as well as in FFs and TFs can result in autofluorescence (AF) signal, which can act as a source of time-variable noise and limit their application to probe fluorescence lifetime in vivo. In this work, we compare the AF signal of FFs and TFs, highlighting the influence of the cladding composition on AF generation. We show that the autofluorescence signal generated in TFs has a peculiar coupling pattern with guided modes, and that far-field detection can be exploited to separate functional fluorescence from AF. On these bases, we provide evidence that TFs can be employed to implement depth-resolved fluorescence lifetime photometry, potentially enabling the extraction of a new set of information from deep brain regions, as time-correlating single photon counting starts to be applied in freely-moving animals to monitor the intracellular biochemical state of neurons.

Influence of the anatomical features of different brain regions on the spatial localization of fiber photometry signals.

Fiber photometry is widely used in neuroscience labs for in vivo detection of functional fluorescence from optical indicators of neuronal activity with a simple optical fiber. The fiber is commonly placed next to the region of interest to both excite and collect the fluorescence signal. However, the path of both excitation and fluorescence photons is altered by the uneven optical properties of the brain, due to local variation of the refractive index, different cellular types, densities and shapes. Nonetheless, the effect of the local anatomy on the actual shape and extent of the volume of tissue that interfaces with the fiber has received little attention so far. To fill this gap, we measured the size and shape of fiber photometry efficiency field in the primary motor and somatosensory cortex, in the hippocampus and in the striatum of the mouse brain, highlighting how their substructures determine the detected signal and the depth at which photons can be mined. Importantly, we show that the information on the spatial expression of the fluorescent probes alone is not sufficient to account for the contribution of local subregions to the overall collected signal, and it must be combined with the optical properties of the tissue adjacent to the fiber tip.

3D-microenvironments initiate TCF4 expression rescuing nuclear ß-catenin activity in MCF-7 breast cancer cells.

Mechanical cues sensed by tumor cells in their microenvironment can influence important mechanisms including adhesion, invasion and proliferation. However, a common mechanosensitive protein and/or pathway can be regulated in different ways among diverse types of tumors. Of particular interest are human breast epithelial cancers, which markedly exhibit a heterogeneous pattern of nuclear ß-catenin localization, a protein known to be involved in both mechanotransduction and tumorigenesis. ß-catenin can be aberrantly accumulated in the nucleus wherein it binds to and activates lymphoid enhancer factor/T cell factor (LEF/TCF) transcription factors. At present, little is known about how mechanical cues are integrated into breast cancer cells harboring impaired mechanisms of ß-catenin's nuclear uptake and/or retention. This prompted us to investigate the influence of mechanical cues on MCF-7 human breast cancer cells which are known to fail in relocating ß-catenin into the nucleus due to very low baseline levels of LEF/TCFs. Exploiting three-dimensional (3D) microscaffolds realized by two-photon lithography, we show that surrounding MCF-7 cells have not only a nuclear pool of ß-catenin, but also rescue from their defective expression of TCF4 and boost invasiveness. Together with heightened amounts of vimentin, a ß-catenin/TCF-target gene regulator of proliferation and invasiveness, such 3D-elicited changes indicate an epithelial-to-mesenchymal phenotypic switch of MCF-7 cells. This is also consistent with an increased in situ MCF-7 cell proliferation that can be abrogated by blocking ß-catenin/TCF-transcription activity. Collectively, these data suggest that 3D microenvironments are per se sufficient to prime a TCF4-dependent rescuing of ß-catenin nuclear activity in MCF-7 cells. The employed methodology could, therefore, provide a mechanism-based rationale to dissect further aspects of mechanotranscription in breast cancerogenesis, somewhat independent of ß-catenin's nuclear accumulation. More importantly, by considering the heterogeneity of ß-catenin signaling pathway in breast cancer patients, these data may open alternative avenues for personalized disease management and prevention. STATEMENT OF SIGNIFICANCE: Mechanical cues play a critical role in cancer pathogenesis. Little is known about their influence in breast cancer cells harboring impaired mechanisms of ß-catenin's nuclear uptake and/or retention, involved in both mechanotransduction and tumorigenesis. We engineered 3D scaffold, by two-photon lithography, to study the influence of mechanical cues on MCF-7 cells which are known to fail in relocating ß-catenin into the nucleus. We found that 3D microenvironments are per se sufficient to prime a TCF4-dependent rescuing of ß-catenin nuclear activity that boost cell proliferation and invasiveness. Thus, let us suggest that our system could provide a mechanism-based rationale to further dissect key aspects of mechanotranscription in breast cancerogenesis and progression, somewhat independent of ß-catenin's nuclear accumulation.

Studying Cell Mechanobiology in 3D: The Two-Photon Lithography Approach.

Two-photon lithography is a laser writing technique that can produce 3D microstructures with resolutions below the diffraction limit. This review focuses on its applications to study mechanical properties of cells, an emerging field known as mechanobiology. We review 3D structural designs and materials in the context of new experimental designs, including estimating forces exerted by single cells, studying selective adhesion on substrates, and creating 3D networks of cells. We then focus on emerging applications, including structures for assessing cancer cell invasiveness, whose migration properties depend on the cell mechanical response to the environment, and 3D architectures and materials to study stem cell differentiation, as 3D structure shape and patterning play a key role in defining cell fates.

Anxiolytic- and antidepressant-like activities of H-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512), a novel selective delta opioid receptor agonist.

Knockout and pharmacological studies have shown that delta opioid peptide (DOP) receptor signalling regulates emotional responses. In the present study, the in vitro and in vivo pharmacological profile of the DOP ligand, H-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512) was investigated. In receptor binding experiments performed on membranes of CHO cells expressing the human recombinant opioid receptors, UFP-512 displayed very high affinity (pKi 10.20) and selectivity (>150-fold) for DOP sites. In functional studies ([35S]GTP gamma S binding in CHOhDOP membranes and electrically stimulated mouse vas deferens) UFP-512 behaved as a DOP selective full agonist showing potency values more than 100-fold higher than DPDPE. In vivo, in the mouse forced swimming test, UFP-512 reduced immobility time both after intracerebroventricular (i.c.v.) and intraperitoneal (i.p.) administration. Similar effects were recorded in rats. Moreover, UFP-512 evoked anxiolytic-like effects in the mouse elevated plus maze and light-dark aversion assays. All these in vivo actions of UFP-512 were fully prevented by the selective DOP antagonist naltrindole (3 mg/kg, s.c.). In conclusion, the present findings demonstrate that UFP-512 behaves as a highly potent and selective agonist at DOP receptors and corroborate the proposal that the selective activation of DOP receptors elicits robust anxiolytic- and antidepressant-like effects in rodents.

Tailoring light delivery for optogenetics by modal demultiplexing in tapered optical fibers.

Optogenetic control of neural activity in deep brain regions ideally requires precise and flexible light delivery with non-invasive devices. To this end, Tapered Optical Fibers (TFs) represent a versatile tool that can deliver light over either large brain volumes or spatially confined sub-regions, while being sensibly smaller than flat-cleaved optical fibers. In this work, we report on the possibility of further extending light emission length along the taper in the range 0.4 mm-3.0 mm by increasing the numerical aperture of the TFs to NA = 0.66. We investigated the dependence between the input angle of light (θin) and the output position along the taper, finding that for θin > 10° this relationship is linear. This mode-division demultiplexing property of the taper was confirmed with a ray tracing model and characterized for 473 nm and 561 nm light in quasi-transparent solution and in brain slices, with the two wavelengths used to illuminate simultaneously two different regions of the brain using only one waveguide. The results presented in this manuscript can guide neuroscientists to design their optogenetic experiments on the base of this mode-division demultiplexing approach, providing a tool that potentially allow for dynamic targeting of regions with diverse extension, from the mouse VTA up to the macaque visual cortex.

Tapered Fibers Combined With a Multi-Electrode Array for Optogenetics in Mouse Medial Prefrontal Cortex.

Optogenetics offers many advantages in terms of cell-type specificity, allowing to investigate functional connectivity between different brain areas at high spatial and neural population selectivity. In order to obtain simultaneous optical control and electrical readout of neural activity, devices called "optrodes" are employed. They are typically composed of a linear array of microelectrodes integrated on a slender probe shafts combined with flat-cleaved optical fibers (FF) placed above the recording sites. However, due to tissue absorption and scattering, light delivered by the FF unevenly illuminates the region of interest. This issue is of particular relevance when cellular populations are disposed along the dorso-ventral axis, such as in medial prefrontal cortex (mPFC) where cortical layers are aligned vertically. The study presented here aims at using tapered optical fibers (TFs) in combination with a 16-electrode neural probe to better access neural populations distributed along the dorso-ventral axis in the mPFC of newborn mice, restricting light delivery over a specific portion of the cortical layer of interest. Half of the TF surface is coated with a reflecting metal blocking the light to enable light delivery from one side of the probe's shaft only, with the probe base being designed to host the fiber without interfering with the wire-bonds that connect the recording sites to a printed circuit board. Monte-Carlo simulations have been implemented to define the relative TF-probe position and to identify the light intensity distribution above the recording sites. In vivo recordings indicate that simultaneous optical stimulation and electrical readout of neural activity in the mPFC benefit from the use of the engineered TF-based optrode in terms of a more uniform light distribution along the dorso-ventral axis and the possibility of restricting light delivery to a subset of electrical recording sites of interest.

In vitro and in vivo studies on UFP-112, a novel potent and long lasting agonist selective for the nociceptin/orphanin FQ receptor.

[(pF)Phe(4)Aib(7)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-112) has been designed as a novel ligand for the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) by combining into the same peptide different chemical modifications reported to increase N/OFQ potency. In vitro data obtained in the electrically stimulated mouse vas deferens demonstrated that UFP-112 behaved as a high potency (pEC(50) 9.43) full agonist at the NOP receptor. UFP-112 effects were sensitive to the NOP antagonist UFP-101 but not to naloxone and no longer evident in tissues taken from NOP(-/-) mice. In vitro half life of UFP-112 in mouse plasma and brain homogenate was 2.6- and 3.5-fold higher than that of N/OFQ. In vivo, in the mouse tail withdrawal assay, UFP-112 (1-100pmol, i.c.v.) mimicked the actions of N/OFQ producing pronociceptive effects after i.c.v. administration and antinociceptive effects when given i.t.; in both cases, UFP-112 was approximately 100-fold more potent than the natural peptide and produced longer lasting effects. UFP-112 also mimicked the hyperphagic effect of N/OFQ producing a bell shaped dose response curve with the maximum reached at 10pmol. The hyperphagic effects of N/OFQ and UFP-112 were absent in NOP(-/-) mice. Equi-effective high doses of UFP-112 (0.1nmol) and N/OFQ (10nmol) were injected i.c.v. in mice and spontaneous locomotor activity recorded for 16h. N/OFQ produced a clear inhibitory effect which lasted for 60min while UFP-112 elicited longer lasting effects (>6h). In conscious rats, UFP-112 (0.1 and 10nmol/kg, i.v.) produced a marked and sustained decrease in heart rate, blood pressure, and urinary sodium excretion and a profound increase in urine flow. Collectively, these findings demonstrate that UFP-112 behaves in vitro and in vivo as a highly potent and selective ligand able to produce full and long lasting activation of NOP receptors.

Microenvironmental Stiffness of 3D Polymeric Structures to Study Invasive Rates of Cancer Cells.

Cells are highly dynamic elements, continuously interacting with the extracellular environment. Mechanical forces sensed and applied by cells are responsible for cellular adhesion, motility, and deformation, and are heavily involved in determining cancer spreading and metastasis formation. Cell/extracellular matrix interactions are commonly analyzed with the use of hydrogels and 3D microfabricated scaffolds. However, currently available techniques have a limited control over the stiffness of microscaffolds and do not allow for separating environmental properties from biological processes in driving cell mechanical behavior, including nuclear deformability and cell invasiveness. Herein, a new approach is presented to study tumor cell invasiveness by exploiting an innovative class of polymeric scaffolds based on two-photon lithography to control the stiffness of deterministic microenvironments in 3D. This is obtained by fine-tuning of the laser power during the lithography, thus locally modifying both structural and mechanical properties in the same fabrication process. Cage-like structures and cylindric stent-like microscaffolds are fabricated with different Young's modulus and stiffness gradients, allowing obtaining new insights on the mechanical interplay between tumor cells and the surrounding environments. In particular, cell invasion is mostly driven by softer architectures, and the introduction of 3D stiffness "weak spots" is shown to boost the rate at which cancer cells invade the scaffolds. The possibility to modulate structural compliance also allowed estimating the force distribution exerted by a single cell on the scaffold, revealing that both pushing and pulling forces are involved in the cell-structure interaction. Overall, exploiting this method to obtain a wide range of 3D architectures with locally engineered stiffness can pave the way for unique applications to study tumor cell dynamics.

Single-cell-based evaluation of sperm progressive motility via fluorescent assessment of mitochondria membrane potential.

Sperm cells progressive motility is the most important parameter involved in the fertilization process. Sperm middle piece contains mitochondria, which play a critical role in energy production and whose proper operation ensures the reproductive success. Notably, sperm progressive motility is strictly related to mitochondrial membrane potential (MMP) and consequently to mitochondrial functionality. Although previous studies presented an evaluation of mitochondrial function through MMP assessment in entire sperm cells samples, a quantitative approach at single-cell level could provide more insights in the analysis of semen quality. Here we combine laser scanning confocal microscopy and functional fluorescent staining of mitochondrial membrane to assess MMP distribution among isolated spermatozoa. We found that the sperm fluorescence value increases as a function of growing progressive motility and that such fluorescence is influenced by MMP disruptors, potentially allowing for the discrimination of different quality classes of sperm cells in heterogeneous populations.

Dynamic illumination of spatially restricted or large brain volumes via a single tapered optical fiber.

Optogenetics promises precise spatiotemporal control of neural processes using light. However, the spatial extent of illumination within the brain is difficult to control and cannot be adjusted using standard fiber optics. We demonstrate that optical fibers with tapered tips can be used to illuminate either spatially restricted or large brain volumes. Remotely adjusting the light input angle to the fiber varies the light-emitting portion of the taper over several millimeters without movement of the implant. We use this mode to activate dorsal versus ventral striatum of individual mice and reveal different effects of each manipulation on motor behavior. Conversely, injecting light over the full numerical aperture of the fiber results in light emission from the entire taper surface, achieving broader and more efficient optogenetic activation of neurons, compared to standard flat-faced fiber stimulation. Thus, tapered fibers permit focal or broad illumination that can be precisely and dynamically matched to experimental needs.

Dmt-Tic-NH-CH2-Bid (UFP-502), a potent DOP receptor agonist: in vitro and in vivo studies.

Knockout and pharmacological studies demonstrated that the activation of delta opioid peptide (DOP) receptors produces antidepressant-like effects in rodents. Here we report the results obtained with the novel DOP ligand H-Dmt-Tic-NH-CH(2)-Bid (UFP-502). UFP-502 bound with high affinity (pK(i) 9.43) to recombinant DOP receptors displaying moderate selectivity over MOP and KOP. In CHO(hDOP) [(35)S]GTPgammaS binding and mouse vas deferens experiments, UFP-502 behaved as a potent (pEC(50) 10.09 and 10.70, respectively) full agonist. In these preparations, naloxone, naltrindole and N,N(CH(3))(2)Dmt-Tic-OH showed similar pA(2) values against UFP-502 and DPDPE and the same rank order of potency. In vivo in mice, UFP-502 mimicked DPDPE actions, producing a significant reduction of immobility time after intracerebroventricular administration in the forced swimming test and a clear antinociceptive effect after intrathecal injection in the tail withdrawal assay. However, while the effects of DPDPE were fully prevented by naltrindole those evoked by UFP-502 were unaffected (tail withdrawal assay) or only partially reversed (forced swimming test). In conclusion, UFP-502 represents a novel and useful chemical template for the design of selective agonists for the DOP receptor.

In vitro and in vivo pharmacological characterization of the nociceptin/orphanin FQ receptor ligand Ac-RYYRIK-ol.

It was recently reported that the hexapeptide Ac-RYYRIK-ol binds with high affinity nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptors and competitively antagonizes N/OFQ actions in the mouse vas deferens assay. Here we further describe the in vitro and in vivo pharmacological features of this NOP receptor ligand. In mouse brain homogenate the degradation half life of Ac-RYYRIK-ol (2.48 min) was significantly higher than that of the parent compound Ac-RYYRIK-NH2 (1.20 min). In the electrically stimulated mouse vas deferens, Ac-RYYRIK-ol (10-1000 nM) competitively antagonized the inhibitory effect of N/OFQ (pA2=8.46), while in the isolated mouse colon the hexapeptide mimicked N/OFQ contractile effects thus behaving as a NOP receptor agonist (pEC50=9.09). This latter effect was no longer evident in colon tissues taken from mice knock out for the NOP receptor gene (NOP-/-). In vivo in mice, similarly to N/OFQ, Ac-RYYRIK-ol (dose range 0.001-1 nmol) produced: i) pronociceptive effects after intracerebroventricular (i.c.v.) administration and antinociceptive actions when given intrathecally (i.t.) in the tail withdrawal assay; ii) inhibition of locomotor activity and iii) stimulation of food intake after supraspinal administration. Finally, in the forced swimming test, Ac-RYYRIK-ol was inactive per se, but reversed the antidepressant-like effects elicited by the NOP receptor selective antagonist UFP-101 ([Nphe(1),Arg(14),Lys(15)]N/OFQ-NH2). Thus, in all these in vivo assays Ac-RYYRIK-ol mimicked the actions of N/OFQ showing however higher potency. In conclusion, Ac-RYYRIK-ol displayed a complex pharmacological profile which is likely due to the low efficacy agonist nature of this novel ligand of the NOP receptor. The high potency, selectivity of action, and in vivo effectiveness make Ac-RYYRIK-ol a useful pharmacological tool for future studies in the field of N/OFQ and its NOP receptor.

Three-dimensional cage-like microscaffolds for cell invasion studies.

Cancer cell motility is one of the major events involved in metastatic process. Tumor cells that disseminate from a primary tumor can migrate into the vascular system and, being carried by the bloodstream, transmigrate across the endothelium, giving rise to a new tumor site. However, during the invasive process, tumor cells must pass through the extracellular matrix, whose structural and mechanical properties define the parameters of the migration process. Here, we propose 3D-complex cage-like microstructures, realized by two-photon (TP) direct laser writing (DLW), to analyze cell migration through pores significantly smaller than the cell nucleus. We found that the ability to traverse differently sized pores depends on the metastatic potential and on the invasiveness of the cell lines, allowing to establish a pore-area threshold value able to discriminate between non-tumorigenic and tumorigenic human breast cells.