microfluidic-co-culture-renal-healthy-tumor-epithelium-to-model

Microfluidic Co-culture of Renal Healthy and Tumor Epithelium to Model Kidney Cancer Progression

Microphysiological systems (MPS) have enabled the introduction of more complex and relevant physiological elements into in vitro models, recreating ...

University Medicine Greifswald

The activity of drug transporters is central to the secretory function of the kidneys and a defining feature of renal proximal tubule epithelial cells (RPTECs). The expression, regulation, and function of these membrane-bound proteins is well understood under normal renal physiological conditions. However, the impact of drug transporters on the pathophysiology of kidney cancer is still elusive. In the present study, we employed different renal cell carcinoma (RCC) cell lines and a prototypical non-malignant RPTEC cell line to characterize the activity, expression, and potential regulatory mechanisms of relevant renal drug transporters in RCC in vitro. An analysis of the uptake and efflux activity, the expression of drug transporters, and the evaluation of cisplatin cytotoxicity under the effects of methylation or epidermal growth factor receptor (EGFR) inhibition showed that the RCC cells retained substantial drug transport activity. In RCC cells, P-glycoprotein was localized in the nucleus and its pharmacological inhibition enhanced cisplatin toxicity in non-malignant RPTECs. On the other hand, methylation inhibition enhanced cisplatin toxicity by upregulating the organic cation uptake activity in RCC cells. Differential effects of methylation and EGFR were observed in transporter expression, showing regulatory heterogeneity in these cells. Interestingly, the non-malignant RPTEC cell line that was used lacked the machinery responsible for organic cation transport, which reiterates the functional losses that renal cells undergo in vitro.

In Vitro Characterization of Renal Drug Transporter Activity in Kidney Cancer.

MicroRNAs (miRNA) are ubiquitous non-coding RNAs that have a prominent role in cellular regulation. The expression of many miRNAs is often found deregulated in prostate cancer (PCa) and castration-resistant prostate cancer (CRPC). Although their expression can be associated with PCa and CRPC, their functions and regulatory activity in cancer development are poorly understood. In this study, we used different proteomics tools to analyze the activity of hsa-miR-3687-3p (miR-3687) and hsa-miR-4417-3p (miR-4417), two miRNAs upregulated in CRPC. PCa and CRPC cell lines were transfected with miR-3687 or miR-4417 to overexpress the miRNAs. Cell lysates were analyzed using 2D gel electrophoresis and proteins were subsequently identified using mass spectrometry (Maldi-MS/MS). A whole cell lysate, without 2D-gel separation, was analyzed by ESI-MS/MS. The expression of deregulated proteins found across both methods was further investigated using Western blotting. Gene ontology and cellular process network analysis determined that miR-3687 and miR-4417 are involved in diverse regulatory mechanisms that support the CRPC phenotype, including metabolism and inflammation. Moreover, both miRNAs are associated with extracellular vesicles, which point toward a secretory mechanism. The tumor protein D52 isoform 1 (TD52-IF1), which regulates neuroendocrine trans-differentiation, was found to be substantially deregulated in androgen-insensitive cells by both miR-3687 and miR-4417. These findings show that these miRNAs potentially support the CRPC by truncating the TD52-IF1 expression after the onset of androgen resistance.

Identification of the Regulatory Targets of miR-3687 and miR-4417 in Prostate Cancer Cells Using a Proteomics Approach.

Cisplatin is extensively used in the treatment of head and neck carcinomas. Cetuximab combination therapy is employed in recurrent and metastatic settings. Sunitinib showed positive results in the treatment of head and neck carcinomas, both as monotherapy or in combination with cetuximab. Nonetheless, the mechanism governing these pharmacological interactions is largely unresolved. This study investigates the impact of cetuximab on the cytotoxicity of cisplatin and sunitinib using cells representative of head and neck carcinoma and the oral epithelium.

Recapitulating the Pharmacological Interactions of Cetuximab with Sunitinib and Cisplatin in Head and Neck Carcinoma Cells in vitro.

Accurate prediction of pharmacokinetic parameters, such as renal clearance, is fundamental to the development of effective and safe new treatments for patients. However, conventional renal models have a limited ability to predict renal drug secretion, a process that is dependent on transporters in the proximal tubule. Improvements in microphysiological systems (MPS) have extended our in vitro capabilities to predict pharmacokinetic parameters. In this study a kidney-MPS model was developed that successfully recreated renal drug secretion. Human proximal tubule cells grown in the kidney-MPS, resem­bling an in vivo phenotype, actively secreted the organic cation drug metformin and organic anion drug cidofovir, in contrast to cells cultured in conventional culture formats. Metformin and cidofovir renal secretory clearance were predicted from kid­ney-MPS data within 3.3- and 1.3-fold, respectively, of clinically reported values by employing a semi-mechanistic drug distribution model using kidney-MPS drug transport parameters together with in vitro to in vivo extrapolation. This approach introduces an effective application of a kidney-MPS model coupled with pharmacokinetic modelling tools to evaluate and predict renal drug clearance in humans. Kidney-MPS renal clearance predictions can potentially complement pharma-cokinetic animal studies and contribute to the reduction of pre-clinical species use during drug development.

Amplifying the impact of kidney microphysiological systems: predicting renal drug clearance using mechanistic modelling based on reconstructed drug secretion.

The treatment of advanced renal cell carcinoma has been substantially improved by the introduction of targeted and immune therapies and their respective combinations. Unleashing the activity of the immune system opened a new and successful front in the fight against cancers. Despite the benefits, drug resistance phenomena and adverse side effects can compromise efficacy. The development of new modalities of drugs and therapies with properties and mechanisms of action that break away from conventional medicines was expanded in recent years. This perspective discusses the prospects of these innovative and highly potent novel treatments in overcoming much of the current issues surrounding the resistance to approved renal cell carcinoma treatments and the challenges facing their introduction.

The Potential Impact of New Drug and Therapeutic Modalities on Drug Resistance to Renal Cell Carcinoma.

The study of prostate cancer in vitro relies on established cell lines that lack important physiological characteristics, such as proper polarization and expression of relevant biomarkers. Microphysiological systems (MPS) can replicate cancer microenvironments and lead to cellular phenotypic changes that better represent organ physiology in vitro. In this study, we developed an MPS model comprising conventional prostate cancer cells to evaluate their activity under dynamic culture conditions. Androgen-sensitive (LNCaP) and androgen-insensitive (PC3) cells were grown in conventional and 3D cultures, both static and dynamic. Cell morphology, the secretion of prostate-specific antigen, and the expression of key prostate markers and microRNAs were analyzed. LNCaP formed spheroids in 3D and MPS cultures, with morphological changes supported by the upregulation of cytokeratins and adhesion proteins. LNCaP also maintained a constant prostate-specific antigen secretion in MPS. PC3 cells did not develop complex structures in 3D and MPS cultures. PSA expression at the gene level was downregulated in LNCaP-MPS and considerably upregulated in PC3-MPS. MicroRNA expression was altered by the 3D static and dynamic culture, both intra- and extracellularly. MicroRNAs associated with prostate cancer progression were mostly upregulated in LNCaP-MPS. Overall dynamic cell culture substantially altered the morphology and expression of LNCaP cells, arguably augmenting their prostate cancer phenotype. This novel approach demonstrates that microRNA expression in prostate cancer cells is sensitive to external stimuli and that MPS can effectively promote important physiological changes in conventional prostate cancer models.

Microfluidic-based prostate cancer model for investigating the secretion of prostate-specific antigen and microRNAs in vitro.

Organic anion transporters 1 and 3 (OAT1 and OAT3) play a crucial role in kidney function by regulating the secretion of multiple renally cleared small molecules and toxic metabolic by-products. Assessing the activity of these transporters is essential for drug development purposes as they can significantly impact drug disposition and safety. OAT1 and OAT3 are amongst the most abundant drug transporters expressed in human renal proximal tubules. However, their expression is lost when cells are isolated and cultured in vitro, which is a persistent issue across all human and animal renal proximal tubule cell models, including primary cells and cell lines. Although it is well known that the overall expression of drug transporters is affected in vitro, the underlying reasons for the loss of OAT1 and OAT3 are still not fully understood. Nonetheless, research into the regulatory mechanisms of these transporters has provided insights into the molecular pathways underlying their expression and activity. In this review, we explore the regulatory mechanisms that govern the expression and activity of OAT1 and OAT3 and investigate the physiological changes that proximal tubule cells undergo and that potentially result in the loss of these transporters. A better understanding of the regulation of these transporters could aid in the development of strategies, such as introducing microfluidic conditions or epigenetic modification inhibitors, to improve their expression and activity in vitro and to create more physiologically relevant models. Consequently, this will enable more accurate assessment for drug development and safety applications.

Renal Organic Anion Transporters 1 and 3 In Vitro: Gone but Not Forgotten.

The advent of micro-physiological systems (MPS) in biomedical research has enabled the introduction of more complex and relevant physiological into in vitro models. The recreation of complex morphological features in three-dimensional environments can recapitulate otherwise absent dynamic interactions in conventional models. In this study we developed an advanced in vitro Renal Cell Carcinoma (RCC) that mimics the interplay between healthy and malignant renal tissue. Based on the TissUse Humimic platform our model combines healthy renal proximal tubule epithelial cells (RPTEC) and RCC. Co-culturing reconstructed RPTEC tubules with RCC spheroids in a closed micro-perfused circuit resulted in significant phenotypical changes to the tubules. Expression of immune factors revealed that interleukin-8 (IL-8) and tumor necrosis factor-alfa (TNF-α) were upregulated in the non-malignant cells while neutrophil gelatinase-associated lipocalin (NGAL) was downregulated in both RCC and RPTEC. Metabolic analysis showed that RCC prompted a shift in the energy production of RPTEC tubules, inducing glycolysis, in a metabolic adaptation that likely supports RCC growth and immunogenicity. In contrast, RCC maintained stable metabolic activity, emphasizing their resilience to external factors. RNA-seq and biological process analysis of primary RTPTEC tubules demonstrated that the 3D tubular architecture and MPS conditions reverted cells to a predominant oxidative phosphorylate state, a departure from the glycolytic metabolism observed in 2D culture. This dynamic RCC co-culture model, approximates the physiology of healthy renal tubules to that of RCC, providing new insights into tumor-host interactions. Our approach can show that an RCC-MPS can expand the complexity and scope of pathophysiology and biomarker studies in kidney cancer research.

Integrating tumor and healthy epithelium in a micro-physiology multi-compartment approach to study renal cell carcinoma pathophysiology.

Author Correction: Integrating tumor and healthy epithelium in a micro-physiology multi-compartment approach to study renal cell carcinoma pathophysiology.

Renal cell carcinoma (RCC) is the most common form of kidney cancer, known for its immune evasion and resistance to chemotherapy. Evidence indicates that the SARS-CoV-2 virus may worsen outcomes for RCC patients, as well as patients with diminished renal function. Evidence suggests that the SARS-CoV-2 virus may exacerbate outcomes in RCC patients and those with impaired renal function. This study explored the unidirectional effects of RCC cells and the SARS-CoV-2 spike protein (S protein) on human renal proximal tubule epithelial cells (RPTECs) using a microphysiological approach. We co-cultured RCC cells (Caki-1) with RPTEC and exposed them to the SARS-CoV-2 S protein under dynamic 3D conditions. The impact on metabolic activity, gene expression, immune secretions, and S protein internalization was evaluated. The SARS-CoV-2 S protein was internalized by RPTEC but poorly interacted with RCC cells. RPTECs exposed to RCC cells and the S protein exhibited upregulated expression of genes involved in immunogenic pathways, particularly those related to antigen processing and presentation via the major histocompatibility complex I (MHCI). Additionally, increased TNF-α secretion suggested a pro-inflammatory response. Metabolic shifts toward glycolysis were observed in RCC co-culture, while the presence of the S protein led to minor changes. The presence of RCC cells amplified the immune-modulatory effects of the SARS-CoV-2 S protein on the renal epithelium, potentially exacerbating renal inflammation and fostering tumor-supportive conditions. These findings suggest that COVID-19 infections can impact renal function in the presence of kidney cancer.

Article	Year
In Vitro Characterization of Renal Drug Transporter Activity in Kidney Cancer. International journal of molecular sciences\| PubMed ID: 36077583	2022
Identification of the Regulatory Targets of miR-3687 and miR-4417 in Prostate Cancer Cells Using a Proteomics Approach. International journal of molecular sciences\| PubMed ID: 36142477	2022
Recapitulating the Pharmacological Interactions of Cetuximab with Sunitinib and Cisplatin in Head and Neck Carcinoma Cells in vitro. Pharmacology\| PubMed ID: 36273461	2023
Amplifying the impact of kidney microphysiological systems: predicting renal drug clearance using mechanistic modelling based on reconstructed drug secretion. ALTEX\| PubMed ID: 36343109	2023
The Potential Impact of New Drug and Therapeutic Modalities on Drug Resistance to Renal Cell Carcinoma. Anticancer research\| PubMed ID: 36854518	2023
Microfluidic-based prostate cancer model for investigating the secretion of prostate-specific antigen and microRNAs in vitro. Scientific reports\| PubMed ID: 37468746	2023
Renal Organic Anion Transporters 1 and 3 In Vitro: Gone but Not Forgotten. International journal of molecular sciences\| PubMed ID: 37895098	2023
Integrating tumor and healthy epithelium in a micro-physiology multi-compartment approach to study renal cell carcinoma pathophysiology. Scientific reports\| PubMed ID: 38653823	2024
Author Correction: Integrating tumor and healthy epithelium in a micro-physiology multi-compartment approach to study renal cell carcinoma pathophysiology. Scientific reports\| PubMed ID: 38918453	2024
SARS-CoV-2 Spike Protein Amplifies the Immunogenicity of Healthy Renal Epithelium in the Presence of Renal Cell Carcinoma. Cells\| PubMed ID: 39768130	2024

Pedro Caetano Pinto

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