Characterization of Renal Cell Carcinoma Heterotypic 3D Co-Cultures with Immune Cell Subsets.
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ABSTRACT:
SUBMITTER: Rausch M
PROVIDER: S-EPMC8197009 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Background: Head and neck squamous cell carcinomas (HNSCC) are phenotypically and molecularly heterogeneous and frequently develop therapy resistance. Reliable patient-derived 3D tumor models are urgently needed to further study the complex pathogenesis of these tumors and to overcome treatment failure. Methods: We developed a three-dimensional organotypic co-culture (3D-OTC) model for HNSCC that maintains the architecture and cell composition of the individual tumor. A dermal equivalent (DE), composed of healthy human-derived fibroblasts and viscose fibers, served as a scaffold for the patient sample. DEs were co-cultivated with 13 vital HNSCC explants (non-human papillomavirus (HPV) driven, n = 7; HPV-driven, n = 6). Fractionated irradiation was applied to 5 samples (non-HPV-driven, n = 2; HPV-driven n = 3). To evaluate expression of ki-67, cleaved caspase-3, pan-cytokeratin, p16INK4a, CD45, ∝smooth muscle actin and vimentin over time, immunohistochemistry and immunofluorescence staining were performed Patient checkup data were collected for up to 32 months after first diagnosis. Results: All non-HPV-driven 3D-OTCs encompassed proliferative cancer cells during cultivation for up to 21 days. Proliferation indices of primaries and 3D-OTCs were comparable and consistent over time. Overall, tumor explants displayed heterogeneous growth patterns (i.e., invasive, expansive, silent). Cancer-associated fibroblasts and leukocytes could be detected for up to 21 days. HPV DNA was detectable in both primary and 3D-OTCs (day 14) of HPV-driven tumors. However, p16INK4a expression levels were varying. Morphological alterations and radioresistant tumor cells were detected in 3D-OTC after fractionated irradiation in HPV-driven and non-driven samples. Conclusions: Our 3D-OTC model for HNSCC supports cancer cell survival and proliferation in their original microenvironment. The model enables investigation of invasive cancer growth and might, in the future, serve as a platform to perform sensitivity testing upon treatment to predict therapy response.
| S-EPMC7463661 | biostudies-literature
Project description:Invasive lobular carcinoma (ILC) is the second most common subtype of breast cancer following invasive ductal carcinoma (IDC) and characterized by the loss of E-cadherin-mediated adherens junctions. Despite displaying unique histologic and clinical features, ILC still remains a chronically understudied disease, with limited knowledge gleaned from available laboratory research models. Here we report a comprehensive 2D and 3D phenotypic characterization of four estrogen receptor-positive human ILC cell lines: MDA-MB-134, SUM44, MDA-MB-330, and BCK4. Compared with the IDC cell lines MCF7, T47D, and MDA-MB-231, ultra-low attachment culture conditions revealed remarkable anchorage independence unique to ILC cells, a feature not evident in soft-agar gels. Three-dimensional Collagen I and Matrigel culture indicated a generally loose morphology for ILC cell lines, which exhibited differing preferences for adhesion to extracellular matrix proteins in 2D. Furthermore, ILC cells were limited in their ability to migrate and invade in wound-scratch and transwell assays, with the exception of haptotaxis to Collagen I. Transcriptional comparison of these cell lines confirmed the decreased cell proliferation and E-cadherin-mediated intercellular junctions in ILC while uncovering the induction of novel pathways related to cyclic nucleotide phosphodiesterase activity, ion channels, drug metabolism, and alternative cell adhesion molecules such as N-cadherin, some of which were differentially regulated in ILC versus IDC tumors. Altogether, these studies provide an invaluable resource for the breast cancer research community and facilitate further functional discoveries toward understanding ILC, identifying novel drug targets, and ultimately improving the outcome of patients with ILC.Significance: These findings provide the breast cancer research community with a comprehensive assessment of human invasive lobular carcinoma (ILC) cell line signaling and behavior in various culture conditions, aiding future endeavors to develop therapies and to ultimately improve survival in patients with ILC. Cancer Res; 78(21); 6209-22. ©2018 AACR.
| S-EPMC6507416 | biostudies-literature
Project description:ObjectivesReproducing human hair follicles in vitro is often limited by various reasons such as the lack of a systematic approach to culture distinct hair follicle cell types to reproduce their spatial relationship. Here, we reproduce hair follicle-like constructs resembling the spatial orientation of different cells in vivo, to study the role of keratinocytes in maintaining cellular compartmentalization among hair follicle-related cells.Materials and methodsDermal papilla (DP) cells, HaCaT keratinocytes and human dermal fibroblast (HDF) cells were seeded sequentially into three-dimensional (3D) microwells fabricated from polyethylene glycol diacrylate hydrogels. Quantitative polymerase chain reaction was used to compare inductive gene expression of 3D and two-dimensional (2D) DP. DP and HaCaT cells were transfected with green fluorescent protein and red fluorescent protein lentivirus, respectively, to enable cell visualization using confocal microscopy.ResultsThe 3D DP cultures showed significantly enhanced expression of essential DP genes as compared 2D cultures. Core-shell configurations containing keratinocytes forming the outer shell and DP forming the core were observed. Migratory polarization was mediated by cell-cell interaction between the keratinocytes and HDF cells, while preserving the aggregated state of the DP cells.ConclusionsKeratinocytes may play a role in maintaining compartmentalization between the DP and the surrounding HDF residing in the dermis, and therefore maintains the aggregative state of the DP cells, necessary for hair follicle development and function.
| S-EPMC6797517 | biostudies-literature
Project description:Immune infiltration of tumors is closely associated with clinical outcome in renal cell carcinoma (RCC). Tumor-infiltrating immune cells (TIICs) regulate cancer progression and are appealing therapeutic targets. The purpose of this study was to determine the composition of TIICs in RCC and further reveal the independent prognostic values of TIICs. CIBERSORT, an established algorithm, was applied to estimate the proportions of 22 immune cell types based on gene expression profiles of 891 tumors. Cox regression was used to evaluate the association of TIICs and immune checkpoint modulators with overall survival (OS). We found that CD8+ T cells were associated with prolonged OS (hazard ratio [HR] = 0.09, 95% confidence interval [CI].01-.53; P = 0.03) in chromophobe carcinoma (KICH). A higher proportion of regulatory T cells was associated with a worse outcome (HR = 1.59, 95% CI 1.23-.06; P < 0.01) in renal clear cell carcinoma (KIRC). In renal papillary cell carcinoma (KIRP), M1 macrophages were associated with a favorable outcome (HR = .43, 95% CI .25-.72; P < 0.01), while M2 macrophages indicated a worse outcome (HR = 2.55, 95% CI 1.45-4.47; P < 0.01). Moreover, the immunomodulator molecules CTLA4 and LAG3 were associated with a poor prognosis in KIRC, and IDO1 and PD-L2 were associated with a poor prognosis in KIRP. This study indicates TIICs are important determinants of prognosis in RCC meanwhile reveals potential targets and biomarkers for immunotherapy development.
| S-EPMC6501001 | biostudies-literature
Project description:The treatment of advanced renal cell carcinoma remains a challenge. To develop novel therapeutic approaches, primary cell cultures as an in vitro model are considered more representative than commercial cell lines. In this study, we analyzed the gene expression of previously established primary cell cultures of clear cell renal cell carcinoma by bulk (3'm)RNA sequencing and compared it to the tissue of origin. The objectives were the identification of dysregulated pathways under cell culture conditions. Furthermore, we assessed the suitability of primary cell cultures for studying crucial biological pathways, including hypoxia, growth receptor signaling and immune evasion. RNA sequencing of primary cell cultures of renal cell carcinoma and a following Enrichr database analysis revealed multiple dysregulated pathways under cell culture conditions. 444 genes were significantly upregulated and 888 genes downregulated compared to the tissue of origin. The upregulated genes are crucial in DNA repair, cell cycle, hypoxia and metabolic shift towards aerobic glycolysis. A downregulation was observed for genes involved in pathways of immune cell differentiation and cell adhesion. We furthermore observed that 7275 genes have a similar mRNA expression in cell cultures and in tumor tissue, including genes involved in the immune checkpoint signaling or in pathways responsible for tyrosine kinase receptor resistance. Our findings confirm that primary cell cultures are a representative tool for specified experimental approaches. The results presented in this study give further valuable insights into the complex adaptation of patient-derived cells to a new microenvironment, hypoxia and other cell culture conditions, which are often neglected in daily research, and allow new translational and therapeutic approaches.
| S-EPMC9130782 | biostudies-literature
Project description:We present a pair-wise co-culturing technique that creates large numbers of heterotypic cell pairs in patterned arrays. Lithographic patterning produces arrays with thousands of traps, each designed to accommodate only two cells and confine them at these sites for co-culturing. Two variants are introduced: a random seeding method that sediments a mixture of two cell types onto the array, and an approach that incorporates ferromagnetic thin films into the arrays and attracts cells that have been attached to ferromagnetic nanowires to the array sites through dipole interactions. The array technique includes the utilization of custom image analysis software that extracts data from multi-channel fluorescence images and records information about the cells in every trap, enabling the acquisition of accurate, high-statistics data. The applicability of the technique was demonstrated in experiments examining proliferation rates in pairs of bovine pulmonary artery endothelial and smooth muscle cells. Results demonstrated that heterotypic interactions favored smooth muscle cell proliferation while disfavoring endothelial cell proliferation. This is one example of a variety of cell-cell interactions that could be probed with this method.
| S-EPMC3444241 | biostudies-literature
Project description:The composition of tumor-infiltrating lymphocytes (TIL) reflects biology and immunogenicity of cancer. Here, we characterize T-cell subsets and expression of immune checkpoint molecules in head and neck squamous cell carcinoma (HNSCC). We analyzed TIL subsets in primary tumors (n = 34), blood (peripheral blood mononuclear cells (PBMC); n = 34) and non-cancerous mucosa (n = 7) of 34 treatment-naïve HNSCC patients and PBMC of 15 healthy controls. Flow cytometry analyses revealed a highly variable T-cell infiltration mainly of an effector memory phenotype (CD45RA-/CCR7-). Naïve T cells (CD45RA+/CCR7+) were decreased in the microenvironment compared to PBMC of patients, while regulatory T cells (CD4+/CD25+/CD127low and CD4+/CD39+) were elevated. Furthermore, we performed digital image analyses of entire cross sections of HNSCC to define the 'Immunoscore' (CD3+ and CD8+ cell infiltration in tumor core and invasive margin) and quantified MHC class I expression on tumor cells by immunohistochemistry. Immune checkpoint molecules cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1) were increased in TILs compared to peripheral T cells in flow-cytometric analysis. Human papillomavirus (HPV) positive tumors showed higher numbers of TILs, but a similar composition of T-cell subsets and checkpoint molecule expression compared to HPV negative tumors. Taken together, the tumor microenvironment of HNSCC is characterized by a strong infiltration of regulatory T cells and high checkpoint molecule expression on T-cell subsets. In view of increasingly used immunotherapies, a detailed knowledge of TILs and checkpoint molecule expression on TILs is of high translational relevance.
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Project description:BackgroundSLFN11 has been found to regulate the development and progression of a variety of cancers and is associated with drug therapy, while its role in clear cell renal cell carcinoma (ccRCC) remains unclear; therefore, the aim of this study was to investigate SLFN11 expression in ccRCC patients and its correlations with clinicopathological and immunological features.MethodsGene profiles of ccRCC and the clinicopathological information of patients were downloaded from the TCGA database. Microarrays from the GEO database were used as a validation set for SLFN11 expression, which was experimentally verified in renal cancer cell lines by quantitative polymerase chain reaction (qPCR); protein expression and methylation levels were obtained from the HPA database and the UALCAN database. ROC curves, Kaplan-Meier survival analysis and Cox analysis were used to assess the diagnostic and predictive value of SLFN11 in ccRCC. Protein-protein interaction (PPI) networks for SLFN11 were obtained from the STRING website, and the TISIDB and TIMER 2.0 databases were used to study the relationship between SLFN11 and immune infiltration in the tumour microenvironment (TME).ResultsSLFN11 was significantly overexpressed in ccRCC tissues and renal cancer cell lines, which may be closely related to its hypermethylation status (P < 0.001). SLFN11 was positively correlated with a highly aggressive disease state, with the ROC curve showing an AUC value of 0.910 for SLFN11 in diagnosing ccRCC, and Kaplan-Meier and Cox analyses also revealed that upregulation of SLFN11 predicted a poor prognosis for ccRCC patients (P < 0.05). In addition, enrichment analysis showed that SLFN11 was closely associated with immune-related signalling pathways, and further exploration comprehensively demonstrated strong positive correlations with tumour immune lymphocytes, immune checkpoint genes, chemokines and chemokine receptors.ConclusionOverall, our data analysis shows that SLFN11 is a strong diagnostic and prognostic biomarker for ccRCC and is also associated with immune infiltration in the TME.
| S-EPMC8521339 | biostudies-literature
Project description:The immune system has long been known to play a critical role in the body's defence against cancer, and there have been multiple attempts to harness it for therapeutic gain. Renal cancer was, historically, one of a small number of tumour types where immune manipulation had been shown to be effective. The current generation of immune checkpoint inhibitors are rapidly entering into routine clinical practice in the management of a number of tumour types, including renal cancer, where one drug, nivolumab, an anti-programmed death-1 (PD-1) monoclonal antibody (mAb), is licensed for patients who have progressed on prior systemic treatment. Ongoing trials aim to maximize the benefits that can be gained from this new class of drug by exploring optimal timing in the natural course of the disease as well as combinations with other checkpoint inhibitors and drugs from different classes.
| S-EPMC5869245 | biostudies-literature
Project description:Immune checkpoint blockade (ICB) is the foundation of current first-line therapies in patients with metastatic renal cell carcinoma (mRCC) with the potential for eliciting long-lasting remissions. With the expanding arsenal of ICB-based therapies, biomarkers of response are urgently needed to guide optimal therapeutic selection. We review the data behind ICB therapy in RCC, emerging biomarkers of response, and the evolving role of surgery in patients with mRCC.
| S-EPMC8364550 | biostudies-literature