In-Vivo Fusion of Human Cancer and Hamster Stromal Cells Permanently Transduces and Transcribes Human DNA
ABSTRACT: The heterotransplantation of human tumors to immune-deprived rodents has become an important preclinical tool for evaluating human tumor biology and responsiveness to therapeutic agents, based on the supposition that they reflect their original human origin. However, we demonstrate that such tumors can in fact change genetically, either by becoming tumors of the animal recipient or by becoming a hybrid of the human tumor and the animal host’s cells. Three human tumors transplanted to the cheek pouch of golden hamsters resulted in permanently transplantable tumors that metastasize in the hamsters. Based on diverse lines of evidence, these transplants from a human glioblastoma multiforme and two Hodgkin lymphomas were malignant hybrids, showing both human and hamster genes in the malignant tumor cells, while retaining the morphology of the original human tumors. We demonstrate that even after being preserved for over 40 years in paraffin blocks, microarray analysis of RNA from these transplants disclosed over 3000 human genes derived from all 23 human chromosome pairs amongst these tumor transplants. A total of 3759 probe sets (ranging from 1040 to 1303 in each transplant) detected human gene transcripts in formalin-fixed, paraffin-embedded sections of the 3 hybrid tumors stored for over 40 years; the probe sets unambiguously map to 3107 unique Human Entrez Gene IDs and are representative of all human chromosomes, although, 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 transcripts from 33 human genes , were detected in all hybrid tumor samples. Five of these 33 genes encode transcription factors that regulate cell growth and differentiation, five encode cell adhesion and transmigration-associated proteins known to participate in oncogenesis and/or metastasis and invasion, and additional genes encode components of pathways involved with signal transduction, regulation of apoptosis, DNA repair, and multidrug resistance. We posit that in-vivo fusion may disclose genes implicated in tumor progression, as well as 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 functions of the donor DNA. Human gene expression profiles were determined by microarray analysis for 3 different Human-Hamster hybrid tumors, two Hodgkin lymphomas (GW-532, GW-584) and a glioblastoma multiforme (GB-749), that were first generated in the hamster cheek pouch after human tumor grafting and then propagated in hamsters and in cell cultures for years. Human gene expression was assessed using total RNA isolated from FFPE samples from GW-532 generations 2 and 34, GW-584 generation 28, and GB-749, in comparison to that for a Hamster control RNA sample isolated from a Hamster melanoma cell line (ATCC CCL-49). Expressed human genes were identified using MAS 5.0 signal expression values and detection P-values in the following manner: 1) Unannotated probe sets, as well as probe sets with no signal value greater than the median signal for AFFX spike-in controls with all Absent Detection Calls, were omitted from further analysis; 2) All remaining signal values for the hamster cell line sample were multiplied by the ratio of the median signal in all FFPE hybrid samples for AFFX spike-in control probe sets called present in all samples divided by the median signal for the same probe sets in the hamster CCL-49 sample; 3) Human transcripts were considered positive in a human-hamster hybrid FFPE sample if (a) a probe set signal exhibited a 2-fold or greater increase in any FFPE hybrid sample compared to the CCL-49 coontrol sample, (b) the fold change was greater than 2 standard deviations for that probe set across the FFPE samples, and (c) was called present (P) or marginal (M) for at least one or more FFPE samples.
Project description:After demonstrating, with karyotyping, polymerase chain reaction (PCR) and fluorescence in-situ hybridization, the retention of certain human chromosomes and genes following the spontaneous fusion of human tumor and hamster cells in-vivo, 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 P-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 (?)-satellite DNA and the F11R transcripts in additional tumor transplant generations. We posit that in-vivo 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 in-vivo cell fusion, genes encoding oncogenic and organogenic traits may be identified.
Project description:We report the in-vivo fusion of two Hodgkin lymphomas with golden hamster cheek pouch cells, resulting in serially-transplanted (over 5-6 years) GW-532 and GW-584 heterosynkaryon tumor cells displaying both human and hamster DNA (by FISH), lymphoma-like morphology, aggressive metastasis, and retention of 7 human genes (CD74, CXCR4, CD19, CD20, CD71, CD79b, and VIM) out of 24 tested by PCR. The prevalence of B-cell restricted genes (CD19, CD20, and CD79b) suggests that this uniform population may be the clonal initiating (malignant) cells of Hodgkin lymphoma, despite their not showing translation to their respective proteins by immunohistochemical analysis. This is believed to be the first report of in-vivo cell-cell fusion of human lymphoma and rodent host cells, and may be a method to disclose genes regulating both organoid and metastasis signatures, suggesting that the horizontal transfer of tumor DNA to adjacent stromal cells may be implicated in tumor heterogeneity and progression. The B-cell gene signature of the hybrid xenografts suggests that Hodgkin lymphoma, or its initiating cells, is a B-cell malignancy.
Project description:Using a human-hamster radiation hybrid panel (G3), we have mapped loci regulating gene expression due to copy number increase. Copy number from radiation hybrid clones were measured relative to the A23 hamster recipient line. Expression from 79 radiation hybrid clones were measured in duplicate and compared to expression of the A23 hamster recipient line (GA_illumina_expression1 and GA_illumina_expression2). Expression of liver, kidney and heart from human and hamster are also included (GA_illumina_expression_R).
Project description:Using a human-hamster radiation hybrid panel (G3), we have mapped loci regulating gene expression due to copy number increase. Overall design: Copy number from radiation hybrid clones were measured relative to the A23 hamster recipient line. Expression from 79 radiation hybrid clones were measured in duplicate and compared to expression of the A23 hamster recipient line (GA_illumina_expression1 and GA_illumina_expression2). Expression of liver, kidney and heart from human and hamster are also included (GA_illumina_expression_R).
Project description:BACKGROUND: We have recently demonstrated that expression profiling is a more accurate and objective method to classify gliomas than histology. Similar to most expression profiling studies, our experiments were performed using fresh frozen (FF) glioma samples whereas most archival samples are fixed in formalin and embedded in paraffin (FFPE). Identification of the same, expression-based intrinsic subtypes in FFPE-stored samples would enable validation of the prognostic value of these subtypes on these archival samples. In this study, we have therefore determined whether the intrinsic subtypes identified using FF material can be reproduced in FFPE-stored samples. METHODS: We have performed expression profiling on 55 paired FF-FFPE glioma samples using HU133 plus 2.0 arrays (FF) and Exon 1.0 ST arrays (FFPE). The median time in paraffin of the FFPE samples was 14.1 years (range 6.6-26.4 years). RESULTS: In general, the correlation between FF and FFPE expression in a single sample was poor. We then selected the most variable probe sets per gene (n=17,583), and of these, the 5000 most variable probe sets on FFPE expression profiles. This unsupervised selection resulted in a better concordance (R(2)=0.54) between expression of FF and FFPE samples. Importantly, this probe set selection resulted in a correct assignment of 87% of FFPE samples into one of seven intrinsic subtypes identified using FF samples. Assignment to the same molecular cluster as the paired FF tissue was not correlated to time in paraffin. CONCLUSION: We are the first to examine a large cohort of paired FF and FFPE samples. We show that expression data from FFPE material can be used to assign samples to intrinsic molecular subtypes identified using FF material. This assignment allows the use of archival material, including material derived from large-randomised clinical trials, to determine the predictive and/or prognostic value of 'intrinsic glioma subtypes' on Exon arrays. This would enable clinicians to provide patients with an objective and accurate diagnosis and prognosis, and a personalised treatment strategy.
Project description:MicroRNAs (miRNAs) are excellent tumor biomarkers because of their cell-type specificity and abundance. However, many miRNA detection methods, such as real-time PCR, obliterate valuable visuospatial information in tissue samples. To enable miRNA visualization in formalin-fixed paraffin-embedded (FFPE) tissues, we developed multicolor miRNA FISH. As a proof of concept, we used this method to differentiate two skin tumors, basal cell carcinoma (BCC) and Merkel cell carcinoma (MCC), with overlapping histologic features but distinct cellular origins. Using sequencing-based miRNA profiling and discriminant analysis, we identified the tumor-specific miRNAs miR-205 and miR-375 in BCC and MCC, respectively. We addressed three major shortcomings in miRNA FISH, identifying optimal conditions for miRNA fixation and ribosomal RNA (rRNA) retention using model compounds and high-pressure liquid chromatography (HPLC) analyses, enhancing signal amplification and detection by increasing probe-hapten linker lengths, and improving probe specificity using shortened probes with minimal rRNA sequence complementarity. We validated our method on 4 BCC and 12 MCC tumors. Amplified miR-205 and miR-375 signals were normalized against directly detectable reference rRNA signals. Tumors were classified using predefined cutoff values, and all were correctly identified in blinded analysis. Our study establishes a reliable miRNA FISH technique for parallel visualization of differentially expressed miRNAs in FFPE tumor tissues.
Project description:Gene expression profiling of tumors is a successful tool for the discovery of new cancer biomarkers and potential targets for the development of new therapeutic strategies. Reliable profiling is preferably performed on fresh frozen (FF) tissues in which the quality of nucleic acids is better preserved than in formalin-fixed paraffin-embedded (FFPE) material. However, since snap-freezing of biopsy materials is often not part of daily routine in pathology laboratories, one may have to rely on archival FFPE material. Procedures to retrieve the RNAs from FFPE materials have been developed and therefore, datasets obtained from FFPE and FF materials need to be made compatible to ensure reliable comparisons are possible.To develop an efficient method to compare gene expression profiles obtained from FFPE and FF samples using the same platform.Twenty-six FFPE-FF sample pairs of the same tumors representing various cancer types, and two FFPE-FF sample pairs of breast cancer cell lines, were included. Total RNA was extracted and gene expression profiling was carried out using Illumina's Whole-Genome cDNA-mediated Annealing, Selection, extension and Ligation (WG-DASL) V3 arrays, enabling the simultaneous detection of 24,526 mRNA transcripts. A sample exclusion criterion was created based on the expression of 11 stably expressed reference genes. Pearson correlation at the probe level was calculated for paired FFPE-FF, and three cut-off values were chosen. Spearman correlation coefficients between the matched FFPE and FF samples were calculated for three probe lists with varying levels of significance and compared to the correlation based on all measured probes. Unsupervised hierarchical cluster analysis was performed to verify performance of the included probe lists to compare matched FPPE-FF samples.Twenty-seven FFPE-FF pairs passed the sample exclusion criterion. From the profiles of 27 FFPE and FF matched samples, the best correlating probes were identified for various levels of significance (Pearson P<0.01, n = 1,432; P<0.05, n = 2,530; and P<0.10, n = 3,351 probes). Unsupervised hierarchical clustering of the 27 pairs using the resulting probes yielded 25, 21, and 19 correctly clustered pairs, respectively, compared to 1 pair when all probes were used.The proposed method enables comparison of gene expression profiles of FFPE and/or FF origin measured on the same platform.
Project description:DNA from archival formalin-fixed and paraffin embedded (FFPE) tissue is an invaluable resource for genome-wide methylation studies although concerns about poor quality may limit its use. In this study, we compared DNA methylation profiles of breast tumors using DNA from fresh-frozen (FF) tissues and three types of matched FFPE samples.For 9/10 patients, correlation and unsupervised clustering analysis revealed that the FF and FFPE samples were consistently correlated with each other and clustered into distinct subgroups. Greater than 84% of the top 100 loci previously shown to differentiate ER+ and ER- tumors in FF tissues were also FFPE DML. Weighted Correlation Gene Network Analyses (WCGNA) grouped the DML loci into 16 modules in FF tissue, with ~85% of the module membership preserved across tissue types.Restored FFPE and matched FF samples were profiled using the Illumina Infinium HumanMethylation450K platform. Methylation levels (?-values) across all loci and the top 100 loci previously shown to differentiate tumors by estrogen receptor status (ER+ or ER-) in a larger FF study, were compared between matched FF and FFPE samples using Pearson's correlation, hierarchical clustering and WCGNA. Positive predictive values and sensitivity levels for detecting differentially methylated loci (DML) in FF samples were calculated in an independent FFPE cohort.FFPE breast tumors samples show lower overall detection of DMLs versus FF, however FFPE and FF DMLs compare favorably. These results support the emerging consensus that the 450K platform can be employed to investigate epigenetics in large sets of archival FFPE tissues.
Project description:We compared the reproducibility of multiple reaction monitoring (MRM) mass spectrometry-based peptide quantitation in tryptic digests from formalin-fixed, paraffin-embedded (FFPE) and frozen clear cell renal cell carcinoma tissues. The analyses targeted a candidate set of 114 peptides previously identified in shotgun proteomic analyses, of which 104 were detectable in FFPE and frozen tissue. Although signal intensities for MRM of peptides from FFPE tissue were on average 66% of those in frozen tissue, median coefficients of variation (CV) for measurements in FFPE and frozen tissues were nearly identical (18-20%). Measurements of lysine C-terminal peptides and arginine C-terminal peptides from FFPE tissue were similarly reproducible (19.5% and 18.3% median CV, respectively). We further evaluated the precision of MRM-based quantitation by analysis of peptides from the Her2 receptor in FFPE and frozen tissues from a Her2 overexpressing mouse xenograft model of breast cancer and in human FFPE breast cancer specimens. We obtained equivalent MRM measurements of HER2 receptor levels in FFPE and frozen mouse xenografts derived from HER2-overexpressing BT474 cells and HER2-negative Sum159 cells. MRM analyses of 5 HER2-positive and 5 HER-negative human FFPE breast tumors confirmed the results of immunohistochemical analyses, thus demonstrating the feasibility of HER2 protein quantification in FFPE tissue specimens. The data demonstrate that MRM analyses can be performed with equal precision on FFPE and frozen tissues and that lysine-containing peptides can be selected for quantitative comparisons, despite the greater impact of formalin fixation on lysine residues. The data further illustrate the feasibility of applying MRM to quantify clinically important tissue biomarkers in FFPE specimens.
Project description:Targeting of the HER2 protein in human breast cancer represents a major advance in oncology but relies on measurements of total HER2 protein and not HER2 signaling network activation. We used reverse-phase protein microarrays (RPMA) to measure total and phosphorylated HER2 in the context of HER family signaling to understand correlations between phosphorylated and total levels of HER2 and downstream signaling activity.Three independent study sets, comprising a total of 415 individual patient samples from flash-frozen core biopsy samples and formalin-fixed and paraffin-embedded (FFPE) surgical and core samples, were analyzed via RPMA. The phosphorylation and total levels of the HER receptor family proteins and downstream signaling molecules were measured in laser capture microdissected (LCM) enriched tumor epithelium from 127 frozen pretreatment core biopsy samples and whole-tissue lysates from 288 FFPE samples and these results were compared with FISH and immunohistochemistry (IHC).RPMA measurements of total HER2 were highly concordant (>90% all sets) with FISH and/or IHC data, as was phosphorylation of HER2 in the FISH/IHC(+) population. Phosphorylation analysis of HER family signaling identified HER2 activation in some FISH/IHC(-) tumors and, identical to that seen with FISH/IHC(+) tumors, the HER2 activation was concordant with EGF receptor (EGFR) and HER3 phosphorylation and downstream signaling endpoint activation.Molecular profiling of HER2 signaling of a large cohort of human breast cancer specimens using a quantitative and sensitive functional pathway activation mapping technique reveals IHC(-)/FISH(-)/pHER2(+) tumors with HER2 pathway activation independent of total HER2 levels and functional signaling through HER3 and EGFR.