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

Optimization of the L/VP4 Substrate A.

<p>Alignment of SVV L/VP4 cleavage site (shown in italics) with L/VP4 junction sequences of closely related cardioviruses. The amino acids in each sequence that diverge from the consensus sequence (shown in bold) are highlighted. <b>B.</b> Conversion of modified L/VP4.1 FRET substrates by purified SVV-001 3C^pro over time. Data points represent the average of three replicates at each time point. The endogenous L/VP4 substrate and optimized L/VP4.1 substrate are shown for reference. L/VP4.3 and L/VP4.4 are P4 Y→F substitution and P4 Y→M substitution substrates, respectively. Lines of the same color correspond to curve fits from GraphPad. Error bars on data points were removed for figure clarity. <b>C.</b> Conversion of truncated L/VP4.1 FRET substrates by purified SVV-001 3C^pro over time. Data points represent the average of three replicates at each time point. The endogenous L/VP4 substrate and optimized L/VP4.1 substrate are shown for comparison. L/VP4.5 and L/VP4.6 are P6 and P5/P6 truncations of L/VP4.1, respectively. Lines of the same color correspond to curve fits from GraphPad. Error bars on data points were removed for figure clarity.</p

Conceptual schematic of use of an SVV 3C^pro activated peptide prodrug in combination with SVV virotherapy as a novel form of VDEPT.

<p>SVV infects a fraction of tumor cells (1), producing 3C^pro during the viral life cycle. Upon cell lysis, new SVV virions and 3C^pro are released into nearby tissue (2). Administered peptide prodrug would be excluded from cells by the presence of the attached peptide (3), sparing normal tissues, which are non-permissive and therefore cannot express 3C^pro. The 3C^pro present at high concentration exclusively within the tumor microenvironment cleaves this peptide sequence (4), allowing the cytotoxic moiety to enter both infected and adjacent uninfected cells within the tumor, resulting in a powerful local bystander effect (5).</p

Summary of second order rate constants (k_cat/K_M) for the two endogenous substrates, controls and L/VP4 amino acid substitution/truncation mutants.

<p>The altered substrates are shown with the amino acid(s) substitution(s) or truncations shown in bold. Reported values are the average <i>k</i>_cat/<i>K</i>_M values of 5–7 experiments. Uncertainty is expressed by standard deviation.</p><p>Summary of second order rate constants (k_cat/K_M) for the two endogenous substrates, controls and L/VP4 amino acid substitution/truncation mutants.</p

SVV-001 3C^pro substrates are cleaved in the context of a cellular infection.

<p><b>A.</b> Conversion of FRET substrates by SVV-001 3C^pro produced by a cellular SVV infection of permissive SCLC line, NCI-H446. Data points represent the average of three replicates at each time point. The L/VP4.1 FP substrate incubated with uninfected and infected cells, using similar incubations of NHL FP substrate as a negative control. Lines of the same color correspond to curve fits from GraphPad. Error bars on data points were removed for figure clarity. <b>B.</b> Initial reaction rates of L/VP4.1 peptide cleavage by recombinant SVV-001 3C^pro. Data points represent the initial rate of reaction at each concentration of L/VP4.1 peptide calculated from three replicate experiments. Data points were fit to a Michaelis-Menten nonlinear regression from GraphPad and the kinetic constants determined by the curve fit were reported. Standard deviation values of the kinetics constants were calculated by the GraphPad software and propagated through second order rate constant calculations. <b>C.</b> Proteolysis of CPQ2/5-FAM peptides by native SVV-001 3C^pro in a cellular assay with NCI-H446. Data points represent the average relative fluorescence units (RFUs) increase of three replicates at each time point relative to fluorescence at time zero. The L/VP4.1 FQ peptide was incubated with uninfected and infected cells, using similar incubations of NHL FQ peptide as a negative control. Lines of the same color correspond to connecting line between points.</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

Log scale distribution of GLI1 mRNA expression in a) already published MB cases, along with 1 new case of MB from our repository b) distribution of GLI1 mRNA expression (RNA-Seq data) of the TCGA-GBM sub-cohort (N = 149), c) distribution of GLI1 mRNA expression in NIBMG-GBM cases (N = 19), d) distribution of GLI1 mRNA expression in GBM patient-derived early passage neurospheres (N = 6) and e) comparison of median GLI1 mRNA expression levels of high-Hh-MB (N = 13), low-Hh-MB (N-44), NIBMG-GBM (N = 19) and GBM patient-derived neurospheres (N = 6).

<p>Log scale distribution of GLI1 mRNA expression in a) already published MB cases, along with 1 new case of MB from our repository b) distribution of GLI1 mRNA expression (RNA-Seq data) of the TCGA-GBM sub-cohort (N = 149), c) distribution of GLI1 mRNA expression in NIBMG-GBM cases (N = 19), d) distribution of GLI1 mRNA expression in GBM patient-derived early passage neurospheres (N = 6) and e) comparison of median GLI1 mRNA expression levels of high-Hh-MB (N = 13), low-Hh-MB (N-44), NIBMG-GBM (N = 19) and GBM patient-derived neurospheres (N = 6).</p