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

TRANSLATIONAL STUDIES OF TARGETING THE THROMBOXANE A2 RECEPTOR IN CANCER.

Thromboxane A2 (TXA2) is an eicosanoid formed by the action of thromboxane A2 synthase (TXS) utilizing the cyclooxygenase product, prostaglandin H2, as the substrate. This prostanoid acts mainly by binding to its cognate thromboxane receptor (TP), a G protein coupled receptor (GPCR). GPCRs convey the majority of signal transduction across cell membranes. They are heptahelical serpentine receptors with many clinical implications. Aberrant expression or deregulated activity of GPCRs contributes to some of the most prevalent human diseases. TP activation can cause platelet activation, vasoconstriction, thrombosis and mitogenesis. A single copy of the TP gives rise to TPα and TPβ isoforms, which share the first 328 amino acids but differ at the carboxy-terminal domains. Previous studies in our lab have shown that the TXA2-TPβ signaling axis pathway regulates tumor cell cytoskeleton by inducing cell contraction during migration. The cytoskeleton reorganization observed was mediated through small RhoA GTPase activation. Metastasis is the major cause of death from cancer and acquisition of motility by cancer cells is a detrimental component in metastasis. Therefore, it is important to identify a natural TP antagonist that will serve as a therapeutic regimen for cancer by inhibiting the TPβ induced cell contraction. Our work has shown that SIU1315, a phytoestrogen, blocks cell contraction induced by the TP agonist U46619. The SIU1315 inhibitory concentration (IC50) of U46619 induced contraction was determined to be 10 uM and treatment with the compound alleviated RhoA activation. Moreover, SIU1315 reduces the migratory capacity and invasiveness of PC3 cells. Utilization of the compound to inhibit TP-mediated tumor cell motility and metastatic progression in an in vivo model of experimental metastasis had an unexpected outcome. Administration of SIU1315 in female SCID mice, that received tumor cells via tail vein injection, did not prevent the metastatic spread of malignant cells. On the contrary, mice that had been treated with SIU1315 were indicative of a higher number of metastatic lung lesions than the non-treated control group. Similar evidence was observed by luciferase readouts in other organs. It is suspected that SIU1315 stimulates the vasodilatory nitric oxide that further interferes with the metastatic process. SIU1315 could be used as an anti-metastatic agent in combination with compounds that would suppress activation of nitric oxide. Furthermore, the quest for TPβ specific antibodies, that would have the potential to distinguish between thromboxane receptor isoforms, led to investigation of a number of purified antibodies. Validation of TPβ specific antibodies demonstrated that none of the examined antibodies was specific for TPβ. Determination of the specific TPβ domain that is responsible for U46619 induced cell contraction proved rather difficult. Generation and utilization of TPβ mutants requires additional optimization studies. The function of TP receptors in lung cancer has not been extensively studied. Kaplan-Meier analysis indicated that patients with high levels of TP expression had significantly shorter disease free survival than patients with low TP expression. Generation of stable lung cancer cell lines expressing TP receptors revealed a higher proliferative advantage of cells expressing TPα when compared to the vector control. U46619 induced activation of TP receptors designated a time dependent stimulation of phosphorylated Erk in stable lung cancer cell lines. Evidence suggested the involvement of phosphorylated Akt in U46619 mediated TP activation. Data indicated differential activation of phosphorylated Erk isoforms. The significance of this observation requires further investigation. TP activation seemed to be positively correlated with β-catenin expression. In conclusion, translational studies on the thromboxane receptor reveal major functions in cancer pathogenesis. Therapeutic targeting of the TP receptor could prove beneficial for cancer patients

Quantitation of Murine Stroma and Selective Purification of the Human Tumor Component of Patient-Derived Xenografts for Genomic Analysis.

Patient-derived xenograft (PDX) mouse models are increasingly used for preclinical therapeutic testing of human cancer. A limitation in molecular and genetic characterization of PDX tumors is the presence of integral murine stroma. This is particularly problematic for genomic sequencing of PDX models. Rapid and dependable approaches for quantitating stromal content and purifying the malignant human component of these tumors are needed. We used a recently developed technique exploiting species-specific polymerase chain reaction (PCR) amplicon length (ssPAL) differences to define the fractional composition of murine and human DNA, which was proportional to the fractional composition of cells in a series of lung cancer PDX lines. We compared four methods of human cancer cell isolation: fluorescence-activated cell sorting (FACS), an immunomagnetic mouse cell depletion (MCD) approach, and two distinct EpCAM-based immunomagnetic positive selection methods. We further analyzed DNA extracted from the resulting enriched human cancer cells by targeted sequencing using a clinically validated multi-gene panel. Stromal content varied widely among tumors of similar histology, but appeared stable over multiple serial tumor passages of an individual model. FACS and MCD were superior to either positive selection approach, especially in cases of high stromal content, and consistently allowed high quality human-specific genomic profiling. ssPAL is a dependable approach to quantitation of murine stromal content, and MCD is a simple, efficient, and high yield approach to human cancer cell isolation for genomic analysis of PDX tumors

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

Protocol to dissociate, process, and analyze the human lung tissue using single-cell RNA-seq

We report a protocol for obtaining high-quality single-cell transcriptomics data from human lung biospecimens acquired from core needle biopsies, fine-needle aspirates, surgical resection, and pleural effusions. The protocol relies upon the brief mechanical and enzymatic disruption of tissue, enrichment of live cells by fluorescence-activated cell sorting (FACS), and droplet-based single-cell RNA sequencing (scRNA-seq). The protocol also details a procedure for analyzing the scRNA-seq data. For complete details on the use and execution of this protocol, please refer to Chan et al. (2021)

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 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