17 research outputs found
Rat stromal cells or a combination of tumor and stromal cells have a growth advantage in serum free cultures.
<p><b>(a)</b> FISH with an <i>EGFR</i>/Chromosome 7 probe in red and green, respectively, and immunohistochemical staining with antibodies against EGFR. In P8 cultures, both EGFR amplification and the chromosome 7 probe are not detectable after two months. In P6 cultures, EGFR amplification is preserved in spheroids (dotted line), while both EGFR amplification and the chromosome 7 probe are not detectable in single cells. EGFR expression is lost in P8 cultures, while it is still detectable at a low expression level in spheroids from P6 cultures. Quantification of EGFR expressing cells in three random high power (400×) microscopic view fields (HPF) in each group. Asterix indicates 0%. Values represent mean ± s.d. <b>(b)</b> Immunohistochemical staining with antibodies against human-specific and rat-specific nestin. In P8 cultures, rat-nestin positive stromal cells have a growth advantage over human cells. In P6 cultures, a mix of human and rat cells survive. Human cells form spheroids, while single cells are rat cells. Scale bars 50 µm. Quantification of rat and human nestin expressing cells in three random high power (400×) microscopic view fields (HPF) in each group. Asterix indicates 0%. Values represent mean ± s.d.</p
Tumor versus Stromal Cells in Culture—Survival of the Fittest?
<div><p>Two of the signature genetic events that occur in human gliomas, <i>EGFR</i> amplification and <i>IDH</i> mutation, are poorly represented in experimental models <i>in vitro</i>. <i>EGFR</i> amplification, for example, occurs in 40 to 50% of GBM, and yet, <i>EGFR</i> amplification is rarely preserved in cell cultures derived from human tumors. To analyze the fate of <i>EGFR</i> amplified and <i>IDH</i> mutated cells in culture, we followed the development over time of cultures derived from human xenografts in nude rats enriched for tumor cells with <i>EGFR</i> amplification and of cultures derived from patient samples with <i>IDH</i> mutations, in serum monolayer and spheroid suspension culture, under serum and serum free conditions. We observed under serum monolayer conditions, that nestin positive or nestin and SMA double positive rat stromal cells outgrew <i>EGFR</i> amplified tumor cells, while serum spheroid cultures preserved tumor cells with <i>EGFR</i> amplification. Serum free suspension culture exhibited a more variable cell composition in that the resultant cell populations were either predominantly nestin/SOX2 co-expressing rat stromal cells or human tumor cells, or a mixture of both. The selection for nestin/SMA positive stromal cells under serum monolayer conditions was also consistently observed in human oligodendrogliomas and oligoastrocytomas with <i>IDH</i> mutations. Our results highlight for the first time that serum monolayer conditions can select for stromal cells instead of tumor cells in certain brain tumor subtypes. This result has an important impact on the establishment of new tumor cell cultures from brain tumors and raises the question of the proper conditions for the growth of the tumor cell populations of interest.</p></div
EGFR amplification and expression is preserved in serum spheroid cultures and “lost” in monolayer cultures.
<p>Cultured spheroids or single cells were coagulated, fixed with 4% formalin, and embedded in paraffin. FISH with an <i>EGFR</i>/Chromosome 7 probe in red and green, respectively, and immunohistochemical staining with antibodies against EGFR. In monolayer culture, both EGFR amplification and the chromosome 7 probe are not detectable after two months in cultures from both xenografts. Immunhistochemical staining for EGFR is also negative. Scale bars 50 µm. Quantification of EGFR expressing cells in three random high power (400×) microscopic view fields (HPF) in each group. Asterix indicates 0%. Values represent mean ± s.d.</p
Rat stromal cells have a selective growth advantage over EGFR amplified cells in serum monolayer culture.
<p>Cultured spheroids or single cells were coagulated, fixed with 4% formalin, and embedded in paraffin. Immunohistochemical staining was performed with antibodies against human-specific and rat-specific nestin. After two months, nestin-expressing rat cells take over at expense of human tumor cells in serum monolayer culture. As expected, nestin-expressing human cells are predominant in serum spheroid cultures. Scale bars 50 µm. Quantification of human and rat nestin expressing cells in three random high power (400×) microscopic view fields (HPF) in each group. Asterix indicates 0%. Values represent mean ± s.d.</p
Characterization of rat stromal cells <i>in vitro</i> and <i>in vivo</i>.
<p><b>(a)</b> Rat stromal cells from P8 NBM, and P8 and P6 monolayer cultures were stained with antibodies against GFAP, beta-tubulin III, SOX2 and SMA. Rat cells from P8 NBM cultures are positive for SOX2 and negative for SMA, while monolayer cells are negative for SOX2 and positive for SMA. <b>(b)</b> Xenografts were stained with antibodies against rat nestin, SOX2 and SMA. Rat nestin is positive on tumor vessels and single cells in human <i>EGFR</i> amplified GBM xenografts <i>in vivo</i>. Double staining for rat nestin and SOX2 shows rat nestin+/SOX- vessels and rat nestin+/SOX2+ single cells (arrow). SMA is positive on vessels. Scale bars 50 µm.</p
Genomic profiles are normal in monolayer cell populations derived from human oligodendrogliomas.
<p><b>(a)</b> Sanger sequencing demonstrates that the <i>IDH1</i> mutation of the primary tumor is not preserved in the derived monolayer cell population. <b>(b)</b> Immunostaining with antibodies against IDH1 R132H confirms that monolayer cells are negative and the primary biopsy strongly positive. <b>(c)</b> aCGH profiles for the derived cell populations and the matching human oligodendroglioma are shown. Data is shown in log<sub>2</sub> space across the genome where log<sub>2</sub> 0 (at the midline) indicates the normal diploid copy number. Asterisks (*) highlight 1p and 19q losses that are typical of oligodendroglioma.</p
Characterization of stromal cells derived from human oligodendroglioma <i>in vitro</i> and <i>in vivo</i>.
<p><b>(a)</b> Human stromal cells from oligodendrogliomas, cultured under monolayer conditions are positive for nestin and SMA. Only few single cells are positive for GFAP. <b>(b)</b> Nestin is localized to reactive astrocytes and the vasculature in human oligodendroglioma samples <i>in vivo</i>. SMA is positive on the vasculature. Scale bars 50 µm.</p
Oligodendroglioma cells produce 2HG in culture.
<p>Concentrations of 2HG in the medium from spheres and non-dissociated tissue from the xenograft relative to spheres from a freshly dissociated GBM without <i>IDH</i> mutation were measured over time. Measurements were made in triplicate. The decrease in 2HG concentrations is concomitant with the removal of approximately 50% of the spheres for implantation in between the 21- and 28-day time points.</p
Astrocytes intermingled in the tumor bed of both the primary tumor and the xenograft.
<p>(A) Sections of the primary human tumor and xenograft were immunostained for nestin, Iba1, and GFAP to observe infiltration by neural stem or progenitor cells, microglia/macrophages, and astrocytes, respectively. Inset in Iba1 staining displays morphology of microglia on the contra-lateral side of the mouse xenograft at the same magnification. CXCL12 staining was performed on sections from the xenograft shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059773#pone-0059773-g003" target="_blank">Figure 3B and E</a>. Vessels from the primary tumor and xenograft are shown as well as (B) the subventricular zone and (C) the hippocampus of the xenograft. Scale bars 100 μm.</p
Serial passaging of the xenograft.
<p>(A) T2 weighted MRI of affected first generation animal; (B) spheres obtained from dissociated tumor from a eGFP NOD/SCID positive mouse; (C) Kaplan-Meier survival curve of animals with oligodendroglioma when sacrificed in days. All animals were allowed to live until symptomatic or death. Animals without any evidence of oligodendroglioma were not included in the curve. The animal at 374 days in the first generation was included as a censured event because the animal was sacrificed due to the positive MRI rather than being symptomatic. White arrows highlight the implantation site in A and individual eGFP positive cells within spheres in B. Black arrow in A highlights the midline. Blue and black boxes in C represent animals from first and second generations, respectively. Scale bars 100 μm.</p