Project description: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 hostâs 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:We treated 6-8 week old mice that had floxed alleles of both Apc and Pten (for both alleles in each case) that also carry an Ovgp1-iCre-ERT2 transgene, with one of two treatments; a third group received neither treatment. The Ovgp1-iCre-ERT2 expresses Cre recombinase fused to a tamoxifen-inducible fragment of the estrogen receptor, in tissues where the Ovgp1 gene (oviductal glycoprotein 1) is expressed, which is almost exclusively in mouse oviductal epithelium (equivalent to human fallopian tube epithelium = FTE). Treating the mice with tamoxifen permits the Cre recombinase to enter the cell nucleus and inactivate the Apc and Pten genes. Six of the mice were treated with intraperitoneal injection of tamoxifen (0.1g/kg of body weight) dissolved in corn oil on days 1 and 3 and developed oviductal tumors (OdT) yielding 6 of the samples. Four mice (yielding 5 samples) were instead injected with 50 million plaque-forming units of replication-incompetent AdCre into both ovarian bursal cavities on day 1, which inactivated Apc and Pten in the ovarian surface epithelium (OSE), and lead to ovarian tumors (OT). Ovaries were also harvested from four untreated 6-8 week old mice with the same genotype, with two ovaries from each mouse comprising one control sample. RNA was purified from tumor or normal tissue, and targets for Affymetrix arrays synthesized from the mRNAs. We used Affymetrix Mouse Gene 2.1 ST arrays, which hold 41345 probe-sets, but we largely analyzed just those 25216 probe-sets that were mapped to Entrez gene IDs. Raw data was processed with Robust Multi-array Average algorithm (RMA). Data is log2-transformed transcript abundance estimates. We fit a one-way ANOVA model to the three groups of samples. We supply a supplementary excel workbook that holds the same data as the data matrix file, but also holds the probe-set annotation at the time we analyzed the data, and some simple statistical calculations, which select subsets of the probe-sets as differentially expressed. Consumers should consider obtaining more up-to-date probe-set annotation for the array platform. We also provide supplementary excel files that show our simple analysis of GSE6008, which consists of 99 human ovarian tumor samples of 4 types, and 4 normal ovary samples, where we fit an ANOVA model to the 5 groups. In yet another supplementary file we show the correlation between each human tumor and mouse tumor, where we correlate the difference in log2-transformed values of each tumor from the average of the normals for the same species, for just those genes that were 1-to-1 best homologs according to build 68 of NCBI's Homologene, in order to see how much the human tumors resemble the mouse tumors.

Project description:We treated 6-8 week old mice that had floxed alleles of both Apc and Pten (for both alleles in each case) that also carry an Ovgp1-iCre-ERT2 transgene, with one of two treatments; a third group received neither treatment. The Ovgp1-iCre-ERT2 expresses Cre recombinase fused to a tamoxifen-inducible fragment of the estrogen receptor, in tissues where the Ovgp1 gene (oviductal glycoprotein 1) is expressed, which is almost exclusively in mouse oviductal epithelium (equivalent to human fallopian tube epithelium = FTE). Treating the mice with tamoxifen permits the Cre recombinase to enter the cell nucleus and inactivate the Apc and Pten genes. Six of the mice were treated with intraperitoneal injection of tamoxifen (0.1g/kg of body weight) dissolved in corn oil on days 1 and 3 and developed oviductal tumors (OdT) yielding 6 of the samples. Four mice (yielding 5 samples) were instead injected with 50 million plaque-forming units of replication-incompetent AdCre into both ovarian bursal cavities on day 1, which inactivated Apc and Pten in the ovarian surface epithelium (OSE), and lead to ovarian tumors (OT). Ovaries were also harvested from four untreated 6-8 week old mice with the same genotype, with two ovaries from each mouse comprising one control sample. RNA was purified from tumor or normal tissue, and targets for Affymetrix arrays synthesized from the mRNAs. We used Affymetrix Mouse Gene 2.1 ST arrays, which hold 41345 probe-sets, but we largely analyzed just those 25216 probe-sets that were mapped to Entrez gene IDs. Raw data was processed with Robust Multi-array Average algorithm (RMA). Data is log2-transformed transcript abundance estimates. We fit a one-way ANOVA model to the three groups of samples. We supply a supplementary excel workbook that holds the same data as the data matrix file, but also holds the probe-set annotation at the time we analyzed the data, and some simple statistical calculations, which select subsets of the probe-sets as differentially expressed. Consumers should consider obtaining more up-to-date probe-set annotation for the array platform. We also provide supplementary excel files that show our simple analysis of GSE6008, which consists of 99 human ovarian tumor samples of 4 types, and 4 normal ovary samples, where we fit an ANOVA model to the 5 groups. In yet another supplementary file we show the correlation between each human tumor and mouse tumor, where we correlate the difference in log2-transformed values of each tumor from the average of the normals for the same species, for just those genes that were 1-to-1 best homologs according to build 68 of NCBI's Homologene, in order to see how much the human tumors resemble the mouse tumors. Six oviductal tumor samples from 6 mice with conditional knockout of Apc and Pten driven by an Ovgp1-iCre-ERT2 transgene , 5 ovarian tumor samples from 4 mice with conditional knockout of Apc and Pten driven by injection of AdCre into both ovarian bursal cavities, and 4 normal ovary samples from 4 mice.

Project description:To identify a prognostic gene signature accounting for the distinct clinical outcomes in ovarian cancer patients Despite the existence of morphologically indistinguishable disease, patients with advanced ovarian tumors display a broad range of survival end points. We hypothesize that gene expression profiling can identify a prognostic signature accounting for these distinct clinical outcomes. To resolve survival-associated loci, gene expression profiling was completed for an extensive set of 185(90 optimal/95 suboptimal) primary ovarian tumors using the Affymetrix human U133A microarray. Cox regression analysis identified probe sets associated with survival in optimally and suboptimally debulked tumor sets at a P value of <0.01. Leave-one-out cross-validation was applied to each tumor cohort and confirmed by a permutation test. External validation was conducted by applying the gene signature to a publicly available array database of expression profiles of advanced stage suboptimally debulked tumors. The prognostic signature successfully classified the tumors according to survival for suboptimally (P = 0.0179) but not optimally debulked (P = 0.144) patients. The suboptimal gene signature was validated using the independent set of tumors (odds ratio, 8.75; P = 0.0146). To elucidate signaling events amenable to therapeutic intervention in suboptimally debulked patients, pathway analysis was completed for the top 57 survival-associated probe sets. For suboptimally debulked patients, confirmation of the predictive gene signature supports the existence of a clinically relevant predictor, as well as the possibility of novel therapeutic opportunities. Ultimately, the prognostic classifier defined for suboptimally debulked tumors may aid in the classification and enhancement of patient outcome for this high-risk population. Gene expression profiling was completed for an extensive set of 185 primary ovarian tumors and 10 normal ovarian surface epithelium using the Affymetrix human U133A microarray

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In-Vivo Fusion of Human Cancer and Hamster Stromal Cells Permanently Transduces and Transcribes Human DNA

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