第647回千葉医学会例会・第5回千葉大学小児外科教室例会 6.

<p>(A) A schematic describing the experimental process used to test the four cell separation techniques. A single tumor from each PDX line was processed using a gentleMACS^™ Octo Dissociator to obtain a single cell suspension comprising a heterogeneous mixture of human cells (blue) and mouse cells (orange). The suspension was divided in five samples. The pre-sort sample received no further processing. The remaining 4 tubes were processed using each method to obtain separate human and mouse cell populations. Each resulting human fraction and the pre-sort sample were analyzed using ssPAL. (B) ssPAL analysis results for primer pair 5 and 43 were averaged together to obtain the human DNA percentage for each separation method. After performing ssPAL analysis, results indicate that on average, MCD yields the highest sample purity over FACS, EpCAM-M and EpCAM-SC. The starting amount of murine stromal contamination also influences the effectiveness of the kit used, with EpCAM-M and EpCAM-SC performing poorly with higher initial murine content. Error bars represent standard error.</p

PDX mouse model tumor transplantation schema and passage over time.

<p>(A) This schematic summarizes our protocol for PDX generation, implantation, and passaging. Tumor tissue is collected from the patient and prepped into a single cell suspension using the gentleMACS^™ Octo Dissociator. Cells are mixed 1:1 with matrigel and implanted single flank in immunosuppressed mice. Once tumor reaches end volume and is ready for passage; it is collected, processed into a single cell suspension, then cells are re-implanted in the next set of mice. (B) ssPAL analysis reveals that PDX tumors do not show significant variation in murine stromal content over successive passages. MSK-LX242 and MSK-LX29 are lung adenocarcinoma PDX lines, MSK-LX95 is a SCLC PDX line, and MSK-LX96 is a mesothelioma PDX line. Error bars represent standard error. P0 = Passage 0, P1 = Passage 1, P2 = Passage 2, etc.</p

ssPAL analysis yields precise measurements with accuracy comparable to FACS.

<p>(A) After performing capillary electrophoresis, the presence of each PCR product (human and murine) for both primer pairs is evaluated. The peak at 206 bp corresponds to the murine fraction (orange), the peak at 211 bp correspond to the human fraction (blue). The resulting peak areas are proportional to the murine and human DNA content in a given sample. (B) ssPAL analysis is performed using two primer pairs (5 and 43) that amplify homologous regions of the mouse and human genome. This technique can accurately detect the percentages of murine DNA in pre-set mixtures of NIH 3T3 and Jurkat cells DNA within a range of 1% to 99%. Sensitivity is lost when analyzing values outside of this range. (C) FACS is the gold standard to separate human and murine cells and quantify the percentage of each population. In this representative plot, a PDX tumor from line MSK-LX29 is sorted using EpCAM and H-2K^d. (D) Murine DNA content determined by ssPAL is proportional to murine cell content measured by FACS.</p

ssPAL analysis detects spontaneous murine tumor formation.

<p>ssPAL analysis detects spontaneous murine tumor formation.</p

ssPAL analysis highlights significant murine stromal content variation across multiple lung PDX lines.

<p>(A) The ssPAL analysis results for primer pairs 5 and 43 were averaged. Murine stromal contamination exhibits a wide range between PDX lines. (B) While stromal contamination is variable between PDX lines, it is consistent between tumors of the same cancer subtype. Stromal content in SCLC PDX was significantly lower than in LUAD (two-tailed Student’s <i>t</i>-test). LUAD = lung adenocarcinoma, SCLC = small cell lung cancer, PLMESO = pleural mesothelioma, LUSC = lung squamous carcinoma.</p

Additional file 5 of Genomic and transcriptomic analysis of a diffuse pleural mesothelioma patient-derived xenograft library

Additional file 5: Table S2. Mapping statistics for RNA-Seq datase

Additional file 7 of Genomic and transcriptomic analysis of a diffuse pleural mesothelioma patient-derived xenograft library

Additional file 7: Figure S8. Patient outcomes based on clinical benefit of platinum-based chemotherapy

Additional file 6 of Genomic and transcriptomic analysis of a diffuse pleural mesothelioma patient-derived xenograft library

Additional file 6: Figure S7. Gene expression changes in TCGA mesothelioma tumors as a function of consensus histology

Additional file 3 of Genomic and transcriptomic analysis of a diffuse pleural mesothelioma patient-derived xenograft library

Additional file 3: Figure S2-S5. Detailed annotation of comparative histology of available patient samples and PDX models

Additional file 1 of Genomic and transcriptomic analysis of a diffuse pleural mesothelioma patient-derived xenograft library

Additional file 1: Table S1. Patient demographics at the time of PDX collection by histology