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Supplementary Figure from A Natural Polyphenol Exerts Antitumor Activity and Circumvents Anti–PD-1 Resistance through Effects on the Gut Microbiota

Several approaches to manipulate the gut microbiome for improving the activity of cancer immune-checkpoint inhibitors (ICI) are currently under evaluation. Here, we show that oral supplementation with the polyphenol-rich berry camu-camu (CC; Myrciaria dubia) in mice shifted gut microbial composition, which translated into antitumor activity and a stronger anti–PD-1 response. We identified castalagin, an ellagitannin, as the active compound in CC. Oral administration of castalagin enriched for bacteria associated with efficient immunotherapeutic responses (Ruminococcaceae and Alistipes) and improved the CD8+/FOXP3+CD4+ ratio within the tumor microenvironment. Moreover, castalagin induced metabolic changes, resulting in an increase in taurine-conjugated bile acids. Oral supplementation of castalagin following fecal microbiota transplantation from ICI-refractory patients into mice supported anti–PD-1 activity. Finally, we found that castalagin binds to Ruminococcus bromii and promoted an anticancer response. Altogether, our results identify castalagin as a polyphenol that acts as a prebiotic to circumvent anti–PD-1 resistance.Significance:The polyphenol castalagin isolated from a berry has an antitumor effect through direct interactions with commensal bacteria, thus reprogramming the tumor microenvironment. In addition, in preclinical ICI-resistant models, castalagin reestablishes the efficacy of anti–PD-1. Together, these results provide a strong biological rationale to test castalagin as part of a clinical trial.This article is highlighted in the In This Issue feature, p. 873

Venous thrombotic events (VTEs) are a frequent complication of non-small cell lung cancer (NSCLC) and are associated with increased morbidity. Immune checkpoint inhibitors (ICIs) are revolutionizing the management of NSCLC, but little is known about their impact on thrombosis. This study aims to define the incidence and clinical relevance of VTEs in NSCLC patients receiving these treatments.A retrospective multicentric cohort study including 593 patients from three centers in Canada and France was performed. The cumulative incidence of VTEs after ICIs was estimated using competing risk analysis, and the association of these events with survival and response to treatment was determined. Finally, univariate and multivariate tests were performed to identify VTE risk factors.The cumulative incidence of VTEs in the cohort was 14.8% (95% CI = 7.4-22.2%) for an incidence rate of 76.5 (95% CI = 59.9-97.8) thrombosis per 1000 person-years, with most thromboses occurring rapidly after treatment initiation. VTEs were not correlated with overall survival, progression-free survival, or objective response to ICIs. Age ˂ 65 years old (HR = 2.00; 95% CI = 1.11-3.59) and tumors with PD-L1 1-49% (HR = 3.36; 95% CI = 1.19-9.50) or PD-L1 ≥ 50% (HR = 3.22; 95% CI = 1.21-8.57) were associated with more VTEs after 12 months of ICI initiation. Also, a delay of less than 12 months from diagnosis to the first ICI treatment (HR = 2.06; 95% CI = 1.09-3.89) and active smoking (HR = 2.00; 95% CI = 1.12-3.58) are probable risk factors of VTEs.This study suggests that the incidence of VTEs in NSCLC patients treated with ICIs is comparable to what is reported in other cohorts of patients treated with chemotherapy. In our cohort, VTEs were not associated with a decreased survival or response to therapy. Patient age 65 and tumors with PD-L1 ≥ 1% were associated with a higher risk of VTEs under ICIs.

The PACIFIC trial demonstrated that durvalumab therapy following chemoradiation (CRT) was associated with improved overall survival (OS) in patients with stage III non-small cell lung cancer (NSCLC). It is unclear whether the results obtained as part of randomised controlled trials are a reflection of real-world (RW) data. Several questions remain unanswered with regard to RW durvalumab use, such as optimal time to durvalumab initiation, incidence of pneumonitis and response in PD-L1 subgroups.In this multicentre retrospective analysis, 147 patients with stage III NSCLC treated with CRT followed by durvalumab were compared with a historical cohort of 121 patients treated with CRT alone. Survival curves were estimated using the Kaplan-Meier method and compared with the log-rank test in univariate analysis. Multivariate analysis was performed to evaluate the effect of standard prognostic factors for durvalumab use.Median OS was not reached in the durvalumab group, compared with 26.9 months in the historical group (hazard ratio [HR]: 0.56, 95% confidence interval [CI]: 0.37-0.85, p = 0.001). In the durvalumab group, our data suggest improved 12-month OS in patients with PD-L1 expression ≥50% (100% vs 86%, HR: 0.25, 95% CI: 0.11-0.58, p = 0.007). There was no difference in OS between patients with a PD-L1 expression of 1-49% and patients with PD-L1 expression1%. Delay in durvalumab initiation beyond 42 days did not impact OS. Incidence of pneumonitis was similar in the durvalumab and historical groups. In the durvalumab group, patients who experienced any-grade pneumonitis had a lower 12-month OS than patients without pneumonitis (85% vs 95%, respectively; HR: 3.3, 95% CI: 1.2-9.0, p = 0.006).This multicentre analysis suggests that PD-L1 expression ≥50% was associated with favourable OS in patients with stage III NSCLC treated with durvalumab after CRT, whereas the presence of pneumonitis represented a negative prognostic factor.

Immune checkpoint inhibitors (ICI) represent the backbone treatment for advanced non-small cell lung cancer (NSCLC). Emerging data suggest that increased gut microbiome diversity is associated with favorable response to ICI and that antibiotic-induced dysbiosis is associated with deleterious outcomes. 18F-FDG physiologic colonic uptake on PET/CT increases following treatment with antibiotics (ATB) and could act as a surrogate marker for microbiome composition and predict prognosis. The aim of this study was to determine if 18F-FDG physiologic colonic uptake prior to ICI initiation correlates with gut microbiome profiling and clinical outcomes in patients with advanced NSCLC. Seventy-one patients with advanced NSCLC who underwent a PET/CT prior to ICI were identified. Blinded colonic contouring was performed for each colon segment and patients were stratified according to the median of the average colon SUVmax as well as for each segment in low vs. high SUVmax groups. Response rate, progression-free survival (PFS), and overall survival (OS) were compared in the low vs. high SUVmax groups. Gut microbiome composition was analyzed for 23 patients using metagenomics sequencing. The high colon SUVmax group had a higher proportion of non-responders (p = 0.033) and significantly shorter PFS (4.1 vs. 11.3 months, HR 1.94, 95% CI 1.11–3.41, p = 0.005). High caecum SUVmax correlated with numerically shorter OS (10.8 vs. 27.6 months, HR 1.85, 95% CI 0.97–3.53, p = 0.058). Metagenomics sequencing revealed distinctive microbiome populations in each group. Patients with low caecum SUVmax had higher microbiome diversity (p = 0.046) and were enriched with Bifidobacteriaceae, Lachnospiraceae, and Bacteroidaceae. Lower colon physiologic 18F-FDG uptake on PET/CT prior to ICI initiation was associated with better clinical outcomes and higher gut microbiome diversity in patients with advanced NSCLC. Here, we propose that 18F-FDG physiologic colonic uptake on PET/CT could serve as a potential novel marker of gut microbiome composition and may predict clinical outcomes in this population.

Several approaches to manipulate the gut microbiome for improving the activity of cancer immune-checkpoint inhibitors (ICI) are currently under evaluation. Here, we show that oral supplementation with the polyphenol-rich berry camu-camu (CC; Myrciaria dubia) in mice shifted gut microbial composition, which translated into antitumor activity and a stronger anti–PD-1 response. We identified castalagin, an ellagitannin, as the active compound in CC. Oral administration of castalagin enriched for bacteria associated with efficient immunotherapeutic responses (Ruminococcaceae and Alistipes) and improved the CD8+/FOXP3+CD4+ ratio within the tumor microenvironment. Moreover, castalagin induced metabolic changes, resulting in an increase in taurine-conjugated bile acids. Oral supplementation of castalagin following fecal microbiota transplantation from ICI-refractory patients into mice supported anti–PD-1 activity. Finally, we found that castalagin binds to Ruminococcus bromii and promoted an anticancer response. Altogether, our results identify castalagin as a polyphenol that acts as a prebiotic to circumvent anti–PD-1 resistance. Significance: The polyphenol castalagin isolated from a berry has an antitumor effect through direct interactions with commensal bacteria, thus reprogramming the tumor microenvironment. In addition, in preclinical ICI-resistant models, castalagin reestablishes the efficacy of anti–PD-1. Together, these results provide a strong biological rationale to test castalagin as part of a clinical trial. This article is highlighted in the In This Issue feature, p. 873

Immune checkpoint inhibitors (ICI) represent the backbone treatment for advanced non-small cell lung cancer (NSCLC). Emerging data suggest that increased gut microbiome diversity is associated with favorable response to ICI and that antibiotic-induced dysbiosis is associated with deleterious outcomes.Seventy-one patients with advanced NSCLC who underwent a PET/CT prior to ICI were identified. Blinded colonic contouring was performed for each colon segment and patients were stratified according to the median of the average colon SUVThe high colon SUVLower colon physiologic

Introduction Studies suggest that patients with cancer are more likely to experience severe outcomes from COVID-19. Therefore, cancer centers have undertaken efforts to care for patients with cancer in COVID-free units. Nevertheless, the frequency and relevance of nosocomial transmission of COVID-19 in patients with cancer remain unknown. The goal of this study was to determine the incidence and impact of hospital-acquired COVID-19 in this population and identify predictive factors for COVID-19 severity in patients with cancer. Methods Patients with cancer and a laboratory-confirmed diagnosis of COVID-19 were prospectively identified using provincial registries and hospital databases between March 3rd and May 23rd, 2020 in the provinces of Quebec and British Columbia in Canada. Patient’s baseline characteristics including age, sex, comorbidities, cancer type, and type of anti-cancer treatment were collected. The exposure of interest was incidence of hospital-acquired infection defined by diagnosis of SARS-CoV-2 ≥ 5 days after hospital admission for COVID-unrelated cause. Co-primary outcomes were death or composite outcomes of severe illness from COVID-19 such as hospitalization, supplemental oxygen, intensive-care unit (ICU) admission and/or mechanical ventilation. Results A total of 252 patients (N=249 adult, and N=3 pediatric) with COVID-19 and cancer were identified, and the majority were residents of Quebec (N=233). One-hundred-and-six patients (42.1%) received active anti-cancer treatment in the last 3 months prior to COVID-19 diagnosis. During a median follow-up of 25 days, 33 (13.1%) required admission to the ICU, and 71 (28.2%) died. Forty-seven (19.1%) had a diagnosis of hospital-acquired COVID-19. Median overall survival was shorter in those with hospital-acquired infection, compared to a contemporary community-acquired population (27 days vs unreached, HR 2.3, 95% CI 1.2-4.4, p=0.0006). Multivariate analysis demonstrated that hospital-acquired COVID-19, age, ECOG status, and advanced stage of cancer were independently associated with death. Interpretation Our study demonstrates a high rate of nosocomial transmission of COVID-19, associated with increased mortality in both univariate and multivariate analysis in the cancer population, reinforcing the importance of treating patients with cancer in COVID-free units. We also validated that age and advanced cancer were negative predictive factors for COVID-19 severity in patients with cancer., Highlights • We found a high rate of nosocomial transmission of COVID-19 in patients with cancer. • Nosocomial COVID-19 increased mortality compared to community-acquired infection. • Nosocomial COVID-19, age, ECOG, and stage were independently associated with death. • COVID-free units and infection control procedures are required to protect patients.

e21198 Background: Venous thromboembolism is a frequent complication of non-small cell lung cancer (NSCLC) and is associated with a worse prognosis, a reduced quality of life, and increased healthcare costs. Immune checkpoint inhibitors (ICI) are revolutionizing the management of NSCLC, but little is known about their impact on thrombosis. This study aims to define the incidence and clinical relevance of thrombosis in NSCLC patients receiving these treatments. Methods: A retrospective multicentric cohort study including 593 patients from three centers in Canada and France was performed. The cumulative incidence of venous thrombotic events after ICIs was calculated, and the impact of these events on survival and response to treatment was determined. Finally, univariate log-rank tests were performed to identify thrombosis risk factors. Results: The incidence of venous thrombosis in the cohort was 9.9% for an incidence rate of 76.5 thrombosis per 1000 person-years, with most thromboses occurring rapidly after treatment initiation. Thrombosis was not correlated with overall survival, progression-free survival, or objective response to ICIs (summarized in the table below). Age ˂ 65 years old (HR = 1.66; 95 % CI = 1.00 – 2.82) and a delay of less than 12 months from diagnosis to the first ICI treatment (HR = 1.74; 95 % CI = 1.03 – 2.87) were associated with an increased risk of thrombosis. Tumors with PD-L1 > 1% were associated with more thrombosis in the first year since the beginning of therapy (HR = 3.06; 95 % CI = 1.19 – 4.76, p=0.015). Conclusions: This study suggests that the time distribution and incidence of thrombotic events in NSCLC patients treated with ICI are comparable to what is reported in other cohorts of patients treated with chemotherapy. In our cohort, thrombosis was not a prognostic factor for survival or response to therapy. Patient age < 65 and tumors with PD-L1 > 1% were associated to a higher risk of thrombotic events.[Table: see text]

e21010 Background: ICIs changed the way NSCLC is treated, but not all patients benefit from it. PD-L1 level is used to predict response to therapy, but its performance is sub-optimal. KRAS is important in NSCLC tumorigenesis, but the impact of its mutations in patients treated with ICIs is unclear. Similarly, studies evaluating co-mutations in TP53, STK11 and KEAP1 as well as the NLR showed that they may predict the benefit of ICIs. Methods: We conducted a retrospective study including all consenting patients with NSCLC treated with ICIs at the CHUM between July 2015 and June 2020. OS and PFS were compared in co-mutation subgroups using Kaplan-Meier and logrank methods. Co-mutations in TP53, STK11 and KEAP1 as well as the NLR were accounted for. Overall response rate (ORR) and safety data was also compared in subgroups and will be detailed at the meeting. Results: We included 100 patients with known KRAS status. From these, 50 were wild-type ( KRASWT) and 50 were mutated ( KRASMut). The most frequent mutation was G12C (54%). Co-mutation status for TP53, STK11 and KEAP1 were known for, respectively, 40, 39 and 38 patients. Co-mutations for these genes were present in respectively 19 (47.5%), 8 (20.5%) and 4 (10.5%). Data comparing KRASMut and KRASWT showed non-significant differences in survival (median OS of respectively 21.1 vs. 17.7 months, p = 0.27). The presence of STK11 and/or KEAP1 mutations was associated with a negative impact on survival when compared with wild-type (median OS 7.4 vs 20.4 months, p = 0.001). When the presence of a KRAS mutation was compounded with STK11 and KEAP1, KRASMut (vs KRASWT) trended to a better prognosis in STK11+KEAP1WT tumors (median OS of 21.1 for KRASMut vs 15.8 for KRASWT, p = 0.15), but not in STK11+/-KEAP1Mut tumors (7.4 for KRASMut vs 7.0 for KRASWT). No influence on survival was seen in relationship to the TP53 co-mutation. Interestingly, the NLR was significantly higher with STK11 mutations (6.66Mut vs 3.59WT, p = 00012), slightly lower with TP53 mutations (3.23Mut vs 4.82WT, p = 0.047) but not impacted by KEAP1 (3.72Mut vs 4.20WT, p = 0.72) or KRAS mutations (4.32Mut vs 5.21WT, p = 0.34). Conclusions: The STK11 and KEAP1 mutations are significant adverse predictors of ICI therapy benefit. The NLR is strongly impacted by STK11 mutations but not by KEAP1 mutations suggesting marked differences in the resistance mechanism for both mutations. In STK11-KEAP1WT tumors, KRAS mutations seems to be associated with improved survival in NSCLC patient treated with ICIs.

Background: Studies suggest that patients with cancer are more likely to experience severe outcomes from COVID-19. Therefore, cancer centers have undertaken efforts to care for patients with cancer in COVID-free zones. Nevertheless, nosocomial transmission of COVID-19 in patients with cancer likely occurs, but the frequency and relevance of these events remain unknown. The goal of this study was to determine the incidence and impact of hospital-acquired COVID-19 in this population and identify prognostic factors for COVID-19 severity in patients with cancer. Methods: Patients with cancer and a laboratory-confirmed or presumed diagnosis of COVID-19 were prospectively identified using provincial registries and hospital databases between March 3rd and May 23rd, 2020, in the provinces of Quebec and British Columbia. Patients’ baseline characteristics including age, sex, comorbidities, cancer type, and type of anticancer treatment were collected. The primary outcome was incidence of hospital-acquired infection defined by diagnosis of SARS-CoV-2 5 days after hospital admission for COVID-unrelated cause. Co-primary outcomes were death or composite outcomes of severe illness from COVID-19 such as hospitalization, supplemental oxygen, intensive-care unit (ICU) admission, and/or mechanical ventilation. Results: A total of 253 patients (N=250 adult and N=3 pediatric) with COVID-19 and cancer were identified, and the majority were residents of Quebec (N=236). Ninety patients (35.6%) received active anticancer treatment in the last 3 months prior to COVID-19 diagnosis. During a median follow-up of 23 days, 209 (82.6%) required hospitalization, 38 (15%) required admission to ICU, and 71 (28%) died. Forty-seven (19%) had a diagnosis of hospital-acquired COVID-19. Median overall survival was shorter in those with hospital-acquired infection, compared to a contemporary community-acquired population (27 days vs. 71 days, HR 2.2, 95% CI 1.2-4.0, p=0.002). Multivariate analysis demonstrated that hospital-acquired COVID-19, age, ECOG status, and advanced stage of cancer were independently associated with death. Conclusion: Our study demonstrates a high rate of nosocomial transmission of COVID-19, associated with increased mortality in both univariate and multivariate analysis in the cancer population, reinforcing the importance of treating patients with cancer in COVID-free zones. We also validated that age, poor ECOG, and advanced cancer were negative prognostic factors for COVID-19 in patients with cancer. Citation Format: Arielle Elkrief, Antoine Desilets, Neha Papneja, Lena Cvetkovic, Catherine Groleau, Yahia Abdelali Lakehal, Layla Shbat, Corentin Richard, Julie Malo, Wiam Belkaid, Erin Cook, Stephane Doucet, Thai Hoa Tran, Patrice Savard, Kevin Jao, Nathalie Daaboul, Eric Bhang, Jonathan Loree, Wilson Miller, Donald Vinh, Nathaniel Bouganim, Gerald Batist, Caroline Letendre, Bertrand Routy. High mortality among hospital-acquired COVID-19 infection in patients with cancer: An observational cohort study from Quebec and British Columbia [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2020 Jul 20-22. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(18_Suppl):Abstract nr S12-01.

9600 Background: Immune checkpoint inhibitors (ICI) represent the backbone treatment of advanced non-small cell lung cancer (aNSCLC) patients. Emerging evidence suggests increased gut microbiome (GM) diversity is associated with favorable response. Conversely, antibiotic-induced dysbiosis may be associated with deleterious outcomes in patients receiving ICI in multiple retrospective studies and one prospective study. 18F-FDG physiologic colonic uptake on PET/CT increases following treatment with antibiotics and could be a surrogate marker for GM diversity and therefore clinical response. The aim of this study was to determine if 18F-FDG physiologic colonic uptake prior to ICI initiation correlates with outcomes and GM metagenomics in patients with advanced NSCLC. Methods: 71 patients with aNSCLC who underwent PET/CT prior to ICI were identified. For each patient, the colon was manually contoured, SUVmax was measured in each segment of the colon by a nuclear medicine specialist and average SUVmax was calculated for the whole colon. Patients were stratified in two groups according to median colon SUVmax (low vs high uptake). 18F-FDG physiologic colonic uptake was then compared to overall survival (OS), objective response (ORR), and progression-free survival (PFS). For patients with available stool samples (n = 10), GM composition was defined using metagenomics sequencing. Results: 71 patients (54% men, median age: 68 years) with aNSCLC were included in the study and ICI was the first line of therapy in 38% of those patients. The mean colon SUV for the low and high uptake groups were 1.41 (CI 95% 1.35-1.47) and 2.18 (CI 95% 1.90-2.46) respectively. The high uptake group had a higher proportion of non-responders (p = 0.033) and significant shorter PFS (4.1 months vs 11.3 months, p = 0.005). In the caecum, high uptake also correlated with numerically shorter OS (10.82 vs 27.56 months, p = 0.058) compared to low uptake group. Despite the low number of samples, metagenomics sequencing revealed that PLS-DA (Partial Least Squares Discriminant Analysis) for diversity was lower in the high SUV group (p = 0.008). Conclusions: Higher colon SUVmax on pre-ICI FDG PET/CT is associated with worse clinical outcomes and lower baseline GM diversity in patients with advanced NSCLC. Here, we propose that 18F-FDG physiologic colonic uptake on PET/CT could serve as a surrogate marker of GM diversity and predicts clinical outcomes.

Background: The gut microbiota has been shown to be an important determinant of the efficacy of immune checkpoint inhibitions (ICI) in cancer. Several lines of evidence suggest that antibiotic (ATB) usage prior to or within the first month of ICI initiation negatively impacts clinical outcomes. Methods: We examined patients with advanced melanoma treated with an anti-PD-1 monoclonal antibody (mAb) or an anti-CTLA-4 mAb alone or in combination with chemotherapy. Those receiving ATB within 30 days of beginning ICI were compared with those who did not receive ATB. Response rates as determined by RECIST 1.1, progression-free survival (PFS), overall survival (OS) and immune-related toxicities were assessed. Results: Of these 74 patients analyzed, a total of 10 patients received ATB (13.5%) within 30 days of initiation of ICI. Patients who received ATB 30 days prior to the administration of ICI experienced more primary resistance (progressive disease) (0% of the objective response rate compared to 34%), and progression-free survival (PFS) was significantly shorter (2.4 vs 7.3 months, HR 0.28, 95% CI (0.10–0.76) p = 0.01). Overall survival (OS) was also shorter; however, this was not statistically significant (10.7 vs 18.3 months, HR:0.52, 95% CI (0.21–1.32) p = 0.17). The multivariate analysis further supported that ATB administration was associated with worse PFS (HR 0.32 (0.13–0.83) 95% CI, p = 0.02). Conclusion: These findings suggest that ATB use within 30 days prior to ICI initiation in patients with advanced melanoma may adversely affect patient outcomes.

Axonal degeneration after traumatic brain injury and nerve compression is considered a common underlying cause of temporary as well as permanent disability. Because a proper functioning of neural network requires phase coherence of all components, even subtle changes in circuitry may lead to network failure. However, it is still not possible to determine which axons will recover or degenerate after injury. Several groups have studied the pressure threshold for axonal injury within a nerve, but difficulty accessing the injured region; insufficient imaging methods and the extremely small dimensions involved have prevented the evaluation of the response of individual axons to injury. We combined microfluidics with atomic force microscopy and in vivo imaging to estimate the threshold force required to 1), uncouple axonal transport without impairing axonal survival, and 2), compromise axonal survival in both individual and bundled axons. We found that rat hippocampal axons completely recover axonal transport with no detectable axonal loss when compressed with pressures up to 65 ± 30 Pa for 10 min, while dorsal root ganglia axons can resist to pressures up to 540 ± 220 Pa. We investigated the reasons for the differential susceptibility of hippocampal and DRG axons to mechanical injury and estimated the elasticity of live axons. We found that dorsal root ganglia axons have a 20% lower elastic modulus than hippocampal axons. Our results emphasize the importance of the integrity of the axonal cytoskeleton in deciding the axonal fate after damage and open up new avenues to improve injury diagnosis and to identify ways to protect axons.

The formation of functional synapses on artificial substrates is a very important step in the development of engineered in vitro neural networks. Spherical supported bilayer lipid membranes (SS-BLMs) are used here as a novel substrate to demonstrate presynaptic vesicle accumulation at an in vitro synaptic junction. Confocal fluorescence microscopy, cryo-transmission electron microscopy (cryo-TEM), and fluorescence recovery after photobleaching (FRAP) experiments have been used to characterize the SS-BLMs. Conventional immunocytochemistry combined with confocal fluorescence microscopy was used to observe the formation of presynaptic vesicles at the neuron-SS-BLM contacts. These results indicate that lipid phases may play a role in the observed phenomenon, in addition to the chemical and electrostatic interactions between the neurons and SS-BLMs. The biocompatibility of lipid bilayers along with their membrane tunability makes the suggested approach a useful "toolkit" for many neuroengineering applications including artificial synapse formation and synaptogenesis in vivo.

Nodes of Ranvier are regularly placed, nonmyelinated axon segments along myelinated nerves. Here we show that nodal membranes isolated from the central nervous system (CNS) of mammals restricted neurite outgrowth of cultured neurons. Proteomic analysis of these membranes revealed several inhibitors of neurite outgrowth, including the oligodendrocyte myelin glycoprotein (OMgp). In rat spinal cord, OMgp was not localized to compact myelin, as previously thought, but to oligodendroglia-like cells, whose processes converge to form a ring that completely encircles the nodes. In OMgp-null mice, CNS nodes were abnormally wide and collateral sprouting was observed. Nodal ensheathment in the CNS may stabilize the node and prevent axonal sprouting.

Background Normal development and the response to injury both require cell growth, migration and morphological remodeling, guided by a complex local landscape of permissive and inhibitory cues. A standard approach for studying by such cues is to culture cells on uniform substrates containing known concentrations of these molecules, however this method fails to represent the molecular complexity of the natural growth environment. Results To mimic the local complexity of environmental conditions in vitro, we used a contact micropatterning technique to examine cell growth and differentiation on patterned substrates printed with the commonly studied growth permissive and inhibitory substrates, poly-L-lysine (PLL) and myelin, respectively. We show that micropatterning of PLL can be used to direct adherence and axonal outgrowth of hippocampal and cortical neurons as well as other cells with diverse morphologies like Oli-neu oligodendrocyte progenitor cell lines and fibroblast-like COS7 cells in culture. Surprisingly, COS7 cells exhibited a preference for low concentration (1 pg/mL) PLL zones over adjacent zones printed with high concentrations (1 mg/mL). We demonstrate that micropatterning is also useful for studying factors that inhibit growth as it can direct cells to grow along straight lines that are easy to quantify. Furthermore, we provide the first demonstration of microcontact printing of myelin-associated proteins and show that they impair process outgrowth from Oli-neu oligodendrocyte precursor cells. Conclusion We conclude that microcontact printing is an efficient and reproducible method for patterning proteins and brain-derived myelin on glass surfaces in order to study the effects of the microenvironment on cell growth and morphogenesis.

To understand the molecular anatomy of myelin membranes, we performed a large-scale, liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS)-based lipidome and proteome screen on freshly purified human and murine myelin fractions. We identified more than 700 lipid moieties and above 1,000 proteins in the two species, including 284 common lipids and 257 common proteins. This study establishes the first comprehensive map of myelin membrane components in human and mice. Although this study demonstrates many similarities between human and murine myelin, several components have been identified exclusively in each species. Future quantitative validation studies focused on interspecies differences will authenticate the myelin membrane anatomy. The combined lipidome and proteome map presented here can nevertheless be used as a reference library for myelin health and disease.

Classical cadherins, which are adhesion molecules functioning at the CNS synapse, are synthesized as adhesively inactive precursor proteins in the endoplasmic reticulum (ER). Signal sequence and prodomain cleavage in the ER and Golgi apparatus, respectively, activates their adhesive properties. Here, we provide the first evidence for sorting of nonadhesive precursor N-cadherin (ProN) to the neuronal surface, where it coexists with adhesively competent mature N-cadherin (N-cad), generating a spectrum of adhesive strengths. In cultured hippocampal neurons, a high ProN/N-cad ratio downregulates synapse formation. Neurons expressing genetically engineered uncleavable ProN make markedly fewer synapses. The synapse number can be rescued to normality by depleting surface ProN levels through prodomain cleavage by an exogenous protease. Finally, prodomain processing is developmentally regulated in the rat hippocampus. We conclude that it is the ProN/N-cad ratio and not mature N-cad alone that is critical for regulation of adhesion during synaptogenesis.

CNS synapse assembly typically follows after stable contacts between “appropriate” axonal and dendritic membranes are made. We show that presynaptic boutons selectively formde novofollowing neuronal fiber adhesion to beads coated with poly-d-lysine (PDL), an artificial cationic polypeptide. As demonstrated by atomic force and live confocal microscopy, functional presynaptic boutons self-assemble as rapidly as 1 h after bead contact, and are found to contain a variety of proteins characteristic of presynaptic endings. Interestingly, presynaptic compartment assembly does not depend on the presence of a biological postsynaptic membrane surface. Rather, heparan sulfate proteoglycans, including syndecan-2, as well as others possibly adsorbed onto the bead matrix or expressed on the axon surface, are required for assembly to proceed by a mechanism dependent on the dynamic reorganization of F-actin. Our results indicate that certain (but not all) nonspecific cationic molecules like PDL, with presumably electrostatically mediated adhesive properties, can effectively bypass cognate and natural postsynaptic ligands to trigger presynaptic assembly in the absence of specific target recognition. In contrast, we find that postsynaptic compartment assembly depends on the prior presence of a mature presynaptic ending.