Neoadjuvant sintilimab plus chemotherapy in EGFR-mutant NSCLC: Phase 2 trial interim results (NEOTIDE/CTONG2104).

The clinical efficacy of neoadjuvant immunotherapy plus chemotherapy remains elusive in localized epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC). Here, we report interim results of a Simon's two-stage design, phase 2 trial using neoadjuvant sintilimab with carboplatin and nab-paclitaxel in resectable EGFR-mutant NSCLC. All 18 patients undergo radical surgery, with one patient experiencing surgery delay. Fourteen patients exhibit confirmed radiological response, with 44% achieving major pathological response (MPR) and no pathological complete response (pCR). Similar genomic alterations are observed before and after treatment without influencing the efficacy of subsequent EGFR-tyrosine kinase inhibitors (TKIs) in vitro. Infiltration and T cell receptor (TCR) clonal expansion of CCR8+ regulatory T (Treg)hi/CXCL13+ exhausted T (Tex)lo cells define a subtype of EGFR-mutant NSCLC highly resistant to immunotherapy, with the phenotype potentially serving as a promising signature to predict immunotherapy efficacy. Informed circulating tumor DNA (ctDNA) detection in EGFR-mutant NSCLC could help identify patients nonresponsive to neoadjuvant immunochemotherapy. These findings provide supportive data for the utilization of neoadjuvant immunochemotherapy and insight into immune resistance in EGFR-mutant NSCLC.

Synaptic mechanisms for motor variability in a feedforward network.

Behavioral variability often arises from variable activity in the behavior-generating neural network. The synaptic mechanisms underlying this variability are poorly understood. We show that synaptic noise, in conjunction with weak feedforward excitation, generates variable motor output in the Aplysia feeding system. A command-like neuron (CBI-10) triggers rhythmic motor programs more variable than programs triggered by CBI-2. CBI-10 weakly excites a pivotal pattern-generating interneuron (B34) strongly activated by CBI-2. The activation properties of B34 substantially account for the degree of program variability. CBI-10- and CBI-2-induced EPSPs in B34 vary in amplitude across trials, suggesting that there is synaptic noise. Computational studies show that synaptic noise is required for program variability. Further, at network state transition points when synaptic conductance is low, maximum program variability is promoted by moderate noise levels. Thus, synaptic strength and noise act together in a nonlinear manner to determine the degree of variability within a feedforward network.

Newly Identified Aplysia SPTR-Gene Family-Derived Peptides: Localization and Function.

When individual neurons in a circuit contain multiple neuropeptides, these peptides can target different sets of follower neurons. This endows the circuit with a certain degree of flexibility. Here we identified a novel family of peptides, the Aplysia SPTR-Gene Family-Derived peptides (apSPTR-GF-DPs). We demonstrated apSPTR-GF-DPs, particularly apSPTR-GF-DP2, are expressed in the Aplysia CNS using immunohistochemistry and MALDI-TOF MS. Furthermore, apSPTR-GF-DP2 is present in single projection neurons, e.g., in the cerebral-buccal interneuron-12 (CBI-12). Previous studies have demonstrated that CBI-12 contains two other peptides, FCAP/CP2. In addition, CBI-12 and CP2 promote shortening of the protraction phase of motor programs. Here, we demonstrate that FCAP shortens protraction. Moreover, we show that apSPTR-GF-DP2 also shortens protraction. Surprisingly, apSPTR-GF-DP2 does not increase the excitability of retraction interneuron B64. B64 terminates protraction and is modulated by FCAP/CP2 and CBI-12. Instead, we show that apSPTR-GF-DP2 and CBI-12 increase B20 excitability and B20 activity can shorten protraction. Taken together, these data indicate that different CBI-12 peptides target different sets of pattern-generating interneurons to exert similar modulatory actions. These findings provide the first definitive evidence for SPTR-GF's role in modulation of feeding, and a form of molecular degeneracy by multiple peptide cotransmitters in single identified neurons.

Discovery of leucokinin-like neuropeptides that modulate a specific parameter of feeding motor programs in the molluscan model, Aplysia.

A better understanding of neuromodulation in a behavioral system requires identification of active modulatory transmitters. Here, we used identifiable neurons in a neurobiological model system, the mollusc Aplysia, to study neuropeptides, a diverse class of neuromodulators. We took advantage of two types of feeding neurons, B48 and B1/B2, in the Aplysia buccal ganglion that might contain different neuropeptides. We performed a representational difference analysis (RDA) by subtraction of mRNAs in B48 versus mRNAs in B1/B2. The RDA identified an unusually long (2025 amino acids) peptide precursor encoding Aplysia leucokinin-like peptides (ALKs; e.g. ALK-1 and ALK-2). Northern blot analysis revealed that, compared with other ganglia (e.g. the pedal-pleural ganglion), ALK mRNA is predominantly present in the buccal ganglion, which controls feeding behavior. We then used in situ hybridization and immunohistochemistry to localize ALKs to specific neurons, including B48. MALDI-TOF MS on single buccal neurons revealed expression of 40 ALK precursor-derived peptides. Among these, ALK-1 and ALK-2 are active in the feeding network; they shortened the radula protraction phase of feeding motor programs triggered by a command-like neuron. We also found that this effect may be mediated by the ALK-stimulated enhancement of activity of an interneuron, which has previously been shown to terminate protraction. We conclude that our multipronged approach is effective for determining the structure and defining the diverse functions of leucokinin-like peptides. Notably, the ALK precursor is the first verified nonarthropod precursor for leucokinin-like peptides with a novel, marked modulatory effect on a specific parameter (protraction duration) of feeding motor programs.

A clarifying method that improves imaging of Aplysia ganglia.

Improvements in the imaging of neural circuits are essential for studies of network function in both invertebrates and vertebrates. Therefore, CLARITY, a new imaging enhancement technique developed for mouse brains has attracted broad interest from researchers working on other species. We studied the potential of a modified version of CLARITY to enhance the imaging of ganglia in an invertebrate Aplysia. For example, we have modified the hydrogel solution and designed a small container for the Aplysia ganglia. The ganglia were first processed for immunohistochemistry, and then for CLARITY. We examined the compatibility of these techniques and the extent to which the imaging of fluorescence improved using confocal microscopy. We found that CLARITY did indeed enhance the imaging of CP2 immunopositive neurons in Aplysia ganglia. For example, it improved visualization of small, weak immunoreactive neurons deep in the ganglia. Our modifications of CLARITY make this new method suitable for future use in Aplysia experiments. Furthermore, our techniques are likely to facilitate imaging in other invertebrate ganglia.

miR-96 promotes cell proliferation, migration and invasion by targeting PTPN9 in breast cancer.

microRNAs (miRNAs) have emerged as major regulators of the initiation and progression of human cancers, including breast cancer. The aim of this study is to determine the expression pattern of miR-96 in breast cancer and to investigate its biological role during tumorigenesis. We showed that miR-96 was significantly upregulated in breast cancer. We then investigated its function and found that miR-96 significantly promoted cell proliferation, migration and invasion in vitro and enhanced tumor growth in vivo. Furthermore, we explored the molecular mechanisms by which miR-96 contributes to breast cancer progression and identified PTPN9 (protein tyrosine phosphatase, non-receptor type 9) as a direct target gene of miR-96. Finally, we showed that PTPN9 had opposite effects to those of miR-96 on breast cancer cells, suggesting that miR-96 may promote breast tumorigenesis by silencing PTPN9. Taken together, this study highlights an important role for miR-96 in the regulation of PTPN9 in breast cancer cells and may provide insight into the molecular mechanisms of breast carcinogenesis.

A d-Amino Acid-Containing Neuropeptide Discovery Funnel.

A receptor binding class of d-amino acid-containing peptides (DAACPs) is formed in animals from an enzymatically mediated post-translational modification of ribosomally translated all-l-amino acid peptides. Although this modification can be required for biological actions, detecting it is challenging because DAACPs have the same mass as their all-l-amino acid counterparts. We developed a suite of mass spectrometry (MS) protocols for the nontargeted discovery of DAACPs and validated their effectiveness using neurons from Aplysia californica. The approach involves the following three steps, with each confirming and refining the hits found in the prior step. The first step is screening for peptides resistant to digestion by aminopeptidase M. The second verifies the presence of a chiral amino acid via acid hydrolysis in deuterium chloride, labeling with Marfey's reagent, and liquid chromatography-mass spectrometry to determine the chirality of each amino acid. The third involves synthesizing the putative DAACPs and comparing them to the endogenous standards. Advantages of the method, the d-amino acid-containing neuropeptide discovery funnel, are that it is capable of detecting the d-form of any common chiral amino acid, and the first two steps do not require peptide standards. Using these protocols, we report that two peptides from the Aplysia achatin-like neuropeptide precursor exist as GdYFD and SdYADSKDEESNAALSDFA. Interestingly, GdYFD was bioactive in the Aplysia feeding and locomotor circuits but SdYADSKDEESNAALSDFA was not. The discovery funnel provides an effective means to characterize DAACPs in the nervous systems of animals in a nontargeted manner.

Aplysia Locomotion: Network and Behavioral Actions of GdFFD, a D-Amino Acid-Containing Neuropeptide.

One emerging principle is that neuromodulators, such as neuropeptides, regulate multiple behaviors, particularly motivated behaviors, e.g., feeding and locomotion. However, how neuromodulators act on multiple neural networks to exert their actions remains poorly understood. These actions depend on the chemical form of the peptide, e.g., an alternation of L- to D-form of an amino acid can endow the peptide with bioactivity, as is the case for the Aplysia peptide GdFFD (where dF indicates D-phenylalanine). GdFFD has been shown to act as an extrinsic neuromodulator in the feeding network, while the all L-amino acid form, GFFD, was not bioactive. Given that both GdFFD/GFFD are also present in pedal neurons that mediate locomotion, we sought to determine whether they impact locomotion. We first examined effects of both peptides on isolated ganglia, and monitored fictive programs using the parapedal commissural nerve (PPCN). Indeed, GdFFD was bioactive and GFFD was not. GdFFD increased the frequency with which neural activity was observed in the PPCN. In part, there was an increase in bursting spiking activity that resembled fictive locomotion. Additionally, there was significant activity between bursts. To determine how the peptide-induced activity in the isolated CNS is translated into behavior, we recorded animal movements, and developed a computer program to automatically track the animal and calculate the path of movement and velocity of locomotion. We found that GdFFD significantly reduced locomotion and induced a foot curl. These data suggest that the increase in PPCN activity observed in the isolated CNS during GdFFD application corresponds to a reduction, rather than an increase, in locomotion. In contrast, GFFD had no effect. Thus, our study suggests that GdFFD may act as an intrinsic neuromodulator in the Aplysia locomotor network. More generally, our study indicates that physiological and behavioral analyses should be combined to evaluate peptide actions.

Functional Characterization of a Vesicular Glutamate Transporter in an Interneuron That Makes Excitatory and Inhibitory Synaptic Connections in a Molluscan Neural Circuit.

Understanding circuit function requires the characterization of component neurons and their neurotransmitters. Previous work on radula protraction in the Aplysia feeding circuit demonstrated that critical neurons initiate feeding via cholinergic excitation. In contrast, it is less clear how retraction is mediated at the interneuronal level. In particular, glutamate involvement was suggested, but was not directly confirmed. Here we study a suspected glutamatergic retraction interneuron, B64. We used the representational difference analysis (RDA) method to successfully clone an Aplysia vesicular glutamate transporter (ApVGLUT) from B64 and from a glutamatergic motor neuron B38. Previously, RDA was used to characterize novel neuropeptides. Here we demonstrate its utility for characterizing other types of molecules. Bioinformatics suggests that ApVGLUT is more closely related to mammalian VGLUTs than to Drosophila and Caenorhabditis elegans VGLUTs. We expressed ApVGLUT in a cell line, and demonstrated that it indeed transports glutamate in an ATP and proton gradient-dependent manner. We mapped the ApVGLUT distribution in the CNS using in situ hybridization and immunocytochemistry. Further, we demonstrated that B64 is ApVGLUT positive, supporting the idea that it is glutamatergic. Although glutamate is primarily an excitatory transmitter in the mammalian CNS, B64 elicits inhibitory PSPs in protraction neurons to terminate protraction and excitatory PSPs in retraction neurons to maintain retraction. Pharmacological data indicated that both types of PSPs are mediated by glutamate. Thus, glutamate mediates the dual function of B64 in Aplysia. More generally, our systematic approaches based on RDA may facilitate analyses of transmitter actions in small circuits with identifiable neurons.

Complementary interactions between command-like interneurons that function to activate and specify motor programs.

Motor activity is often initiated by a population of command-like interneurons. Command-like interneurons that reliably drive programs have received the most attention, so little is known about how less reliable command-like interneurons may contribute to program generation. We study two electrically coupled interneurons, cerebral-buccal interneuron-2 (CBI-2) and CBI-11, which activate feeding motor programs in the mollusk Aplysia californica. Earlier work indicated that, in rested preparations, CBI-2, a powerful activator of programs, can trigger ingestive and egestive programs. CBI-2 reliably generated ingestive patterns only when it was repeatedly stimulated. The ability of CBI-2 to trigger motor activity has been attributed to the two program-promoting peptides it contains, FCAP and CP2. Here, we show that CBI-11 differs from CBI-2 in that it contains FCAP but not CP2. Furthermore, it is weak in its ability to drive programs. On its own, CBI-11 is therefore less effective as a program activator. When it is successful, however, CBI-11 is an effective specifier of motor activity; that is, it drives mostly ingestive programs. Importantly, we found that CBI-2 and CBI-11 complement each other's actions. First, prestimulation of CBI-2 enhanced the ability of CBI-11 to drive programs. This effect appears to be partly mediated by CP2. Second, coactivation of CBI-11 with CBI-2 makes CBI-2 programs immediately ingestive. This effect may be mediated by specific actions that CBI-11 exerts on pattern-generating interneurons. Therefore, different classes of command-like neurons in a motor network may make distinct, but potentially complementary, contributions as either activators or specifiers of motor activity.