The Relevance of Testing the Efficacy of Anti-Angiogenesis Treatments on Cells Derived from Primary Tumors: A New Method for the Personalized Treatment of Renal Cell Carcinoma

<div><p>Despite the numerous available drugs, the most appropriate treatments for patients affected by common or rare renal cell carcinomas (RCC), like those associated with the Xp11.2 translocation/transcription factor for immunoglobulin heavy-chain enhancer 3 (<i>TFE3</i>) gene fusion (TFE3 RCC), are not clearly defined. We aimed to make a parallel between the sensitivity to targeted therapies on living patients and on cells derived from the initial tumor. Three patients diagnosed with a metastatic RCC (one clear cell RCC [ccRCC], two TFE3 RCC) were treated with anti-angiogenesis drugs. The concentrations of the different drugs giving 50% inhibition of cell proliferation (IC50) were determined with the Thiazolyl Blue Tetrazolium Bromide (MTT) assay on cells from the primary tumors and a reference sensitive RCC cell line (786-O). We considered the cells to be sensitive if the IC50 was lower or equal to that in 786-O cells, and insensitive if the IC50 was higher to that in 786-O cells (IC 50 of 6±1 µM for sunitinib, 10±1 µM for everolimus and 6±1 µM for sorafenib). Based on this standard, the response in patients and in cells was equivalent. The efficacy of anti-angiogenesis therapies was also tested in cells obtained from five patients with non-metastatic ccRCC, and untreated as recommended by clinical practice in order to determine the best treatment in case of progression toward a metastatic grade. <i>In vitro</i> experiments may represent a method for evaluating the best first-line treatment for personalized management of ccRCC during the period following surgery.</p></div

Clinical and genetic characteristics of the metastatic and non-metastatic patients.

<p>Metastatic patients are presented on a black background; non metastatic patients are presented on a grey background.</p

Structural and functional analysis of coral Hypoxia Inducible Factor

<div><p>Tissues of symbiotic Cnidarians are exposed to wide, rapid and daily variations of oxygen concentration. Indeed, during daytime, intracellular O₂ concentration increases due to symbiont photosynthesis, while during night, respiration of both host cells and symbionts leads to intra-tissue hypoxia. The Hypoxia Inducible Factor 1 (HIF-1) is a heterodimeric transcription factor used for maintenance of oxygen homeostasis and adaptation to hypoxia. Here, we carried out a mechanistic study of the response to variations of O₂ concentrations of the coral model <i>Stylophora pistillata</i>. <i>In silico</i> analysis showed that homologs of HIF-1 α (SpiHIF-1α) and HIF-1β (SpiHIF-1β) exist in coral. A specific SpiHIF-1 DNA binding on mammalian Hypoxia Response Element (HRE) sequences was shown in extracts from coral exposed to dark conditions. Then, we cloned the coral HIF-1α and β genes and determined their expression and transcriptional activity. Although HIF-1α has an incomplete Oxygen-dependent Degradation Domain (ODD) relative to its human homolog, its protein level is increased under hypoxia when tested in mammalian cells. Moreover, co-transfection of SpiHIF-1α and β in mammalian cells stimulated an artificial promoter containing HRE only in hypoxic conditions. This study shows the strong conservation of molecular mechanisms involved in adaptation to O₂ concentration between Cnidarians and Mammals whose ancestors diverged about 1,200–1,500 million years ago.</p></div

<i>In vitro</i> sensitivity of the TF, CC and M cells to targeted therapies.

<p>The IC50 of the different drugs was determined using the MTT test with concentrations of the drugs ranging from 0.1 µM to 100 µM in 786-O (786), TF, CC and M cells. The means SEMs and p-values (Student t test), in comparison with the IC50 for 786-O cells, the reference value, are shown. *p<0.05; **p<0.01 (Student t test).</p

Level of expression of the different receptors targeted by sunitinib and sorafenib.

<p>(−) indicates that no or very low levels of detectable amplified cDNA were observed on an agarose gel; (+) indicates amplified cDNA. The <i>Score/8</i> indicates the presence of receptorsargeted by sunitinib or sorafenib. The <i>E Score</i> indicates the relative expression level as detected by quantitative and semi-quantitative PCR. A level of sensitivity to sunitinib similar to that of 786-O cells is indicated by a grey background; sensitivity only to higher concentrations of the drug is indicated by a black background.</p

Characterization of TFE3 expression and <i>TFE3</i> rearrangement in the initial tumor and in TF cells.

<p>A) Immunohistochemical staining for TFE3 of the initial tumor. Labeling with anti-TFE3 antibodies was also performed on cells from passages 14 and 16 (P14 and P16 TFE3 cells) embedded in paraffin. TFE3 labeling was also performed on ccRCC cells cultured under the same conditions as TF cells. ccRCC cells served as a negative control. Note the cytoplasmic background instead of only nuclear labeling. B) <b>Image a</b>: An uncultured cell suspension from the renal cell tumor hybridized with a dual-color break-apart FISH probe framing <i>TFE3</i>. A rearrangement of <i>TFE3</i> in the upper nucleus (tumor cell) is observed with BAC probes CTD-2534B7 (red signal; 3′ side of <i>TFE3</i>) and CTD-3009K20 (green signal; 5′ side of TFE3). The red and green signals are clearly separated as a result of the pericentric inversion of chromosome X (thin arrows). Two rearranged signals are observed because the abnormal chromosome X is duplicated in tumor cells. In contrast, red and green signals are closely juxtaposed in the two normal nuclei that contain one X chromosome, respectively (thick arrows). <b>Image b</b>: A normal partial metaphase cell hybridized with a dual color break-apart BAC FISH probe framing <i>TFE3</i>. BAC probes CTD-2534B7 (red signal; 3′ side of <i>TFE3</i>) and CTD-3009K20 (green signal; 5′ side of <i>TFE3</i>) are closely juxtaposed at Xp11.23 on the short arm of the X chromosome. <b>Image c</b>: A partial abnormal tumor metaphase cell (cell line, passage 9) hybridized with a dual color break-apart FISH probe framing <i>TFE3</i>. As a result of the X chromosome pericentric inversion, BAC probe CTD-3009K20 (green signal; 5′ side of <i>TFE3</i>) is translocated from its normal location at Xp11.23 to <i>NONO</i> locus at Xq13.1 on the long arm of the X chromosome. BAC probes CTD-2534B7 (red signal; 3′ side of <i>TFE3</i>) remains at the original <i>TFE3</i> locus at Xp11.23. <b>Image d</b>: A partial abnormal tumor metaphase cell (cell line, passage 9) hybridized with a dual-color break-apart FISH probe framing <i>NONO</i>. As a result of the X chromosome pericentric inversion, BAC probe RP11-753F2 (green signal; 5′ side of <i>NONO</i>) is translocated from its normal location at Xq13.1 to <i>TFE3</i> locus at Xp11.23 on the short arm of the X chromosome. BAC probes RP11-624G23 (red signal; 3′ side of <i>NONO</i>) remains at the original <i>NONO</i> locus at Xq13.1. C) Western blot analysis of the presence of TFE3 in cells from the “TFE3” tumor, in ccRCC 786-O cells and ccRCC cells obtained from an independent tumor. 786-O and ccRCC cells served as negative controls. ERK served as a loading control.</p

SpiHIF promotes human HRE transcriptional activation.

<p>HEK293 were transfected either with luciferase reporter plasmid together with empty, <i>spiHIF</i>α, <i>spiHIFβ</i>, or a combination of both <i>spiHIF</i> expression vectors. In all cases, a control Renilla expression vector was co-transfected to normalize for transfection efficiency. 24h after transfection, cells were maintained under normoxic or hypoxic conditions in an anaerobic workstation for 24h. Cells were lyzed and firefly and renilla luciferase activity were measured. A ratio of firefly/renilla luciferase activity is shown. Results are representative of three independent experiments performed in triplicate. Values are means ± SD (n = 3). * P< 0.05.</p

Wild-type C1845-induced gene expression

<p>Cells were infected for four hours with Afa/Dr DAEC wild-type strain C1845 and total RNA extracted. Selected gene expression was assayed by q-PCR. The oligonucleotides used in this study were designed by Applied Biosystems, the manufacturer's references are given in brackets. Abbreviations are as follows: VEGF: Vascular Endothelial Growth Factor, IL8: interleukin 8, PlGF: Placental growth factor, TGFβ R2: Transforming growth factor Receptor 2, EGF R1: Epithelial growth factor Receptor 1. Results (means±SD) are representative of independent experiments (where n represents the number of experiments). *, p<0.001 versus non infected cells.</p

Proliferative response, Erk activation and tube like structure formation in HUVEC in the presence of culture medium of wild-type C1845-infected T84 cells.

<p>³H-thymidine incorporation into DNA and Erk activation were essayed in HUVEC incubated with the following. In A, ³H-thymidine incorporation into DNA. Control (1), VEGF at 10 ng/ml (2), culture medium of non-infected T84 cells (3), culture medium of wild-type C1845-infected T84 cells (1×) (4), culture medium of wild-type C1845-infected T84 cells (0.5×) (5). In B, Western blot showing phospho-Erk1/2 and total Erk1/2 visualized with respectively anti-phospho-Erk and anti-Erk antibodies. Control (1), VEGF at 10 ng/ml (23), culture medium of wild-type C1845-infected T84 cells (1×) (3), culture medium of wild-type C1845-infected T84 cells (0.5×) (4). The results are representative of at least three independent experiments. *, p<0,01. In C, Endothelial cells were seeded on matrigel and incubated with growing medium (control) alone or supplemented with T84 conditionned medium (CM), C1845–condition medium (C1845) or VEGF. Images show that only C1845-conditionned medium (C1845) and VEGF allow the alignment of endothelial cells in tube like structure.</p

C1845 bacteria increase VEGF expression in cultured intestinal epithelial cells.

<p>In A, increase in VEGF mRNA expression. Confluent serum-starved T84 cells (5×10⁶ cells/well) were infected with wild-type C1845 bacteria and total RNA was prepared as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001359#s4" target="_blank">Materials and Methods</a> section. Results for non-infected (NI) or cells infected with 5×10⁷ CFU/ml C1845 bacteria for the indicated time are shown on the left panel. The dose response effect is presented on the right. Cells were infected for four hours with the indicated number of bacteria. Results of Northern blots are presented in the upper part of the Figure and q-PCR shown in the lower part. These results are representative of three independent experiments. In B, confluent serum-starved LS174, Caco-2/TC7 or INT407 cells were infected with 5×10⁷ CFU/ml wild-type C1845 bacteria for four hours. VEGF mRNA expression was assayed by q-PCR. The signal corresponding to VEGF and 36B4 transcripts was quantified using a phosphoImager. Under each condition the signal was normalized to the 36B4 probe. Results are expressed as arbitrary units corresponding to the fold stimulation of treated versus non-treated conditions. In C, increase in the VEGF protein in the culture medium of wild-type C1845-infected T84 cells. Cells were infected with 5×10⁷ CFU/ml wild-type C1845 bacteria for four hours and the supernatant were collected as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001359#s4" target="_blank">Materials and Methods</a> section. The VEGF protein level was then quantified using an ELISA. Quantification of the results from two independent experiments (means±SD) is shown. *, p<0,01</p