Development of extracellular matrix supported 3D culture of renal cancer cells and renal cancer stem cells

Novel experimental conditions of cancer cell line culture have evolved throughout the recent years, with significantly growing interest in xeno-free, serum-free and three-dimensional culture variants. The choice of proper culture media may enable to mimic tumor microenvironment and promotion of cancer stem cells proliferation. To assess whether stem-like phenotype inducing media may be applied in renal cancer stem cell research, we performed a widespread screening of 13 cell culture media dedicated for mesenchymal cells, stem cells as well as mesenchymal stem cells. We have also screened extracellular matrix compounds and selected optimal RCC 3D—ECM supported culture model. Our results revealed that 786-O as well as HKCSCs cell line cultures in xeno-free media (NutriStem/StemXvivo) and laminin coated plates provide a useful tool in RCC cancer biology research and at the same time enable effective drug toxicity screening. We propose bio-mimic 3D RCC cell culture model with specific low-serum and xeno-free media that promote RCC cell viability and stem-like phenotype according to the tested genes encoding stemness factors including E-cadherin, N-cadherin, HIF1, HIF2, VEGF, SOX2, PAX2 and NESTIN

TFIIS-Dependent Non-coding Transcription Regulates Developmental Genome Rearrangements.

Because of their nuclear dimorphism, ciliates provide a unique opportunity to study the role of non-coding RNAs (ncRNAs) in the communication between germline and somatic lineages. In these unicellular eukaryotes, a new somatic nucleus develops at each sexual cycle from a copy of the zygotic (germline) nucleus, while the old somatic nucleus degenerates. In the ciliate Paramecium tetraurelia, the genome is massively rearranged during this process through the reproducible elimination of repeated sequences and the precise excision of over 45,000 short, single-copy Internal Eliminated Sequences (IESs). Different types of ncRNAs resulting from genome-wide transcription were shown to be involved in the epigenetic regulation of genome rearrangements. To understand how ncRNAs are produced from the entire genome, we have focused on a homolog of the TFIIS elongation factor, which regulates RNA polymerase II transcriptional pausing. Six TFIIS-paralogs, representing four distinct families, can be found in P. tetraurelia genome. Using RNA interference, we showed that TFIIS4, which encodes a development-specific TFIIS protein, is essential for the formation of a functional somatic genome. Molecular analyses and high-throughput DNA sequencing upon TFIIS4 RNAi demonstrated that TFIIS4 is involved in all kinds of genome rearrangements, including excision of ~48% of IESs. Localization of a GFP-TFIIS4 fusion revealed that TFIIS4 appears specifically in the new somatic nucleus at an early developmental stage, before IES excision. RT-PCR experiments showed that TFIIS4 is necessary for the synthesis of IES-containing non-coding transcripts. We propose that these IES+ transcripts originate from the developing somatic nucleus and serve as pairing substrates for germline-specific short RNAs that target elimination of their homologous sequences. Our study, therefore, connects the onset of zygotic non coding transcription to the control of genome plasticity in Paramecium, and establishes for the first time a specific role of TFIIS in non-coding transcription in eukaryotes

Metastatic renal cell carcinoma cells growing in 3D on poly‑D‑lysine or laminin present a stem‑like phenotype and drug resistance

3D spheroids are built by heterogeneous cell types in different proliferative and metabolic states and are enriched in cancer stem cells. The main aim of the study was to investigate the usefulness of a novel metastatic renal cell carcinoma (RCC) 3D spheroid culture for in vitro cancer stem cell physiology research and drug toxicity screening. RCC cell lines, Caki-1 (skin metastasis derived) and ACHN (pleural effusion derived), were efficiently cultured in growth-factor/serum deprived, defined, StemXvivo and Nutristem medium on laminin-coated or poly-D-lysine-coated plates. In optimal 3D culture conditions, ACHN cells (StemXVivo/poly-D-lysine) formed small spheroids with remaining adherent cells of an epithelial phenotype, while Caki-1 cells (StemXVivo/laminin) formed large dark spheroids with significantly reduced cell viability in the center. In the 3D structures, expression levels of genes encoding stem transcription factors (OCT4, SOX2, NES) and RCC stem cell markers (CD105, CD133) were deregulated in comparison to these expression levels in traditional 2D culture. Sunitinib, epirubicin and doxycycline were more toxic to cells cultured in monolayers than for cells in 3D spheroids. High numbers of cells arrested in the G0/G1 phase of the cell cycle were found in spheroids under sunitinib treatment. We showed that metastatic RCC 3D spheroids supported with ECM are a useful model to determine the cancer cell growth characteristics that are not found in adherent 2D cultures. Due to the more complex architecture, spheroids may mimic in vivo micrometastases and may be more appropriate to investigate novel drug candidate responses, including the direct effects of tyrosine kinase inhibitor activity against RCC cells

Development of extracellular matrix supported 3D culture of renal cancer cells and renal cancer stem cells

Novel experimental conditions of cancer cell line culture have evolved throughout the recent years, with significantly growing interest in xeno-free, serum-free and three-dimensional culture variants. The choice of proper culture media may enable to mimic tumor microenvironment and promotion of cancer stem cells proliferation. To assess whether stem-like phenotype inducing media may be applied in renal cancer stem cell research, we performed a widespread screening of 13 cell culture media dedicated for mesenchymal cells, stem cells as well as mesenchymal stem cells. We have also screened extracellular matrix compounds and selected optimal RCC 3DECM supported culture model. Our results revealed that 786-O as well as HKCSCs cell line cultures in xeno-free media (NutriStem/StemXvivo) and laminin coated plates provide a useful tool in RCC cancer biology research and at the same time enable effective drug toxicity screening. We propose bio-mimic 3D RCC cell culture model with specific low-serum and xeno-free media that promote RCC cell viability and stem-like phenotype according to the tested genes encoding stemness factors including E-cadherin, N-cadherin, HIF1, HIF2, VEGF, SOX2, PAX2 and NESTIN

Detection of IES-containing (IES+) transcripts.

<p>(A) RT-PCR and Southern blot detection of IES-containing transcripts (IES+) in a control culture (cells silenced for <i>ICL7</i> gene expression) and in <i>TFIIS4</i>-silenced cells. Autogamy stages are marked as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005383#pgen.1005383.s006" target="_blank">S6 Fig</a>: V–vegetative cells, -2.5 –cells during meiosis, 0 to 56 –autogamy stages in hours. Time-window when IES excision take place based on PCR shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005383#pgen.1005383.g004" target="_blank">Fig 4</a> is indicated. PCR primers were located within each tested IESs: 51G4404, 51A6649 and 51A4404. The TMP1b panel shows the RT-PCR signal obtained for the constitutively expressed gene encoding trichocyst matrix protein TMP1b. (B) Histograms showing the normalization of IES+ signals shown in (A) with TMP1b mRNA. (C) Detection of IES-containing transcripts (IES+) with PCR primers located within IES 51G4404 in a control experiment, in which the <i>ND7</i> gene was silenced, and in <i>PiggyMac</i>-silenced cells. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005383#pgen.1005383.s008" target="_blank">S8 Fig</a>, panel B for details about autogamy stages.</p

Localization of GFP fusion proteins forTFIIS1a, TFIIS2a, TFIIS3 and TFIIS4.

<p>For each transgene, representative images illustrate different developmental stages observed in a population of cells derived from a single injected caryonide. Panels a, f, l and s show vegetative cells (note that one vegetative cell is also present in the middle of panel b and on the left of panel h). All other panels show successive stages of autogamy: panels g, m and t–meiotic crescent stage; panels b and n–first meiotic division; panels h and u–cells with 8 haploid nuclei resulting from meiosis II; panels c, i, o and v–fragmentation of old MAC; panels d, j, p, and w–early MAC development; panels e, k, r and x–late MAC development. Note that panel b contains not only one meiotic cell (on the left) but also one vegetative cell (in the middle) and two cells with their fragmented old MAC (at the top and on the right). In all panels, white arrows point at MICs (some were omitted when MICs were not clearly distinguishable by DAPI staining), white arrowheads indicate new MACs. Yellow arrowheads in panels i and o point to division products of the zygotic nucleus. (A) A GFP-TFIIS1a fusion localizes to old, then new MACs. (B) A GFP-TFIIS2a fusion localizes to old MAC during meiosis, then to new MACs and is present in meiotic MICs. (C) As in B for a GFP-TFIIS3 fusion. GFP-TFIIS3 cannot be seen in division products of the zygotic nucleus. (D) A GFP-TFIIS4 fusion is essentially restricted to the new MACs specifically during early MAC development. Very weak GFP signal is visible in the old MAC during meiosis.</p

Analysis of IES excision in <i>TFIIS4</i>-silenced cells.

<p>(A) PCR analysis of the excision of IESs located in the surface antigen gene <i>A</i>^<i>51</i> using primers located around each IES. In each panel, the larger fragment corresponds to the non-excised form (IES+), the smaller fragment to the excised form (IES-). Known maternally controlled IESs are labeled with an asterisk. The autogamy time-course experiment was performed using a strain harboring a somatic (macronuclear) deletion of part of surface antigen gene <i>A</i>^<i>51</i>, which overlaps 3 tested IESs – 51A1835, 51A4404, 51A2591 and partially 51A4578. In this experiment, we obtained 93% lethality in post-autogamous progeny of <i>TFIIS4</i>-silenced cells. (B) As in A for IESs located in other regions. The PCR products corresponding to each IES- form are amplified mostly from the fragments of the old MAC. Oligonucleotide sequences are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005383#pgen.1005383.s013" target="_blank">S2 Table</a>. (C) IES retention scores calculated from the genome-wide sequencing of DNA extracted from purified nuclei of cells silenced for <i>TFIIS4</i> during an independent RNAi experiment (87% lethality in post-autogamous progeny). (D) Superimposed histogram of TFIIS4 retention scores for all IESs (dark blue) and for IESs that are significantly retained in TFIIS4-depleted cells (light blue). Around 25,000 IESs are not significantly affected by the inactivation of <i>TFIIS4</i> and a large fraction of IESs exhibits a retention score equal to 0. For TFIIS4-dependent IESs, retention scores are almost uniformly distributed between 0.1 and 0.7. (E) The graph shows a positive correlation between IES size and retention score in <i>TFIIS4</i> RNAi. The box plot displays the IES size distribution for all IESs and for each of <i>TFIIS4</i> retention score (RS) quartiles. The median retention score (horizontal line inside the box) and the first (top of box) and third (bottom of box) quartiles are shown. Range of RS for particular quartiles are as follows: Q1: [0–0.01[; Q2: [0.01–0.12[; Q3: [0.12–0.39[; Q4: [0.39–1.00]. The medians are significantly different between all the groups (p < 2e-40). (F) Venn diagram of significantly retained IESs after <i>TFIIS4</i>, <i>DCL5</i> or <i>DCL2/3</i> silencing. Almost all IESs that are dependent upon Dcl2/3 or Dcl5 for their excision are also dependent upon TFIIS4.</p

Localization of GFP fusion proteins forTFIIS1a, TFIIS2a, TFIIS3 and TFIIS4.

<p>For each transgene, representative images illustrate different developmental stages observed in a population of cells derived from a single injected caryonide. Panels a, f, l and s show vegetative cells (note that one vegetative cell is also present in the middle of panel b and on the left of panel h). All other panels show successive stages of autogamy: panels g, m and t–meiotic crescent stage; panels b and n–first meiotic division; panels h and u–cells with 8 haploid nuclei resulting from meiosis II; panels c, i, o and v–fragmentation of old MAC; panels d, j, p, and w–early MAC development; panels e, k, r and x–late MAC development. Note that panel b contains not only one meiotic cell (on the left) but also one vegetative cell (in the middle) and two cells with their fragmented old MAC (at the top and on the right). In all panels, white arrows point at MICs (some were omitted when MICs were not clearly distinguishable by DAPI staining), white arrowheads indicate new MACs. Yellow arrowheads in panels i and o point to division products of the zygotic nucleus. (A) A GFP-TFIIS1a fusion localizes to old, then new MACs. (B) A GFP-TFIIS2a fusion localizes to old MAC during meiosis, then to new MACs and is present in meiotic MICs. (C) As in B for a GFP-TFIIS3 fusion. GFP-TFIIS3 cannot be seen in division products of the zygotic nucleus. (D) A GFP-TFIIS4 fusion is essentially restricted to the new MACs specifically during early MAC development. Very weak GFP signal is visible in the old MAC during meiosis.</p

RNAi-screening for essential <i>TFIIS</i> genes.

<p>Survival test of post-autogamous cells submitted to RNAi against all <i>TFIIS</i> genes and control non-essential genes—<i>ICL7</i> and <i>ND7</i>. The last column “none” corresponds to the control grown in standard non-feeding <i>K</i>. <i>pneumoniae</i> medium. For each condition, the number of replicate experiments is indicated in the last line. In one replicate experiment, wild-type survivors were systematically tested for MAC regeneration (as previously described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005383#pgen.1005383.ref052" target="_blank">52</a>]) and all turned out to be true postautogamous cells.</p><p>RNAi-screening for essential <i>TFIIS</i> genes.</p