<i>In-Vivo</i> Fusion of Human Cancer and Hamster Stromal Cells Permanently Transduces and Transcribes Human DNA

<div><p>After demonstrating, with karyotyping, polymerase chain reaction (PCR) and fluorescence <i>in-situ</i> hybridization, the retention of certain human chromosomes and genes following the spontaneous fusion of human tumor and hamster cells <i>in-vivo</i>, it was postulated that cell fusion causes the horizontal transmission of malignancy and donor genes. Here, we analyzed gene expression profiles of 3 different hybrid tumors first generated in the hamster cheek pouch after human tumor grafting, and then propagated in hamsters and in cell cultures for years: two Hodgkin lymphomas (GW-532, GW-584) and a glioblastoma multiforme (GB-749). Based on the criteria of MAS 5.0 detection <i>P</i>-values ≤0.065 and at least a 2-fold greater signal expression value than a hamster melanoma control, we identified 3,759 probe sets (ranging from 1,040 to 1,303 in each transplant) from formalin-fixed, paraffin-embedded sections of the 3 hybrid tumors, which unambiguously mapped to 3,107 unique Entrez Gene IDs, representative of all human chromosomes; however, by karyology, one of the hybrid tumors (GB-749) had a total of 15 human chromosomes in its cells. Among the genes mapped, 39 probe sets, representing 33 unique Entrez Gene IDs, complied with the detection criteria in all hybrid tumor samples. Five of these 33 genes encode transcription factors that are known to regulate cell growth and differentiation; five encode cell adhesion- and transmigration-associated proteins that participate in oncogenesis and/or metastasis and invasion; and additional genes encode proteins involved in signaling pathways, regulation of apoptosis, DNA repair, and multidrug resistance. These findings were corroborated by PCR and reverse transcription PCR, showing the presence of human alphoid (α)-satellite DNA and the <i>F11R</i> transcripts in additional tumor transplant generations. We posit that <i>in-vivo</i> fusion discloses genes implicated in tumor progression, and gene families coding for the organoid phenotype. Thus, cancer cells can transduce adjacent stromal cells, with the resulting progeny having permanently transcribed genes with malignant and other gene functions of the donor DNA. Using heterospecific <i>in-vivo</i> cell fusion, genes encoding oncogenic and organogenic traits may be identified.</p></div

Clustered heat map of the 39 human probe sets detected in all four hybrid tumor samples.

<p>The heat map depicts expression signal values for 39 Affymetrix Human U133_X3P probe sets detected in FFPE sections from all four hybrids tested (IMM001-004) and a hamster control (IMM006). Prior to unsupervised hierarchical clustering, the MAS 5.0 signal values were log2-transformed and row mean centered. Samples were clustered by Complete Linkage based on Pearson correlation; probe sets were clustered by Complete Linkage based on Euclidean distance. Criteria for detectable human gene expression included MAS 5.0 Detection p-values ≤0.065 in the hybrid sample and >0.065 in the hamster control, and ≥2-fold increased signal in the hybrid sample vs. the hamster control.</p

Characteristics of test articles used in the microarray study.

a<p>Prepared from FFPE specimens as indicated, except IMM006, which was prepared from CCL-49, a Syrian golden hamster melanoma cell line acquired from ATCC.</p>b<p>Human genes of <i>CD74</i>, <i>CXCR4</i>, <i>CD19</i>, <i>CD79b</i>, and <i>VIM</i> were detected by PCR (Ref. 37).</p>c<p>Human genes of <i>CD74</i>, <i>CXCR4</i>, <i>CD20</i>, and <i>CD79b</i> were detected by PCR (Ref. 37).</p>d<p>The expression of CD74, CXCR4 and PLAGL2 were detected by IHC staining (Ref. 36).</p>e<p>Not applicable.</p><p>Characteristics of test articles used in the microarray study.</p

The 39 probe sets determined to be positive in all hybrid FFPE specimens.

<p>The 39 probe sets determined to be positive in all hybrid FFPE specimens.</p

One-step reverse transcription PCR.

<p>The mRNA transcripts of <i>the F11R</i> gene were detectable in GW-532 generation 11 (lane 1), GW-584 generation 3 (lane 2), and the positive control of human HepG2 cells (lane 6), but not in the negative control hamster spleen cells (lane 5). The experimental conditions and the nominal amount of RNA used for each sample are indicated.</p

PCR of human alpha satellite DNA.

<p>The presence of human DNA was demonstrated by the detection of the 171-bp product in GW-532 generation 52 (lane 2), GW-532 generation 82 (lane 3), GB-749 generation 2 (lane 5), and GW-584 generation 3 (lane 6), but not in the negative control of hamster melanoma, CCL-49 (lane 8). The 171-bp and its higher oligomers were detected in the positive control of human Raji lymphoma cells (lane 7). The experimental conditions and the nominal amount of DNA used for each sample are indicated.</p