Autologous humanized mouse models of iPSC-derived tumors enable characterization and modulation of cancer-immune cell interactions.

Modeling the tumor-immune cell interactions in humanized mice is complex and limits drug development. Here, we generated easily accessible tumor models by transforming either primary skin fibroblasts or induced pluripotent stem cell-derived cell lines injected in immune-deficient mice reconstituted with human autologous immune cells. Our results showed that fibroblastic, hepatic, or neural tumors were all efficiently infiltrated and partially or totally rejected by autologous immune cells in humanized mice. Characterization of tumor-immune infiltrates revealed high expression levels of the dysfunction markers Tim3 and PD-1 in T cells and an enrichment in regulatory T cells, suggesting rapid establishment of immunomodulatory phenotypes. Inhibition of PD-1 by Nivolumab in humanized mice resulted in increased immune cell infiltration and a slight decrease in tumor growth. We expect that these versatile and accessible cancer models will facilitate preclinical studies and the evaluation of autologous cancer immunotherapies across a range of different tumor cell types.

Long-term Outcome of Kidney Recipients Transplanted for Aristolochic Acid Nephropathy.

BACKGROUND: Aristolochic acids (AA) are nephrotoxic and carcinogenic. The aim of this study was to assess the long-term outcome of patients with AA nephropathy (AAN) after kidney transplantation. METHODS: Observational study. Patients' characteristics, long-term surveillance and follow-up data, patient and graft survival, as well as outcomes with respect to rejection, cardiovascular complications, infections, and cancers with a focus on urothelial carcinomas, are reported. RESULTS: Twenty patients transplanted for AAN were included. All were submitted to prophylactic bilateral ureteronephrectomy and annual surveillance of the bladder. Median duration of posttransplant follow-up was 12.5 (3-19) years. Time from diagnosis of AAN to renal replacement therapy was relatively short (1 [0-15] years). Immunosuppression consisted of a triple therapy in the majority of patients. Nineteen patients had upper urinary tract multifocal atypia. Eleven patients presented with urothelial carcinomas of the upper tract; 2 of them with additional bladder urothelial carcinomas. Of these 2 patients, one required radical cystectomy. One patient developed a hepatocarcinoma. Patient survival was 100% in AAN patients at 5, 10, and 15 years after transplantation. Graft survival at 5, 10, and 15 years was 95%, 83%, and 75%. CONCLUSIONS: Despite a high prevalence of urothelial carcinoma and the risk of bladder carcinoma, the long-term patient and kidney graft survival is excellent in patients with AAN, provided that prophylactic bilateral ureteronephrectomy and lifelong surveillance of the bladder are performed.

Dedifferentiation and aberrations of the endolysosomal compartment characterize the early stage of nephropathic cystinosis.

Nephropathic cystinosis, a lysosomal storage disease caused by mutations in the CTNS gene encoding the lysosomal cystine transporter cystinosin, is characterized by generalized proximal tubule (PT) dysfunction that progresses, if untreated, to end-stage renal disease. The pathogenesis of defective PT cellular transport in nephropathic cystinosis remains unclear. We characterized a recently generated line of C57BL/6 Ctns mice and analyzed endocytic uptake, lysosome function, and dedifferentiation and proliferation markers using primary cultures of PT epithelial cells derived from Ctns(-/-) and Ctns(+/+) littermates. Metabolic studies revealed that Ctns(-/-) mice show a progressive PT dysfunction characterized by low-molecular-weight (LMW) proteinuria, glucosuria and phosphaturia, before structural damage and in the absence of renal failure. These changes are related to decreased expression of the multi-ligand receptors megalin and cubilin and to increased dedifferentiation (ZONAB transcription factor) and proliferation (PCNA and Cyclin D1) rates. Studies on PT cells derived from Ctns(-/-) kidneys confirmed cystine overload, with accumulation of enlarged, dysfunctional lysosomes and reduced expression of endocytic receptors reflected by decreased uptake of specific ligands. These changes were related to a loss of integrity of tight junctions with a nuclear translocation of ZONAB and increased proliferation, as observed in Ctns(-/-) kidneys. These data reveal that the absence of cystinosin in PT cells triggers aberrations of the endolysosomal compartment, transport defects and an abnormal transcription program in the early stage of nephropathic cystinosis. Insights into the early manifestations of cystinosis may offer new targets for intervention, before irreversible renal damage.

Decreased renal accumulation of aminoglycoside reflects defective receptor-mediated endocytosis in cystic fibrosis and Dent's disease.

The clinical use of aminoglycoside (AG) antibiotics is limited by their renal toxicity, which is caused by drug accumulation in proximal tubule (PT) cells. Clinical studies reported that renal clearance of AG is enhanced in cystic fibrosis (CF) patients, which might reflect the role of CFTR in PT cell endocytosis. In order to assess the role of chloride transporters on the renal handling of AG, we investigated gentamicin uptake and renal accumulation in mice lacking functional CFTR (Cftr ( ∆F/∆F)) or knock-out for the Cl(-)/H(+) exchanger ClC-5 (Clcn5 ( Y/- )). The latter represent a paradigm of PT dysfunction and defective receptor-mediated endocytosis. As compared with controls, Cftr ( ∆F/∆F) and Clcn5 ( Y/- ) mice showed a 15% to 85% decrease in gentamicin accumulation in the kidney, respectively, in absence of renal failure. Studies on primary cultures of Cftr ( ∆F/∆F) and Clcn5 ( Y/- ) mouse PT cells confirmed the reduction in gentamicin uptake, although colocalization with endosomes and lysosomes was maintained. Quantification of endocytosis in PT cells revealed that gentamicin, similar to albumin, preferentially binds to megalin. The functional loss of ClC-5 or CFTR was reflected by a decrease of the endocytic uptake of gentamicin, with a more pronounced effect in cells lacking ClC-5. These results support the concept that CFTR, as well as ClC-5, plays a relevant role in PT cell endocytosis. They also demonstrate that the functional loss of these two chloride transporters is associated with impaired uptake of AG in PT cells, reflected by a decreased renal accumulation of the drug.

Influence of 17q gain and promoter polymorphisms on mRNA expression of somatostatin receptor type 2 in neuroblastoma.

BACKGROUND: Neuroblastoma, the most frequent solid extracranial tumor in children, is characterized by a wide spectrum of clinical behaviours. We previously reported that high expression of somatostatin receptor type-2 (sst2) mRNA is associated to increased overall and event free survival. Several genetic abnormalities are detected in neuroblastomas, frequently involving balanced and/or unbalanced gain on the long arm on chromosome 17, the same region containing sst2 gene. METHODS: In this study we detected balanced and/or unbalanced 17q gain in 50 neuroblastomas. Since two polymorphisms in sst2 promoter (-57 C>G and -83 A>G) were previously described as responsible for an in vitro reduction of sst2 mRNA expression, promoter sequencing was also performed in the same samples. The results were compared to sst2 mRNA expression, measured by real-time RT-PCR. RESULTS: The frequency of 17q gain (14/50 neuroblastomas) was significantly associated to sst2 mRNA over-expression (Fischer's exact test: p=0.0012). The sst2 expression was significant higher both in balance and unbalance 17q amplifications (ANOVA: p=0.04). Conversely, we found a reduction of sst2 mRNA in neuroblastomas with -57 C>G promoter polymorphism (ANOVA: p=0.03). CONCLUSION: We highlighted that 17q gain and promoter polymorphisms can play a role into the regulation of sst2 expression in neuroblastomas.

Early evidence of lymphoproliferative disorder: post-transplant monitoring of Epstein-Barr infection in adult and pediatric patients.

Transplant patients are at high risk of post-transplant lymphoproliferative disorder (PTLD). A strong correlation between Epstein-Barr virus (EBV) and PTLD is observed in pediatric patients with primary infection after transplant. Because many patients have responded to reversal of immunosuppressive therapy, an early identification of EBV is essential for the reduction of immunosuppression and/or introduction of antiviral therapy to prevent PTLD. Polymerase chain reaction (PCR) is a specific and sensitive method to identify EBV DNA in blood. The aim of our study was to establish a protocol for monitoring EBV infection in transplanted patients for early identification those at high risk of PTLD. Viral presence in peripheral blood leukocytes (PBL) and serum samples was revealed by Nested PCR; positive specimens were quantified with Real Time PCR (RT-PCR). DNA in PBL was observed in 12 cases and 6 showed EBV in sera. Quantitative analysis showed a wide range of EBV DNA copies in leukocytes that were higher than in sera. Two patients displayed high viral load values in both PBL and sera associated with clinical evidence of PTLD. Our data suggest that the study of the EBV load represents an essential approach in the diagnosis of PTLD and the analysis of serum samples could provide useful information in the post-transplant monitoring of high-risk patients.

Prognostic value of somatostatin receptor subtype 2 expression in colorectal cancer.

The clinical relevance of the somatostatin receptor subtype 2 (sst2) is well defined in neuroendocrine tumors but it is still a matter of debate whether its expression may have a role also in other tumors not arising from the neuroectoderm. We investigated the prognostic value of the expression levels of sst2 mRNA in a consistent group of patients affected by colorectal cancer. Survival analysis of cancer-related death showed that patients with a high sst2 mRNA expression had an unfavourable outcome (p=0.037) and a significantly shorter disease-free survival (p=0.008). Surprisingly, our findings suggest that sst2 gene overexpression is a feature of colorectal tumors that have a negative outlook; in addition, it may allow additional insight into conventional therapeutic approaches for more aggressive tumors, whose prognosis needs to be improved.

Tankyrase, a positive regulator of telomere elongation, is over expressed in human breast cancer.

Tankyrase promotes telomere elongation by interaction with the telomeric protein binding factor TRF1, a negative regulator of telomere extension. We measured tankyrase mRNA by real-time RT-PCR in 66 breast cancers and in paired normal tissues. Results were compared with hTERT mRNA expression. The levels of tankyrase in breast cancers were significantly higher in comparison to normal tissues (P<0.0001) and significantly related to the status of progesterone receptors. No relationship was found between tankyrase and hTERT mRNA expression in breast cancers. According to our results, tankyrase expression appeared up regulated in breast cancers.

Quantitative evaluation of somatostatin receptor subtype 2 expression in sporadic colorectal tumor and in the corresponding normal mucosa.

PURPOSE: The somatostatin (SS) receptor subtype 2 (sst2) is the principal mediator of the antiproliferative effects of SS and has the highest affinity for the commercially available SS analogues. The purpose of this study was to evaluate sst2 mRNA expression by quantitative reverse transcription-PCR (RT-PCR) in colon cancers and in corresponding normal tissues. EXPERIMENTAL DESIGN: The expression of sst2 mRNA was measured with a quantitative method based on real time RT-PCR with TaqMan assay in 100 colon cancers and in the corresponding normal tissues. In a limited number of patients, these results were compared with those obtained by in situ hybridization (n = 26) and by in vivo imaging with (111)In-pentetreotide (n = 17). RESULTS: Results obtained by quantitative RT-PCR on sst2 expression in colorectal cancer were significantly related to those obtained by in situ hybridization and (111)In-pentetreotide scintigraphy. Sst2 was expressed in all of the tumors investigated without any relationship with localization, grading, and stage of disease. Although the paired, unaffected mucosa tends to express a higher abundance of sst2 than the corresponding cancer samples, this difference did not reach a statistical significance. However, in patients with elevated carcinoembryonic antigen levels (>5 ng/ml) there was a significant loss of sst2 mRNA in the tumor when compared with its paired normal tissue. CONCLUSIONS: In this study we confirmed, by a quantitative method, that colorectal cancer does not express higher concentrations of sst2 mRNA than the corresponding unaffected tissue. Conversely, a loss of sst2 was found in patients with elevated preoperative concentrations of carcinoembryonic antigen, an unfavorable prognostic marker for colorectal cancer.