We present an in vitro micropatterning approach in which the density and spatial presentation of two separate protein layers can be independently controlled to form cell stripe assays through (1) the simultaneous application of microcontact printing (μCP) and microfluidic network patterning (μFN) to generate alternating stripes of pure single protein layers or (2) through μCP onto a pre-adsorbed homogeneous protein layer to generate alternating single and dual protein stripes. This approach enabled the creation of choice boundaries in which protein-protein interactions were limited and the effects of spatially segregated or colocalized dual protein signals on model primary neuronal behavior could be readily interrogated and compared on both glass and tissue culture polystyrene substrates. Dorsal root ganglion (DRG) cell body attachment was dictated largely by non-specific cell adhesion interactions and interactions between the guidance molecules laminin and aggrecan were insufficient to explain aggrecan inhibition on neurite outgrowth. The presentation of a specific laminin epitope stabilized by interactions with aggrecan and destabilized by μCP was a strong predictor of neurite promoting activity. These observations provide evidence that aggrecan is intrinsically inhibitory and that laminin-aggrecan interactions do not diminish laminin growth promoting properties.

We have investigated the influence of micrometer-and sub-micrometer-scale surface heterogeneities in patterned octadecyltrichlorosilane (OTS) films on human serum albumin (HSA) adsorption and its spatial distribution. 5-μm-wide OTS patterns were created on glass substrates by micro-contact printing and in some instances subsequent annealing was used to alter OTS molecule distribution within the patterns. Scanning force microscopy (SFM), advancing water contact angles and water vapor condensation figures were used to characterize the OTS films and to assess the nature of the heterogeneities within the various surface areas. High-resolution fluorescence microscopy was used to record images of fluorescently labeled albumin on OTS patterned films and fluorescence intensity was quantified and converted into the adsorbed amount. Adsorbed albumin was also characterized through SFM measurements. Combined SFM topography and lateral force microscopy (LFM) imaging revealed that micro-contact printing of OTS onto glass both replicated the stamp pattern and created small islands within the non-stamped regions between the patterns. The OTS coverage within stamped regions was not fully continuous but improved with subsequent annealing. Annealing also resulted in OTS island growth within the non-stamped regions and decreased average wettability on both the stamped and non-stamped areas. The extent of albumin adsorption was not proportional to OTS coverage, but correlated with the sub-μm distribution of OTS chains. We inferred that the surface distribution of ligands such as OTS on a sub-μm length scale determines the nature of albumin adsorption and its kinetics.

Injured neurons intrinsically adapt to and partially overcome inhibitory proteoglycan expression in the central nervous system by upregulating integrin expression. It remains unclear however, to what extent varying proteoglycan concentrations influence the strength of this response, how rapidly neurons adapt to proteoglycans, and how pathfinding dynamics are altered over time as integrin expression is modulated in response to proteglycan signals. To investigate these quandaries, we created well-defined substrata in which postnatal DRG neuron pathfinding dynamics and growth cone integrin expression were interrogated as a function of proteoglycan substrata density. DRGs responded by upregulating integrin expression in a proteoglycan dose dependent fashion and exhibited robust outgrowth over all proteoglycan densities at initial time frames. However, after prolonged proteoglycan exposure, neurons exhibited decreasing velocities associated with increasing proteoglycan densities, while neurons growing on low proteoglycan levels exhibited robust outgrowth at all time points. Additionally, DRG outgrowth over proteoglycan density step boundaries, and a brief β1 integrin functional block proved that regeneration was integrin dependent and that DRGs exhibit delayed slowing and loss in persistence after even transient encounters with dense proteoglycan boundaries. These findings demonstrate the complexity of proteoglycan regulation on integrin expression and regenerative pathfinding.

Proteoglycan expression patterns in the central nervous system guide neuronal pathfinding during development, but also disrupt regeneration after injuries. To deepen our understanding of the molecular level effects of proteoglycan spatial arrangements on neuronal pathfinding, we designed micropatterning stamps for the precise placement of near single molecule chondroitin sulfate proteoglycan (CSPG) clusters into regularly spaced arrays. Actin ultrastructural analysis in dorsal root ganglion neurons grown on laminin-coated substrata patterned with aggrecan cluster arrays revealed filopodial and lamellapodial edge contact avoidance of individual clusters, while growth cone lamellapodia and central domains were able to span multiple clusters over a range of cluster densities. Total internal reflection fluorescence microscopy interrogation of growth cone substratum morphology further revealed persistence of integrin mediated substratum adhesion and local out-ofplane membrane bending over clusters on the height scale of aggrecan glycosaminoglycan side chains. Direct imaging of cell adhesion molecule CD44 expression in growth cones revealed an aggrecan dose dependent upregulation in CD44 molecules. Evidence of CD44 clustering coinciding with underlying aggrecan molecules imply CSPG-CD44 interactions. The results reveal the limited local repulsive effect of CSPGs on neuronal structures and provide evidence that CD44 upregulation in neurons is affected by local CSPG expression.

Protein micropatterning techniques are increasingly applied in cell choice assays to investigate fundamental biological phenomena that contribute to the host response to implanted biomaterials, and to explore the effects of protein stability and biological activity on cell behavior for in vitro cell studies. In the area of neuronal regeneration the protein micropatterning and cell choice assays are used to improve our understanding of the mechanisms directing nervous system during development and regenerative failure in the central nervous system (CNS) wound healing environment. In these cell assays, protein micropatterns need to be characterized for protein stability, bioactivity, and spatial distribution and then correlated with observed mammalian cell behavior using appropriate model system for CNS development and repair. This review provides the background on protein micropatterning for cell choice assays and describes some novel patterns that were developed to interrogate neuronal adaptation to inhibitory signals encountered in CNS injuries. Rationale for studying the role of proteoglycans in CNS injuriesNeurons from the central nervous system (CNS) possess a limited capacity to regenerate beyond scar tissue formation barriers in injuries even in the presence of minimal trauma to tissue organization [1,2]. A major culprit in CNS neuronal regenerative failure are the inihibitory proteoglycans (PGs), which are upregulated at the site of CNS injuries. PGs are composed of a core protein with varying numbers of attached glycosaminoglycan (GAG) side chains [3,4]. Proteoglycans are first translated intracellularly into proteins in the endoplasmic reticulum, and then GAG chains are later attached in the Golgi apparatus before secretion into the extracellular space [5][6][7][8]. The study of PGs has particularly been pronounced in the field of neurobiology and development of the nervous system. Numerous studies have concluded that PGs act as barriers that direct the migration of neural crest cells and the outgrowth direction of pathfinding neurons during development [9][10][11][12][13][14][15][16][17]. A number of these studies have additionally shown that cell guidance by PGs is based on an inhibitory signal located within the chondroitin sulfate GAG chains [9,18,19], with the result that the artificial addition of chondroitin sulfate (CS) sugars to the developing nervous system disrupts normal pathfinding [20] as well as does the removal of chondroitin sulfate chains through enzymatic treatment [9,19]. Davies et al. found that chondroitin sulfate proteoglycans (CSPG) are a major constituent of the glial scar and that dorsal root ganglion (DRG) outgrowth termination coincided with an increase in CSPG expression density [21]. Other sources have also shown that CSPGs are upregulated after CNS injuries [22][23][24][25].Interestingly, native adult CNS neurons actually posses the intrinsic ability to regenerate when the wound healing environment is prepared by eliminating inhibitory factors [23,26,27]. In additi...

e20579 Background: Recently, the PORT-C (China) and Lung ART (Europe) trials have been reported for non-small cell lung cancer patients (NSCLC) with surgically resected N2 nodal disease subsequently randomized to post-operative radiation (PORT). The two studies noted widely different locoregional relapse (LR) rates in the control arms, 18.3% in PORT-C and 28.1%(46% of recurrences) in Lung ART. We performed a meta-analysis of patients with N0-N2 disease to better understand risk factors for LR, and the possible differences in risk and rates between Asian (AP) vs. non-Asian populations (NAP). Methods: The present systematic review and meta-analysis identified all original studies of curative NSCLC surgical resections which reported risk and rates of LR between January 1st, 2000 and January 10th, 2021. Studies were excluded if patient number was less than 10, if metastatic disease was present, or if any neo-adjuvant chemotherapy and/or radiation was given. Eighty-seven studies were included; of these, 56 were of high quality (HQ) based on the Newcastle-Ottawa Scale (ratings 7-9). For each risk factor, we derived pooled relative risk (RR) and rate estimates using random-effects models. Results: Overall, the three highest pooled RRs for LR were N2 vs. N0 (RR 3.01), lymphovascular invasion (LVI; 1.92), and advanced T stage (T3-T4) vs. T1 (1.86). For HQ studies, the highest RRs for LR were LVI (1.94), sublobar vs. lobar resection (1.86), and N1 vs. N0 (1.84), but N2 vs N0 was no longer significant (RR 3.0 (95% confidence interval 0.57 -15.61) based on only 2 studies. The RRs for LR were consistent for most factors across geographic areas, although the RRs for male vs. female sex were higher in AP (1.44) than in NAP (1.09). The pooled rate of LR at 5-years was lower in the AP 12.00% (6.92-17.09) vs. NAP 22.66% (17.06 - 28.26), despite similar overall recurrence rates (both LR and distal) at 5 years in both populations: 38.03% (25.15-50.90) in AP and 37.30% (32.44-42.17) in NAP. However, a lower 5-year mortality rate was noted in AP (24.30%, 15.56 -33.03) than in NAP (45.87%, 41.23-50.50). Conclusions: Our meta-analysis found that N2 nodal disease is not a risk factor for LR when considering HQ studies based upon scant data, and confirmed that LR is lower in AP. We propose that prospective evaluation of LR risk factors and rates should be undertaken prior to any other prospective evaluation of PORT because LR may not be dependent upon N2 node status and because LR rates may differ in AP.