Direct Reprogramming of Hepatocytes Into JAK/Stat-Dependent LGR5+ Liver Cells Able to Initiate Intrahepatic Cholangiocarcinoma.

Somatic cells that have been partially reprogrammed by the factors Oct4, Sox2, Klf4, and cMyc (OSKM) have been demonstrated to be potentially tumorigenic in vitro and in vivo due to the acquisition of cancer-associated genomic alterations and the absence of OSKM clearance over time. In the present study, we obtained partially reprogrammed, SSEA1-negative cells by transducing murine hepatocytes with Δ1Δ3-deleted adenoviruses that expressed the 4 OSKM factors. We observed that, under long-term 2D and 3D culture conditions, hepatocytes could be converted into LGR5-positive cells with self-renewal capacity that was dependent on 3 cross-signaling pathways: IL6/Jak/Stat3, LGR5/R-spondin, and Wnt/ß-catenin. Following engraftment in syngeneic mice, LGR5-positive cells that expressed the cancer markers CD51, CD166, and CD73 were capable of forming invasive and metastatic tumors reminiscent of intrahepatic cholangiocarcinoma (ICC): they were positive for CK19 and CK7, featured associations of cord-like structures, and contained cuboidal and atypical cells with dissimilar degrees of pleomorphism and mitosis. The LGR5+-derived tumors exhibited a highly vascularized stroma with substantial fibrosis. In addition, we identified pro-angiogenic factors and signaling pathways involved in neo-angiogenesis and vascular development, which represent potential new targets for anti-angiogenic strategies to overcome tumor resistance to current ICC treatments.

Human virome profiling identified CMV as the major viral driver of a high accumulation of senescent CD8+ T cells in patients with advanced NSCLC.

Circulating senescent CD8+ T (T8sen) cells are characterized by a lack of proliferative capacities but retain cytotoxic activity and have been associated to resistance to immunotherapy in patients with advanced non-small cell lung cancer (aNSCLC). We aimed to better characterize T8sen and to determine which factors were associated with their accumulation in patients with aNSCLC. Circulating T8sen cells were characterized by a higher expression of SA-ßgal and the transcription factor T-bet, confirming their senescent status. Using whole virome profiling, cytomegalovirus (CMV) was the only virus associated with T8sen. CMV was necessary but not sufficient to explain high accumulation of T8sen (T8senhigh status). In CMV+ patients, the proportion of T8sen cells increased with cancer progression. Last, CMV-induced T8senhigh phenotype but not CMV seropositivity itself was associated with worse progression-free and overall survival in patients treated with anti-PD-(L)1 therapy but not with chemotherapy. Overall, CMV is the unique viral driver of T8sen-driven resistance to anti-PD-(L)1 antibodies in patients with aNSCLC.

Chimeric antigen-receptor (CAR) engineered natural killer cells in a chronic myeloid leukemia (CML) blast crisis model.

During the last two decades, the introduction of tyrosine kinase inhibitors (TKIs) to the therapy has changed the natural history of CML but progression into accelerated and blast phase (AP/BP) occurs in 3-5% of cases, especially in patients resistant to several lines of TKIs. In TKI-refractory patients in advanced phases, the only curative option is hematopoietic stem cell transplantation. We and others have shown the relevance of the expression of the Interleukin-2-Receptor α subunit (IL2RA/CD25) as a biomarker of CML progression, suggesting its potential use as a therapeutic target for CAR-based therapies. Here we show the development of a CAR-NK therapy model able to target efficiently a blast crisis cell line (K562). The design of the CAR was based on the scFv of the clinically approved anti-CD25 monoclonal antibody (Basiliximab). The CAR construct was integrated into NK92 cells resulting in the generation of CD25 CAR-NK92 cells. Target K562 cells were engineered by lentiviral gene transfer of CD25. In vitro functionality experiments and in vivo leukemogenicity experiments in NSG mice transplanted by K562-CD25 cells showed the efficacy and specificity of this strategy. These proof-of-concept studies could represent a first step for further development of this technology in refractory/relapsed (R/R) CML patients in BP as well as in R/R acute myeloblastic leukemias (AML).

Chimeric antigen-receptor (CAR)-engineered natural killer (NK) cells targeting chronic myeloid Leukemia (CML) blast crisis

Background The use of tyrosine kinase inhibitors (TKIs) has dramatically modified the therapy of chronic myeloid leukemia (CML), generating durable remissions and prolonging survival in TKI-responders. However, progression to blast crisis (BC) still occurs especially in TKI-resistant patients and represents a clinical challenge. We and others have identified IL2RA/CD25 as a typical cell surface marker of BC-CML and reported that its overexpression is correlated with the progression of CML from CP-CML to BC-CML (Imeri et al, Cells 2023). Here we show the experimental development of a third-generation CAR-NK therapy strategy against the CD25 based on the scFV of the clinically approved monoclonal humanized antibody, Basiliximab. Methods: As NK cell model, we have used the NK92 cell line which has a well-established and clinically demonstrated NK cell activity. We have lentivirally transduced NK92 cells with the CAR construct containing a selectable gene (GFP). After FACS-sorting of GFP-positive cells, phenotypical characterization was performed by FACS. The expression of the CAR-CD25 at the surface of the cells was demonstrated an anti-Fab antibody and double-positive (Fab/GFP) cells were further purified. The functionality of the cells was evaluated using CD107a degranulation assay after 3h co-culture and ELISA for IFN-gamma release. We have in parallel engineered K562 cells expressing CD25 by lentiviral transduction (K562-CD25) as well as also a second target cell line (RAJI) using the same strategy. Annexin V staining of target K562 cells was used for in vitro cytotoxicity assessments. For in vivo assays, NSG mice were intraperitoneally (IP) injected with K562-CD25 cells expressing Luciferase at Day-3 (3.10 6 cells/mouse, n = 13). At Days 0, 3, and 7, mice were treated by IP injection of either irradiated CD25 CAR-NK92 cells (10 .10 6/mouse n=6) or irradiated Wild-type (WT)-NK92 cells (n=5). The clinical evolution of mice transplanted mice was followed weekly by luminescence (IVIS 200). Results: After cell sorting, we obtained more than 90% of double-positive NK92 CAR+/GFP+ cells. Lentiviral transduction did not affect the activatory or inhibitory signals of NK92 cells. No statistical differences were observed between CD25 CAR-NK92 and WT- NK92 cells for the expression of NKp30, NKp46, KirDl2-3, TIGIT, and DNAM. However, we observed a strong increase in the Granzyme B and Perforin in CD25 CAR-NK92 cells after co-culture with K562-CD25 as compared to WT NK92 cells (p<0.001). Importantly, we have found increased levels of degranulation after co-culture of target K562-CD25 with CD25-CAR-NK92 cells (40%) as compared to cells co-cultured with WT NK92 alone (20% ) suggesting strongly the occurrence of an additional specific effect due to CAR-CD25. IFN-gamma levels after co-culture of CAR CD25 NK92 cells were also found to be significantly increased ( 400 pg/ml) in as compared to co-cultures of target cells with WT-NK92 (200 pg/ml)(p<0.0001). Similarly,in vitro cytotoxicity assays showed induction of higher levels of apoptosis in target cells (K562-CD25 and Raji-CD25) when co-cultured with CD25 CAR-NK92 as compared to NK92 WT (p<0.0001). In in vivo experiments, we have analyzed K562-CD25 leukemia-bearing mice treated with CAR-NK92 cells (n=6) or WT-NK92 cells (n=5). These experiments analyzed at D+30 post-transplant showed stronger anti-leukemia effect of CAR-NK therapy by IVIS imaging with a survival rate of 84% for mice treated with CD25 CAR-NK92 versus 40% for those treated with WT-NK92. All control mice transplanted with K562-CD25 cells and left untreated died by D+20. Conclusion: We show here for the first time the potential use of an NK cell-mediated CAR therapy strategy targeting CD25 which has been shown to be upregulated in CML blast crisis. The experimental data show a significantly increased and selective in vitro and in vivo cytotoxicity of CD25 CAR-NK92 cells against CD25-expressing leukemia cells as compared to WT-NK92 cells. These results suggest that targeting CD25 by a CD25 CAR based on Basilixiamb's scFV might be an interesting tool in BC-CML and in all acute leukemias overexpressing CD25. In order to translate these findings to NK cells derived from induced pluripotent stem cells (iPSCs), we have produced iPSCs expressing CAR-CD25 constructs and experiments are underway to evaluate the therapeutic potential of iPSC-derived CD25 CAR-NK cells in CML blast crisis or AML models.

Patient-Derived iPSCs Reveal Evidence of Telomere Instability and DNA Repair Deficiency in Coats Plus Syndrome.

Coats plus (CP) syndrome is an inherited autosomal recessive condition that results from mutations in the conserved telomere maintenance component 1 gene (CTC1). The CTC1 protein functions as a part of the CST protein complex, a protein heterotrimer consisting of CTC1-STN1-TEN1 which promotes telomere DNA synthesis and inhibits telomerase-mediated telomere elongation. However, it is unclear how CTC1 mutations may have an effect on telomere structure and function. For that purpose, we established the very first induced pluripotent stem cell lines (iPSCs) from a compound heterozygous patient with CP carrying deleterious mutations in both alleles of CTC1. Telomere dysfunction and chromosomal instability were assessed in both circulating lymphocytes and iPSCs from the patient and from healthy controls of similar age. The circulating lymphocytes and iPSCs from the CP patient were characterized by their higher telomere length heterogeneity and telomere aberrations compared to those in control cells from healthy donors. Moreover, in contrast to iPSCs from healthy controls, the high levels of telomerase were associated with activation of the alternative lengthening of telomere (ALT) pathway in CP-iPSCs. This was accompanied by inappropriate activation of the DNA repair proteins γH2AX, 53BP1, and ATM, as well as with accumulation of DNA damage, micronuclei, and anaphase bridges. CP-iPSCs presented features of cellular senescence and increased radiation sensitivity. Clonal dicentric chromosomes were identified only in CP-iPSCs after exposure to radiation, thus mirroring the role of telomere dysfunction in their formation. These data demonstrate that iPSCs derived from CP patients can be used as a model system for molecular studies of the CP syndrome and underscores the complexity of telomere dysfunction associated with the defect of DNA repair machinery in the CP syndrome.

Evidence of Antitumor and Antimetastatic Potential of Induced Pluripotent Stem Cell-Based Vaccines in Cancer Immunotherapy.

Cancer is maintained by the activity of a rare population of self-renewing "cancer stem cells" (CSCs), which are resistant to conventional therapies. CSCs over-express several proteins shared with induced pluripotent stem cells (iPSCs). We show here that allogenic or autologous murine iPSCs, combined with a histone deacetylase inhibitor (HDACi), are able to elicit major anti-tumor responses in a highly aggressive triple-negative breast cancer, as a relevant cancer stemness model. This immunotherapy strategy was effective in preventing tumor establishment and efficiently targeted CSCs by inducing extensive modifications of the tumor microenvironment. The anti-tumoral effect was correlated with the generation of CD4+, CD8+ T cells, and CD44+ CD62L- CCR7low CD127low T-effector memory cells, and the reduction of CD4+ CD25+FoxP3+ Tregs, Arg1+ CD11b+ Gr1+, and Arg1+ and CD11b+ Ly6+ myeloid-derived suppressor cell populations within the tumor. The anti-tumoral effect was associated with a reduction in metastatic dissemination and an improvement in the survival rate. These results demonstrate for the first time the clinical relevance of using an off-the-shelf allogeneic iPSC-based vaccine combined with an HDACi as a novel pan-cancer anti-cancer immunotherapy strategy against aggressive tumors harboring stemness features with high metastatic potential.

iPSC-Derived Organoids as Therapeutic Models in Regenerative Medicine and Oncology.

Progress made during the last decade in stem cell biology allows currently an unprecedented potential to translate these advances into the clinical applications and to shape the future of regenerative medicine. Organoid technology is amongst these major developments, derived from primary tissues or more recently, from induced pluripotent stem cells (iPSC). The use of iPSC technology offers the possibility of cancer modeling especially in hereditary cancers with germline oncogenic mutations. Similarly, it has the advantage to be amenable to genome editing with introduction of specific oncogenic alterations using CRISPR-mediated gene editing. In the field of regenerative medicine, iPSC-derived organoids hold promise for the generation of future advanced therapeutic medicinal products (ATMP) for organ repair. Finally, it appears that they can be of highly useful experimental tools to determine cell targets of SARS-Cov-2 infections allowing to test anti-Covid drugs. Thus, with the possibilities of genomic editing and the development of new protocols for differentiation toward functional tissues, it is expected that iPSC-derived organoid technology will represent also a therapeutic tool in all areas of medicine.

HLA-dependent heterogeneity and macrophage immunoproteasome activation during lung COVID-19 disease.

BACKGROUND: The worldwide pandemic caused by the SARS-CoV-2 virus is characterized by significant and unpredictable heterogeneity in symptoms that remains poorly understood. METHODS: Transcriptome and single cell transcriptome of COVID19 lung were integrated with deeplearning analysis of MHC class I immunopeptidome against SARS-COV2 proteome. RESULTS: An analysis of the transcriptomes of lung samples from COVID-19 patients revealed that activation of MHC class I antigen presentation in these tissues was correlated with the amount of SARS-CoV-2 RNA present. Similarly, a positive relationship was detected in these samples between the level of SARS-CoV-2 and the expression of a genomic cluster located in the 6p21.32 region (40 kb long, inside the MHC-II cluster) that encodes constituents of the immunoproteasome. An analysis of single-cell transcriptomes of bronchoalveolar cells highlighted the activation of the immunoproteasome in CD68 + M1 macrophages of COVID-19 patients in addition to a PSMB8-based trajectory in these cells that featured an activation of defense response during mild cases of the disease, and an impairment of alveolar clearance mechanisms during severe COVID-19. By examining the binding affinity of the SARS-CoV-2 immunopeptidome with the most common HLA-A, -B, and -C alleles worldwide, we found higher numbers of stronger presenters in type A alleles and in Asian populations, which could shed light on why this disease is now less widespread in this part of the world. CONCLUSIONS: HLA-dependent heterogeneity in macrophage immunoproteasome activation during lung COVID-19 disease could have implications for efforts to predict the response to HLA-dependent SARS-CoV-2 vaccines in the global population.

Detection of Hematopoietic Stem Cell Transcriptome in Human Fetal Kidneys and Kidney Organoids Derived From Human Induced Pluripotent Stem Cells.

BACKGROUND: In mammalians, hematopoietic stem cells (HSCs) arise in the dorsal aorta from the hemogenic endothelium, followed by their migration to the fetal liver and to the bone marrow. In zebrafish, the kidney is the site of primary hematopoiesis. In humans, the presence of HSCs in the fetal or adult kidney has not been established. METHODS: We analyzed the presence of HSC markers in the human fetal kidneys by analysis of single-cell datasets. We then analyzed in kidney organoids derived from induced pluripotent stem cells (iPSCs) the presence of hematopoietic markers using transcriptome analyses. RESULTS: Twelve clusters were identified as stromal, endothelial, and nephron cell type-specific markers in the two fetal stage (17 weeks) kidney datasets. Among these, the expression of hematopoietic cells in cluster 9 showed an expression of primitive markers. Moreover, whole transcriptome analysis of our iPSC-derived kidney organoids revealed induction of the primitive hematopoietic transcription factor RUNX1 as found in the human fetal kidney cortex. CONCLUSION: These finding support the presence of cells expressing HSC transcriptome in the human kidney. The mechanisms of the appearance of the cells with the same transcriptional features during iPSC-derived kidney organoid generation require further investigation.

Metabolomic analyses of COVID-19 patients unravel stage-dependent and prognostic biomarkers.

The circulating metabolome provides a snapshot of the physiological state of the organism responding to pathogenic challenges. Here we report alterations in the plasma metabolome reflecting the clinical presentation of COVID-19 patients with mild (ambulatory) diseases, moderate disease (radiologically confirmed pneumonitis, hospitalization and oxygen therapy), and critical disease (in intensive care). This analysis revealed major disease- and stage-associated shifts in the metabolome, meaning that at least 77 metabolites including amino acids, lipids, polyamines and sugars, as well as their derivatives, were altered in critical COVID-19 patient's plasma as compared to mild COVID-19 patients. Among a uniformly moderate cohort of patients who received tocilizumab, only 10 metabolites were different among individuals with a favorable evolution as compared to those who required transfer into the intensive care unit. The elevation of one single metabolite, anthranilic acid, had a poor prognostic value, correlating with the maintenance of high interleukin-10 and -18 levels. Given that products of the kynurenine pathway including anthranilic acid have immunosuppressive properties, we speculate on the therapeutic utility to inhibit the rate-limiting enzymes of this pathway including indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase.

iPSC-Derived Hereditary Breast Cancer Model Reveals the BRCA1-Deleted Tumor Niche as a New Culprit in Disease Progression.

Tumor progression begins when cancer cells recruit tumor-associated stromal cells to produce a vascular niche, ultimately resulting in uncontrolled growth, invasion, and metastasis. It is poorly understood, though, how this process might be affected by deletions or mutations in the breast cancer type 1 susceptibility (BRCA1) gene in patients with a lifetime risk of developing breast and/or ovarian cancer. To model the BRCA1-deleted stroma, we first generated induced pluripotent stem cells (iPSCs) from patients carrying a germline deletion of exon 17 of the BRCA1 gene (BRCA1+/- who, based on their family histories, were at a high risk for cancer. Using peripheral blood mononuclear cells (PBMCs) of these two affected family members and two normal (BRCA1+/+) individuals, we established a number of iPSC clones via non-integrating Sendai virus-based delivery of the four OCT4, SOX2, KLF4, and c-MYC factors. Induced mesenchymal stem cells (iMSCs) were generated and used as normal and pathological stromal cells. In transcriptome analyses, BRCA1+/- iMSCs exhibited a unique pro-angiogenic signature: compared to non-mutated iMSCs, they expressed high levels of HIF-1α, angiogenic factors belonging to the VEGF, PDGF, and ANGPT subfamilies showing high angiogenic potential. This was confirmed in vitro through the increased capacity to generate tube-like structures compared to BRCA1+/+ iMSCs and in vivo by a matrigel plug angiogenesis assay where the BRCA1+/- iMSCs promoted the development of an extended and organized vessel network. We also reported a highly increased migration capacity of BRCA1+/- iMSCs through an in vitro wound healing assay that correlated with the upregulation of the periostin (POSTN). Finally, we assessed the ability of both iMSCs to facilitate the engraftment of murine breast cancer cells using a xenogenic 4T1 transplant model. The co-injection of BRCA1+/- iMSCs and 4T1 breast cancer cells into mouse mammary fat pads gave rise to highly aggressive tumor growth (2-fold increase in tumor volume compared to 4T1 alone, p = 0.01283) and a higher prevalence of spontaneous metastatic spread to the lungs. Here, we report for the first time a major effect of BRCA1 haploinsufficiency on tumor-associated stroma in the context of BRCA1-associated cancers. The unique iMSC model used here was generated using patient-specific iPSCs, which opens new therapeutic avenues for the prevention and personalized treatment of BRCA1-associated hereditary breast cancer.

Molecular investigation of adequate sources of mesenchymal stem cells for cell therapy of COVID-19-associated organ failure.

The use of mesenchymal stem cells (MSC) derived from several sources has been suggested as a major anti-inflammation strategy during the recent outbreak of coronavirus-19 (COVID-19). As the virus enters the target cells through the receptor ACE2, it is important to determine if the MSC population transfused to patients could also be a target for the virus entry. We report here that ACE2 is highly expressed in adult bone marrow, adipose tissue, or umbilical cord-derived MSC. On the other hand, placenta-derived MSC express low levels of ACE2 but only in early passages of cultures. MSC derived from human embryonic stem cell or human induced pluripotent stem cells express also very low levels of ACE2. The transcriptome analysis of the MSCs with lowest expression of ACE2 in fetal-like MSCs is found to be associated in particularly with an anti-inflammatory signature. These results are of major interest for designing future clinical MSC-based stem cell therapies for severe COVID-19 infections.

PPARγ Cistrome Repression during Activation of Lung Monocyte-Macrophages in Severe COVID-19.

The molecular mechanisms of cytokine storm in patients with severe COVID-19 infections are poorly understood. To uncover these events, we performed transcriptome analyses of lung biopsies from patients with COVID-19, revealing a gene enrichment pattern similar to that of PPARγ-knockout macrophages. Single-cell gene expression analysis of bronchoalveolar lavage fluids revealed a characteristic trajectory of PPARγ-related disturbance in the CD14+/CD16+ cells. We identified a correlation with the disease severity and the reduced expression of several members of the PPARγ complex such as EP300, RXRA, RARA, SUMO1, NR3C1, and CCDC88A. ChIP-seq analyses confirmed repression of the PPARγ-RXRA-NR3C1 cistrome in COVID-19 lung samples. Further analysis of protein-protein networks highlighted an interaction between the PPARγ-associated protein SUMO1 and a nucleoprotein of the SARS virus. Overall, these results demonstrate for the first time the involvement of the PPARγ complex in severe COVID-19 lung disease and suggest strongly its role in the major monocyte/macrophage-mediated inflammatory storm.

Elevated Calprotectin and Abnormal Myeloid Cell Subsets Discriminate Severe from Mild COVID-19.

Blood myeloid cells are known to be dysregulated in coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2. It is unknown whether the innate myeloid response differs with disease severity and whether markers of innate immunity discriminate high-risk patients. Thus, we performed high-dimensional flow cytometry and single-cell RNA sequencing of COVID-19 patient peripheral blood cells and detected disappearance of non-classical CD14LowCD16High monocytes, accumulation of HLA-DRLow classical monocytes (Human Leukocyte Antigen - DR isotype), and release of massive amounts of calprotectin (S100A8/S100A9) in severe cases. Immature CD10LowCD101-CXCR4+/- neutrophils with an immunosuppressive profile accumulated in the blood and lungs, suggesting emergency myelopoiesis. Finally, we show that calprotectin plasma level and a routine flow cytometry assay detecting decreased frequencies of non-classical monocytes could discriminate patients who develop a severe form of COVID-19, suggesting a predictive value that deserves prospective evaluation.

Single-Cell Transcriptome in Chronic Myeloid Leukemia: Pseudotime Analysis Reveals Evidence of Embryonic and Transitional Stem Cell States.

Recent experimental data suggest that the heterogeneity of chronic myeloid leukemia (CML) stem cells may be the result of the development of unique molecular events generating functional consequences in terms of the resistance and persistence of leukemic stem cells. To explore this phenomenon, we designed a single-cell transcriptome assay evaluating simultaneously the expression of 87 genes. Highly purified CD34+ cells from three CML patients at diagnosis were immobilized in microfluidic chips, and the expression of 87 genes was evaluated in each cell. This analysis identified a group of 13 highly connected genes including NANOG, POU5F1, LIN28A, and SOX2, representing on average 8.59% of the cell population analyzed. Bioinformatics analysis with the corrected matrix and t-distributed stochastic neighbor embedding (tSNE) algorithm identified four distinct clusters, and the pseudotime analysis confirmed the presence of seven stem cell states in the four clusters identified. ALOX5 expression was associated with the group of cells expressing the pluripotency markers. In in vitro analyses, two genes that were predicted to undergo similar regulation using pseudotime analysis (ALOX5 and FGFR) were found to be similarly inhibited by ponatinib, an FGFR inhibitor. Finally, in an independent cohort of CML patients, we found that pluripotency gene expression is a common feature of CD34+ CML cells at diagnosis. Overall, these experiments allowed identification of individual CD34+ cells expressing high levels of pluripotency genes at diagnosis, in which a continuum of transitional states were identified using pseudotime analysis. These results suggest that leukemic stem cell persistence in CML needs to be targeted simultaneously rather than using a single pathway.

A novel neuronal organoid model mimicking glioblastoma (GBM) features from induced pluripotent stem cells (iPSC).

BACKGROUND: Current experimental models using either human or mouse cell lines, are not representative of the complex features of GBM. In particular, there is no model to study patient-derived iPSCs to generate a GBM model. Overexpression of c-met gene is one of the molecular features of GBM leading to increased signaling via STAT3 phosphorylation. We generated an iPSC line from a patient with c-met mutation and we asked whether we could use it to generate neuronal-like organoids mimicking features of GBM. METHODS: We have generated iPSC-aggregates differentiating towards organoids. We analyzed them by gene expression profiling, immunostaining and transmission electronic microscopy analyses (TEM). RESULTS: Herein we describe that c-met-mutated iPSC aggregates spontaneously differentiate into dopaminergic neurons more rapidly than control iPSC aggregates in culture. Gene expression profiling of c-met-mutated iPSC aggregates at day +90 showed neuronal- and GBM-related genes, reproducing a genomic network described in primary human GBM. Comparative TEM analyses confirmed the enrichment of these structures in intermediate filaments and abnormal cilia, a feature described in human GBM. The c-met-mutated iPSC-derived organoids, as compared to controls expressed high levels of glial fibrillary acidic protein (GFAP), which is a typical marker of human GBM, as well as high levels of phospho-MET and phospho-STAT3. The use of temozolomide (TMZ) showed a preferential cytotoxicity of this drug in c-met-mutated neuronal-like organoids. GENERAL SIGNIFICANCE: This study shows the feasibility of generating "off-the shelf" neuronal-like organoid model mimicking GBM using c-met-mutated iPSC aggregates and its potential future use in research.

Determinants of the outcomes of patients with cancer infected with SARS-CoV-2: results from the Gustave Roussy cohort.

Patients with cancer are presumed to be at increased risk of severe COVID-19 outcomes due to underlying malignancy and treatment-induced immunosuppression. Of the first 178 patients managed for COVID-19 at the Gustave Roussy Cancer Centre, 125 (70.2%) were hospitalized, 47 (26.4%) developed clinical worsening and 31 (17.4%) died. An age of over 70 years, smoking status, metastatic disease, cytotoxic chemotherapy and an Eastern Cooperative Oncology Group score of ≥2 at the last visit were the strongest determinants of increased risk of death. In multivariable analysis, the Eastern Cooperative Oncology Group score remained the only predictor of death. In contrast, immunotherapy, hormone therapy and targeted therapy did not increase clinical worsening or death risk. Biomarker studies found that C-reactive protein and lactate dehydrogenase levels were significantly associated with an increased risk of clinical worsening, while C-reactive protein and D-dimer levels were associated with an increased risk of death. COVID-19 management impacted the oncological treatment strategy, inducing a median 20 d delay in 41% of patients and adaptation of the therapeutic strategy in 30% of patients.

iPSC-Derived Embryoid Bodies as Models of c-Met-Mutated Hereditary Papillary Renal Cell Carcinoma.

Hereditary cancers with cancer-predisposing mutations represent unique models of human oncogenesis, as a driving oncogenic event is present in germline. Currently, there are no satisfactory models to study these malignancies. We report the generation of IPSC from the somatic cells of a patient with hereditary c-met-mutated papillary renal cell carcinoma (PRCC). From these cells we have generated spontaneous aggregates organizing in structures which expressed kidney markers such as PODXL and Six2. These structures expressed PRCC markers both in vitro and in vivo in NSG mice. Gene-expression profiling showed striking molecular similarities with signatures found in a large cohort of PRCC tumor samples. This analysis, applied to primary cancers with and without c-met mutation, showed overexpression of the BHLHE40 and KDM4C only in the c-met-mutated PRCC tumors, as predicted by c-met-mutated embryoid bodies transcriptome. These data therefore represent the first proof of concept of "hereditary renal cancer in a dish" model using c-met-mutated iPSC-derived embryoid bodies, opening new perspectives for discovery of novel predictive progression markers and for drug-screening for future precision-medicine strategies.

Genomic landscape analyses of reprogrammed cells using integrative and non-integrative methods reveal variable cancer-associated alterations.

Recent development of cell reprogramming technologies brought a major hope for future cell therapy applications by the use of these cells or their derivatives. For this purpose, one of the major requirements is the absence of genomic alterations generating a risk of cell transformation. Here we analyzed by microarray-based comparative genomic hybridization human iPSC generated by two non-integrative and one integrative method at pluripotent stage as well as in corresponding teratomas. We show that all iPSC lines exhibit copy number variations (CNV) of several genes deregulated in oncogenesis. These cancer-associated genomic alterations were more pronounced in virally programmed hiPSCs and their derivative teratoma as compared to those found in iPSC generated by mRNA-mediated reprogramming. Bioinformatics analysis showed the involvement of these genes in human leukemia and carcinoma. We conclude that genetic screening should become a standard procedure to ensure that hiPSCs are free from cancer-associated genomic alterations before clinical use.