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Organotypic primary blood vessel models of clear cell renal cell carcinoma for single-patient clinical trials.

ABSTRACT: Clear cell renal cell carcinoma (ccRCC) is a common genitourinary cancer associated with the development of abnormal tumor angiogenesis. Although multiple anti-angiogenic therapies have been developed, responses to individual treatment are highly variable between patients. Thus, the use of one-patient clinical trials has been suggested as an alternative to standard trials. We used a microfluidic device to generate organotypic primary patient-specific blood vessel models using normal (NEnC) and tumor-associated primary CD31+ selected cells (TEnC). Our model was able to recapitulate differences in angiogenic sprouting and vessel permeability that characterize normal and tumor-associated vessels. We analyzed the expression profile of vessel models to define vascular normalization in a patient-specific manner. Using this data, we identified actionable targets to normalize TEnC vessel function to a more NEnC-like phenotype. Finally, we tested two of these drugs in our patient-specific models to determine the efficiency in restoring vessel function showing the potential of the model for single-patient clinical trials.

SUBMITTER: Virumbrales-Munoz M

PROVIDER: S-EPMC8743028 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundPatients with clear cell renal cell carcinoma (ccRCC) have few therapeutic options, as ccRCC is unresponsive to chemotherapy and is highly resistant to radiation. Recently targeted therapies have extended progression-free survival, but responses are variable and no significant overall survival benefit has been achieved. Commercial ccRCC cell lines are often used as model systems to develop novel therapeutic approaches, but these do not accurately recapitulate primary ccRCC tumors at the genomic and transcriptional levels. Furthermore, ccRCC exhibits significant intertumor genetic heterogeneity, and the limited cell lines available fail to represent this aspect of ccRCC. Our objective was to generate accurate preclinical in vitro models of ccRCC using tumor tissues from ccRCC patients.MethodsccRCC primary single cell suspensions were cultured in fetal bovine serum (FBS)-containing media or defined serum-free media. Established cultures were characterized by genomic verification of mutations present in the primary tumors, expression of renal epithelial markers, and transcriptional profiling.ResultsThe apparent efficiency of primary cell culture establishment was high in both culture conditions, but genotyping revealed that the majority of cultures contained normal, not cancer cells. ccRCC characteristically shows biallelic loss of the von Hippel Lindau (VHL) gene, leading to accumulation of hypoxia-inducible factor (HIF) and expression of HIF target genes. Purification of cells based on expression of carbonic anhydrase IX (CA9), a cell surface HIF target, followed by culture in FBS enabled establishment of ccRCC cell cultures with an efficiency of >80 %. Culture in serum-free conditions selected for growth of normal renal proximal tubule epithelial cells. Transcriptional profiling of ccRCC and matched normal cell cultures identified up- and down-regulated networks in ccRCC and comparison to The Cancer Genome Atlas confirmed the clinical validity of our cell cultures.ConclusionsThe ability to establish primary cultures of ccRCC cells and matched normal kidney epithelial cells from almost every patient provides a resource for future development of novel therapies and personalized medicine for ccRCC patients.

Project description:Patient-derived xenografts (PDX) are high-fidelity cancer models typically credentialled by genomics, transcriptomics and proteomics. Characterization of metabolic reprogramming, a hallmark of cancer, is less frequent. Dysregulated metabolism is a key feature of clear cell renal cell carcinoma (ccRCC) and authentic preclinical models are needed to evaluate novel imaging and therapeutic approaches targeting metabolism. We characterized 5 PDX from high-grade or metastatic ccRCC by multiparametric magnetic resonance imaging (MRI) and steady state metabolic profiling and flux analysis. Similar to MRI of clinical ccRCC, T2-weighted images of orthotopic tumors of most PDX were homogeneous. The increased hyperintense (cystic) areas observed in one PDX mimicked the cystic phenotype typical of some RCC. The negligible hypointense (necrotic) areas of PDX grown under the highly vascularized renal capsule are beneficial for preclinical studies. Mean apparent diffusion coefficient (ADC) values were equivalent to those of ccRCC in human patients. Hyperpolarized (HP) [1-13C]pyruvate MRI of PDX showed high glycolytic activity typical of high-grade primary and metastatic ccRCC with considerable intra- and inter-tumoral variability, as has been observed in clinical HP MRI of ccRCC. Comparison of steady state metabolite concentrations and metabolic flux in [U-13C]glucose-labeled tumors highlighted the distinctive phenotypes of two PDX with elevated levels of numerous metabolites and increased fractional enrichment of lactate and/or glutamate, capturing the metabolic heterogeneity of glycolysis and the TCA cycle in clinical ccRCC. Culturing PDX cells and reimplanting to generate xenografts (XEN), or passaging PDX in vivo, altered some imaging and metabolic characteristics while transcription remained like that of the original PDX. These findings show that PDX are realistic models of ccRCC for imaging and metabolic studies but that the plasticity of metabolism must be considered when manipulating PDX for preclinical studies.

Project description:Genetically engineered mouse models (GEMMs) are important immunocompetent models for research into the roles of individual genes in cancer and the development of novel therapies. Here we use inducible CRISPR-Cas9 systems to develop two GEMMs which aim to model the extensive chromosome p3 deletion frequently observed in clear cell renal cell carcinoma (ccRCC). We cloned paired guide RNAs targeting early exons of Bap1, Pbrm1, and Setd2 in a construct containing a Cas9D10A (nickase, hSpCsn1n) driven by tetracycline (tet)-responsive elements (TRE3G) to develop our first GEMM. The founder mouse was crossed with two previously established transgenic lines, one carrying the tet-transactivator (tTA, Tet-Off) and one with a triple-mutant stabilized HIF1A-M3 (TRAnsgenic Cancer of the Kidney, TRACK), both driven by a truncated, proximal tubule-specific γ-glutamyltransferase 1 (ggt or γGT) promoter, to create triple-transgenic animals. Our results indicate that this model (BPS-TA) induces low numbers of somatic mutations in Bap1 and Pbrm1 (but not in Setd2), known tumor suppressor genes in human ccRCC. These mutations, largely restricted to kidneys and testis, induced no detectable tissue transformation in a cohort of 13 month old mice (N = 10). To gain insights into the low frequencies of insertions and deletions (indels) in BPS-TA mice we analyzed wild type (WT, N = 7) and BPS-TA (N = 4) kidneys by RNAseq. This showed activation of both DNA damage and immune response, suggesting activation of tumor suppressive mechanisms in response to genome editing. We then modified our approach by generating a second model in which a ggt-driven, cre-regulated Cas9WT(hSpCsn1) was employed to introduce Bap1, Pbrm1, and Setd2 genome edits in the TRACK line (BPS-Cre). The BPS-TA and BPS-Cre lines are both tightly controlled in a spatiotemporal manner with doxycycline (dox) and tamoxifen (tam), respectively. In addition, whereas the BPS-TA line relies on paired guide RNAs (gRNAs), the BPS-Cre line requires only single gRNAs for gene perturbation. In the BPS-Cre we identified increased Pbrm1 gene-editing frequencies compared to the BPS-TA model. Whereas we did not detect Setd2 edits in the BPS-TA kidneys, we found extensive editing of Setd2 in the BPS-Cre model. Bap1 editing efficiencies were comparable between the two models. Although no gross malignancies were observed in our study, this is the first reported GEMM which models the extensive chromosome 3p deletion frequently observed in kidney cancer patients. Further studies are required (1) to model more extensive 3p deletions, e.g. impacting additional genes, and (2) to increase the cellular resolution, e.g. by employing single-cell RNAseq to ascertain the effects of specific combinatorial gene inactivation.

Project description:BackgroundClinicopathological scores are used to predict the likelihood of recurrence-free survival for patients with clear cell renal cell carcinoma (ccRCC) after surgery. These are fallible, particularly in the middle range. This inevitably means that a significant proportion of ccRCC patients who will not develop recurrent disease enroll into clinical trials. As an exemplar of using digital pathology, we sought to improve the predictive power of "recurrence free" designation in localized ccRCC patients, by precise measurement of ccRCC nuclear morphological features using computational image analysis, thereby replacing manual nuclear grade assessment.Materials and methodsTNM 8 UICC pathological stage pT1-pT3 ccRCC cases were recruited in Scotland and in Singapore. A Leibovich score (LS) was calculated. Definiens Tissue studio® (Definiens GmbH, Munich) image analysis platform was used to measure tumor nuclear morphological features in digitized hematoxylin and eosin (H&E) images.ResultsReplacing human-defined nuclear grade with computer-defined mean perimeter generated a modified Leibovich algorithm, improved overall specificity 0.86 from 0.76 in the training cohort. The greatest increase in specificity was seen in LS 5 and 6, which went from 0 to 0.57 and 0.40, respectively. The modified Leibovich algorithm increased the specificity from 0.84 to 0.94 in the validation cohort.ConclusionsCcRCC nuclear mean perimeter, measured by computational image analysis, together with tumor stage and size, node status and necrosis improved the accuracy of predicting recurrence-free in the localized ccRCC patients. This finding was validated in an ethnically different Singaporean cohort, despite the different H and E staining protocol and scanner used. This may be a useful patient selection tool for recruitment to multicenter studies, preventing some patients from receiving unnecessary additional treatment while reducing the number of patients required to achieve adequate power within neoadjuvant and adjuvant clinical studies.

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Organotypic primary blood vessel models of clear cell renal cell carcinoma for single-patient clinical trials.

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