292 Ex vivo profiling of PD-1 blockade using an organotypic tissue slice model in solid tumors

BackgroundTumor explant models provide a powerful ex vivo tool to evaluate complex biological mechanisms in a controlled environment. Ex vivo models retain much of the original tumor biology, heterogeneity, and tumor microenvironment, and therefore provide a useful preclinical platform and functional approach to assess drug responses rapidly and directly.MethodsTo explore mechanisms of resistance to cancer immunotherapy, we established an organotypic tissue slice Air-Liquid Interface (ALI) ex vivo system utilizing surgical tumor specimens from patients to assess the impact of the clinically utilized anti-PD-1 antibody nivolumab (OPDIVO). In the present study, we built a real-world patient cohort comprised of six tumor types: non-small cell lung cancer, melanoma, pancreatic ductal adenocarcinoma, breast cancer, prostate cancer, and colorectal cancer. We assessed tissue morphology, histology, PD-L1 IHC (CPS and TPS), CD8 T cell topology, proliferation in the tumor and stromal compartments, and secretome profiling.ResultsOur tumor slice model highly recapitulated features of the original tumor, including tumor architecture, immune phenotypes, and the prognostic markers. To identify responses to aPD-1 treatment, we compared baseline values for the cultured tumor slices with values at different timepoints post treatment. Secretome profiling of tissue explant supernatants using a panel of 94 analytes, revealed alterations to cytokines produced in the tumor microenvironment in response to aPD-1 treatment. We found that soluble expression patterns were associated with T-cell patterns (inflamed, excluded and desert) and PD-L1 score (CPS and TPS) in tumor tissues. These cytokines mediate critical functions across the immune cell cycle. Ongoing efforts to characterize T cell activation, exhaustion, tumor intrinsic responses and microenvironment composition using Imaging Mass Cytometry will be presented.ConclusionsIn this study, we demonstrated the feasibility of using fresh, surgically resected human tumors to test aPD-1 responses in an ex vivo system. Further, this model system has the potential to drive discovery and translational efforts by evaluating mechanisms of resistance to cancer immunotherapy and evaluate new single agent or combination therapies in the ex vivo setting.

Using Organotypic Tissue Slices to Investigate the Microenvironment of Pancreatic Cancer: Pharmacotyping and Beyond

Organotypic tissue slices prepared from patient tumors are a semi-intact ex vivo preparation that recapitulates many aspects of the tumor microenvironment (TME). While connections to the vasculature and nervous system are severed, the integral functional elements of the tumor remain intact for many days during the slice culture. During this window of time, the slice platforms offer a suite of molecular, biomechanical and functional tools to investigate PDAC biology. In this review, we first briefly discuss the development of pancreatic tissue slices as a model system. Next, we touch upon using slices as an orthogonal approach to study the TME as compared to other established 3D models, such as organoids. Distinct from most other models, the pancreatic slices contain autologous immune and other stromal cells. Taking advantage of the existing immune cells within the slices, we will discuss the breakthrough studies which investigate the immune compartment in the pancreas slices. These studies will provide an important framework for future investigations seeking to exploit or reprogram the TME for cancer therapy.

Maturation-Dependent Differences in the Re-innervation of the Denervated Dentate Gyrus by Sprouting Associational and Commissural Mossy Cell Axons in Organotypic Tissue Cultures of Entorhinal Cortex and Hippocampus

Sprouting of surviving axons is one of the major reorganization mechanisms of the injured brain contributing to a partial restoration of function. Of note, sprouting is maturation as well as age-dependent and strong in juvenile brains, moderate in adult and weak in aged brains. We have established a model system of complex organotypic tissue cultures to study sprouting in the dentate gyrus following entorhinal denervation. Entorhinal denervation performed after 2 weeks postnatally resulted in a robust, rapid, and very extensive sprouting response of commissural/associational fibers, which could be visualized using calretinin as an axonal marker. In the present study, we analyzed the effect of maturation on this form of sprouting and compared cultures denervated at 2 weeks postnatally with cultures denervated at 4 weeks postnatally. Calretinin immunofluorescence labeling as well as time-lapse imaging of virally-labeled (AAV2-hSyn1-GFP) commissural axons was employed to study the sprouting response in aged cultures. Compared to the young cultures commissural/associational sprouting was attenuated and showed a pattern similar to the one following entorhinal denervation in adult animals in vivo. We conclude that a maturation-dependent attenuation of sprouting occurs also in vitro, which now offers the chance to study, understand and influence maturation-dependent differences in brain repair in these culture preparations.

The actin-modulating protein synaptopodin mediates long-term survival of dendritic spines

Large spines are stable and important for memory trace formation. The majority of large spines also contains synaptopodin (SP), an actin-modulating and plasticity-related protein. Since SP stabilizes F-actin, we speculated that the presence of SP within large spines could explain their long lifetime. Indeed, using 2-photon time-lapse imaging of SP-transgenic granule cells in mouse organotypic tissue cultures we found that spines containing SP survived considerably longer than spines of equal size without SP. Of note, SP-positive (SP+) spines that underwent pruning first lost SP before disappearing. Whereas the survival time courses of SP+ spines followed conditional two-stage decay functions, SP-negative (SP-) spines and all spines of SP-deficient animals showed single-phase exponential decays. This was also the case following afferent denervation. These results implicate SP as a major regulator of long-term spine stability: SP clusters stabilize spines, and the presence of SP indicates spines of high stability.

Biotechnological approach for substantiation of differentiated application of neuroprotective therapy in patients with hereditary neuromuscular diseases

Hereditary neuromuscular diseases are a group of genetic diseases characterized by an onset of the disease in most cases in childhood, having a steadily progressive course of the pathological process, leading to more rapid disability of patients and having a high mortality rate at the age of 18–20 years.Objective. To study a condition of the intra-organ structure in patients with hereditary muscular atrophy and muscular dystrophy using testing of nerve tissue in the organotypic environment in order to justify the prescription of symptomatic neuroprotective therapy.Materials and methods. Ninety patients with hereditary neuromuscular diseases (spinal muscular atrophy types 1, 2 and 3 [n = 30], Duchenne muscular dystrophy [n = 60]) were examined; the control group consisted of 30 healthy people. In vitro – explants of sensory ganglia of 10–12-day-old chicken embryos. A comprehensive clinical, laboratory and experimental study was conducted. Concentrations of neurotrophic factors (Brain Growth Factor, Nerve Growth Factor, Ciliary Neurotrophic Factor) were determined by the enzyme immunoassay method in blood plasma samples using RayBiotech kits and in accordance with the manufacturer's instructions. The experimental study included 300 explants of sensory ganglia of 10–12-day chicken embryos cultured in Petri dishes on collagen substrates in a CO2 incubator (Sanyo, Japan) for 3 days at 36.5 °C and 5% CO2. In order to clarify the biochemical mechanisms involved in pathological cascades in patients with hereditary neuromuscular diseases, a test system was developed that included a sequential analysis of the patient's blood plasma in an organotypic tissue culture at a dilution of 1: 70, followed by an addition of reagents to the medium: synthetic nerve growth factor (NGF) (100 pg/ml). Explant cultivation was carried out according to the method developed at Institute for Physiology n.a. I.P. Pavlov (Saint Petersburg, Russia). Visualization of the objects was made using Axiostar Plus microscope (Carl Zeiss, Germany). The resulting images were analyzed with the help of ImageJ software. A morphometric method was used to quantify the growth of explants. The area index (AI) was calculated as the ratio of the area of the explant growth zone to the initial area. AI reference value was 100%.Results. Brain Growth Factor concentration was at the highest level in patients with progressive amyotrophy, while in patients with progressive myodystrophy, the blood concentra tion of this factor was at a level comparable to the control data, and in some patients the concentration of Brain Growth Factor was lower than normal. NGF concentration showed the highest values in the group of patients with progressive amyotrophies. Blood plasma of patients with progressive amyotrophy dose dependently inhibits the growth of neurites of the spinal ganglia, and blood plasma of patients with myodystrophy has a neurite-weakening effect on the growth of neurites. Introduction of synthetic NGF (100 pg/ml) to organotypic tissue culture containing blood plasma of patients with myodystrophy increased the area index value of 114.0 [111.0; 116.0]%; in explants containing blood plasma of patients with progressive amyotrophy, increased growth of neurites was not observed AI = 80.0 [74.5; 83.0]%.Conclusion. The data obtained are indicative of features of neurotrophic regulation in patients with hereditary muscular atrophy and muscular dystrophy, which should be taken into account when conducting symptomatic treatment aimed at stimulating reparative processes in the nervous tissue. We recommend patients with the neurite-weakening effect of blood plasma to have neuroprotective drugs therapy, and in case of patients with neuritis-inhibiting effect on neurites in organotypic culture of nervous tissue we recommend choosing a drug in vitro individually using pharmacological analysis.

Amyloid-beta mediates homeostatic synaptic plasticity

ABSTRACTThe physiological role of the amyloid-precursor protein (APP) is insufficiently understood. Recent work has implicated APP in the regulation of synaptic plasticity. Substantial evidence exists for a role of APP and its secreted ectodomain APPsα in Hebbian plasticity. Here, we addressed the relevance of APP in homeostatic synaptic plasticity using organotypic tissue cultures of APP−/− mice. In the absence of APP, dentate granule cells failed to strengthen their excitatory synapses homeostatically. Homeostatic plasticity is rescued by amyloid-β (Aβ) and not by APPsα, and it is neither observed in APP+/+ tissue treated with β- or γ-secretase inhibitors nor in synaptopodin-deficient cultures lacking the Ca2+-dependent molecular machinery of the spine apparatus. Together, these results suggest a role of APP processing via the amyloidogenic pathway in homeostatic synaptic plasticity, representing a function of relevance for brain physiology as well as for brain states associated with increased Aβ levels.

The actin-modulating protein Synaptopodin mediates long-term survival of dendritic spines

Large spines are stable and important for memory trace formation. The majority of large spines also contains Synaptopodin (SP), an actin-modulating and plasticity-related protein. Since SP stabilizes F-actin, we speculated that the presence of SP within large spines could explain their long lifetime. Indeed, using time-lapse 2-photon-imaging of SP-transgenic granule cells in mouse organotypic tissue cultures we found that spines containing SP survived considerably longer than spines of equal size without SP. Of note, SP-positive spines that underwent pruning first lost SP before disappearing. Whereas the survival time courses of SP-positive (SP+) spines followed conditional two-phase decay functions, SP-negative (SP-) spines and all spines of SP-deficient animals showed single exponential decays. These results implicate SP as a major regulator of long-term spine stability: SP clusters stabilize spines and the presence of SP indicates spines of high stability.