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Prion-like low-complexity domains (PLCDs) are involved in the formation and regulation of distinct biomolecular condensates that form via coupled associative and segregative phase transitions. We previously deciphered how evolutionarily conserved sequence features drive phase separation of PLCDs through homotypic interactions. However, condensates typically encompass a diverse mixture of proteins with PLCDs. Here, we combine simulations and experiments to study mixtures of PLCDs from two RNA binding proteins namely, hnRNPA1 and FUS. We find that 1:1 mixtures of the A1-LCD and FUS-LCD undergo phase separation more readily than either of the PLCDs on their own. The enhanced driving forces for phase separation of mixtures of A1-LCD and FUS-LCD arise partly from complementary electrostatic interactions between the two proteins. This complex coacervation-like mechanism adds to complementary interactions among aromatic residues. Further, tie line analysis shows that stoichiometric ratios of different components and their sequence-encoded interactions jointly contribute to the driving forces for condensate formation. These results highlight how expression levels might be tuned to regulate the driving forces for condensate formationin vivo. Simulations also show that the organization of PLCDs within condensates deviates from expectations based on random mixture models. Instead, spatial organization within condensates will reflect the relative strengths of homotypic versus heterotypic interactions. We also uncover rules for how interaction strengths and sequence lengths modulate conformational preferences of molecules at interfaces of condensates formed by mixtures of proteins. Overall, our findings emphasize the network-like organization of molecules within multicomponent condensates, and the distinctive, composition-specific conformational features of condensate interfaces.Significance StatementBiomolecular condensates are mixtures of different protein and nucleic acid molecules that organize biochemical reactions in cells. Much of what we know about how condensates form comes from studies of phase transitions of individual components of condensates. Here, we report results from studies of phase transitions of mixtures of archetypal protein domains that feature in distinct condensates. Our investigations, aided by a blend of computations and experiments, show that the phase transitions of mixtures are governed by a complex interplay of homotypic and heterotypic interactions. The results point to how expression levels of different protein components can be tuned in cells to modulate internal structures, compositions, and interfaces of condensates, thus affording distinct ways to control the functions of condensates.

Recent investigations have suggested that biomolecular condensates are viscoelastic materials. This implies that material properties of condensates are governed by internal microstructures. Furthermore, computations show that the internal organization in protein condensates is spatially inhomogeneous, featuring hub-and-spoke-like percolated networks of molecules. Here, we test these predictions using imaging of single fluorogenic dyes that are turned-on in response to specific chemical microenvironments. We deployed Nile blue (NB), Nile red (NR), and merocyanine 540 (MC540) for epifluorescence and single-molecule localization microscopy imaging of condensates formed by intrinsically disordered, low-complexity domains of proteins. Imaging with NB reveals internal environments that are uniformly hydrophobic, whereas NR shows preferential binding to hubs that are more hydrophobic than the surrounding background within condensates. Finally, imaging with MC540 suggests that interfaces of condensates are unique chemical environments. Overall, the high spatiotemporal resolution and environmental sensitivity of single-fluorogen imaging reveals spatially inhomogeneous organization of molecules within condensates.

Biomolecular condensates form via phase transitions of condensate-specific biomacromolecules. Intrinsically disordered regions (IDRs) featuring the appropriate sequence grammar can contribute homotypic and heterotypic interactions to the driving forces for phase separation of multivalent proteins. At this juncture, experiments and computations have matured to the point where the concentrations of coexisting dense and dilute phases can be quantified for individual IDRs in complex milieus bothin vitroandin vivo. For a macromolecule such as a disordered protein in a solvent, the locus of points that connects concentrations of the two coexisting phases defines a phase boundary or binodal. Often, only a few points along the binodal, especially in the dense phase, are accessible for measurement. In such cases and for quantitative and comparative analysis of parameters that describe the driving forces for phase separation, it is useful to fit measured or computed binodals to well-known mean-field free energies for polymer solutions. Unfortunately, the non-linearity of the underlying free energy functions makes it challenging to put mean-field theories into practice. Here, we present FIREBALL, a suite of computational tools designed to enable efficient construction, analysis, and fitting to experimental or computed data of binodals. We show that depending on the theory being used, one can also extract information regarding coil-to-globule transitions of individual macromolecules. Here, we emphasize the ease-of-use and utility of FIREBALL using examples based on data for two different IDRs.Statement of SignificanceMacromolecular phase separation drives the assembly of membraneless bodies known as biomolecular condensates. Measurements and computer simulations can now be brought to bear to quantify how the concentrations of macromolecules in coexisting dilute and dense phases vary with changes to solution conditions. These mappings can be fit to analytical expressions for free energies of solution to extract information regarding parameters that enable comparative assessments of the balance of macromolecule-solvent interactions across different systems. However, the underlying free energies are non-linear and fitting them to actual data is non-trivial. To enable comparative numerical analyses, we introduce FIREBALL, a user-friendly suite of computational tools that allows one to generate, analyze, and fit phase diagrams and coil-to-globule transitions using well-known theories.

Recent investigations have suggested that biomolecular condensates are viscoelastic materials. This implies that material properties of condensates are governed by internal microstructures. Furthermore, computations show that the internal organization in protein condensates is spatially inhomogeneous, featuring hub-and-spoke-like percolated networks of molecules. Here, we test these predictions using imaging of single fluorogenic dyes that are turned-on in response to specific chemical microenvironments. We deployed Nile blue (NB), Nile red (NR), and merocyanine 540 (MC540) for epifluorescence and single-molecule localization microscopy imaging of condensates formed by intrinsically disordered, low-complexity domains of proteins. Imaging with NB reveals internal environments that are uniformly hydrophobic, whereas NR shows preferential binding to hubs that are more hydrophobic than the surrounding background within condensates. Finally, imaging with MC540 suggests that interfaces of condensates are unique chemical environments. Overall, the high spatiotemporal resolution and environmental sensitivity of single-fluorogen imaging reveals spatially inhomogeneous organization of molecules within condensates.

Biomolecular condensates form via spontaneous and driven phase transitions of multivalent proteins and nucleic acids. These macromolecules can be organized in spatially inhomogeneous ways that lead to multiple coexisting dense phases with distinct macromolecular interfaces. While considerable attention has focused on the physical driving forces that give rise to phase separation from bulk solutions, the interactions that underlie adsorption driven wetting transitions remain unclear. Here, we report that pyrimidine-rich RNAs function as adsorbents that enable cascades of wetting transitions that include partial and complete wetting of condensates formed by purine-rich RNAs. Computations show that macromolecules that are scaffolds of condensates are oriented perpendicular to condensate interfaces whereas adsorbents are oriented parallel to interfaces. Our results yield heuristics for the design of synthetic materials that can be based on RNA-rich condensates featuring bespoke interfaces and distinct local microenvironments created by the interplay between scaffolds versus adsorbents.

Prion-like low-complexity domains (PLCDs) have distinctive sequence grammars that determine their driving forces for phase separation. Here we uncover the physicochemical underpinnings of how evolutionarily conserved compositional biases influence the phase behaviour of PLCDs. We interpret our results in the context of the stickers-and-spacers model for the phase separation of associative polymers. We find that tyrosine is a stronger sticker than phenylalanine, whereas arginine is a context-dependent auxiliary sticker. In contrast, lysine weakens sticker-sticker interactions. Increasing the net charge per residue destabilizes phase separation while also weakening the strong coupling between single-chain contraction in dilute phases and multichain interactions that give rise to phase separation. Finally, glycine and serine residues act as non-equivalent spacers, and thus make the glycine versus serine contents an important determinant of the driving forces for phase separation. The totality of our results leads to a set of rules that enable comparative estimates of composition-specific driving forces for PLCD phase separation.

The nucleolus is a multilayered structure. Each layer is thought to be a compositionally distinct phase, although how these phases form and interface with one another remains unclear. Using computational, proteomics,in vitro, andin vivostudies, we uncover distinct molecular grammars within intrinsically disordered regions (IDRs) of nucleolar proteins that localize to fibrillar centers (FCs) and dense fibrillar components (DFCs). FC and DFC proteins feature two distinct types of IDRs namely those with long tracts of acidic residues and those with blocks of lysines interspersed by acid-rich-regions. We find that phase separation driven by complex coacervation in mixtures of nucleolar proteins, with their distinctive IDRs, and ribosomal DNA and RNA molecules is sufficient to drive the formation of structural facsimiles of FCs and DFCs.One-Sentence SummaryFacsimiles of core nucleolar substructures were reconstituted via phase separation of key protein and nucleic acid mixtures.

Introduction: Radical cystectomy is the standard of care for muscle invasive bladder cancer (MIBC). Radiotherapy (RT) is a bladder preserving option that offers patients comparable survival rates. However, up to 30% of patients require salvage cystectomy. Emerging evidence points to an important yet poorly understood link between response to RT and the tumor microenvironment (TME). Notably, tertiary lymphoid structures (TLS) are being investigated as a potential local hub for mounting an immune response. These lymph-node-like structures are highly organized with a B cell zone surrounded by T cells. Mature TLS also have an active germinal center (GC) with follicular dendritic cells. A number of studies correlated TLS with improved outcomes in several types of solid cancers. Here, we explore the use of TLS and its associated immune TME as a predictive biomarker for response to RT in MIBC. Methods: Hematoxylin and Eosin (H&E) stained formalin-fixed-paraffin-embedded sections of pre-RT biopsy from 147 MIBC patients were examined to identify TLS presence with confirmation from a pathologist. Subsequent multiplex immunohistochemistry (mIHC) staining with CD21 and CD23 characterized TLS maturity. To investigate the interaction between TLS and TME, tissue microarrays (TMAs) were constructed, and gene expression profiles were obtained for 106 patients using NanoString’s Digital Spatial technology. Moreover, the TMAs were stained with CD20, Neutrophil Elastase, CD68, CD8, CD4, and FoxP3 by mIHC. Image analysis was performed on the Halo platform. TLS gene signature was correlated to the immune infiltration and the patient outcomes. Results: H&E revealed that 19.7% of patients (n=68) had TLS with a GC, 17.0% (n=25) had TLS without a GC, and 12.9% (n=19) had no TLS. In the remaining 50.3% of cases (n=74), confirmation of TLS presence required staining with CD3 and CD20. Gene expression of TLS marker CXCL13 was higher among complete responders to RT (p-value=0.0440). This trend was weakened when we examined a previously described 12-chemokine TLS signature (p= 0.1506). When segregating patients into TLS high/TLS low groups based on the gene signature, we observed significantly higher infiltration of CD8+ T cells (p Citation Format: Nour Hassan, José Joao Mansure, Mina Farag, Ronald Kool, Eva Michaud, Mohanned Alessa, Luis Souhami, Fabio Cury, Fadi Brimo, Wassim Kassouf. The predictive power of tertiary lymphoid structures in assessing response to trimodal therapy in muscle invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 609.

The formation of membraneless biomolecular condensates is driven by macromolecules with sticker-and-spacer architectures that undergo phase separation coupled to percolation (PSCP). Driving forces for PSCP are governed by the interplay between reversible inter-sticker crosslinks and solvation preferences of spacers. Here, we introduce molecular and mesoscale descriptions of structures within, outside, and at the interfaces of condensates that are formed by prion-like low complexity domains (PLCDs), which are exemplars of intrinsically disordered, linear multivalent proteins. Our studies are based on simulations that accurately describe sequence-specific phase behaviors of PLCDs. We find that networks of reversible, intermolecular, inter-sticker crosslinks organize PLCDs into small-world topologies within condensates. These topologies result from distinct conformational preferences within dense, dilute, and interfacial regions. Specifically, the degree of conformational expansion varies non-monotonically, being most expanded at the interface and most compact in the dilute phase with molecules preferring to be oriented perpendicular to condensate interfaces. This contrasts with dense and dilute phases where molecules are randomly oriented relative to one another. Our results demonstrate that even simple condensates, with only one type of macromolecule, feature inhomogeneous spatial organizations of molecules and interfacial features that likely prime them for being locations of biochemical activity.

Not too sticky There is increasing evidence for a role of liquid-liquid phase separation (LLPS) in many cellular processes. Many proteins that undergo LLPS include prionlike domains (PLDs), which are enriched in polar amino acids and often interspersed with aromatic residues. Combining experimental data with simulations, Martin et al. quantified concentrations of PLDs in coexisting dilute and dense phases as a function of temperature and show that the phase behavior is determined by the number of aromatic residues and their patterning, with uniform patterning of aromatic residues promoting LLPS and inhibiting aggregation. They developed a sticker-and-spacers model that can predict the phase behavior of PLDs on the basis of their sequence. Science , this issue p. 694

With sunitinib treatment of metastatic renal cell carcinoma, most patients end up developing resistance over time. Recent clinical trials have shown that individualizing treatment protocols could delay resistance and result in better outcomes. We developed an in vivo xenograft tumor model and compared tumor growth rate, morphological, and transcriptomic differences between alternative and traditional treatment schedules. Our results show that the alternative treatment regime could delay/postpone cancer progression. Additionally, we identified distinct morphological changes in the tumor with alternative and traditional treatments, likely due to the significantly dysregulated signaling pathways between the protocols. Further investigation of the signaling pathways underlying these morphological changes may lead potential therapeutic targets to be used in a combined treatment with sunitinib, which offers promise in postponing/reversing the resistance of sunitinib.

Background: Transcatheter aortic valve implantation is a feasible alternative to conventional aortic valve replacement with expanding indication extending to low-risk patients. Sutureless and rapid-deployment aortic valves were developed to decrease procedural risks in conventional treatment. This paired-match analysis aims to compare patients undergoing surgical transcatheter aortic valve implantation to sutureless and rapid-deployment aortic valve implantation. Methods: Retrospective database analysis between 2010 and 2016 revealed 214 patients undergoing transcatheter aortic valve implantation procedures through surgical access (predominantly transapical) and 62 sutureless and rapid-deployment aortic valve procedures including 26 patients in need of concomitant coronary artery bypass surgery. After matching, 52 pairs of patients were included and analyzed. Results: In-hospital death (5.8% vs. 3.8%, p = 0.308) was comparable between transcatheter aortic valve implantation (mean age 77 ± 4.3 years) and sutureless and rapid-deployment aortic valve implantation groups (mean age 75 ± 4.0 years), including 32 females in each group. The logistic EuroSCORE was similar (19 ± 12 vs. 17 ± 10, p = 0.257). Postoperative renal failure (p = 0.087) and cerebrovascular accidents (p = 0.315) were without significant difference. The incidence of complete heart block requiring permanent pacemaker treatment was relatively low for both groups (1.9% vs. 7.7%, p = 0.169) for TAVI and sutureless and rapid-deployment valves respectively. Intraoperative use of blood transfusion was higher in the sutureless and rapid-deployment aortic valve implantation group (0.72 U vs. 1.46 U, p = 0.014). Estimated survival calculated no significant difference between both groups after 6 months (transcatheter aortic valve implantation: 74 ± 8% vs. sutureless and rapid-deployment aortic valve implantation: 92 ± 5%, log rank p = 0.097). Conclusion: Since sutureless and rapid-deployment aortic valve implantation is as safe and effective as transapical transcatheter aortic valve implantation, combining the advantage of standard diseased-valve removal with shorter procedural times, sutureless and rapid-deployment aortic valve replacement may be considered as an alternative for patients with elevated operative risk considered to be in the “gray zone” between transcatheter aortic valve implantation and conventional surgery, especially if concomitant myocardial revascularization is required.

Papillary renal cell carcinoma (PRCC) is the most common type of RCC in end-stage kidney disease (ESKD). Papillary adenoma (PA) is a small benign lesion morphologically similar to PRCC and is suggested to be its precursor. PA is also prevalent in ESKD. The evolution of PAs to PRCCs and their relationship to ESKD are poorly understood. A total of 140 PAs, normal kidneys, ESKDs, and PRCCs were analyzed. Previously described markers of renal tubular progenitor cells were analyzed using immunohistochemistry and quantified with digital analysis. Progenitor cells were significantly increased in ESKD (P < 0.0001) and PAs (P = 0.02) in comparison with the normal kidney. Pathway analysis using global miRNA and chromosomal copy number variations revealed a common developmental theme between PA and the PRCCs. Whole exome sequencing showed a KMT2C-specific pathogenic mutation among all PAs and PRCCs. KMT2C is a chromosome 7 epigenetic regulator implicated in development and oncogenesis. Collectively, results show possible connection of PRCCs to PA and the progenitor-like cell population, which are increased in response to renal tubular injury. In addition, each PRCC histologic subtype had its own set of mutational changes, indicating divergence from a common precursor. The study reports previously unknown biological aspects of PRCC development and could influence current surveillance criteria and early detection strategies of PRCC tumors.

Aims The occurrence of hyperbilirubinaemia after heart surgery using cardiopulmonary bypass or post‐operative heart failure is fairly common. We investigated the incidence, predictive value, and post‐operative outcome of hyperbilirubinaemia after cardiac surgery in an effort to identify potential risk factors and significance on clinical outcome. Methods and results Between 2006 and 2016, 1272 (10.1%) out of 12 556 patients developed hyperbilirubinaemia, defined as bilirubin concentration >3 mg/dL, during post‐operative course at our institution. All patients who were operated using cardiopulmonary bypass were included. Hepatic dysfunction was diagnosed preoperatively in 200 patients (15.7%), whereas mean model of end‐stage liver disease score was 11.22 ± 4.99. Early mortality was 17.4% with age [hazard ratio (HR) 1.019, 95% confidence interval (CI) 1.008–1.029; P = 0.001], diabetes (HR 1.115, CI 1.020–1.220; P = 0.017), and emergent procedures (HR 1.315, CI 1.012–1.710) as multivariate predictors. Post‐operative predictors were low‐output syndrome (HR 3.193, 95% CI 2.495–4.086; P

Acute aortic Stanford type A dissection remains a frequent and life-limiting event for patients with Marfan syndrome. Outcome results in this high-risk group are limited.The German Registry for Acute Aortic Dissection Type A collected the data of 56 centers between July 2006 and June 2015. Of 3385 patients undergoing operations for acute aortic Stanford type A dissection, 117 (3.5%) were diagnosed with Marfan syndrome. We performed a propensity score match comparing patients with Marfan syndrome with patients without Marfan syndrome in a 1:2 fashion.Patients with Marfan syndrome were significantly younger (42.9 vs 62.2 years; P .001), predominantly male (76.9% vs 62.9%; P = .002), and less catecholamine dependent (9.4% vs 20.3%; P = .002) compared with the unmatched cohort. They presented with aortic regurgitation (41.6% vs 23.0%; P .001) and involvement of the supra-aortic vessels (50.4% vs 39.5%; P = .017) more often. Propensity matching revealed 82 patients with Marfan syndrome (21 female) with no significant differences in baseline characteristics compared with patients without Marfan syndrome (n = 159, 36 female; P = .607). Although root preservation was more frequent in patients with Marfan syndrome, procedure types did not differ significantly (18.3% vs 10.7%; P = .256). Aortic arch surgery was performed more frequently in matched patients (87.5% vs 97.8%; P = .014). Thirty-day mortality did not differ between patients with and without Marfan syndrome (19.5% vs 20.1%; P = .910). Multivariate regression showed no influence of Marfan syndrome on 30-day mortality (odds ratio, 0.928; 95% confidence interval, 0.346-2.332; P = .876).Marfan syndrome does not adversely affect 30-day outcomes after surgical repair for acute aortic Stanford type A dissection compared with a matched cohort. Long-term outcome analysis is needed to account for the influence of further downstream interventions.

Although international medical graduates (IMGs) constitute considerable percentage of doctors in the National Health Service (NHS), they face several challenges in acclimatizing to the NHS health system. Communication skills, language subtleties, and career progression difficulties are among the most important challenges that overseas doctors face. Some resources are already available to support these doctors and several trusts across the UK have developed local interventions and educational programs to help their doctors bridge the gaps in their knowledge. However, there is no proof of the external validity of these programs and none are identified as effective on a national level. Senior IMGs are leading very popular and inspiring projects using digital platforms, especially social media. We identified several social media pages, groups, and websites subscribed to by hundreds of thousands of doctors in the UK and around the world, including doctors who are planning a future career in the UK. These platforms provide information, resources, support, and answers to questions posed by junior IMGs. Inspired by these projects and also by an Australian project that transformed a local IMG education program, we studied whether using digital platforms and transforming evidence-based local programs to national ones would be the best way forward to support IMGs.

OBJECTIVES Our goal was to evaluate the use of the frozen elephant trunk (FET) technique for the treatment of penetrating aortic ulcers involving the aortic arch. METHODS Between January 2008 and January 2020, a total of 34 patients had the FET technique at 3 aortic centres. The indication for the FET technique was unsuitability for thoracic endovascular aortic repair due to the lack of a sufficient proximal landing zone even after supra-aortic rerouting (subclavian transposition, double transposition), ectasia of the ascending aorta/aortic arch (>40 mm) and/or a shaggy proximal thoracic aorta. RESULTS Additional cardiac procedures were performed in 14 patients (41%), and the beating heart technique was used in 7 patients (21%). Perioperative mortality was 18% (n = 6); 3 of these patients had a major stroke (9%). No case of spinal cord ischaemia was observed, and 2 patients (6%) developed a non-disabling stroke. After a median follow-up of 7 (first quartile: 1; third quartile 29) months, 2 patients (6%) died (1 of malignant disease and 1 of an unclear cause); 10 additional aortic interventions in all aortic segments (29%; endovascular: n = 8 [24%] and conventional surgical: n = 2 [6%]) were performed in 8 patients. CONCLUSIONS The FET technique is a good treatment option for patients with penetrating aortic ulcers involving the aortic arch unsuitable for thoracic endovascular aortic repair. However, the high obliterative atherosclerotic load in these patients is accompanied by an un-neglectable risk of perioperative neurological injury. Concomitant cardiac surgical procedures are frequently needed. Patients commonly require secondary aortic procedures in all aortic segments, emphasizing the need for thorough primary conceptual planning and stringent follow-up.

The pandemic of type 2 diabetes mellitus (T2DM) has been largely attributed to the increasing prevalence of worldwide obesity at a geometric rate. However, the number of non-obese patients with T2DM is also on the rise, and it is as high as 60-80% in some Asian countries. These non-obese individuals have certain peculiarities and have a higher mortality rate compared with obese individuals. The pathophysiology of T2DM in non-obese individuals remains poorly understood, and this has an impact on defining its management. This review discusses the current understanding of the pathophysiology of T2DM in non-obese individuals. The definition of T2DM in non-obese individuals remains controversial because of the limited clinical measurements, and the current definition of obesity using body mass index (BMI) is not very helpful as these individuals have BMIs of

Aims: Acute right heart failure (RHF) is a severe complication of right ventricular infarction. The management of acute RHF poses a number of challenges, such as providing haemodynamic support. Temporary circulatory support (TCS) may be required upon failing medical therapy. The ProtekDuo® dual lumen cannula provides a minimally invasive option for (TCS) through a groin-free internal jugular vein approach. We present the largest patient series to date using the ProtekDuo® cannula as temporary right ventricular assist device (t-RVAD) in RHF after acute myocardial infarction (MI). Methods and results: From July 2016 to November 2019, 10 patients underwent t-RVAD implantation for RHF following acute MI. Transthoracic and transoesophageal echocardiography were performed in all patients to assess cardiac function, with a particular focus on RV function. Cumulative 30-day survival was 60%. Mean TAPSE was 6.4 ± 3.1 mm, mean fractional area change was 12.1 ± 4.2%, and mean right ventricular end diastolic area was 19.8 ± 2.7 cm². Mean implantation time was 32.8 ± 8.3 min. Mean interval after first cardiac intervention was 4.6 ± 5.8 days. Mean t-RVAD time was 10.0 ± 7.4 days with a significant reduction in central venous pressure 19.3 ± 2.7 vs. 8.2 ± 2.6 mmHg, P

Papillary renal cell carcinoma (PRCC) is the most common nonclear cell RCCs and is known to comprise two histological subtypes. PRCC2 is more aggressive and is molecularly distinct from the other subtypes. Despite this, PRCCs are treated together as one entity, and they show poor response to the current therapies that do not target pathways implicated in their pathogenesis. We have previously detected ABCC2 (an ABC transporter), VEGF, and mTOR pathways to be enriched in PRCC2. In this study, we assess the therapeutic potential of targeting these pathways in PRCC2. Twenty RCC cell lines from the Cancer Cell Encyclopedia were compared to the Cancer Genome Atlas PRCC cohort (290), to identify representative PRCC2 cell lines. Cell lines were further validated in xenograft models. Selected cell lines were treated in vitro and in vivo (mice models) under five different conditions, untreated, anti‐VEGF (sunitinib), ABCC2 blocker (MK571), mTOR inhibitor (everolimus) and sunitinib + MK571. Sunitinib +ABCC2 blocker group showed a significant response to therapy compared to the other treatment groups both in vitro (P ≤ 0.0001) and in vivo (P = 0.0132). ABCC2 blockage resulted in higher sunitinib uptake, both in vitro (P = 0.0016) and in vivo (P = 0.0031). Everolimus group demonstrated the second best response in vivo. The double‐treatment group showed the highest apoptotic rate and lowest proliferation rate. There is an urgent need for individualized therapies of RCC subtypes that take into account their specific biology. Our results demonstrate that combined targeted therapy with sunitinib and ABCC2 blocker in PRCC2 has therapeutic potential. The results are likewise potentially significant for other ABCC2 high tumors. However, the results are preliminary and clinical trials are needed to confirm these effects in PRCC2 patients.