Biology subjects

Rao, S.

Publications and source records attributed to Rao, S..

At least 127 records · Page 7Linked to original sources

bioRxiv2019

Cytoplasm localized ARID1B promotes oncogenesis in pancreatic cancer by activating RAF-ERK signaling

The ARID1B/BAF250b subunit of the human SWI/SNF chromatin remodeling complex is a canonical nuclear tumor suppressor. Immunohistochemistry on a pancreatic cancer tissue microarray revealed significant ARID1B cytoplasmic localization that correlated with advanced tumor stage and lymph node positivity. Identification of the nuclear localization signal (NLS) using in silico prediction and subcellular localization studies facilitated evaluation of a possible cytoplasmic function for ARID1B. A cytoplasm-restricted ARID1B-NLS mutant was significantly compromised to regulate transcription activation and tumor suppression functions, as expected. Surprisingly however, cytoplasm-localized ARID1B could bind c-RAF and PPP1CA causing stimulation of RAS-RAF-ERK signaling and {beta}-catenin transcription activity in pancreatic cancer cells. More importantly, cytoplasmic ARID1B resulted in an induction of cell growth and migration in pancreatic cancer cell lines that was dependent on ERK signaling and caused increased tumorigenesis in nude mice. NLS peptides representing mutations identified from pancreatic cancer samples exhibiting ARID1B cytoplasmic localization or curated from cancer somatic mutation database were significantly compromised to effect nuclear localization of a reporter protein. ARID1B cytoplasmic localization correlated significantly with active forms of ERK and {beta}-catenin in primary pancreatic tumor samples. ARID1B may therefore promote oncogenesis through non-canonical cytoplasm-based gain of function mechanisms in addition to dysregulation in the nucleus.

cell biology↗

bioRxiv2019

Novel insights into expansion and functional diversification of MIR169 family in tomato

MIR169 family is an evolutionarily conserved miRNA family in plants. A systematic in-depth analysis of MIR169 family in tomato is lacking. We report eighteen miR169 precursors, annotating new loci for MIR169a, b and d, as well as four novel mature isoforms (MIR169f/g/h/i). The family has expanded by both tandem- and segmental-duplication events during evolution. A tandem-pair MIR169b/b-1 and MIR169b-2/h is polycistronic in nature coding for three MIR169b isoforms and a new variant miR169h, that is evidently absent in the wild relatives S. pennellii and S. pimpinellifolium. Seven novel miR169 targets including RNA-binding-protein, protein-phosphatase, aminotransferase, chaperone, tetratricopeptide-repeat-protein, and transcription factors ARF-9B and SEPELLATA-3 were established by efficient target cleavage in presence of specific precursors as well as increased target abundance upon miR169 chelation by short-tandem-target-mimic construct in transient assays. Comparative antagonistic expression profiles of MIR169:target pairs suggest MIR169 family as ubiquitous regulator of various abiotic stresses (heat, cold, dehydration and salt) and developmental pathways. This regulation is partly brought about by acquisition of new promoters as demonstrated by promoterMIR169:GUS-reporter assays as well as differential processivity of different precursors and miRNA cleavage efficiencies. Thus, the current study augments the functional horizon of MIR169 family with applications for stress tolerance in crops.\n\nHighlightExpansion of MIR169 members by duplication and new mature forms, acquisition of new promoters, differential precursor-miRNA processivity and engaging novel targets increases the functional diversification of MIR169 in tomato. (29/30)

genomics↗

bioRxiv2019

High resolution, dynamic imaging of early mouse and human liver bud morphogenesis in three dimensions

BackgroundLiver organogenesis has thus far served as a paradigm for solid organ formation. The developing liver bud is a well established model of organogenesis, and murine genetic studies demonstrate key molecules involved in key morphogenetic changes. However, the analysis of the liver bud is typically limited to 2D tissue sections, which precludes extensive visualization, quantitation, and analysis. Further, the lack of human liver bud data has further hindered our understanding of human liver organogenesis. Therefore, new analytical and visualization approaches are needed to elicit further morphogenetic details of liver organogenesis and to elucidate differences between mouse and human liver bud growth. ResultsTo address this need, we focused on high resolution imaging, visualization, and analysis of early liver growth by using available online databases for both mouse (EMAP, Toronto Phenogenomics center) and human (3D Atlas of Human Embryology), noninvasive multimodality imaging studies of the murine embryo, and mouse/human liver weight data. First, we performed three-dimensional (3D reconstructions) of stacked, digital tissue sections that had been initially segmented for the liver epithelium and the septum transversum mesenchyme (STM). 3D reconstruction of both mouse and human data sets enabled visualization and analysis of the dynamics of liver bud morphogenesis, including hepatic cord formation and remodeling, mechanisms of growth, and liver-epithelial STM interactions. These studies demonstrated potentially under-appreciated mechanisms of growth, including rapid exponential growth that is matched at the earliest stages by STM growth, and unique differences between mouse and human liver bud growth. To gain further insight into the exponential liver bud growth that was observed, we plotted volumetric data from 3D reconstruction together with fetal liver growth data from multimodality (optical projection tomography, magnetic resonance imaging, micro-CT) and liver weight data to compose complete growth curves during mouse (E8.5-E18) and human (day 25-300) liver development. For further analysis, we performed curve fitting and parameter estimation, using Gompertzian models, which enables the comparison between mouse and human liver bud growth, as well as comparisons to processes like liver regeneration. To demonstrate the importance of mesenchyme in rapid liver bud growth and morphogenesis in the human liver bud, we performed functional analysis in which human pluripotent stem cell (hPSC)-derived hepatic organoids were used to model collective migration that occurs in vivo, demonstrating that migration is strongly dependent upon mesenchyme. DiscussionOur data demonstrates improved visualization with 3D images, under-appreciated and potentially new mechanisms of growth, complete liver growth curves with quantitative analysis through embryonic and fetal stages, and a new functional human stem cell-derived liver organoid assay demonstrating mesenchyme-driven collective migration. These data enhance our understanding of liver organogenesis.

developmental biology↗

bioRxiv2019

Characterization of novel regulators for heat stress tolerance in tomato from Indian sub-continent

The footprint of tomato cultivation, a cool region crop that exhibits heat stress (HS) sensitivity, is increasing in the tropics/sub-tropics. Knowledge of novel regulatory hot-spots from varieties growing in the Indian sub-continent climatic zones could be vital for developing HS-resilient crops. Comparative transcriptome-wide signatures of a tolerant (CLN1621L) and sensitive (CA4) cultivar-pair short-listed from a pool of varieties exhibiting variable thermo-sensitivity using physiological, survival and yield-related traits revealed redundant to cultivar-specific HS-regulation with more up-regulated genes for CLN1621L than CA4. The anatgonisiticly-expressing genes include enzymes; have roles in plant defense and response to different abiotic stresses. Functional characterization of three antagonistic genes by overexpression and TRV-VIGS silencing established Solyc09g014280 (Acylsugar acyltransferase) and Solyc07g056570 (Notabilis), that are up-regulated in tolerant cultivar, as positive regulators of HS-tolerance and Solyc03g020030 (Pin-II proteinase inhibitor), that is down-regulated in CLN1621L, as negative regulator of thermotolerance. Transcriptional assessment of promoters of these genes by SNPs in stress-responsive cis-elements and promoter swapping experiments in opposite cultivar background showed inherent cultivar-specific orchestration of transcription factors in regulating transcription. Moreover, overexpression of three ethylene response transcription factors (ERF.C1/F4/F5) also improved HS-tolerance in tomato. This study identifies several novel HS-tolerance genes and provides proof of their utility in tomato-thermotolerance.\n\nHighlightNovel heat stress regulatory pathways uncovered by comparative transcriptome profiling between contrasting tomato cultivars from Indian sub-continent for improving thermotolerance. (20/30)

genomics↗

bioRxiv2019

Mechanisms of activation and desensitization of full-length glycine receptor in membranes

Glycinergic synapses play a central role in motor control and pain processing in the central nervous system. Glycine receptors (GlyR) are key players in mediating fast inhibitory neurotransmission at these synapses. While previous high-resolution structural studies have provided insights into the molecular architecture of GlyR, several mechanistic questions pertaining to channel function are still unknown. Here, we present Cryo-EM structures of the full-length GlyR protein reconstituted into lipid nanodiscs that are captured in the unliganded (closed) and glycine-bound (open and desensitized) conformations. A comparison of the three states reveals global conformational changes underlying GlyR channel gating. The functional state assignments were validated by molecular dynamics simulations of the structures incorporated in a lipid bilayer. Observed permeation events are in agreement with the anion selectivity of the channel and the reported single-channel conductance of GlyR. These studies establish the structural basis for gating, selectivity, and single-channel conductance of GlyR in a physiological environment.

biophysics↗

bioRxiv2019

Adaptive thermogenesis in mice requires adipocyte light-sensing via Opsin 3

Almost all life forms can decode light information for adaptive advantage. Examples include the visual system, where photoreceptor signals are interpreted as images, and the circadian system, where light entrains a physiological clock. Here we describe a local, non-visual light response in mice that employs encephalopsin (OPN3, a 480 nm, blue light responsive opsin) to regulate the function of adipocytes. Germ line null and adipocyte-specific conditional null mice show a deficit in thermogenesis that is phenocopied in mice under blue-light deficient conditions. We show that blue light stimulation of adipocytes activates hormone sensitive lipase, the rate limiting enzyme in the lipolysis pathway, and that this is OPN3-dependent. Opn3 adipocyte conditional null mice also use reduced levels of fat mass when fasted and cold exposed further suggesting a lipolysis deficit. These data suggest the hypothesis that in mice, a local, OPN3-dependent light response in adipocytes is a mechanism for regulation of energy homeostasis.

physiology↗

bioRxiv2019

The CIViC knowledge model and standard operating procedures for curation and clinical interpretation of variants in cancer

Manually curated variant knowledgebases and their associated knowledge models are serving an increasingly important role in distributing and interpreting variants in cancer. These knowledgebases vary in their level of public accessibility, and the complexity of the models used to capture clinical knowledge. CIViC (Clinical Interpretations of Variants in Cancer - www.civicdb.org) is a fully open, free-to-use cancer variant interpretation knowledgebase that incorporates highly detailed curation of evidence obtained from peer-reviewed publications. Currently, the CIViC knowledge model consists of four main components: Genes, Variants, Evidence Items, and Assertions. Each component has an associated knowledge model and methods for curation. Gene and Variant data contextualize the genomic region(s) involved in the clinical statement. Evidence Items provide structured associations between variants and their clinically predictive/therapeutic, prognostic, diagnostic, predisposing, and functional implications. Finally, CIViC Assertions summarize collections of CIViC Evidence Items for a specific Disease, Variant, and Clinical Significance with incorporation of clinical and technical guidelines. Here we present the CIViC knowledge model, curation standard operating procedures, and detailed examples to support community-driven curation of cancer variants.

bioinformatics↗

bioRxiv2019

Evaluating the ALERT algorithm for local outbreak onset detection in seasonal infectious disease surveillance data

Estimation of epidemic onset timing is an important component of controlling the spread of seasonal infectious dis-eases within community healthcare sites. The Above Local Elevated Respiratory Illness Threshold (ALERT) algorithm uses a threshold-based approach to suggest incidence levels that historically have indicated the transition from endemic to epidemic activity. In this paper, we present the first detailed overview of the computational approach underlying the algorithm. In the motivating example section, we evaluate the performance of ALERT in determining the onset of increased respiratory virus incidence using laboratory testing data from the Childrens Hospital of Colorado. At a threshold of 10 cases per week, ALERT-selected intervention periods performed better than the observed hospital site periods (2004/2005-2012/2013) and a CUSUM method. Additional simulation studies show how data properties may effect ALERT performance on novel data. We found that the conditions under which ALERT showed ideal performance generally included high seasonality and low off-season incidence.

epidemiology↗

bioRxiv2019

An evidence-based network approach to recommending targeted cancer therapies

In this work, we introduce CDGnet, an evidence-based network approach for recommending targeted cancer therapies, available as a user-friendly informatics tool. Our approach can be used to expand the range of options of targeted therapies for cancer patients who undergo molecular profiling. It considers biological pathway information specifically by looking at downstream targets of oncogenes and is personalized for individual patients via the user-inputted molecular alterations and cancer type. CDGnet integrates disparate sources of knowledge and provides results in a number of easily-accessible and usable forms, while separating targeted cancer therapies into categories in an evidence-based manner.

bioinformatics↗

bioRxiv2019

An Interscholastic Network to Generate LexA Enhancer Trap Lines in Drosophila

Binary expression systems like the LexA-LexAop system provide a powerful experimental tool kit to study gene and tissue function in developmental biology, neurobiology and physiology. However, the number of well-defined LexA enhancer trap insertions remains limited. In this study, we present the molecular characterization and initial tissue expression analysis of nearly 100 novel StanEx LexA enhancer traps, derived from the StonEx1 index line. This includes 76 insertions into novel, distinct gene loci not previously associated with enhancer traps or targeted LexA constructs. Additionally, our studies revealed evidence for selective transposase-dependent replacement of a previously-undetected KP element on chromosome III within the StanEx1 genetic background during hybrid dysgenesis, suggesting a molecular basis for the over-representation of LexA insertions at the NK7.1 locus in our screen. Production and characterization of novel fly lines were performed by students and teachers in experiment-based genetics classes within a geographically diverse network of public and independent high schools. Thus, unique partnerships between secondary schools and university-based programs have produced and characterized novel genetic and molecular resources in Drosophila for open-source distribution, and provide paradigms for development of science education through experience-based pedagogy.

genetics↗

bioRxiv2019

Genome-wide copy number variation-, validation- and screening study implicates a novel copy number polymorphism associated with suicide attempts in major depressive disorder

BackgroundThe genetic basis of suicide attempts (SA) remained unclear, especially for the copy number variations (CNVs) involved. The present study aimed to identify the susceptibility variants associated with SA among major depressive disorder (MDD) patients in Chinese, covering both single-nucleotide polymorphisms and CNVs. MethodsWe conducted GWAS on MDD patients with or without SA and top results were tested in a replication study. A genome-wide CNV study was performed. Subsequently, a validation assay using the qRT-PCR technology was performed to confirm the existence of the associated CNV and then applied to the entire cohort to examine the association. ResultsIn CNV analysis, we found that the global rate of CNV was higher in SA compared to non-SA subjects (p=0.023). The genome-wide CNV study revealed a SA-associated CNV region that achieved genome-wide significance (corrected p-value=0.014). The associated CNV was successfully validated and identified to be a common variant in this cohort and its deletion rate was higher in suicide attempters (OR=2.05). Based on the GTEx database, genetic variants that probe this CNV was significantly associated with the expression level of ZNF33B in two brain regions (p-value<4.2e-05). Besides, there was a significant interaction between neuroticism and the CNV in affecting suicidal risk; the CNV showed a significant effect (OR=2.58) in subjects with high neuroticism only. ConclusionsWe identified a new common CNV that may be involved in the etiology of SA. These findings imply an important role of common CNVs in the etiology of SA, which suggests a new promising avenue for investigating the genetic architecture of SA.

genetics↗

bioRxiv2019

ADAMDEC1 maintains a novel growth factor signaling loop in cancer stem cells

Glioblastomas (GBM) are lethal brain tumors where poor outcome is attributed to cellular heterogeneity, therapeutic resistance, and a highly infiltrative nature. These characteristics are preferentially linked to GBM cancer stem cells (GSCs), but how GSCs maintain their stemness is incompletely understood and the subject of intense investigation. Here, we identify a novel signaling loop that induces and maintains GSCs. This loop consists of an atypical metalloproteinase, a disintegrin and metalloproteinase domain-like protein decysin 1 (ADAMDEC1), secreted by GSCs. ADAMDEC1 rapidly solubilizes fibroblast growth factor-2 (FGF2) to stimulate FGF receptor 1 (FGFR1) expressed on GSCs. This signaling axis induces upregulation of Zinc finger E-box-binding homeobox 1 (ZEB1) that regulates ADAMDEC1 expression, creating a positive feedback loop. Genetic or pharmacological targeting of components of this axis attenuates self-renewal and tumor growth. These findings reveal a new signaling axis for GSC maintenance and highlight ADAMDEC1 and FGFR1 as potential therapeutic targets in GBM. Statement of SignificanceCancer stem cells (CSC) drive tumor growth in many cancers including glioblastoma. We identified a novel sheddase, a disintegrin and metalloproteinase domain-like protein decysin 1, that initiates a fibroblast growth factor autocrine loop to promote stemness in CSCs. This loop can be targeted to reduce glioblastoma growth.

cancer biology↗

bioRxiv2019

CHAP: a versatile tool for the structural and functional annotation of ion channel pores

The regulation of ion channel and transporter function requires the modulation of energetic barriers or gates within their transmembrane pathways. However, despite the ever-increasing number of available structures, our understanding of these barriers is often simply determined from calculating the physical dimensions of the pore. Such approaches (e.g. the HOLE program) have worked very well in the past, but there is now considerable evidence that the unusual behaviour of water within the narrow hydrophobic spaces found within many ion channel pores can also produce energetic barriers to ion conduction without requiring physical occlusion of the permeation pathway. Several different classes of ion channels have now been shown to exploit this principle of hydrophobic gating to regulate ion flow. However, measurement of pore radius alone is unable to identify such barriers and new tools are required for more accurate functional annotation of an exponentially increasing number of ion channel structures. We have previously shown how molecular dynamics simulations of water behaviour can be used as a proxy to accurately predict hydrophobic gates. Here we now present a new and highly versatile computational tool, the Channel Annotation Package (CHAP) that implements this methodology to predict the conductive status of new ion channel structures.

biophysics↗

bioRxiv2019

From the clinic to the bench and back again in one dog year: identifying new treatments for sarcoma using a cross-species personalized medicine pipeline

Cancer drug discovery is an inefficient process, with more than 90% of newly-discovered therapies failing to gain regulatory approval. Patient-derived models of cancer offer a promising new approach to identifying personalized treatments; however, for rare cancers, such as sarcomas, access to patient samples can be extremely limited, which precludes development of patient-derived models. To address the limited access to patient samples, we have turned to pet dogs with naturally-occurring sarcomas. Although sarcomas make up less than 1% of all cancers in humans, sarcomas represent at least 15% of all cancers in dogs. Dogs with naturally-occurring sarcomas also have intact immune systems, an accelerated pace of cancer progression, and share the same environment as humans, making them ideal models that bridge key gaps between mouse models and human sarcomas.\n\nHere, we develop a framework for a personalized medicine pipeline that integrates drug screening, validation, and genomics to identify new therapies. We tested this paradigm through the study of a pet dog, Teddy, who presented with six synchronous leiomyosarcomas. By integrating patient-derived cancer models, in vitro drug screens, and in vivo validation we identified proteasome inhibitors as a potential therapy for Teddy. After showing an initial response to the proteasome inhibitor, bortezomib, Teddy developed rapid resistance, and tumor growth resumed. Whole exome sequencing revealed substantial genetic heterogeneity across Teddys multiple recurrent tumors and metastases, suggesting that intra-patient heterogeneity was responsible for the heterogeneous clinical response. Ubiquitin proteomics coupled with exome sequencing revealed multiple candidate driver mutations in proteins related to the proteasome pathway. Together, our results demonstrate how the comparative study of canine sarcomas can offer rapid insights into the process of developing personalized medicine approaches that can lead to new treatments for sarcomas in both humans and canines.

cancer biology↗

bioRxiv2018

Identification of Hydrophobic Gates by Simulation-Enabled Analysis of the Ion Channel Structural Proteome

Ion channel proteins control ionic flux across biological membranes through conformational changes in their transmembrane pores. An exponentially increasing number of channel structures captured in different conformational states are now being determined. However, these newly-resolved structures are commonly classified as either open or closed based solely on the physical dimensions of their pore and it is now known that more accurate annotation of their conductive state requires an additional assessment of the effect of pore hydrophobicity. A narrow hydrophobic gate region may disfavour liquid-phase water, leading to local de-wetting which will form an energetic barrier to water and ion permeation without steric occlusion of the pore. Here we quantify the combined influence of radius and hydrophobicity on pore de-wetting by applying molecular dynamics simulations and machine learning to nearly 200 ion channel structures. This allows us to propose a simple simulation-free heuristic model that rapidly and accurately predicts the presence of hydrophobic gates. This not only enables the functional annotation of new channel structures as soon as they are determined, but may also facilitate the design of novel nanopores controlled by hydrophobic gates. Significance statementIon channels are nanoscale protein pores in cell membranes. An exponentially increasing number of structures for channels means that computational methods for predicting their functional state are needed. Hydrophobic gates in ion channels result in local de-wetting of pores which functionally closes them to water and ion permeation. We use simulations of water behaviour within nearly 200 different ion channel structures to explore how the radius and hydrophobicity of pores determine their hydration vs. de-wetting behaviour. Machine learning-assisted analysis of these simulations enables us to propose a simple model for this relationship. This allows us to present an easy method for the rapid prediction of the functional state of new channel structures as they emerge.

biophysics↗

bioRxiv2018

Enrichment of short mutant cell-free DNA fragments enhanced detection of pancreatic cancer

Analysis of cell-free DNA (cfDNA) is promising for broad applications in clinical settings, but with significant bias towards late-stage cancers. Although recent studies have discussed the diverse and degraded nature of cfDNA molecules, little is known about its impact on the practice of cfDNA analysis. Here we reported a new targeted sequencing by combining single-strand library preparation and target capture (SLHC-seq). By applying the new technology in plasma cfDNA from pancreatic cancer patients, we achieved higher efficiency in analysis of mutations than previously reported using other detection assays. SLHC-seq rescued short or damaged cfDNA fragments along to increase the sensitivity and accuracy of circulating-tumor DNA detection. Most importantly, we found that the small mutant fragments are prevalent in early-stage patients, which provides strong evidence for fragment size-based early detection of pancreatic cancer. Collectively, the new pipeline enhanced our understanding of cfDNA biology and provide new insights for liquid biopsy.

cancer biology↗

bioRxiv2018

Retrospective Evaluation of Cyclosporine in the Treatment of Chronic Hepatitis in Dogs: 2010-2017

BackgroundIdiopathic chronic hepatitis (CH) in dogs is a prevalent hepatic disorder. The etiology is poorly understood; however, there is evidence to support an immune-mediated pathogenesis. No literature exists investigating the efficacy of cyclosporine (CsA) therapy for CH in dogs.\n\nObjectivesTo retrospectively evaluate the efficacy and adverse side effects of CsA in the treatment of CH in dogs, and to identify factors that impact response to CsA therapy.\n\nAnimals48 client-owned dogs diagnosed with CH treated with CsA for at least 2 weeks.\n\nMethodsRetrospective review of medical records between the years 2010-2017.\n\nResultsTwenty-two breeds of dogs were included of which 54% were spayed females, 42% neutered males and 4% intact males. Median age was 8.5 years (range, 0.7-14 years). Complete remission (normalization of alanine aminotransferase [ALT]) in response to CsA was attained in 79% of dogs (38/48). Median dose of CsA at the time of remission was 7.9 mg/kg/day (range, 2.5-12.7mg/kg/day) and median time to remission was 2.5 months (range, 0.75-18 months). None of the factors evaluated including clinical score, presence of ascites, hypoalbuminemia, hyperbilirubinemia, prolonged coagulation times, dose, or duration of therapy influenced remission. Common side effects were gastrointestinal signs in 38% (18/48) and gingival hyperplasia in 25% (12/48) of dogs.\n\nConclusion and Clinical ImportanceCsA proved to be a tolerated and effective medication in attaining remission in dogs with idiopathic CH based on normalization of serum ALT. None of the evaluated factors were shown to negatively impact response to CsA or remission of disease.

immunology↗

bioRxiv2018

Genomic and transcriptomic determinants of therapy resistance and immune landscape evolution during anti-EGFR treatment in colorectal cancer

Anti-epidermal growth factor receptor (EGFR) antibodies (anti-EGFR-Ab) are effective in a subgroup of patients with metastatic colorectal cancer (CRC). We applied genomic and transcriptomic analyses to biopsies from 35 RAS wild-type CRCs treated with the anti-EGFR-Ab cetuximab in a prospective trial to interrogate the molecular resistance landscape. This validated transcriptomic CRC-subtypes as predictors of cetuximab benefit; identified novel associations of NF1-inactivation and non-canonical RAS/RAF-aberrations with primary progression; and of FGF10- and non-canonical BRAF-aberrations with AR. No genetic resistance drivers were detected in 64% of AR biopsies. The majority of these had switched from the cetuximab-sensitive CMS2-subtype pretreatment to the fibroblast- and growth factor-rich CMS4-subtype at progression. Fibroblast supernatant conferred cetuximab resistance in vitro, together supporting subtype-switching as a novel mechanism of AR. Cytotoxic immune infiltrates and immune-checkpoint expression increased following cetuximab responses, potentially providing opportunities to treat CRCs with molecularly heterogeneous AR with immunotherapy.

cancer biology↗