Plasma Membrane Marker Research Articles

Abstract 4114625: Upregulation of Palmitoylating Enzymes, DHHC2 and DHHC5, in Failing Human Hearts Protects Impulse Propagation but Endangers Myocyte Survival after Ischemia/Reperfusion (I/R) Injury

Background: Palmitoylation 'Palm* catalyzed by DHHC enzymes promotes protein binding to membranes, impacting protein translocation and function. Palm* dysregulation has been linked to cancers and neurogenerative disorders; its impact on cardiac function is unclear. We have used global purification of palmitoylated proteins from human and animal hearts followed by proteomic analysis to build 'cardiac palmitoylome', whose 454 constituents are involved in organizing/stabilizing cardiomyocyte microdomains (intercalated discs 'ICD', dyads, and costameres) critical for myocyte functions/homeostasis. We confirmed 11 of the 23 DHHC genes are expressed in human&animal hearts. In particular, DHHC2 and DHHC5 were upregulated in human failing hearts. DHHC enzymes are membrane-embedded, limiting their substrates to enzymes' 'territories' in cells. Objective: To identify DHHC2 and DHHC5 distribution patterns and substrates in myocytes, and to understand how upregulation of DHHC2 or DHHC5 affects myocyte function. Methods: Track adenovirus-expressed mCherry-DHHC2 and -DHHC5 (mChr-DHHCx, x=2 or 5, Adv-mChr as control) in ventricular myocytes from adult Sprague-Dawley rats. Globally purify palmitoylated proteins for proteomic analysis/quantification. Use high-resolution quantitative imaging to analyze how mChr-DHHCx affects myocyte response to ischemia/reperfusion (I/R) injury and the expression/organization of palmitoylated proteins at ICD and lateral surface. Results: mChr-DHHC2 is mainly present in cytoplasmic vesicles bearing markers of recycling or autophagy, with a minor presence along lateral surface, dyads, and ICD. mChr-DHHC5 is mainly present along the lateral surface, dyads and ICD, with a minor presence in small endosomal vesicles. Palmitoylome normalized by proteome identifies DHHC2 and DHHC5 substrates that overlap with cardiac palmitoylome, and match the enzymes' distribution patterns. I/R injury increases endocytosis in myocytes, quantified by uptake of plasma-membrane marker, wheat germ agglutinin. I/R-induced endocytosis is exacerbated by mChr-DHHC2 but, surprising, alleviated by mChr-DHHC5. mChr-DHHC2 and -DHHC5 increase internalization of ICD and lateral membrane proteins, n-cadherin and NCX1 respectively. Conclusions: DHHC2 and DHHC5 have non-overlapping distribution patterns, consistent with varying substrate groups. DHHC2, but not DHHC5, appears involved in the previously reported palm*-dependent massive endocytosis in I/R-stressed cardiomyocytes.

COBRA-LIKE4 Modulates Cellulose Synthase Velocity and Facilitates Cellulose Deposition in the Secondary Cell Wall.

Cellulose is a critical component of secondary cell walls and woody tissues of plants. Cellulose synthase (CESA) complexes (CSCs) produce cellulose as they move within the plasma membrane, extruding glucan chains into the cell wall that coalesce and crystallize into cellulose fibrils. Here we examine COBRA-LIKE4 (COBL4), a GPI-anchored protein on the outer leaflet of the plasma membrane that is required for normal cellulose deposition in secondary cell walls. Characterization of the Arabidopsis (Arabidopsis thaliana) cobl4 mutant alleles called irregular xylem6, irx6-2 and irx6-3, showed reduced ⍺-cellulose content and lower crystallinity, supporting a role for COBL4 in maintaining cellulose quantity and quality. In live-cell imaging, mNeon Green-tagged CESA7 moved in the plasma membrane at higher speeds in the irx6-2 background compared to wild type. To test conservation of COBL4 function between herbaceous and woody plants, poplar (Populus trichocarpa) COBL4 homologs PtCOBL4a and PtCOBL4b were transformed into, and rescued, the Arabidopsis irx6 mutants. Using the Arabidopsis secondary cell wall-inducible VND7-GR system to study poplar COBL4 dynamics, YFP-tagged PtCOBL4a localized to the plasma membrane in regions of high cellulose deposition in secondary cell wall bands. As predicted for a lipid-linked protein, COBL4 was more mobile in the plane of the plasma membrane than CESA7 or a control plasma membrane marker. Following programmed cell death, COBL4 anchored to the secondary cell wall bands. These data support a role for COBL4 as a modulator of cellulose organization in the secondary cell wall, influencing cellulose production and CSC velocity at the plasma membrane.

UNSEG: unsupervised segmentation of cells and their nuclei in complex tissue samples

Multiplexed imaging technologies have made it possible to interrogate complex tissue microenvironments at sub-cellular resolution within their native spatial context. However, proper quantification of this complexity requires the ability to easily and accurately segment cells into their sub-cellular compartments. Within the supervised learning paradigm, deep learning-based segmentation methods demonstrating human level performance have emerged. However, limited work has been done in developing such generalist methods within the unsupervised context. Here we present an easy-to-use unsupervised segmentation (UNSEG) method that achieves deep learning level performance without requiring any training data via leveraging a Bayesian-like framework, and nucleus and cell membrane markers. We show that UNSEG is internally consistent and better at generalizing to the complexity of tissue morphology than current deep learning methods, allowing it to unambiguously identify the cytoplasmic compartment of a cell, and localize molecules to their correct sub-cellular compartment. We also introduce a perturbed watershed algorithm for stably and automatically segmenting a cluster of cell nuclei into individual nuclei that increases the accuracy of classical watershed. Finally, we demonstrate the efficacy of UNSEG on a high-quality annotated gastrointestinal tissue dataset we have generated, on publicly available datasets, and in a range of practical scenarios.

Increased CD123 + HLA-DR- Granulocytes in Allergic Rhinitis and Influence of Allergens on Expression of Cell Membrane Markers.

It is reported that CD123 + HLA-DR- cells in PBMC are basophils, and CD203c, CD63, and FcεRI molecules are activation markers of basophils. However, little is known of CD123 + HLA-DR-cells in blood granulocytes. To investigate the presence of CD123 + HLA-DR- cells in the blood granulocytes and peripheral PBMC of patients with allergic rhinitis (AR), as well as the impact of allergens on the cell membrane markers of basophils. Flow cytometry was used to detect the expression of the membrane molecules. While CD123 + HLA-DR- PBMCs are representative of basophils, their presence did not significantly change in patients with AR. In contrast, both the percentage and number of CD123 + HLA-DR- granulocytes, which make up only up to 50% of basophils, were significantly increased in patients with seasonal (sAR) and perennial AR (pAR). CD63+, CD203c+, and FcεRIα+ cells within CD123 + HLA-DR- granulocytes also showed enhanced activity in patients with AR. Allergen extracts from house dust mite allergen extract (HDME) and Artemisia sieversiana wild extract further increased the number of CD123 + HLA-DR- cells in granulocytes of sAR and pAR patients, as well as in PBMCs of pAR patients. The use of CD123 + HLA-DR- granulocytes and PBMC may not be sufficient for diagnosing AR. Allergens could potentially contribute to the development of AR by influencing the number of CD123 + HLA-DR- cells, as well as the expression of CD63, CD203c, and FcεRIαin these cells.

TRPV4 Antagonist Treatment of Hydrocephalus Causes Cell and Molecular Changes in Multiple Brain Cell Types

Hydrocephalus is defined by cerebrospinal fluid (CSF) accumulation in the brain’s ventricles causing ventriculomegaly, an imbalance in CSF secretion, flow, and/or absorption. Treatments for hydrocephalus include invasive surgical procedures, which commonly fail throughout a patient’s life. Transient receptor potential vanilloid member 4 (TRPV4) is a mechanosensitive cation channel implicated in osmotic regulation. Our laboratory found that a TRPV4 antagonist, RN1734, ameliorates hydrocephalus in a rat model of congenital hydrocephalus. It was hypothesized that treatment slowed production of CSF by targeting choroid plexus (CP), a contiguous layer of tightly regulated epithelial cells, that is responsible for CSF production. However, hydrocephalus pathology can have various effects on the brain. This study focuses on changes occurring in CP epithelial cells (CPe) and glia, which have roles in brain-barrier maintenance and brain fluid/electrolyte regulation. Interestingly, CPe and glia contain many of the same channels and transporters, including TRPV4, that are important in regulating both CSF and brain interstitial fluid. These cells contain aquaporins (AQP), AQP1 in CPe, and AQP4 in glia. AQPs have been implicated in diseases associated with fluid regulation and may interact with TRPV4. We hypothesize that TRPV4 and AQPs are important in hydrocephalus pathology, and that changes may be attenuated with RN1734 treatment. A point mutation in TMEM67 results in hydrocephalus (hydro). Changes in cell morphology, gene transcription, protein expression, localization of TRPV4, AQP1, and AQP4 were observed. Postnatal day 15 (P15) untreated (unt) and RN1734-treated (RN;2mg/ml DMSO; 4mg/kg intraperitoneal injection daily P7-P14) wildtype and mutant ( Tmem67−/−) rats were utilized in this study. CPe and periventricular (PV) glia were visualized using fluorescent immunohistochemistry (IHC) of GFAP and a plasma membrane marker. Cell volume of CPe’s and process morphology of glia were altered in hydro rats. RN returned these changes to relatively normal in CPe, but not glia. Next, we used IHC to examine localization of TRPV4 and the AQPs. Increased immunoreactivity of TRPV4 and AQP1 was observed in CPe of unt and RN Tmem67−/− rats. AQP4 and TRPV4 immunoreactivity increased in the subventricular zone and PV white matter of hydro rats. TRPV4 immunoreactivity, but not AQP4, was normalized with RN. We then utilized RTqPCR to inspect transcriptional changes. The data showed increased AQP1 and decreased TRPV4 mRNA in PV cortex and CP of unt and RN Tmem67−/− rats. Previous data from our laboratory found no change in TRPV4 protein in unt Tmem67−/− CPe, but we were curious about AQP expression. The amount of AQP1 protein was increased in the CP of unt and RN Tmem67−/− rats. In PV cortex, AQP4, but not TRPV4, protein was increased in hydro rats. With RN, AQP4 protein was not normalized. In summary, TRPV4 changes in the Tmem67−/− rats returned to normal with RN, glial morphology and AQP changes did not. This may indicate residual inflammation in the Tmem67−/− rats, which we aim to address in future studies. To further elucidate TRPV4’s role in hydrocephalus pathology, future studies will explore post-translational and membrane localization changes. Overall, investigating cell and molecular changes that occur in hydrocephalus will help find better pharmacological targets for this disease. Funding: United States Department of Defense Congressionally Directed Medical Research Program Award; Hydrocephalus Association. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The use of salmonid epithelial cells to characterize the toxicity of Tenacibaculum maritimum soluble extracellular products.

This study assessed how the etiological agent of mouth rot in farmed Atlantic salmon, Tenacibaculum maritimum, induces toxicity in host salmonid barrier cells, and determined whether environmental changes are relevant for these effects. Tenacibaculum maritimum soluble extracellular products (ECPs) were collected and used to treat Atlantic salmon and rainbow trout intestinal barrier cell lines as a comparative model of bacterial-salmonid cell interactions. Cellular assays that examine cell membrane integrity, marker expression, and metabolic activity revealed that T. maritimum ECPs induced salmonid epithelial cell death through an apoptosis mechanism. Changes in salinity (25, 29, and 33 ppt) and temperature (12°C, 18°C, and 24°C) within the natural ranges observed in Pacific Northwest aquaculture facilities affected bacterial growth and cytotoxicity of T. maritimum ECPs. Our results suggest epithelial barriers as targets of T. maritimum-mediated toxicity in farmed mouth rot-infected Atlantic salmon. The induction of apoptosis by T. maritimum soluble ECPs may also help to explain the absence of overt inflammation typically reported for these fish.

Immunophenotyping of hemocytes from infected Galleria mellonella larvae as an innovative tool for immune profiling, infection studies and drug screening

In recent years, there has been a considerable increasing interest in the use of the greater wax moth Galleria mellonella as an animal model. In vivo pharmacological tests, concerning the efficacy and the toxicity of novel compounds are typically performed in mammalian models. However, the use of the latter is costly, laborious and requires ethical approval. In this context, G. mellonella larvae can be considered a valid option due to their greater ease of use and the absence of ethical rules. Furthermore, it has been demonstrated that the immune system of these invertebrates has similarity with the one of mammals, thus guaranteeing the reliability of this in vivo model, mainly in the microbiological field. To better develop the full potential of this model, we present a novel approach to characterize the hemocyte population from G. mellonella larvae and to highlight the immuno modulation upon infection and treatments. Our approach is based on the detection in isolated hemocytes from G. mellonella hemolymph of cell membrane markers typically expressed by human immune cells upon inflammation and infection, for instance CD14, CD44, CD80, CD163 and CD200. This method highlights the analogies between G. mellonella larvae and humans. Furthermore, we provide an innovative tool to perform pre-clinical evaluations of the efficacy of antimicrobial compounds in vivo to further proceed with clinical trials and support drug discovery campaigns.

Plant elicitor peptide induces endocytosis of plasma membrane proteins in Arabidopsis.

In plants, the regulation of plasma membrane (PM) dynamics through endocytosis plays a crucial role in responding to external environmental cues and defending against pathogens. The Arabidopsis plant elicitor peptides (Peps), originating from precursor proteins called PROPEPs, have been implicated in various aspects of plant immunity. This study delves into the signaling pathway of Peps, particularly Pep1, and its effect on PM protein internalization. Using PIN2 and BRI1 as PM markers, we demonstrated that Pep1 stimulates the endocytosis of these PM-localized proteins through clathrin-mediated endocytosis (CME). CLC2 and CLC3, two light chains of clathrin, are vital for Pep1-induced PIN2-GFP and BRI1-GFP internalization.The internalized PIN2 and BRI1 are subsequently transported to the vacuole via the trans-Golgi network/early endosome (TGN/EE) and prevacuolar compartment (PVC) pathways. Intriguingly, salicylic acid (SA) negatively regulates the effect of Pep1 on PM endocytosis. This study sheds light on a previously unknown signaling pathway by which danger peptides like Pep1 influence PM dynamics, contributing to a deeper understanding of the function of plant elicitor peptide.

Clonal Heterogeneity and Evolution during Treatment in High Hyperdiploid B-Cell Acute Lymphoblastic Leukemia

Introduction: Acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, is caused by the accumulation of multiple genomic aberrations in developing lymphocytes. In children, high hyperdiploidy (HeH) is the most prevalent subtype of B-cell ALL (B-ALL), which is associated with a superior outcome. However, because of the high incidence of HeH B-ALL in children, this subtype is responsible for about 25% of all relapses. The prediction of relapse remains difficult, especially in these good risk subtypes. Recent studies indicate that ALL is a heterogeneous disease, with multiple leukemia clones at diagnosis, each having a distinct set of mutations. We hypothesize that these different subclones have a different sensitivity to treatment and that samples with more heterogeneity are more prone to relapse. We used single-cell sequencing to determine the clonal heterogeneity of HeH B-ALL at diagnosis and followed the clonal evolution during chemotherapy treatment. Methods: Bone marrow samples of 11 patients with HeH B-ALL were collected at diagnosis, during treatment (for 9 cases) and at relapse (for 1 case) (Table 1). Samples during treatment were enriched for leukemia cells using magnetic antibodies targeting cell membrane markers (CD10, CD19, CD34) with the MARS® gentle sorter. We performed targeted single-cell DNA sequencing with the Mission Bio Tapestri® Platform, using our custom amplicon panel of 414 amplicons across 39 commonly mutated genes. This method detected single nucleotide variants (SNVs) and small insertions/deletions (indels) based on targeted PCR amplification in about 4000 cells per sample. Results: We sequenced diagnostic samples from 11 HeH B-ALL patients. For 9 patients we also tested samples during treatment, either at day 15 of treatment (1 case) or at the end of induction (EOI) (8 cases), and for 3 cases an additional sample at the end of consolidation (EOC). Furthermore, for one patient, we analyzed samples of 3 subsequent relapses. In total, 117,148 single cells were processed, with an average of 4500 cells per sample. We detected multiple subclones in almost all diagnostic samples (9/11), ranging from 2 to 10 subclones per case. Most frequently mutated genes were KRAS, NRAS, PTPN11 and FLT3, as well as NSD2. Four cases had 2 or more different KRAS and/or NRAS subclones. Three cases had a subclone with a KRAS/ NRAS mutation co-occurring with a NSD2 mutation. For the patients with multiple subclones at diagnosis, we subsequently performed single-cell analysis on one or more follow-up samples, either during treatment or at relapse. In the samples taken at EOI, we detected residual leukemia cells in all 8 cases: multiple subclones were detected in 5 cases and only one subclone persisted in 3 cases. Residual leukemia cells were also detected in all samples collected at EOC, though in a lesser amount as compared to the previous timepoint sequenced. Interestingly, in the matched diagnosis-relapse case, we found that a minor subclone with a NSD2, PTPN11and KRAS mutation became the dominant clone at first relapse (Figure 1). Conclusions: Single-cell DNA sequencing reveals a strong genetic heterogeneity in HeH B-ALL based on a variety of mutations in KRAS, NRAS, PTPN11, FLT3 and NSD2. RAS mutations and FLT3 mutations are mutually exclusive in subclones, whereas NSD2 mutations and RAS mutations are frequently co-occurring in the same subclone. This cooperation is highlighted in our relapse patient, having a minor subclone with a KRAS and NSD2 mutation at diagnosis that became the largest subclone at relapse. Even though few leukemia cells persisted during treatment, we were able to genetically identify the residual subclones with a potentially higher therapy resistance.

Reducing the Invasiveness of Low- and High-Grade Endometrial Cancers in Both Primary Human Cancer Biopsies and Cell Lines by the Inhibition of Aquaporin-1 Channels.

Aquaporin (AQP) channels in endometrial cancer (EC) cells are of interest as pharmacological targets to reduce tumor progression. A panel of compounds, including AQP1 ion channel inhibitors (AqB011 and 5-(phenoxymethyl) furan-2-carbaldehyde, PMFC), were used to test the hypothesis that inhibition of key AQPs can limit the invasiveness of low- and high-grade EC cells. We evaluated the effects on transwell migration in EC cell lines (Ishikawa, MFE-280) and primary EC cells established from surgical tissues (n = 8). Quantitative PCR uncovered classes of AQPs not previously reported in EC that are differentially regulated by hormonal signaling. With estradiol, Ishikawa showed increased AQPs 5, 11, 12, and decreased AQPs 0 and 4; MFE-280 showed increased AQPs 0, 1, 3, 4, 8, and decreased AQP11. Protein expression was confirmed by Western blot and immunocytochemistry. AQPs 1, 4, and 11 were colocalized with plasma membrane marker; AQP8 was intracellular in Ishikawa and not detectable in MFE-280. AQP1 ion channel inhibitors (AqB011; PMFC) reduced invasiveness of EC cell lines in transwell chamber and spheroid dispersal assays. In Ishikawa cells, transwell invasiveness was reduced ~41% by 80 µM AqB011 and ~55% by 0.5 mM 5-PMFC. In MFE-280, 5-PMFC inhibited invasion by ~77%. In contrast, proposed inhibitors of AQP water pores (acetazolamide, ginsenoside, KeenMind, TGN-020, IMD-0354) were not effective. Treatments of cultured primary EC cells with AqB011 or PMFC significantly reduced the invasiveness of both low- and high-grade primary EC cells in transwell chambers. We confirmed the tumors expressed moderate to high levels of AQP1 detected by immunohistochemistry, whereas expression levels of AQP4, AQP8, and AQP11 were substantially lower. The anti-invasive potency of AqB011 treatment for EC tumor tissues showed a positive linear correlation with AQP1 expression levels. In summary, AQP1 ion channels are important for motility in both low- and high-grade EC subtypes. Inhibition of AQP1 is a promising strategy to inhibit EC invasiveness and improve patient outcomes.

GreenGate 2.0: Backwards compatible addons for assembly of complex transcriptional units and their stacking with GreenGate.

Molecular cloning is a crucial technique in genetic engineering that enables the precise design of synthetic transcriptional units (TUs) and the manipulation of genomes. GreenGate and several other modular molecular cloning systems were developed about ten years ago and are widely used in plant research. All these systems define grammars for assembling transcriptional units from building blocks, cloned as Level 0 modules flanked by four-base pair overhangs and recognition sites for a particular Type IIs endonuclease. Modules are efficiently assembled into Level 1 TUs in a hierarchical assembly process, and Level 2 multigene constructs are assembled by stacking Level 1 TUs. GreenGate is highly popular but has three main limitations. First, using ad-hoc overhangs added by PCR and classical restriction/ligation prevents the efficient use of a one-pot, one-step reaction to generate entry clones and domesticate internal sites; second, a Level 1 TU is assembled from a maximum of six modules, which may be limiting for applications such as multiplex genome editing; third, the generation of Level 2 assemblies is sequential and inefficient. GreenGate 2.0 (GG2.0) expands GreenGate features. It introduces additional overhangs, allowing for the combination of up to 12 Level 0 modules in a Level 1 TU. It includes a Universal Entry Generator plasmid (pUEG) to streamline the generation of Level 0 modules. GG2.0 introduces GreenBraid, a convenient method for stacking transcriptional units iteratively for multigene assemblies. Importantly, GG2.0 is backwards compatible with most existing GreenGate modules. Additionally, GG2.0 includes Level 0 modules for multiplex expression of guide RNAs for CRISPR/Cas9 genome editing and pre-assembled Level 1 vectors for dexamethasone-inducible gene expression and ubiquitous expression of plasma membrane and nuclear fluorescent markers. GG2.0 streamlines and increases the versatility of assembling complex transcriptional units and their combination.

The NEB-P2ry1 lung-brainstem axis and the control of breathing in a mouse model of bronchopulmonary dysplasia

Breathing control problems, like apnea of prematurity, are a common complication in preterm infants with bronchopulmonary dysplasia (BPD), but the underlying factors remain incompletely understood. A subpopulation of pulmonary vagal afferent nerve fibers (P2ry1+) innervate CO2 and O2 sensitive clusters of lung neuroendocrine cells (neuroepithelial bodies (NEBs)) located along conducting airways. P2ry1 fibers provide direct sensory input to the brainstem and inhibit breathing upon stimulation. NEBs are hyperplastic in BPD infants and may increase activity of inhibitory P2ry1 vagal fibers. Herein, we sought to determine if the NEB-P2ry1 lung-brainstem axis is altered in association with breathing control in BPD. BPD in mice was induced by exposure to chronic neonatal (postnatal day (P) 0-14) hyperoxia (Hx; 70% O2; n=20) or normoxia (Nx; n=17). Consistent with human BPD, Hx caused alveolar simplification supported by a reduction in alveolar density (p<0.05) and alveoli (p<0.05) in P22 H&E-stained lung sections. Hx mice spent more time apneic at P14 and P17 (p<0.05) in room air and acute hypoxia. Overall (main effect), room air minute ventilation (VE) was reduced in HX mice (p<0.05). Further, VE was reduced during acute hypoxia at P14 and P17 (not P21) with an overall reduction of breathing in Hx mice (p<0.05). From a subset of mice (n=4-5/group) VCO2, VO2, and heat production at rest were greater in P21 Hx mice during room air, suggesting the Hx mice were hypoventilating. Immunofluorescent staining of CGRP (hallmark NEB marker), Ki67 (proliferation), and SEZ6L2 (new NEB cell-membrane marker) in Hx and Nx lungs were imaged and analyzed by a blinded investigator. Consistent with clinical findings, Hx mice had hyperplastic NEBs (8.6 ± 0.45 vs 10.7 ± 1.3 cells/NEB; p<0.05) while only 2 of 143 NEBs expressed Ki67, suggesting no proliferation at P22. CGRP co-localized with SEZ6L2 in both Nx and Hx lungs. A subset of mice received intratracheal delivery of 1ug SEZ6L2-Saporin toxin to lesion NEBs and assess their function. 4 days after SEZ-Saporin treatment breathing frequency was elevated by 11% ± 5% in room air and by 7% ± 5% during acute hypoxia but not in PBS-treated, adult mice (n=3/group) suggesting NEBs have a mild inhibitory influence under normal conditions. Lastly, wireless optogenetic stimulation of P2ry1 vagal fibers in unanesthetized adult control mice (n=3) increased room air breathing by 20% ± 1% and drive to breathe by 36% ± 19% compared to pre-stim breathing. Together, our data demonstrate that hyperoxia-induced BPD alters the control of breathing which may in part be mediated via a unique lung-brainstem axis comprised of hyperplastic NEBs causing greater inhibition of excitatory P2ry1 vagal fibers. The NEB-P2ry1 axis may serve as a novel target for improving breathing in BPD. GCM: Advancing Healthier Wisconsin REAC Award, AHA 20CDA35310121; MRH: HL122358; JLG: DK133121, HL134850, and HL084207; JLS: DK133121 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Full Spectrum 40-Parameter Single-Tube Flow Cytometry Analysis for Longitudinal Immunophenotyping and Tracing of Clonal Heterogeneity in AML

Acute Myeloid Leukemia (AML) remains notoriously difficult to treat. AMLs manifest themselves within individual patients with remarkable heterogeneity. Heterogeneity can be observed within the tumor itself, whereby multiple genetically distinct subclones can co-exist. We have shown that such differences in genetics translate into differences at the cell biological level as well (epigenome, transcriptome, proteome, metabolome and drug sensitivities) (de Boer et al. 2018) (Erdem et al. 2022). More recently, we began to dissect the heterogeneity within the tumor immune microenvironment. For instance, we identified a subgroup of AML patients that harbor a large population of tumor supportive M2 polarized macrophages in the bone marrow and these patients have the poorest prognosis. Reversely, patients with large populations of tumor suppressive M1 polarized macrophages display a much better prognosis. We also uncovered that patients with high levels of CD4+ T cells display a relatively favorable outcome. Little is known about clonal dynamics and microenvironmental changes over time or as a consequence of treatment. With the rise of new mutation-specific treatments targeting specific genetic subclones, it is important to examine the clonal composition of residual cells, especially at Measurable Residual Disease (MRD) timepoints. This can pose challenges as patients in morphologic complete remission have relatively low blast counts and limited material is available. Full spectrum flow cytometry is a novel technique that allows the use of 40 fluorescent markers in a single panel, enabling extensive immunophenotyping requiring relatively low cell numbers. We set up two antibody panels, one for immunophenotyping based on the OMIP-069 panel (39 colors) (Park et al. 2020) and one for the detection of subclones and macrophages (37 colors). We ran both panels on AML bone marrow samples at diagnosis (n=20), after initial induction therapy (n=4) and on normal bone marrow (NBM) (n=5). Full spectrum flow cytometry showed similar results to conventional flow cytometry, although with a higher resolution. With both panels combined we could identify and quantify 105 distinct cell types. UMAP landscapes were created as a template by merging AML samples and NBM samples, on top of which activation markers and aberrant AML-specific plasma membrane (PM) protein expression profiles could be projected. Additionally, density plots of individual patient samples were generated to compare longitudinal alterations in clonal dynamics and the tumor immune microenvironment before and after treatment. Additional univariate and multivariate survival analyses were performed to link observed changes to disease progression. At diagnosis, considerable heterogeneity amongst individual patients was observed, as expected. Patients either had a tumor supportive M2 macrophage phenotype or a tumor suppressive M1 macrophage phenotype. The lymphoid compartment was overall strongly reduced in AML compared to NBM, but was highly heterogeneous between individual patients whereby the strongest heterogeneity was noted in CD4+ and TCR gamma delta (TCRγδ) T cell subsets. Low levels of either CD4+ T cells and TCRγδ showed trends towards poorer prognosis. After induction therapy, the immune landscapes recovered and started to resemble those of NBM, most notably for T cell subsets but less so for B cells that took longer to recover. AMLs with an M2 phenotype at diagnosis shifted towards an M1 phenotype and non-classical monocytes shifted to more classical monocytes. In general, AML-specific PM markers were lost upon treatment reaching complete remission, although in some cases markers remained detectable within CD45+ blast populations. One patient containing NPMcyt (VAF=0.4) and FLT3-ITD (VAF=0.04) mutations was treated with gilteritinib in addition to cytarabine, reached morphologic complete remission whereby the FLT3-ITD mutation was lost, and the immune landscape restored. However, NPM1 droplet PCR analyses revealed the presence of residual mutant cells, which corresponded with residual cell numbers expressing aberrant CKIT/IL1RAP expression. Whether these cells will give rise to relapsed disease is currently under close investigation. We conclude that full spectrum flow cytometry is a powerful approach that allows extensive longitudinal immunophenotyping and subclone detection requiring low cell numbers. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Oral epithelial cell segmentation from fluorescent multichannel cytology images using deep learning

Background and objectivesCytology is a proven, minimally-invasive cancer screening and surveillance strategy. Given the high incidence of oral cancer globally, there is a need to develop a point-of-care, automated, cytology-based screening tool. Oral cytology image analysis has multiple challenges such as, presence of debris, blood cells, artefacts, and clustered cells, which necessitate a skilled expertise for single-cell detection of atypical cells for diagnosis. The main objective of this study is to develop a semantic segmentation model for Single Epithelial Cell (SEC) separation from fluorescent, multichannel, microscopic oral cytology images and classify the segmented images. MethodsWe have used multi-channel, fluorescent, microscopic images (number of images; n = 2730), which were stained differentially for cytoplasm and nucleus. The cytoplasmic and cell membrane markers used in the study were Mackia Amurensis Agglutinin (MAA; n: 2364) and Sambucus Nigra Agglutinin-1 (SNA-1; n: 366) with a nuclear stain DAPI. The cytology images were labelled for SECs, cluster of cells, artefacts, and blood cells. In this study, we used encoder-decoder models based on the well-established U-Net architecture, modified U-Net and ResNet-34 for multi-class segmentation. The experiments were performed with different class combinations of data to reduce imbalance. The derived MAA dataset (n: 14,706) of SEC, cluster, and artefacts/blood cells were used for developing a classification model. InceptionV3 model and a new custom Convolutional-Neural-Network (CNN) model (Artefact-Net) were trained to classify SNA-1 marker stained segmented images (n:6101). For segmentation models, Intersection Over Union (IoU) and F1 score were used as the evaluation matrices, while the classification models were evaluated using the conventional classification metrics like precision, recall and F1-Score. ResultsThe U-Net and the modified U-Net models gave the best IoU overall (0.73–0.76) as well as for SEC segmentation (079). The images segmented using the modified U-Net model were classified by Artefact-Net and Inception V3 model with F1 scores of 0.96 and 0.95 respectively. The Artefact-Net, when compared to InceptionV3, provided a better precision and F1 score in classifying clusters (Precision: 0.91 vs 0.80; F1: 0.91 vs 0.86). ConclusionThis study establishes a pipeline for SEC segmentation with the segmented component containing only single cells. The pipline will enable automated, cytology-based early detection with reduced bias.

A New Set of Golden-Gate-Based Organelle Marker Plasmids for Colocalization Studies in Plants.

In vivo localization of proteins using fluorescence-based approaches by fusion of the protein of interest (POI) to a fluorescent protein is a cost- and time-effective tool to gain insights into its physiological function in a plant cell. Determining the proper localization, however, requires the co-expression of defined organelle markers (OM). Several marker sets are available but, so far, the procedure requires successful co-transformation of POI and OM into the same cell and/or several cloning steps. We developed a set of vectors containing markers for basic cell organelles that enables the insertion of the gene of interest (GOI) by a single cloning step using the Golden Gate cloning approach and resulting in POI–GFP fusions. The set includes markers for plasma membrane, tonoplast, nucleus, endoplasmic reticulum, Golgi apparatus, peroxisomes, plastids, and mitochondria, all labelled with mCherry. Most of them were derived from well-established marker sets, but those localized in plasma membrane and tonoplast were improved by using different proteins. The final vectors are usable for localization studies in isolated protoplasts and for transient transformation of leaves of Nicotiana benthamiana. Their functionality is demonstrated using two enzymes involved in biosynthesis of jasmonic acid and located in either plastids or peroxisomes.

The Culture Dish Surface Influences the Phenotype and Dissociation Strategy in Distinct Mouse Macrophage Populations.

The nature of the culture dish surface and the technique used to detach adherent cells could very likely influence the cell viability and cell membrane protein integrity of harvested macrophages. Several previous studies assessed the detachment efficacies of enzymatic and non-enzymatic methods for harvesting the single cell suspensions of macrophages, but a comprehensive study assessing different dissociation methods and culture conditions for detaching functionally different macrophage populations has not yet been reported. In this study, via the well-established GM-CSF and M-CSF differentiated bone marrow derived macrophage models (GM-BMDMs and M-BMDMs), we compared four commonly used enzymatic (trypsin and accutase) and non-enzymatic (PBS and EDTA) dissociation methods along with necessary mechanical detaching steps (scraping and pipetting) to evaluate the viable cell recovery and cell surface marker integrality of GM-BMDMs and M-BMDMs cultured on standard cell culture dish (TC dish), or on culture dish (noTC dish) that was not conditioned to enhance adherence. The data showed that accutase yielded a better recovery of viable cells comparing with PBS and EDTA, especially for tightly adherent GM-BMDMs on TC dishes, with a relatively higher level of detected cell membrane marker F4/80 than trypsin. An additional gradient centrifugation-based dead cell removal approach could increase the proportion of viable cells for TC cultured GM-BMDMs after accutase dissociation. Furthermore, transcriptome analysis was performed to evaluate the putative influence of culture dishes. At steady state, BMDMs cultured on noTC dishes exhibited more proinflammatory gene expression signatures (e.g. IL6, CXCL2 and ILlβ) and functions (e.g. TNF and IL17 signaling pathways). Similar inflammatory responses were observed upon LPS challenge regardless of culture conditions and differentiation factors. However, in LPS treated samples, the difference of gene expression patterns, signaling pathways and molecular functions between TC and noTC cultured BMDMs were largely dependent on the types of growth factors (M-CSF and GM-CSF). This observation might provide valuable information for in vitro macrophage studies.

Occludin and claudin-1 are potential prognostic biomarkers in patients with oral squamous cell carcinomas: An observational study

The objective of this study was to evaluate the expression of several cell membrane markers in oral squamous cell carcinomas (OSCC) and to examine their prognostic influence. We analyzed the immunohistochemical expression of claudin-1 (CLDN-1), claudin-4 (CLDN-4), claudin-5 (CLDN-5), claudin-7 (CLDN-7), occludin (OCLN), and E-cadherin (CDHE) in 60 patients with OSCC treated in a central hospital Center of Oporto. The prognostic significance of these biomarkers in cancer-specific survival and recurrence-free survival were evaluated using multivariate analysis. Claudin-1 was observed in the membrane of tumor cells in 51 cases (89.5%), CLDN-4 in 36 cases (63.2%), and CLDN-7 in 48 cases (80%). Claudin-5 was detected in the cytoplasm of tumor cells in 46 cases (78%) and OCLN in 40 cases (70.2%). In a multivariate analysis, the combined evaluation of OCLN and CLDN-1 revealed a significant and independent association with cancer-specific survival and recurrence-free survival. We found a low extent score for OCLN and a high intensity score for CLDN-1, presenting the hazard ratios of 15.48 (P=.014) and 9.446 (P=.012), respectively. The CLDN-1 and OCLN proteins could be involved in tumor progression of OSCC. Their combined deregulated expression showed an adverse effect on survival and therefore they could be regarded as important prognostic biomarkers in OSCC.

Abstract 2035: Imaging mass cytometry identifies structural and cellular composition of the mouse tissue microenvironment

Abstract Imaging Mass Cytometry™ (IMC™) is a vital tool to deeply characterize the complexity and diversity of any tissue without disrupting spatial context. The Hyperion™ Imaging System utilizes IMC, based on CyTOF® technology, to simultaneously assess up to 40 individual structural and functional markers in tissues, providing unprecedented insight into the organization and function of tissue microenvironment. We have previously demonstrated the application of IMC in combination with Maxpar® panel kits to highlight cellular composition of human tissues. Here, we showcase the recently released Maxpar OnDemand Antibodies for IMC application on mouse tissue. We introduced 11 additional biomarkers to our existing mouse antibody catalog, providing the basis for the use of high-multiplex imaging in preclinical investigations. To demonstrate the IMC workflow on mouse tissue, we analyzed a normal mouse tissue microarray using IMC spatial proteomic analysis. Tissues were stained with a 20-marker panel designed to highlight tissue architecture and major immune lineage markers combined with our IMC Cell Segmentation Kit*. The IMC Cell Segmentation Kit facilitates identification of cellular borders using plasma membrane markers that lead to improved nucleus and plasma membrane demarcation. We generated a detailed spatial map of the heterogeneous tissue architecture and successfully identified immune, epithelial, and stromal cell populations in various mouse tissues. Additionally, we classified the activation state of immune cell populations, adhesion state of epithelial cells, and molecular composition of the extracellular matrix.Overall, this work demonstrates the capability of IMC to identify subcellular localization of cellular and structural markers in the mouse tissue microenvironment. Information gained from IMC studies will enable in-depth high-throughput phenotypic characterization of the tissue microenvironment in various mouse models of development and disease, and thus accelerate preclinical discoveries. *The IMC Cell Segmentation Kit is part of the Innovative Solutions menu of custom-made reagents and workflows developed and tested by Fluidigm scientists to give faster access to new cutting-edge solutions for high-multiplex single-cell analysis. Innovative Solutions are not part of the Maxpar catalog. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Qanber Raza, Michael Cohen, Smriti Kala, Liang Lim, Geneve Awong, Andrew Quong, Christina Loh. Imaging mass cytometry identifies structural and cellular composition of the mouse tissue microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2035.

Abstract 1930: Unsupervised sub-cellular segmentation of complex tissue and cell samples using highly multiplexed imaging-derived a priori knowledge

Abstract Spatial context of heterocellular interactions within solid tumor microenvironments (TMEs) is important for deciphering the mechanistic underpinnings of malignant TME phenotypes and leveraging that knowledge to improve personalized and precision medicine. Recent development of highly multiplexed imaging approaches, such as co-detection by indexing (CODEX), cyclic immunofluorescence (cycif), and imaging mass cytometry (IMC) has, for the first time, allowed deep interrogation of this heterocellular spatial complexity. Proper quantitation of this complexity, however, requires the ability to easily and accurately segment and localize cells and their sub-cellular compartments within the spatial context of the tumor microenvironment. Seminal approaches based on semi-supervised and supervised learning methods, including deep learning techniques, have been developed to segment cells and their nuclei. However, generalization of these methods to segmenting heterogenous and complex cell and tissue samples with varying resolution, magnification, and dynamic range, remains a persistent bottleneck. In case of deep learning, the requirement of large and accurate annotated datasets further adds to the challenge of seamless integration of systems-based methods in pathology and cancer research. Here, we demonstrate that by leveraging cell-compartment specific a priori knowledge captured by these imaging modalities, we can segment cells in complex tissue and cell samples in an unsupervised manner, without requiring model training. We specifically show that using nucleus and cell-membrane markers, we can accurately segment sub-cellular compartments of a diversity of tissue samples imaged at different resolutions, magnifications and dynamic ranges, and cell samples at varying levels of confluency. We also demonstrate that our method is fast. Given its ease of use, accuracy, robustness, and no requirement of large, annotated datasets, our unsupervised segmentation method fills a much-needed gap toward integration of spatial systems biology and cancer research within the convergence science paradigm. Citation Format: Bogdan Kochetov, Phoenix D. Bell, Rebecca Raphael, Benjamin J. Raymond, Brian J. Leibowitz, Jingshan Tong, Brenda Diergaarde, Jian Yu, Reetesh K. Pai, Robert E. Schoen, Lin Zhang, Aatur Singhi, Shikhar Uttam. Unsupervised sub-cellular segmentation of complex tissue and cell samples using highly multiplexed imaging-derived a priori knowledge [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1930.

Membrane phosphoinositides and renal epithelial cell polarity determination in the Xenopus pronephros in vivo

BackgroundThough they are only minor components of cell membranes, phosphoinositide lipids (PIs) participate in numerous signaling processes and in membrane identity determination. Many studies of cultured MDCK renal epithelial cells found that PIs are distributed with polarity among plasma membrane (PM) domains, and that their polarized distributions are required for the delivery of distinct populations of apical and basolateral membrane proteins. The extent to which PIs drive these processes in actual renal epithelial cells in vivo has never been examined. We investigate the distribution of PIs in vivo in the pronephros of Xenopus laevis tadpoles using MCherry‐tagged Pleckstrin Homology (PH) domains that selectively bind different PIs (PH‐AKT, PH‐PLCD1, which bind to (PI(3,4,5,)P3 and PI(4,5)P2 respectively) with the goal of assessing whether and how PI localizations affect cell polarity determination and the trafficking of proteins to their sites of ultimate functional residence in renal epithelial cells in situ.MethodsmRNA encoding MCherry‐PH‐AKT or MCherry‐PH‐PLCD1 was injected into fertilized oocytes and their distributions were assessed in the developed pronephros at stage NF45 via fluorescence microscopy. Knockdown (KD) of PTEN, a lipid 3’ phosphatase that regulates membrane PI composition, was achieved via injection of targeted morpholinos and confirmed by western blotting. The effects of PTEN KD on MCherry‐PH‐AKT distribution were assessed by fluorescence microscopy.ResultsIn MDCK cells PH‐AKT and PH‐PLCD1 localize to the basolateral and apical PMs, respectively. Their distributions are quite different in the pronephros, with both sensors localizing apically in the proximal portion of the tubule and basolaterally in the distal part. Interestingly, PTEN localization, at least in proximal tubules, is apical and not different than previously reported in cell models. Finally, PH‐AKT staining dramatically re‐distributes to the lateral domain of renal cells upon PTEN KD but this treatment does not alter the localization of protein markers of epithelial PM polarity.ConclusionsThese studies constitute the first effort to assess the role of PIs in establishing PM polarity in renal epithelial cells in vivo. Our data reveal important differences from observations previously made with in vitro systems. These findings highlight the need to explore the processes that produce renal epithelial cell polarity in vivo and in the context of intact renal tubules.