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Ramakrishnan, S.

Publications and source records attributed to Ramakrishnan, S..

At least 37 records · Page 2Linked to original sources

MaizeCODE reveals bi-directionally expressed enhancers that harbor molecular signatures of maize domestication.

Modern maize was domesticated from Teosinte parviglumis, with subsequent introgressions from Teosinte mexicana, yielding increased kernel row number, loss of the hard fruit case and dissociation from the cob upon maturity, as well as fewer tillers. Molecular approaches have identified several transcription factors involved in the development of these traits, yet revealed that a complex regulatory network is at play. MaizeCODE deploys ENCODE strategies to catalog regulatory regions in the maize genome, generating histone modification and transcription factor ChIP-seq in parallel with transcriptomics datasets in 5 tissues of 3 inbred lines which span the phenotypic diversity of maize, as well as the teosinte inbred TIL11. Integrated analysis of these datasets resulted in the identification of a comprehensive set of regulatory regions in each inbred, and notably of distal enhancers which were differentiated from gene bodies by their lack of H3K4me1. Many of these distal enhancers expressed non- coding enhancer RNAs bi-directionally, reminiscent of "super enhancers" in animal genomes. We show that pollen grains are the most differentiated tissue at the transcriptomic level, and share features with endosperm that may be related to McClintocks chromosome breakage- fusion-bridge cycle. Conversely, ears have the least conservation between maize and teosinte, both in gene expression and within regulatory regions, reflecting conspicuous morphological differences selected during domestication. The identification of molecular signatures of domestication in transcriptional regulatory regions provides a framework for directed breeding strategies in maize.

plant biology↗

Convergent evolution of plant prickles is drivenby repeated gene co-option over deep time

An enduring question in evolutionary biology concerns the degree to which episodes of convergent trait evolution depend on the same genetic programs, particularly over long timescales. Here we genetically dissected repeated origins and losses of prickles, sharp epidermal projections, that convergently evolved in numerous plant lineages. Mutations in a cytokinin hormone biosynthetic gene caused at least 16 independent losses of prickles in eggplants and wild relatives in the genus Solanum. Strikingly, homologs promote prickle formation across angiosperms that collectively diverged over 150 million years ago. By developing new Solanum genetic systems, we leveraged this discovery to eliminate prickles in a wild species and an indigenously foraged berry. Our findings implicate a shared hormone-activation genetic program underlying evolutionarily widespread and recurrent instances of plant morphological innovation.

evolutionary biology↗

S-nitrosylation of EZH2 at C329 and C700 interplay with PRC2 complex assembly, methyltransferase activity, and EZH2 stability to regulate endothelial functions

Nitric oxide (NO), a versatile bio-active molecule modulates cellular function through diverse mechanisms including S-nitrosylation of proteins. However, the role of this post-translational modification in regulating epigenetic pathways was very limitedly explored. Herein, we report that NO causes S-nitrosylation of selected cysteine residues of EZH2 in endothelial cells (EC) resulting in SUZ12 dissociation from EZH2 bound PRC2 complex, reduced methyltransferase activity, and diminished nuclear localization eventually hampering its stability. We detected a significant reduction in H3K27me3 upon exposure to NO as contributed by the early dissociation of SUZ12 from the PRC2 complex. Longer exposure to NO donors caused EZH2 cytosolic translocation, its ubiquitination, and further degradation primarily through the autophagosome-lysosome pathway. Through in silico S-nitrosylation prediction analysis and site-directed mutagenesis assay, we identified three cysteine residues namely at locations 260, 329, and 700 in EZH2 and further determined that S-nitrosylation of cysteine 329 induced EZH2 instability while S-nitrosylation of cysteine 700 abrogated EZH2s catalytic activity. A double mutant of EZH2 containing mutations at Cysteine 329 and 700 remained undeterred to NO exposure. Furthermore, reinforcing H3K27me3 in NO exposed EC through the use of an inhibitor of H3K27me3 demethylase, we confirmed a significant contribution of the EZH2-H3K27me3 axis in defining NO-mediated regulation of endothelial gene expression and migration. Molecular dynamics simulation study revealed SUZ12s inability in efficiently binding to the SAL domain of EZH2 upon S-nitrosylation of C329 and C700. Taken together, our study for the first-time reports that S-nitrosylation dependent regulation of EZH2 and its associated PRC2 complex influences endothelial homeostasis.

cell biology↗

Single-Molecule Morphology of Topologically Digested Olympic Networks

The kinetoplast DNA (kDNA) is the archetype of a two-dimensional Olympic network, composed of thousands of DNA minicircles and found in the mitochondrion of certain parasites. The evolution, replication and self-assembly of this structure are fascinating open questions in biology that can also inform us how to realise synthetic Olympic networks in vitro. To obtain a deeper understanding of the structure and assembly of kDNA networks, we sequenced the Crithidia fasciculata kDNA genome and performed high-resolution Atomic Force Microscopy (AFM) and analysis of kDNA networks that had been partially digested by selected restriction enzymes. We discovered that these topological perturbations lead to networks with significantly different geometrical features and morphologies with respect to the unperturbed kDNA, and that these changes are strongly dependent on the class of DNA circles targeted by the restriction enzymes. Specifically, cleaving maxicircles leads to a dramatic reduction in network size once adsorbed onto the surface, whilst cleaving both maxicircles and a minor class of minicircles yields non-circular and deformed structures. We argue that our results are a consequence of a precise positioning of the maxicircles at the boundary of the network, and we discuss our findings in the context of kDNA biogenesis, design of artificial Olympic networks and detection of in vivo perturbations.

biophysics↗

The genome of the Wollemi pine, a critically endangered living fossil unchanged since the Cretaceous, reveals extensive ancient transposon activity.

We present the genome of the living fossil, Wollemia nobilis, a southern hemisphere conifer morphologically unchanged since the Cretaceous. Presumed extinct until rediscovery in 1994, the Wollemi pine is critically endangered with less than 60 wild adults threatened by intensifying bushfires in the Blue Mountains of Australia. The 12 Gb genome is among the most contiguous large plant genomes assembled, with extremely low heterozygosity and unusual abundance of DNA transposons. Reduced representation and genome re-sequencing of individuals confirms a relictual population since the last major glacial/drying period in Australia, 120 ky BP. Small RNA and methylome sequencing reveal conservation of ancient silencing mechanisms despite the presence of thousands of active and abundant transposons, including some transferred horizontally to conifers from arthropods in the Jurassic. A retrotransposon burst 8-6 my BP coincided with population decline, possibly as an adaptation enhancing epigenetic diversity. Wollemia, like other conifers, is susceptible to Phytophthora, and a suite of defense genes, similar to those in loblolly pine, are targeted for silencing by sRNAs in leaves. The genome provides insight into the earliest seed plants, while enabling conservation efforts.

genomics↗

Synaptophysin Chaperones the Assembly of 12 SNAREpins under each Ready-Release Vesicle

The synaptic vesicle protein Synaptophysin has long been known to form a complex with the v-SNARE VAMP, but a more specific molecular function or mechanism of action in exocytosis has been lacking because gene knockouts have minimal effects. Utilizing fully-defined reconstitution and single-molecule measurements, we now report that Synaptophysin functions as a chaperone that determines the number of SNAREpins assembling between a ready-release vesicle and its target membrane bilayer. Specifically, Synaptophysin directs the assembly of 12 {+/-} 1 SNAREpins under each docked vesicle, even in the face of an excess of SNARE proteins. The SNAREpins assemble in successive waves of 6 {+/-} 1 and 5 {+/-} 2 SNAREpins, respectively, tightly linked to oligomerization of and binding to the vesicle Ca++ sensor Synaptotagmin. Templating of 12 SNAREpins by Synaptophysin is likely the direct result of its hexamer structure and its binding of VAMP2 dimers, both of which we demonstrate in detergent extracts and lipid bilayers. Significance StatementSynaptophysin is the most abundant protein and a unique constituent of synaptic vesicles, yet it has no known function, due to minimal genetic phenotypes and the lack of biochemical assays. Here, we directly establish using two independent methods that the synaptic vesicle protein Synaptophysin forms a hexameric complex containing 12 copies of the v-SNARE VAMP2. These v-SNAREs assemble into SNAREpins as ready-release vesicles are formed in a fully-defined cell-free system, and do so in two equal waves organized by oligomerization of the Ca++ sensor Synaptotagmin. In the absence of Synaptophysin, two waves are also observed, but the number of SNAREpins in each varies widely. We suggest that a single Synaptophysin hexamer in each vesicle symmetrically organizes 6 pairs of peripheral and central SNAREpins, the latter being directly bound to the Synaptotagmin ring. This gives rise to the symmetrical ring-like arrangement of densities observed by cryo-EM tomography under each synaptic vesicle (1, 2).

neuroscience↗

Novel Roles for Diacylglycerol in Synaptic Vesicle Priming and Release Revealed by Complete Reconstitution of Core Protein Machinery

Here we introduce the full functional reconstitution of genetically-validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, Complexin) for synaptic vesicle priming and release in a geometry that enables detailed characterization of the fate of docked vesicles both before and after release is triggered with Ca2+. Using this novel setup, we discover new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca2+-triggered release involving the SNARE assembly chaperone Munc13. We find that low concentrations of DAG profoundly accelerate the rate of Ca2+-dependent release, and high concentrations reduce clamping and permit extensive spontaneous release. As expected, DAG also increases the number of ready-release vesicles. Dynamic single-molecule imaging of Complexin binding to ready-release vesicles directly establishes that DAG accelerates the rate of SNAREpin assembly mediated by Munc13 and Munc18 chaperones. The selective effects of physiologically validated mutations confirmed that the Munc18-Syntaxin-VAMP2 template complex is a functional intermediate in the production of primed, ready-release vesicles, which requires the coordinated action of Munc13 and Munc18. SIGNIFICANCE STATEMENTMunc13 and Munc18 are SNARE-associated chaperones that act as "priming" factors, facilitating the formation of a pool of docked, release-ready vesicles and regulating Ca2+-evoked neurotransmitter release. Although important insights into Munc18/Munc13 function have been gained, how they assemble and operate together remains enigmatic. To address this, we developed a novel biochemically-defined fusion assay which enabled us to investigate the cooperative action of Munc13 and Munc18 in molecular terms. We find that Munc18 nucleates the SNARE complex, while Munc13 promotes and accelerates the SNARE assembly in a DAG-dependent manner. The concerted action of Munc13 and Munc18 stages the SNARE assembly process to ensure efficient clamping and formation of stably docked vesicles, which can be triggered to fuse rapidly ([~]10 msec) upon Ca2+ influx.

neuroscience↗

Regulation of shelterin proteins TERF2IP and TRF2 by H3K4me3-p65 axis drives hyperglycemia dependent endothelial senescence

BackgroundEndothelial senescence has been linked to several cardiovascular diseases. Dysregulation of proteins of the shelterin complex including TRF2 and TERF2IP causes senescence as it hampers DNA repair and cell proliferation. However, whether exposure to hyperglycemia interplays with proteins of the shelterin complex thus further dictates the senescent phenotype of endothelial cells (EC) remain to be explored. Approach and ResultsIn this study, we observed elevated levels of p21 and p53 in endothelial cells upon exposure to intermittent hyperglycemia. We also noted hyperglycemia exposure increased the levels of TERF2IP and TRF2, part of the shelterin complex. No change in the level of TRF1 and TPP1 were detected. Furthermore, a robust induction was detected in p65 level upon intermittent hyperglycemia challenge. ChIP-qPCR analysis revealed enhanced H3K4me3 enrichment in the promoter regions of p65, TERF2IP and TRF2. Inhibition of catalysis of H3K4me3 either by pharmacological inhibitor or siRNA-mediated knockdown of MLL2 attenuated increase in p65, TERF2IP and TRF2 levels including reversal of senescence markers p53 and p21. Interestingly, pharmacological inhibition of NF-{kappa}B signaling also diminished abrupt increase in TERF2IP and TRF2 levels thus further reversed intermittent hyperglycemia-induced p53 and p21 levels. More importantly, co-immunoprecipitation and co-localization analysis revealed an interaction between nuclear p65 and MLL2 in EC stimulated with hyperglycemia. Further knockdown of either TERF2IP or TRF2 impaired intermittent hyperglycemia-induced p53 and p21 expression and associated endothelial senescence. ConclusionOverall, the present study describes an interplay of epigenetics in defining NF-{kappa}B signaling and shelterin proteins expression which further govern the biochemical and functional state of endothelial senescence in hyperglycemic milieu.

biochemistry↗

Rapid Quantification of First and Second Phase Insulin Secretion Dynamics using In vitro Platform for Improving Insulin Therapy

High-throughput quantification of the first- and second-phase insulin secretion dynamics is intractable with current methods. The fact that independent secretion phases play distinct roles in metabolism necessitates partitioning them separately and performing high-throughput compound screening to target them individually. We developed an insulin-nanoluc luciferase reporter system to dissect the molecular and cellular pathways involved in the separate phases of insulin secretion. We validated this method through genetic studies, including knockdown and overexpression, as well as small-molecule screening and their effects on insulin secretion. Furthermore, we demonstrated that the results of this method are well correlated with those of single-vesicle exocytosis experiments conducted on live cells. Thus, we can quantitatively determine the number of vesicles that fuse when a stimulus is applied. We have developed a robust methodology for screening small molecules and cellular pathways that target specific phases of insulin secretion, resulting in a better understanding of insulin secretion, which in turn will result in a more effective insulin therapy through the stimulation of endogenous glucose-stimulated insulin secretion.

cell biology↗

Direct determination of oligomeric organization of integral membrane proteins and lipids from intact customizable bilayer

Hierarchical organization of integral membrane proteins (IMP) and lipids at the membrane is essential for regulating myriad downstream signaling. A quantitative understanding of these processes requires both detections of oligomeric organization of IMPs and lipids directly from intact membranes and determination of key membrane components/properties that regulate them. Addressing this, we have developed a platform that enables native mass spectrometry (nMS) analysis of IMP-lipid complexes directly from intact and customizable lipid membranes. Both the lipid composition and membrane properties (such as curvature, tension, fluidity) of these bilayers can be precisely customized to a target membrane. Subsequent direct nMS analysis of these intact proteo-lipid vesicles can yield the oligomeric states of the embedded IMPs, identify bound lipids, and determine the membrane properties that can regulate the observed IMP-lipid organization. Applying this, we show how lipid binding regulates neurotransmitter release and how membrane composition regulates the functional oligomeric state of a transporter.

biophysics↗

Differential roles of extracellular matrix proteins on primary and metastatic pancreatic cancer cells

Pancreatic cancer adenocarcinoma (PDAC) is reported to be the third highest cause of cancer-related deaths in the United States. PDAC is known for its high proportion of stroma, which accounts for 90% of the tumor mass. The stroma is made up of extracellular matrix (ECM) and non-malignant cells such as inflammatory cells, cancer-associated fibroblasts, and lymphatic and blood vessels. Here, we decoupled the effects of the ECM on PDAC cell lines by culturing cells on surfaces coated with different ECM proteins. Our data show that the primary tumor-derived cell lines have different morphology depending on the ECM proteins on which they are cultured, while metastatic lesion-derived PDAC lines morphology does not change with respect to the different ECM proteins. Similarly, ECM proteins modulate the proliferation rate and the gemcitabine sensitivity of the primary tumor PDAC cell lines, but not the metastatic PDAC lines. Lastly, transcriptomics analysis of the primary tumor PDAC cells cultured on different ECM proteins reveals the regulation of various pathways, such as cell cycle, cell adhesion molecules, and focal adhesion, including the regulation of several integrin genes that are essential for ECM recognition.

cancer biology↗

Establishing Physalis as a new Solanaceae model system enables genetic reevaluation of the inflated calyx syndrome

The highly diverse Solanaceae family contains several widely studied model and crop species. Fully exploring, appreciating, and exploiting this diversity requires additional model systems. Particularly promising are orphan fruit crops in the genus Physalis, which occupy a key evolutionary position in the Solanaceae and capture understudied variation in traits such as inflorescence complexity, fruit ripening and metabolites, disease and insect resistance, self-compatibility, and most notable, the striking Inflated Calyx Syndrome (ICS), an evolutionary novelty found across angiosperms where sepals grow exceptionally large to encapsulate fruits in a protective husk. We recently developed transformation and genome editing in Physalis grisea (groundcherry). However, to systematically explore and unlock the potential of this and related Physalis as genetic systems, high-quality genome assemblies are needed. Here, we present chromosome-scale references for P. grisea and its close relative P. pruinosa and use these resources to study natural and engineered variation in floral traits. We first rapidly identified a natural structural variant in a bHLH gene that causes petal color variation. Further, and against expectations, we found that CRISPR-Cas9 targeted mutagenesis of 11 MADS-box genes, including purported essential regulators of ICS, had no effect on inflation. In a forward genetics screen, we identified huskless, which lacks ICS due to mutation of an AP2-like gene that causes sepals and petals to merge into a single whorl of mixed identity. These resources and findings elevate Physalis to a new Solanaceae model system, and establish a new paradigm for the search of factors driving ICS.

plant biology↗

Increased severity of chronic kidney disease in response to high potassium intake is dependent on mineralocorticoid receptor activation

Dietary treatment is seminal for management of chronic kidney disease (CKD). The aim of our project was to assess the effects of potassium intake on the progression of CKD. We used 2 mouse CKD models to analyze the effects of potassium intake on CKD : the unilateral ureteral obstruction (UUO) and the POD-ATTAC models. POD-ATTAC mice display a podocyte-specific apoptosis after the administration of a chemical inducer. We also studied the effect of mineralocorticoid receptor (MR) using UUO in kidney tubule-specific MR knockout mice. In both UUO and POD-ATTAC mice, high potassium diet increased interstitial fibrosis. High potassium diet also increased the abundance of the extracellular matrix protein fibronectin and decreased the abundance of the epithelial marker Na+-K+ ATPase. Consistently, POD-ATTAC mice fed with high potassium diet displayed lower glomerular filtration rate. Spironolactone, a MR antagonist, decreased fibrosis induced by high potassium diet in POD-ATTAC mice. However, kidney tubule-specific MR knockout did not improve the fibrotic lesions induced by UUO under normal or high potassium diets. Macrophages from high potassium-fed POD-ATTAC mice displayed higher mRNA levels of the pro-inflammatory chemokine MCP1. This effect was decreased by spironolactone, suggesting a role of MR signaling in myeloid cells in the pro-fibrotic effect of potassium-rich diet. High potassium intake generates more fibrosis leading to decreased kidney function in experimental CKD. MR signaling plays a pivotal role in this potassium-induced fibrosis. The effect of reducing potassium intake on CKD progression should be assessed in future clinical trials. Translational statementDietetic approach is a cheap and effective therapy to slow down the development of chronic kidney diseases and kidney fibrosis. Potassium-rich diets are protective against renal and cardiovascular events in the general population, albeit some conflicting data were obtained in patients with chronic kidney disease. We showed that potassium-rich diet accelerates fibrosis development, by enhancing kidney inflammation in two mouse models of chronic kidney disease. These data suggest that potassium-rich diets should not be advised in patients with chronic kidney disease, unless future clinical trials demonstrate any beneficial effect in these patients.

physiology↗

Minos: variant adjudication and joint genotyping of cohorts of bacterial genomes

Short-read variant calling for bacterial genomics is a mature field, and there are many widely-used software tools. Different underlying approaches (eg pileup, local or global assembly, paired-read use, haplotype use) lend each tool different strengths, especially when considering non-SNP (single nucleotide polymorphism) variation or potentially distant reference genomes. It would therefore be valuable to be able to integrate the results from multiple variant callers, using a robust statistical approach to "adjudicate" at loci where there is disagreement between callers. To this end, we present a tool, Minos, for variant adjudication by mapping reads to a genome graph of variant calls. Minos allows users to combine output from multiple variant callers without loss of precision. Minos also addresses a second problem of joint genotyping SNPs and indels in bacterial cohorts, which can also be framed as an adjudication problem. We benchmark on 62 samples from 3 species (Mycobacterium tuberculosis, Staphylococcus aureus, Klebsiella pneumoniae) and an outbreak of 385 M. tuberculosis samples. Finally, we joint genotype a large M. tuberculosis cohort (N{approx}15k) for which the rifampicin phenotype is known. We build a map of non-synonymous variants in the RRDR (rifampicin resistance determining region) of the rpoB gene and extend current knowledge relating RRDR SNPs to heterogeneity in rifampicin resistance levels. We replicate this finding in a second M. tuberculosis cohort (N{approx}13k). Minos is released under the MIT license, available at https://github.com/iqbal-lab-org/minos.

bioinformatics↗

Increasing triacylglycerides and lipid storage by unsaturated lipids protects renal proximal tubules

In diabetic patients, dyslipidemia frequently contributes to organ damage such as diabetic kidney disease (DKD). DKD is associated with excessive renal deposition of triacylglycerol (TAG) in lipid droplets (LD). Yet, it is unclear whether LDs play a protective or damaging role and how this might be influenced by dietary patterns. By using a diabetes mouse model, we find here that high fat diet enriched in the unsaturated oleic acid (OA) caused more lipid storage in LDs in renal proximal tubular cells (PTC) but less tubular damage than a corresponding butter diet with the saturated palmitic acid (PA). Mechanistically, we identify endoplasmic reticulum (ER) stress as the main cause of PA-induced PTC injury. ER stress is caused by elevated cellular levels of saturated TAG precursors and to higher membrane order in the ER. The resulting cell death is preceded by a transcriptional rewiring of phospholipid metabolism. Simultaneous addition of OA rescues the cytotoxic effects by normalizing membrane order and by increasing the total TAG amount. The latter also stimulates the formation of LDs that in turn can release unsaturated lipids upon demand by lipolysis. Our study thus clarifies mechanisms underlying PA-induced cell stress in PTCs and emphasizes the importance of olive oil for the prevention of DKD.

cell biology↗

Molecular Determinants of Complexin Clamping in Reconstituted Single-Vesicle Fusion

Previously we reported that Synaptotagmin-1 and Complexin synergistically clamp the SNARE assembly process to generate and maintain a pool of docked vesicles that fuse rapidly and synchronously upon Ca2+ influx (Ramakrishnan et al. 2020). Here using the same in vitro single-vesicle fusion assay, we establish the molecular details of the Complexin clamp and its physiological relevance. We find that a delay in fusion kinetics, likely imparted by Synaptotagmin-1, is needed for Complexin to block fusion. Systematic truncation/mutational analyses reveal that continuous alpha-helical accessory-central domains of Complexin are essential for its inhibitory function and specific interaction of the accessory helix with the SNAREpins, analogous to the trans clamping model, enhances this functionality. The c-terminal domain promotes clamping by locally elevating Complexin concentration through interactions with the membrane. Further, we find that Complexin likely contributes to rapid Ca2+-synchronized vesicular release by preventing un-initiated fusion rather than by directly facilitating vesicle fusion.

biophysics↗

Racial differences in androgen metabolism and receptor signaling in prostate cancer

Dihydrotestosterone (DHT) and testosterone (T) mediated androgen receptor (AR) nuclear translocation initiates transcription of AR target genes that are pivotal for prostate cancer (PrCa) development and progression. Here we provide data indicating that in contrast to European American (EA) men, African American (AA) men with localized PrCa can exploit an alternative progesterone-androsterone-5-androstanedione pathway for DHT biosynthesis. Enzymes that are involved in alternate pathways of DHT biosynthesis are elevated in PrCa tissues from AA men, compared to EA men, and also correlated with increased serum DHT levels. In addition, higher serum DHT levels reflect increased RNA expression of AR target genes in PrCa tissues from AA men. Interestingly, serum T but not DHT levels are significantly lower in AA men compared to EA men with PrCa. Furthermore, serum progesterone and related intermediate metabolites levels that are produced during alternate pathways of DHT biosynthesis are significantly lower in AA men with PrCa and associated with a shorter time to disease progression. These data highlight that androgen biosynthesis is altered in therapy naive localized PrCa in AA men, and can potentially serve as prognostic indicators of disease progression. SignificanceOur work provides a rationale to examine potential pharmacological interventions that target androgen biosynthesis and AR signaling earlier in the disease continuum in AA men with PrCa. Additionally, our study lays the groundwork for developing serum measurements of intermediate androgen metabolites as PrCa prognostic biomarkers. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/437727v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@932dd0org.highwire.dtl.DTLVardef@a4cca0org.highwire.dtl.DTLVardef@1764be4org.highwire.dtl.DTLVardef@1708a63_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

Patient derived models of bladder cancer amplify tumor specific gene expression compared to surgical specimen while maintaining gene expression of molecular subtype and epithelial mesenchymal transition markers

Patient derived models (PDMs) are a powerful tool to study preclinical responses. However, the benefits of each model have not been compared head-to-head when models are derived from the same surgical specimen. PDMs derived from surgical specimens were established as xenografts (PDX), organoids (PDO), and spheroids (PDS). PDMs were molecularly characterized by RNA sequencing. Differential gene expression was determined between the PDMs and surgical specimens. Surgical specimens had the most differentially expressed genes reflecting loss of immune and stromal compartments in PDMs. PDMs and surgical specimens were clustered using the Euclidian distance analysis to test model fidelity. PDMs upregulated a clear, patient-specific bladder cancer signal. Overall, the molecular profiles of PDXs were the most similar to the matching patient surgical specimen than the PDO and PDS from that patient. The epithelial mesenchymal transition (EMT) gene expression profile is maintained in the PDMs showing the persistence of EMT in both in vivo and in vitro model setting. The consensus molecular subtype was determined in order to compare PDMs to each other and their matching surgical specimen, and only surgical specimens with Basal/Squamous or Luminal Papillary molecular subtype established PDMs. Patient derived models reduce tumor heterogeneity and allow analysis of specific tumor compartments while maintaining the gene expression profile representative of the original tumor.

cancer biology↗