Search

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.

Abstract Background Clear cell renal cell carcinoma (ccRCC), the most aggressive renal cancer, is characterized by early lymph node metastases and bad prognosis. Most therapies targeting advanced or metastatic ccRCC are based, as first-line treatment, on the administration of the vascular endothelial growth factor (VEGF) neutralizing antibody termed Bevacizumab. Despite proven benefits, the expected results were not obtained for the majority of patients. The possibility that an intricate interplay between angiogenesis and immune-checkpoints might exist lead us to evaluate tumor angiogenesis, by means of VEGF expression together with the immune checkpoint HLA-G/ILT4. Methods Tumor specimens were obtained from patients from two separate cohorts: One from “Evita Pueblo” Hospital from Berazategui, (Buenos Aires, Argentina) and the second includes patients surgically operated at the Urology Department of Saint-Louis Hospital (Paris, France) with a confirmed ccRCC diagnosis. Immunohistochemistry was performed with specific antibodies directed against HLA-G, VEGF-A, VEGF-C, D240, CD34, ILT4 and Ca-IX. In addition, gene expression levels were measured in a cell line derived from a ccRCC patient by semi-quantitative RT-PCR. Results Our results show that the highly vascularized tumors of ccRCC patients express high levels of VEGF and the immune-checkpoint HLA-G. In addition, ILT4, one of the HLA-G receptors, was detected on macrophages surrounding tumor cells, suggesting the generation of an immune-tolerant microenvironment that might favor tumorigenesis. Notably, RT-qPCR analysis provided the first evidence on the transcriptional relationship between HLA-G/ILT4 and the VEGF family. Namely, in the presence of HLA-G or ILT4, the levels of VEGF-A are diminished whereas those of VEGF-C are increased. Conclusions In an effort to find new therapeutic molecules and fight against metastasis dissemination associated with the poor survival rates of ccRCC patients, these findings provide the rationale for co-targeting angiogenesis and the immune checkpoint HLA-G.

Abstract Background To evaluate long-term IPSS score and urinary quality of life after radiotherapy for prostate cancer, in patients with prior history of surgical treatment for benign prostatic hyperplasia (BPH). Methods In this retrospective study, we reviewed medical records of patients treated in our department, between 2007 and 2013 with surgery for BPH followed by radiotherapy for localized prostate cancer. Patients were contacted to fill in IPSS questionnaire and they were also asked for urinary quality of life. Predictive factors known to be associated with bad urinary function were also analysed. Results Fifty-nine patients were included in our study. Median age was 70 years. Median follow-up was 4.6 years. Median radiotherapy dose was 78 Gy (5 × 2 Gy/week). Thirty patients (48.5%) received hormone therapy in combination with RT. Main surgery indications were urinary symptoms (65%) and urinary retention (20%). Five-year biochemical-disease free survival was 75% and 5-year clinical relapse free survival was 84%. At the time of the study, the IPSS after radiotherapy was as follows: 0–7: 77.6%; 8–19:20.7%; 20–35: 1.7%. Urinary quality of life was satisfactory for 74.2% of patients. After multivariate analysis, a high dose of RT and a medical history of hypertension were associated with a poorer quality of urinary life (p = 0.04). Conclusion External radiotherapy remains an appropriate treatment option without a major risk for deterioration in urinary function in patient with antecedent surgery for BPH. High dose of RT and a medical history of hypertension were associated with a poorer quality of urinary life.

Background: This study explored the French media’s presentation of ideas and medical information about prostate cancer (PC) that may influence men’s understanding, attitudes and behavior. Methods: A qualitative media content analysis centered on PC information delivered by French professional media. The selected data were produced in the aftermath of the High Health Authority's decision in 2008 not to recommend systematic screening by prostate specific antigen(PSA) for men over 50. Source was the Media Archives of the French National Library. Content was analyzed from 15 television programs, 14 radio programs, and 55 articles from 35 popular French newspapers (online and printed, weekly and monthly) and 20 magazines. Audio content was narrated into textual form and submitted to manual coding along with the print content.Results: Television and radio content focused on the nature of PC, screening and treatment,and conveyed a gender-centric position linked to male sexuality and virility. Newspapers and magazines targeted the testing controversy, the lack of consensus among professionals, and scientific advances in screening and treatment.Conclusion: Media participation in the European testing debate is valuable for allowing patients to hear all opinions on PC risk factors. Debate on testing policy contributes to confusion and uncertainty regarding appropriate action.

The establishment and maintenance of anti-tumor immune responses are the objectives of cancer immunotherapy. Despite recent promising advances, the effectiveness of these approaches has been limited by the multiple immunosuppressive mechanisms developed by tumors (checkpoint). The aim of the present study was to demonstrate intratumor heterogeneity at the levels of immune escape strategies and tumor-host relationships. We focused on well-known checkpoints such as PD1/PDL1 and on a new checkpoint involving HLA-G and its receptors ILT2/ILT4. A prospective study was performed on 19 renal-cell carcinoma patients that were included during hospitalization for surgical tumor resection. Different areas of the tumor were collected for each patient and subjected to both immunohistochemical and flow cytometry analysis. Immune cells from peripheral blood were concomitantly analyzed for each patient. Our results show the heterogeneous expression of PD1/PDL1 and HLA-G/ILT in the various areas of the same tumor. Intratumor heterogeneity was found both at tumor cell and infiltrating immune cell levels. From a clinical point of view, this work highlights the functional redundancies of checkpoints and the need to adapt personalized poly-immunotherapy.

Non-AIDS-related malignancies now represent a frequent cause of death among HIV-infected patients. Albeit bladder cancer is one of the most common malignancies worldwide, it has been rarely reported among HIV-infected patients. We wished to assess the prevalence and characteristics of bladder cancer in HIV-infected patients.We conducted a single center retrospective study from 1998 to 2013 in a university hospital in Paris. Cases of bladder cancer among HIV-infected patients were identified using the electronic records of the hospital database and of the HIV-infected cohort. Patient characteristics and outcomes were retrieved from patients charts. A systematic review of published cases of bladder cancers in patients with HIV-infection was also performed.During the study period we identified 15 HIV-infected patients (0.2% of the cohort) with a bladder cancer. Patients were mostly men (73%) and smokers (67%), with a median age of 56 years at cancer diagnosis. Bladder cancer was diagnosed a median of 14 years after HIV-infection. Most patients were on ART (86%) with median current and nadir CD4 cell counts of 506 and 195 cells/mm3, respectively. Haematuria (73%) was the most frequent presenting symptom and HPV-associated lesions were seen in 6/10 (60%) patients. Histopathology showed transitional cell carcinoma in 80% and a high proportion of tumors with muscle invasion (47%) and high histologic grade (73%). One-year survival rate was 74.6%. The systematic review identified 13 additional cases of urothelial bladder cancers which shared similar features.Bladder cancers in HIV-infected patients remain rare but may occur in relatively young patients with a low nadir CD4 cell count, have aggressive pathological features and can be fatal.