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Methods for Establishing a Renal Cell Carcinoma Tumor Spheroid Model With Immune Infiltration for Immunotherapeutic Studies

Tumor spheroids play an increasingly important role in cancer research. Their ability to recapitulate crucial features of tumor biology that are lost in the classically used 2D models along with their relative simplicity and handiness have made them the most studied 3D tumor model. Their application as a theranostic tool or as a means to study tumor-host interaction is now well-established in various cancers. However, their use in the field of Renal Cell Carcinoma (RCC) remains very limited. The aim of this work is to present methods to implement a basic RCC spheroid model. These methods cover the steps from RCC tumor dissociation to spheroid infiltration by immune cells. We present a protocol for RCC dissociation using Liberase TM and introduce a culture medium containing Epithelial Growth Factor and Hydrocortisone allowing for faster growth of RCC primary cells. We show that the liquid overlay technique allows for the formation of spheroids from cell lines and from primary cultures. We present a method using morphological criteria to select a homogeneous spheroid population based on a Fiji macro. We then show that spheroids can be infiltrated by PBMCs after activation with OKT3 or IL-15. Finally, we provide an example of application by implementing an immune spheroid killing assay allowing observing increased spheroid destruction after treatment with PD-1 inhibitors. Thus the straightforward methods presented here allow for efficient spheroid formation for a simple RCC 3D model that can be standardized and infused with immune cells to study immunotherapies

Micromolecular methods for diagnosis and therapeutic strategy: a case study

International audienceAn intraductal carcinoma, 55 mm across, was diagnosed on a total mastectomy in a 45-year-old woman. The 2 micro-invasive areas found were too small for reliable immunostainings for estrogen, progesterone, and HER2 receptors. In the sentinel lymph-node, a subcapsular tumor embole of about 50 cancer cells was identified on the extemporaneous cryo-cut section, but not on further sections after paraffinembedding of the sample. Considering this tumor metastatic potential, we decided to assess HER2 status on the metastatic embole using pathological and molecular micro-methods. We lasermicrodissected the tumor cells, extracted their DNA, and performed droplet-digital-PCR (ddPCR) for HER2 gene copy number variation. The HER2/RNaseP allele ratio was 5.2 in the laser-microdissected tumor cells, similar to the 5.3 ratio in the HER2overexpressing breast cancer cell line BT-474. We thus optimized the adjuvant treatment of our patient and she received a trastuzumab-based adjuvant chemotherapy

Donor-Derived Keratinocytes in Actinic Keratosis and Squamous Cell Carcinoma in Patients with Kidney Transplant

International audienceNo abstrac

Active chronic sarcoidosis is characterized by increased transitional blood B cells, increased IL-10-producing regulatory B cells and high BAFF levels.

BACKGROUND: Sarcoidosis is a multisystemic disease of unknown etiology characterized by a disproportionate Th1 granulomatous immune response in the organs involved. Plasmatic hypergammaglobulinemia and B cell accumulation in granulomatous lesions suggest the possible role of humoral immune responses in the pathogenesis of sarcoidosis. The purpose of this study is to describe B cell peripheral compartment in sarcoidosis. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed blood B cell subsets and BAFF levels in 33 patients with chronic sarcoidosis (active sarcoidosis n = 18; inactive sarcoidosis n = 15) and 18 healthy donors. Active chronic sarcoidosis patients had significantly less circulating memory B cells (p<0.01), more transitional (p<0.01) and increased numbers of IL-10-producing regulatory B cells (p<0.05) compared with healthy donors and patients with inactive sarcoidosis. BAFF serum levels were significantly higher in patients with active sarcoidosis (p<0.01 versus healthy donors and inactive sarcoidosis patients) and strongly correlated with serum hypergammaglobulinemia (r = 0.53, p<0.01) and angiotensin converting enzyme levels (r = 0.61, p = <0.01). CONCLUSIONS/SIGNIFICANCE: These data show that there is an altered B cell homeostasis in active sarcoidosis and suggest BAFF antagonist drugs as potential new treatments of this disease

Tumor metabolism assessed by FDG-PET/CT and tumor proliferation assessed by genomic grade index to predict response to neoadjuvant chemotherapy in triple negative breast cancer

Purpose: Survival is increased when pathological complete response (pCR) is reached after neoadjuvant chemotherapy (NAC), especially in triple-negative breast cancer (TNBC) patients. Positron emission tomography/computed tomography (PET/CT) with 18F-fluorodeoxyglucose (FDG) and the genomic grade index (GGI), each separately, showed good potential to predict pCR. Our study was designed to evaluate the predictive value for the therapeutic response of a combination of parameters based on FDG-PET, histoclinical features and molecular markers of proliferation. Methods: Molecular parameters were measured on pre-treatment biopsy. Tumor metabolic activity was measured using two PET/CT scans, one before and one after 2 cycles of NAC. The pCR was determined on specimen after NAC. Event-free survival (EFS) was estimated using the Kaplan Meier method. Results: Of 55 TNBC patients, 19 (35%) reached pCR after NAC. Tumor grade and Ki67 were not associated with pCR whereas GGI (P = 0.04) and its component KPNA2 (P = 0.04) showed a predictive value. The change of FDG uptake between PET1 and PET2 (ΔSUVmax) was highly associated with pCR (P = 0.0001) but the absolute value of baseline SUVmax was not (P = 0.11). However, the AUC of pCR prediction increased from 0.63 to 0.76 when baseline SUVmax was combined with the GGI (P = 0.016). The only two parameters associated with EFS were ΔSUVmax (P = 0.048) and pathological response (P = 0.014). Conclusions: The early tumor metabolic change during NAC is a powerful parameter to predict pCR and outcome in TNBC patients. The GGI, determined on pretreatment biopsy, is also predictive of pCR and the combination GGI and baseline SUVmax improves the prediction.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Comprehensive landscape of immune-checkpoints uncovered in clear cell renal cell carcinoma reveals new and emerging therapeutic targets.

International audienceClear cell renal cell carcinoma (ccRCC) constitutes the most common renal cell carcinoma subtype and has long been recognized as an immunogenic cancer. As such, significant attention has been directed toward optimizing immune-checkpoints (IC)-based therapies. Despite proven benefits, a substantial number of patients remain unresponsive to treatment, suggesting that yet unreported, immunosuppressive mechanisms coexist within tumors and their microenvironment. Here, we comprehensively analyzed and ranked forty-four immune-checkpoints expressed in ccRCC on the basis of in-depth analysis of RNAseq data collected from the TCGA database and advanced statistical methods designed to obtain the group of checkpoints that best discriminates tumor from healthy tissues. Immunohistochemistry and flow cytometry confirmed and enlarged the bioinformatics results. In particular, by using the recursive feature elimination method, we show that HLA-G, B7H3, PDL-1 and ILT2 are the most relevant genes that characterize ccRCC. Notably, ILT2 expression was detected for the first time on tumor cells. The levels of other ligand-receptor pairs such as CD70:CD27; 4-1BB:4-1BBL; CD40:CD40L; CD86:CTLA4; MHC-II:Lag3; CD200:CD200R; CD244:CD48 were also found highly expressed in tumors compared to adjacent non-tumor tissues. Collectively, our approach provides a comprehensible classification of forty-four IC expressed in ccRCC, some of which were never reported before to be co-expressed in ccRCC. In addition, the algorithms used allowed identifying the most relevant group that best discriminates tumor from healthy tissues. The data can potentially assist on the choice of valuable immune-therapy targets which hold potential for the development of more effective anti-tumor treatments

18FDG-PET/CT and molecular markers to predict response to neoadjuvant chemotherapy and outcome in HER2-negative advanced luminal breast cancers patients

Background: The efficacy of neoadjuvant chemotherapy regimens in advanced luminal breast cancer patients is difficult to predict. Intrinsic properties of breast tumors, including altered gene expression profile and dynamic evaluation of metabolic properties of tumor cells using positron emission tomography/computed tomography (PET/CT) of tumor cells, have been identified to guide patient's prognosis. The aim of this study is to determine if both analyses may improve the prediction of response to neoadjuvant chemotherapy in ER-positive / HER2-negative breast cancers (BCs) patients. Methods: We used metabolic PET parameters, at diagnosis and after two cycles of chemotherapy and proliferation gene expression profile on biopsy at diagnosis, in particular, the genomic grade index (GGI) analyzed by reverse transcription and quantitative polymerase chain reaction (RT-qPCR). The pathological response was the surrogate endpoint. Results: The change of FDG uptake between baseline PET and interim PET after 2 cycles of neoadjuvant chemotherapy (ΔSUVmax) was highly associated with pCR (p=0.008). We also observed an ability of P53 mutated status (p=0.042), in addition to histological grade (p=0. 0004), and PR expression (p=0.01) to predict pCR in ERpositive BCs, whereas no proliferation marker predicted pCR (P=0.39 for GGI). Finally, only ΔSUVmax was significantly associated with event free survival (p=0.047). Conclusions: Our results confirm the predictive and prognostic value of tumor ΔSUVmax in ER-positive /HER2-negative advanced BCs patients. These findings can be helpful to select high-risk patients within trials investigating novel treatment strategies.SCOPUS: ar.jinfo:eu-repo/semantics/publishe