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Cancer microenvironment is now recognized as a critical regulator of all stages of cancer development. Beside the tumor vasculature and tumor-infiltrating immune cells, other stromal cells such as cancer-associated fibroblasts (CAFs) regulate tumor growth. Fibroblasts are ubiquitous cells in connective tissue, where they shape the extracellular matrix (ECM). Fibroblasts are usually quiescent but get activated when tissue homeostasis is disturbed. Then, activated fibroblasts rebuild the ECM and communicate with local cells to participate in wound repair. These repair properties can go awry when being unchecked, which can lead to fibrosis and subsequently cancer development. CAFs can promote cancer development by fostering tumor cell growth, polarizing immune cells to an immunosuppressive phenotype, and crosslinking collagen to enable tumor cell invasion. Molecular mechanisms of CAF activation, thus, need to be understood to target these cells in tumors. Prostanoid prostaglandin E2 (PGE2) is viewed as a pro-tumor lipid mediator as suggested by studies pharmacologically or genetically targeting the enzymes producing PGE2, such as microsomal PGE synthase-1 (mPGES-1) in tumor models. Similar to CAFs, PGE2 drives tumor cell growth and tumor-associated immune suppression. Therefore, I hypothesized that PGE2 may play a role in CAF activation.
This hypothesis was tested in two mouse models of breast cancer (orthotopic grafting model, and polyoma middle T oncogene transgenic model), besides using isolated mammary gland (MG) fibroblasts in vitro. As expected, given the pro-tumor function of PGE2, knocking out mPGES-1 reduced the growth of oncogene-driven and transplanted mammary tumors. Surprisingly, CAF density was markedly increased when mPGES-1 was depleted. Importantly, despite reduced primary tumor growth, I observed enhanced lung metastasis upon mPGES-1depletion. Using MG-derived fibroblasts in vitro furthermore revealed that treatment with PGE2 reduced a TGFβtriggered CAF-like activation state. Importantly, bioinformatics analysis of a human breast cancer patient dataset revealed a negative correlation of a PGE2 production signature with fibroblast marker genes. In a next step I investigated if the increased CAF infiltrate was connected to the reduced tumor growth upon depletion of PGE2. To unravel this, I first asked through which E prostanoid (EP) receptor PGE2 signals in fibroblasts. MG fibroblasts mainly expressed EP3, and EP3 KO fibroblasts showed a hyper-proliferative and activated phenotype, indicating EP3 as the main PGE2 receptor in MG fibroblasts. Co-injecting of EP3 KO MG fibroblasts and tumor cells in WT mice suppressed tumor growth, whereas co-injection of WT fibroblasts with tumor cell in mPGES-1 KO mice increased tumor growth. These data indicate that PGE2 restricts CAF levels through EP3, which supports tumor growth. Whole transcriptome mRNAsequencing of WT and mPGES-1 KO FACS-sorted CAFs combined with immunohistochemical data suggested a role of p38 mitogen-activated protein kinase (MAPK) in the modulation of fibroblast activation by PGE2.
In summary, I showed in two breast cancer models that mPGES-1 depletion delays breast cancer progression, which is probably driven by the EP3-PGE2 signaling axis in host stroma. PGE2 appears to be a potent anti-fibroblast activation agent in tumors via EP3 and downstream p38 MAPK signaling. This study therefore hits the dogmatic perception of the general pro-tumor nature of PGE2; showing that PGE2 might be a double-edged mediator that can promote tumor growth at the primary site by restricting CAF expansion, which may in turn hinder infiltration of tumor cells to a secondary site.
Despite major improvements of the therapy, many B-cell Non-Hodgkin’s lymphoma (B-NHL) entities still have a poor prognosis. New therapeutic options are urgently needed. Therefore this study sets out to investigate oncogenic signalling pathways in the two B-NHL entities mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL) in order to define new potential therapeutic targets.
MCL cells overexpress the anti-apoptotic protein BCL-2, thereby they evade apoptosis. With venetoclax, the first-in-class BCL-2 specific inhibitor was approved and achieved good response rates in MCL. However, some cases display intrinsic or acquired resistance to venetoclax. In order to improve the therapy, this study aimed to identify genes which confer sensitivity or resistance towards venetoclax upon their respective knockout. To this end, a genome-wide CRISPR/Cas9-based loss-of-function screen was conducted in the MCL cell line Maver-1. The E3 ubiquitin
ligase MARCH5 was identified as one of the top hits conferring sensitivity
towards venetoclax upon its knockout. This finding was validated in a competitive growth assay including two more MCL cell lines, Jeko-1 and Mino. MARCH5 knockout also sensitised Jeko-1 cells towards venetoclax even though this cell line was insensitive towards venetoclax in its wild-type form. Using BH3 profiling, an increased dependency on BCL-2 of MARCH5-depleted cells confirmed this finding. The sensitisation was found to be based on induction of apoptosis upon MARCH5 knockout and to an even higher extent upon additional treatment of MARCH5-depleted cells with venetoclax. As already described for epithelial cancer entities, the BCL-2 family members MCL-1 and NOXA were upregulated in MCL cell lines upon MARCH5 knockout. This led to the hypothesis that MARCH5 is a potential
regulator of intrinsic apoptosis with NOXA as a key component. A competitive growth assay with MARCH5 and NOXA co-depleted cells revealed a partial reversion of the BCL-2 sensitisation compared to MARCH5 knockout alone. Furthermore, mass spectrometry-based methods were used to gain more insight into other cellular pathways and networks which might be regulated in a MARCH5-dependent manner. In an interactome analysis, proteins which regulate mitochondrial morphology, such as Drp-1 were identified as MARCH5 interactors. Besides this expected finding, interaction between MARCH5 and several members of the BCL-2 family as well as a potential connection between MARCH5 and vesicular trafficking was discovered. As expected, an ubiquitinome analysis of MARCH5-depleted cells revealed decreased levels of MCL-1 and NOXA ubiquitination. Additionally, a potential role of MARCH5 in the ubiquitination of several members of the cell cycle regulatory
pathway was discovered. Based on the broad spectrum of cellular pathways which seem to be regulated in a MARCH5-dependent manner, it was hypothesised that MARCH5 primarily regulates BCL-2 family members which in turn regulate intrinsic apoptosis on the one hand and additionally are involved in the regulation of various other pathways on the other hand.
In summary, this study provides insight into a MARCH5-dependent MCL1-1/NOXA axis in MCL cells and potential implications into related cellular processes.
In addition to the anti-apoptotic pathways described above, B-cell receptor (BCR) signalling is known to provide a pro-survival signal to both normal and malignant B-cells. Targeting the BCR signalling pathway therefore is a promising therapeutic target for B-cell malignancies. In order to gain more insight into the differential modes of BCR signalling of ABC- and GCB-DLBCL cells, genes/proteins which displayed differential essentiality in ABC- and GCB-DLBCL cells were aimed to be defined. Consequently, data sets from a CRISPR/Cas9-based loss-of-function screen
were re-analysed. SASH3 was identified as a gene which was essential for GCB- but not for ABC-DLBCL cells. Since this protein is known to be involved in T-cell receptor (TCR)-signalling, SASH3 was assumed to play a potential role in BCR signalling as well and was therefore investigated in more detail. A competitive growth assay confirmed that SASH3 knockout was toxic exclusively for GCB-DLBCL cell lines. An interactome analysis in ABC- and GCB-DLBCL cells revealed interaction between SASH3 and many components of the proximal BCR signalling pathway as well as several downstream signalling pathways such as the PI3K or the NF-ΚB pathway.
An integration of the interactome with data from the CRISPR/Cas9-based loss-offunction screen revealed differential essentiality of the SASH3-interacting proteins in ABC- and GCB-DLBCL cells. It was hypothesised that SASH3 might regulate PI3K signalling on which GCB- but not ABC-DLBCL cells are known to dependent. Discontinuation of the regulation of PI3K signalling could therefore be exclusively toxic to GCB-DLBCL cells.
Taken together, this study describes a subtype-specific dependency of GCB-DLBCL cells on SASH3. Furthermore, the SASH3 interactome has been investigated in B-cells for the first time, thereby highlighting a potential role in proximal BCR signalling and involvement in specific BCR-related downstream signalling pathways.
Development of treatment strategies of chronic inflammatory disorders relies on on-going progress in drug discovery approaches and related molecular biologics. This study presents a gene reporter-based approach of phenotypic screening for anti-inflammatory compounds in the context of rheumatoid arthritis (RA).
CEBPD gene, used as the target gene for the screening readout, encodes CCAAT/enhancer binding protein delta (C/EBPδ) transcription factor (TF). Structural and regulatory characteristics of CEBPD gene as well as function of C/EBPδ TF in the context of inflammation satisfied assay requirements. C/EBPδ TF acts as a key regula-tor of inflammatory gene transcription in macrophages (Mϕ) and is observed to con-tribute to disease development in both a rodent model of RA and RA patient biopsies.
Despite well-described pro-inflammatory effects of C/EBPδ TF, it functions as a cell context-specific signal integrator showing also an anti-inflammatory activity. Conse-quently, both activation and inhibition of CEBPD alike may display a desired anti-inflammatory effect. The aim of this study was to develop a high-throughput screening assay for
CEBPD-modulating compounds and confirm hit compounds’ anti-inflammatory effects via gene expression analysis.
Generation and characterization of a multi-gene-reporter cassette 1.0 encoding enzy-matic secreted alkaline phosphatase (SEAP) gene reporter was a priority during the assay development. Chemiluminescent SEAP assay demonstrating high assay sensitivi-ty, broad linear range, high reproducibility and repeatability was chosen to monitor activity of the defined CEBPD promoter (CEBPD::SEAP). PMA-differentiated and M1-polarized THP-1-derived Mϕ stably expressing multi-gene-reporter cassette 1.0 were used as the assay’s cellular system. mRNA expression of both reporter CEBPD::SEAP and endogenous CEBPD mirrored each other in response to a LPS and IFN-g-triggered inflammatory stimulus (M1 treatment), even though the defined CEBPD promoter re-gion, utilized in the assay, contained only the most proximal and known regulatory se-quences. SEAP chemiluminescence in the reporter cells´ supernatant reliably correlat-ed with the M1 treatment-induced CEBPD::SEAP gene expression. The final screening protocol was developed for semi-automatic screening in the 384-well format.
In total, 2054 compounds from LOPAC®1280 and ENZO®774 libraries were screened twice
using the enzymatic SEAP readout with subsequent analysis of 18 selected compounds: nine with the highest and nine with the lowest signals, further characterized by qPCR. Gene expression levels of endogenous CEBPD, CEBPD::SEAP reporter as well as, IL-6,
IL-1β, and CCL2 as inflammatory markers were quantified. qPCR assays failed to corre-late to SEAP readout in 15 compounds within three standard deviations (SDs) from sol-vent control: nine low signal and six high signal compounds. Demonstrating both assay sensitivity and specificity, a correlation between qPCR gene expression and SEAP readout was observed for three hit compounds with signals above three SDs: BET inhib-itors (BETi) GSK 1210151A and Ro 11-1464 as well as an HDAC inhibitor (HDACi) vori-nostat. The control compound trichostatin A (TSA) that reproducibly upregulated SEAP readout is also an HDAC inhibitor with a similar structure to vorinostat and was there-fore included in the anti-inflammatory phenotype analysis.
The observed suppression of IL-6, IL-1ß, and CCL2 gene expression by hit compounds suggested their anti-inflammatory effect in THP-1 reporter Mϕ. mRNA expression of
IL-6 and CCL2 was suppressed by HDACi and BETi at both 4 and 24 hours, while BETi reduced IL-1β mRNA expression 24 hour time point. BETi significantly upregulated gene expression of both reporter CEBPD::SEAP and endogenous CEBPD, 4 hours after M1 treatment. At the same time point, HDACi completely abolished the mRNA expres-sion of the endogenous CEBPD, while simultaneously upregulating mRNA expression of the reporter CEBPD::SEAP. The use of the most proximal 300 base pairs region of en-dogenous CEBPD promoter, making the upstream regulatory elements unavailable in the assay, may account for differential expression levels of SEAP and C/EBPδ TF. This observation corroborated the need to include a longer and more extensive CEBPD´s gene regulatory area. Thus, an improved multi-gene-reporter cassette 2.0 was gener-ated to be used on the basis of a bacterial artificial chromosome (BAC) covering CE-BPD´s genomic area of about 200,000 base pairs.
The generated screening assay is flexible, reliable, and sensitive displaying potential for drug discovery and drug repurposing. The pharmacological modulation of CEBPD gene expression, first reported for GSK 1210151A, Ro 11-1464, and vorinostat, contrib-utes to the understanding of inflammatory responses in Mϕ and may have RA thera-peutic applications.
Attention deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder that typically begins in childhood and is associated with the cardinal symptoms of inattentiveness, hyperactivity, and impulsiveness. In a significant number of cases, ADHD persists into adulthood and leads to profound psychosocial impairment and costs to the population. The course of the disorder and the severity of psychosocial impairment are further influenced by the presence of comorbidities. The risk of developing psychiatric comorbidities such as affective disorders, personality disorders and substance use disorders is increased compared to the general population. Studies also indicate that ADHD is associated with a higher burden of somatic disorders such as obesity, diabetes mellitus, asthma and migraine. In the last decades, there has been a growing body of research that identified sex-related differences in ADHD, but there is still insufficient evidence on specific issues. In addition to the sex-ratio, which is more balanced in adulthood compared to childhood, there are also indications that differences exist at the symptom level and that the comorbid disorders that occur more frequently in ADHD also seem to differ in men and women, although the studies are not yet clear on this. Using resting-state analyses of functional magnetic resonance imaging (fMRI), we aimed to address the question of whether we can detect sex differences in ADHD and selected comorbidities (substance use disorder, depression, obesity) based on altered functional connectivity profiles. A central role for the pathogenesis of ADHD is the dysregulation of dopaminergic neurotransmission, specifically altered reward processing, as an expression of impaired impulse control. In the present study, we focused on a neuroanatomical hub, namely, the external part of the globus pallidus (GPe), which we defined as a "region of interest" for the analyses performed. There is growing evidence that the globus pallidus not only plays a role in the extrapyramidal motor system, but also integrates cognitive and reward-related information, functions that are impaired in ADHD. In a first step, we looked for sex differences in ADHD patients (n=137) and separately in healthy controls (HC) (n=45), then we compared a similar group of HC and ADHD patients to compare sex-differences in ADHD patients and HC. In a second step, we investigated whether the neural basis of comorbidity patterns differed between male and female patients. Analysis of the images of 182 participants was performed using the SPM-based CONN toolbox V 18.b. When comparing subjects with ADHD and HC, we observed an interaction between the GPe and the middle left temporal gyrus, with the effect being more pronounced in healthy subjects. When analyzing the large ADHD sample, an interaction between the GPe and the frontal pole/middle right frontal gyrus was observed. The connectivity between the GPe and the frontal and temporal brain areas appeared to be more pronounced in female ADHD patients than in males, with the sex-effect being reversed and more pronounced in healthy subjects. The results suggest that in patients with ADHD there is a loss of sex-specialization in GPe-connectivity. Males with ADHD and depression showed lower functional connectivity between the GPe and parts of the occipital cortex than females with ADHD and depression. To our knowledge, this is the first study to investigate sex-specific functional connectivity networks using a seed-based connectivity analysis of the external globus pallidus in adult ADHD patients with and without comorbidities. The study serves to improve our knowledge of GPe involvement in ADHD and sex-specific recruitment of this network. Taken as a whole, this study contributes to our understanding of the neurobiological correlates of ADHD and suggests possible differences between males and females with ADHD centered on altered connectivity with the GPe, helping to provide a different perspective on current research and new ideas for further studies.
Smoking tobacco throughout pregnancy is one of the single most important avoidable causes of adverse pregnancy outcomes. If compared with other risk factors in the perinatal period, exposure to tobacco smoke is considered to be amongst the most harmful. It is associated with high rates of long and short term morbidity and mortality for mother and child. Despite this importance until now a scientometric analysis about the development and the state of scientific knowledge about smoking and pregnancy has not been published. In order to close this gap this work was conceived. In this dissertation quantitative and qualitative data on this topic was analyzed using a variety of objective scientometric methods like the number of scientific contributions, the number of citations and the modified Hirsch-index (H-index). A collective volume of 10,043 entries covering a time period from 1900 to December 5, 2012 was obtained from the Web of Science (WoS) data base. Publishing activities of authors, institutions and countries, their cooperation, reception within the international scientific community and its reactions were interpreted and illustrated.
Autism Spectrum Disorder (ASD) is a neurodevelopmental condition with an onset in early development. ASD has varying degrees of severity and thus affects people differently throughout their lives. Early diagnosis of ASD is essential to provide children with individually-tailored support.8 Eye-tracking may contribute to an earlier diagnosis: Several studies showed differences in eye movements between people with autism spectrum disorder (ASD) and typically developing controls (TD). Different eye movements may contribute to different visual perception that perpetuates to problems in attention, communication and social interaction.
Eye movements are divided into: (1) Fixations (2) Saccades (fast and short eye movements) and (3) Smooth Pursuit Eye Movements (SPEM). SPEM follow the target in a continuous manner. The latter are the subject of the present thesis. SPEM consist of two phases: the open loop phase (= phase of initiation, first 50- 100ms) and the closed loop phase (= phase of maintenance, after about 100ms). SPEM are usually measured by a gain index. It is defined as the ratio of smooth pursuit velocity and visual target velocity and ideally equals to 1.2
In young children, corneal-reflection (CR) eye-tracking is usually applied to quantify eye movement. It allows precise measurements without the use of potentially intrusive devices.
Studies in ASD reported deficits in open loop and closed loop pursuit in children and adults with a mean age of 19.32 (TD) and 20.04 (ASD) years. However, SPEM in preschoolers with ASD remain understudied, although this developmental phase is crucial to the development of non-social and social attentional abilities.
In the present study 66 toddlers and preschoolers (18 to 72 months; ASD: n = 33, TD: n = 33) with matched cognitive abilities and sex were assessed. The main objective was to compare the gain index (Smooth Pursuit Gain = SPG). SPEM were compared between groups with gain index as a dependent measure. We hypothesized that participants with ASD show lower average gain compared to the control group.
We could show a significant group influence on the gain when considering interactions between target velocity and group (p = 0.041). The TD group showed a greater dependence on the increasing object speed than the ASD group with a trend of -0.30 ± 0.11 in the TD group and a trend of -0.13 ± 0.12 in the ASD group. Across groups, the gain decreased with increasing target velocity and dropped faster in vertical than in horizontal trials. Additionally, participants showed a lower SPG in vertical sequences than in horizontal sequences. This supports the general validity of the measure.
Toddlers and preschoolers represent a group that has been subject of little research to date. In addition, there has been only a limited number of studies analyzing SPEM in ASD. To check for a possible group difference without interactions a study with a larger sample size at fixed target velocity and target direction should follow.
The carpal tunnel syndrome (CTS) is a chronic compression of the median nerve in the carpal tunnel, a condition in which the nerve is constricted especially under the flexor retinaculum (FR). The disease predominantly appears between 40 and 83 years of age. Women are significantly more often affected than men. The same applies to overweight people in comparison to normal weight people. Abnormal sensations at night, including paresthesias and dysesthesias, are classical CTS symptoms, predominately involving the middle fingers, later also the thumb. Diagnosis of CTS usually proceeds by motor nerve conduction study (mNCS) and determination of the distal motoric latency (DML). In conformity with electrophysiology, peripheral nerve ultrasonography has also attained an important diagnostic informative value. In principle, there is an open surgical procedure and an endoscopic carpal roof cleavage. The goal of therapy is the complete open division of the flexor retinaculum (FR) in order to relieve the median nerve from compression.
This work examines the morphological alterations of the median nerve at the site of the carpal tunnel after surgical decompression by means of high-resolution neurosonography in the scope of a prospective study. More than 100 patients were examined between October and December 2014 for planned decompressions surgery due to CTS. A total of 81 patients were prospectively included, 5 of which could not take part in the follow-up after six months and were excluded from this evaluation. A medical CTS case history, clinical examination findings, as well as a neurographic result were included. Patients with a relapse operation were not considered in this regard. Apart from a clinical examination and questioning of the patient three and six months after surgery, an electrophysiological examination and a high-resolution sonography of the median nerve were also carried out. Electroneurography and nerve sonography of the median nerve were applied to both hands. A prolonged distal motor latency of the median nerve amounting to 4ms, as well as a slowed nerve conduction velocity below the benchmark value of approx. 45m/s, were classified as pathological findings. In sonography, the largest cross-section area (CSA) of the median nerve was measured by applying transversal slicing to the distal transverse creases of the skin on the palmar surface of the wrist (rasceta) as well as 5cm proximal to the rasceta. The highest CSA values were determined visually. In cases of doubt several transversal slices were made until the highest CSA value could be identified.
The average age at which the disease was contracted amounted to 56.9 years. With one exception, all patients complained of nocturnal brachialgia before surgery (74, 96.2%). As far as neurological symptoms were concerned, 72 patients had paresthesias (93.6%) and 29 patients (37.7%) felt permanent numbness. A thenar atrophy of higher degree was diagnosed in two patients (2.6%). These complaints had improved in the patients surveyed in the scope of postoperative evaluations after three and six months.
Patients with motor deficits had a statistically significantly longer preoperative distal motor latency (10.5 ± 2.8ms vs. 6.5 ± 2.3ms). We observed an improvement of distal motor latency in 98% of the patients three months and six months after surgical decompression, displaying a statistically significant DML decrease from 6.6 ± 2.4ms to 4.8 ± 1.0ms and from 6.6 ± 2.4ms to 4.4 ± 1.0ms, respectively. There was a statistically significant correlation between the decrease of the nerve cross-section area and the decrease of distal motor latency.
At the time of the follow-up examination, three months after surgery, we were able to document a decrease in the CSA value in 80% of the patients. The mean CSA value decreased from 14.7 ± 4.4mm² to 12.4 ± 3.4 mm². Six months after surgical decompression the mean CSA value decreased from 14.3 ± 4.4mm² to 9.6 ± 2.3mm². Patients with a preoperative CSA value of ≥ 12mm² displayed a significantly greater relative reduction of their postoperative CSA value. Concerning all preoperative and postoperative parameters in patients who had undergone either open or endoscopic surgery, none revealed significant differences. Neither could an exploratory analysis (i.e. age, diabetic diseases) reveal any significant correlation between the parameters. Prior to surgery, a flattening of the median nerve or a loss of its fascicular structure (texture) had also been seen to exist in patients, apart from the nerve's larger cross-section area. Nerve sonography is an inexpensive and fast method. It is also extraordinarily reliable in the assessment of the CTS diagnosis and suits the necessary demands. We achieved a good efficiency with our sonographic examinations in the study presented here. New and improved developments show that high-resolution sonography will gain more and more significance in future CTS diagnostics.
Aim: The aim of this study was to measure cortico-cortical connectivity in multiple sclerosis (MS) patients by TMS-evoked potential (TEP) latencies in EEG evoked by transcranial magnetic stimulation (TMS) of the hand area of the primary motor cortex of one hemisphere. TEPs were recorded on the stimulated- and at the homologue site in the non-stimulated contralateral hemisphere. Both interhemispheric directions were tested. Interhemispheric latencies of the two main reproducible TEPs, the positive component at 60 ms and the negative component at 100 ms (P60 and N100, respectively), were expected to be significantly prolonged in MS-patients compared to healthy volunteers.
Material and methods: The study compared interhemispheric propagation of P60 and N100 in groups of 12 patients with early-stage relapsing-remitting MS (RRMS) and 16 age- and gender-matched healthy controls. The study was approved by the Ethics Committee of the Medical Faculty of the Goethe-University of Frankfurt/Main and conformed to the latest revision of the Declaration of Helsinki of 2008. TEPs were recorded by means of EEG and their latencies were statistically evaluated in 10 channels around the stimulation site and in 10 corresponding electrodes in the non-stimulated contralateral hemisphere. Interhemispheric conduction time was calculated by the difference of TEP latency in non-stimulated vs. stimulated hemisphere.
Results: An ANOVA on interhemispheric conduction time showed a significant prolongation for the N100 from left to right hemisphere in MS compared to controls, while no group differences were found for the P60 and the N100 from right to left hemisphere.
Conclusion: The results provide first evidence that the N100 may constitute an interesting marker to measure interhemispheric conduction delays in early-stage RRMS. The specificity of the present finding and its relation to fiber tract pathology should be examined in further correlative analyses with diffusion tensor imaging and other structural MRI data.
Slack (sequence like a Ca2+ -activated K + channel; also termed Slo2.2, Kcnt1, or KNa 1.1) is a Na+ -activated K + channel that is highly expressed in the peripheral and central nervous system. Previous studies have shown that Slack is enriched in the isolectin B4binding, non-peptidergic subpopulation of C-fiber sensory neurons and that Slack controls the sensory input in neuropathic pain. Recent single-cell RNA-sequencing studies suggested that Slack is highly co-expressed with transient receptor potential (TRP) ankyrin 1 (TRPA1) in sensory neurons. By using in situ hybridization and immunostaining we confirmed that Slack is highly co-localized with TRPA1 in sensory neurons, but only to a minor extent with TRP vanilloid 1. Mice lacking Slack globally or conditionally in sensory neurons (SNS-Slack─/─ ), but not mice lacking Slack conditionally in neurons of the spinal dorsal horn (Lbx1-Slack─/─ ), displayed increased pain behavior after intraplantar injection of the TRPA1 activator allyl isothiocyanate. Patch-clamp recordings with cultured primary neurons and in a HEK-293 cell line transfected with TRPA1 and Slack revealed that Slack-dependent K + currents are modulated in a TRPA1-dependent manner. Taken together, these findings highlight Slack as a modulator of TRPA1-mediated activation of sensory neurons.
Furthermore, we investigated the contribution of Slack in the spinal dorsal horn to pain processing. Lbx1-Slack ─/─ mice demonstrated normal basal pain sensitivity and Complete Freund’s Adjuvant-induced inflammatory pain. Interestingly, we observed a significantly increased spared nerve injury (SNI)-induced neuropathic pain hypersensitivity in Lbx1-Slack ─/─ mutants compared to control littermates. Moreover, we tested the effects of pharmacological Slack activation in the SNI model. Systemic and intrathecal, but not intraplantar administration of the Slack opener loxapine significantly alleviated SNI-induced hypersensitivity in control mice, but only slightly in Lbx1Slack ─/─ mice, further supporting the inhibitory function of Slack in spinal dorsal horn neurons in neuropathic pain processing.
Altogether, our data suggest that Slack in sensory neurons controls TRPA1-induced pain, whereas Slack in spinal dorsal horn neurons inhibits peripheral nerve injury induced neuropathic pain. These data provide further insights into the molecular mechanisms of pain sensation.
Although immune checkpoint inhibitors such as anti-PD-1 antibodies have shown remarkable clinical success in many different tumor types, the proportion of patients benefiting from this treatment option remains low. Therefore, there is a need to sensitize tumors for immune checkpoint blockade. In this study two approaches were tested, a chemoimmunotherapy approach combining PD-1 checkpoint blockade with doxorubicin (DOX) chemotherapy, and ablation of the sphingosine-1-phosphate (S1P) receptor (S1PR4) based on the following rationale. Chemotherapy was shown to induce immune paralysis which contributes to tumor relapse, while PD-1 signaling was shown to facilitate the acquisition of chemoresistance. Thus, combinatorial chemoimmunotherapy is expected to be beneficial by maintaining or even activating anti-tumor immunity during chemotherapy. S1PR4 is an immune cell specific receptor, whose ablation slowed tumor progression by activating anti-tumor immunity in a mouse model that was previously insensitive to anti-PD-1 monotherapy. This suggested that S1PR4 ablation might pre-activate immunity to sensitize for anti-PD-1 therapy.
To test these combinatorial approaches, two tumor mouse models were employed, namely the MC38 murine adenocarcinoma model as well as the transgenic polyoma middle T oncogene (PyMT) breast cancer model. In the MC38 model, a mild synergistic effect of PD-1 immune checkpoint blockade and S1PR4 ablation was observed, indicated by improved tumor progression and survival as compared to the WT control, and an increased number of tumor-free mice compared to anti-PD-1 therapy alone in WT mice. These observations correlated with an enhanced natural killer (NK) cell infiltrate and increased CXCL9 and CXCL10 production in anti-PD-1 treated S1PR4 KO tumors. As noted before, the PyMT model was largely resistant to anti-PD-1 monotherapy in a therapeutic setting. S1PR4 ablation alone showed significant tumor reduction that was not further enhanced by anti-PD-1 treatment. The same was observed when chemotherapy with DOX was added, where WT tumors relapsed, while S1PR4 KO tumor did not. Addition of anti-PD-1 did only mildly increase tumor control in S1PR4 KO mice, indicating that S1PR4 KO per se very efficiently re-activated anti-tumor immunity. Since S1PR4 KO induces type I 12 interferon (IFN-1) over-production in S1PR4 KO PyMT tumors, a link between high IFN-1 levels and tumor immunity was tested by using mice deficient in the IFN-1 receptor (IFNAR1). Unexpectedly, DOX chemotherapy was most efficient in mice with IFNAR ablation only as compared to WT, S1PR4 KO or S1PR4 and IFNAR1 double KO mice, although deficiency in IFNAR signaling is predominantly regarded as tumor promoting. The underlying mechanisms need to be tested in future studies. Interestingly, chemoimmunotherapy in WT mice prevented tumor relapse to a similar extent than S1PR4 KO and was superior to chemotherapy or immune checkpoint blockade alone. To investigate mechanisms of chemoimmunotherapy success compared to monotherapy, whole transcriptome analysis was used, which identified a set of genes that were upregulated specifically upon chemoimmunotherapy. This gene signature and, more specifically, a condensed four-gene signature predicted favorable survival of human mammary carcinoma patients in the METABRIC cohort.
Moreover, PyMT tumors treated with chemoimmunotherapy contained higher levels of cytotoxic lymphocytes, particularly NK cells. Gene set enrichment analysis and ELISA measurements revealed increased IL-27 production and signaling in PyMT tumors upon chemoimmunotherapy. Moreover, IL-27 improved NK cell cytotoxicity against PyMT cells in vitro. These data supported recent clinical observations indicating a benefit of chemoimmunotherapy compared to monotherapy in breast cancer and suggested potential underlying mechanisms.
Taken together the present work revealed new strategies to reactivate tumor immunity leading to improved chemotherapy response, namely a combination with immune checkpoint blockade and ablation of S1PR4, which activated different lymphocyte compartments within tumors.