Journal: Materials Today Bio

Article Title: Stromal homeostasis-restoring “rocket-like” nanomedicine inhibited pancreatic tumor growth in vivo

doi: 10.1016/j.mtbio.2026.103014

Figure Lengend Snippet: In vitro evaluation of Panc-1 and Pan02 cells after different treatments. (A) Colony formation assay of Panc-1 and Pan02 cells after different treatments. (B) Quantification of colony numbers of Panc-1 and Pan02 cells under the indicated treatments. (C) Representative images of cell migration of Panc-1 and Pan02 cells after different treatments. (D) Quantification of residual area of Panc-1 and Pan02 cells in each group. (E) ROS fluorescence intensity of Panc-1 cells after different treatments. (F) ROS fluorescence intensity of Pan02 cells after different treatments. (G) Viability of Panc-1 cells co-cultured with L929 cells in a transwell system after different treatments. (H) Viability of Pan02 cells co-cultured with L929 cells in a transwell system after different treatments. (I) Representative CLSM images of Panc-1 cells co-stained with Calcein-AM (green) and PI (red) after treatment with different groups (scale bar: 200 μm). (J) Representative CLSM images of Pan02 cells co-stained with Calcein-AM (green) and PI (red) after treatment with different groups. (K) Immunofluorescence staining of uPA in Panc-1 cells after different treatments.(scale bar:100 μm). (L) Immunofluorescence staining of uPA in Pan02 cells after different treatments. Data are presented as mean ± standard deviation (SD), n = 3. Statistical significance was analyzed by one-way ANOVA with t -test; ns, not significant; ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Triethylamine (TEA, Sigma-Aldrich); Cetyltrimethylammonium bromide (CTAB, Xinyanbomei); Sodium salicylate (NaSal, Sigma-Aldrich); Tetraethyl orthosilicate (TEOS, CATO); 1,2-Bis(triethoxysilyl)ethane (BTES, Xinhengyan); Ethanol; Hydrochloric acid; Methanol; Gemcitabine (MedChemExpress); Ammonium bicarbonate (Coolaber); Urokinase-type plasminogen activator (Solarbio); CCK-8 kit (CWBIO); ROS staining kit (Poolyue); Calcein-AM/PI kit (DOJINDO); anti-uPA antibody (HUABIO); Calcium chloride (Supelco); Indocyanine green (zrbiorise); Dulbecco's modified Eagle medium (DMEM, Sigma-Aldrich); Panc-1, Pan02, and L929 cells (ATCC); Fluorescein isothiocyanate (FITC, Qisong); 4′,6-diamidino-2-phenylindole (DAPI, Solarbio); DUTP (Roche); IPR-803 (MCE).

Techniques: In Vitro, Colony Assay, Migration, Fluorescence, Cell Culture, Staining, Immunofluorescence, Standard Deviation

Journal: The Journal of Clinical Investigation

Article Title: Simultaneous CRISPR/Cas9-induced double-strand breaks are lethal in models of pancreatic cancer

doi: 10.1172/JCI190121

Figure Lengend Snippet: ( A ) Clonogenic survival with increased number of CRISPR/Cas9 target sites in the human genome of 2 PC cell lines. Number of target sites in parentheses; “rep” indicates repetitive element-targeting. N = 3; mean ± SEM, normalized to NT. ( B ) Cell survival with increased number of CRISPR/Cas9 target sites as detected by alamar blue cell viability assay. N = 3; mean ± SEM, normalized to NT. ( C ) Representative images of γH2A.X staining in Panc10.05 cells transduced with NT or 715F(5) or 230F(12) multitarget sgRNAs. Images at 40× original magnification; scale bar is 5 μm. N = 3. ( D ) Number of γH2A.X foci as a function of the number of CRISPR/Cas9 target sites. >100 nuclei were analyzed for each condition. Dunnett’s test between NT and each multitarget sgRNA; *** P < 0.001, **** P < 0.0001. N = 3; mean ± SEM. ( E ) Clonogenic and cell survival 21 days after electroporating in CRISPR/Cas9 with multitarget sgRNAs or a pool of 5 sgRNAs targeting different noncoding mutations in the Panc10.05 genome. N = 2/3; mean ± SEM, normalized to NT.

Article Snippet: With the exception of Panc10.05, which is an ATCC line, all other cell lines and their derivatives (including Panc10.05-derived lines) are available through Material Transfer Agreements.

Techniques: CRISPR, Viability Assay, Staining, Transduction

Journal: The Journal of Clinical Investigation

Article Title: Simultaneous CRISPR/Cas9-induced double-strand breaks are lethal in models of pancreatic cancer

doi: 10.1172/JCI190121

Figure Lengend Snippet: ( A – C ) Tumor growth experiment in subcutaneous xenograft models. Panc10.05 Cas9-expressing cells transduced with the following sgRNAs: NT, 715F(5), 230F(12), or a pool of 9 sgRNAs targeting different noncoding mutations unique to Panc10.05 (Panc10.05 pool) were injected into nude mice for tumor growth. ( A ) Percentage of tumors present postxenograft. # indicates absence of 2 data points due to early death around week 5 (33–36 days). ( B ) Tumor volume measurements postxenograft. Dunn-Šidák test between NT and the other treatment groups on week 6, all **** P < 0.0001. N = 10; mean ± SEM. & indicates absence of week 5 and 6 data points of 2 tumors due to early death. $ indicates absence of 2 data points from week 6 due to early death. ( C ) Tumor weight measurements on week 6 postxenograft. Dunnett’s test between NT ( N = 8) and 715F(5): P = 0.0003 ( N = 8), 230F(12): P = 0.0008 ( N = 10), and Panc10.05 pool: P = 0.0004 ( N = 10). *** P < 0.001. Mean ± SEM was shown. ( D and E ) Metastatic growth experiment in hemispleen injection mouse models of liver metastasis. Hematoxylin and eosin (H&E) staining of the liver sections of mice treated with ( D ) NT ( N = 7) or ( E ) 230F(12) ( N = 5) sgRNA-expressing PC cells. Black arrow: tumor growth; green arrow: tumor regression. The top and bottom panels represent liver sections from 2 different mice of the same treatment group. Images at 20× original magnification; scale bar is 100 μm.

Article Snippet: With the exception of Panc10.05, which is an ATCC line, all other cell lines and their derivatives (including Panc10.05-derived lines) are available through Material Transfer Agreements.

Techniques: Expressing, Transduction, Injection, Staining

Journal: The Journal of Clinical Investigation

Article Title: Simultaneous CRISPR/Cas9-induced double-strand breaks are lethal in models of pancreatic cancer

doi: 10.1172/JCI190121

Figure Lengend Snippet: ( A ) Analysis workflow for quantification of on- and off-target sites in resistant colonies from clonogenicity assays. ( B and C ) Comparisons of mutation frequency at 1–2 mismatch (mm) sites detected by WGS and targeted deep NGS in resistant colonies. ( B ) 531F(2) sgRNA–resistant colonies. Four 1 mm sites were sequenced using the same primers. N = 2, mean ± SEM. ( C ) Panc10.05 164R(14) sgRNA–resistant colony. Noncanonical PAMs were indicated in parentheses. N = 1. ( D ) Fold-change of multitarget sgRNAs in 2 PC cell lines 21 days after transduction. Number of target sites in parentheses; “rep” indicates repetitive element targeting. N = 3; mean ± SEM. ( E ) sgRNA tag survival over time. N = 3; mean ± SEM. ( F ) Mutation frequencies of eight 164R(14) sgRNA target sites in Panc10.05 Cas9-expressing cells at various time points. N = 3; mean ± SEM. Bell-shaped least squares regression; R 2 = 0.60–0.74. Relatively low percentages were due to the absence of antibiotic selection of transduced cells.

Article Snippet: With the exception of Panc10.05, which is an ATCC line, all other cell lines and their derivatives (including Panc10.05-derived lines) are available through Material Transfer Agreements.

Techniques: Mutagenesis, Transduction, Expressing, Selection

Journal: The Journal of Clinical Investigation

Article Title: Simultaneous CRISPR/Cas9-induced double-strand breaks are lethal in models of pancreatic cancer

doi: 10.1172/JCI190121

Figure Lengend Snippet: ( A – C ) TS0111 Cas9-expressing cells were transduced with 164R(14) sgRNA and subjected to break-apart FISH assays. ( A ) Break-apart FISH strategy at the 1q41 cut site. Abnormal FISH patterns were shown using cells collected at early time points. DNA was stained with DAPI. N = 1. ( B ) Complex rearrangements were observed in cells 14 and 16 days after transduction. N = 1. ( C ) Percentage of cells with rearrangements at 1q41 detected by break-apart FISH assay over time. ( D ) Shown are Panc10.05 cells transduced with NT2 or 164R(14) and stained with wheat germ agglutinin (WGA; green) and Hoechst 33342 (blue) 14 days after transduction. White arrow: large nucleus; yellow arrows: multiple nuclei in a cell. N = 3. ( E ) Number of TS0111-transduced cells with >6 X chromosomes over time using XY FISH. ( F ) Apoptosis analysis of Panc10.05 cells after treatment with 164R(14) or NT2 using annexin V flow cytometry assay. Šidák’s multiple comparisons test, day 7: ** P = 0.005, day 14: *** P = 0.0008, and day 21: P = 0.53. N = 3; mean ± SEM.

Article Snippet: With the exception of Panc10.05, which is an ATCC line, all other cell lines and their derivatives (including Panc10.05-derived lines) are available through Material Transfer Agreements.

Techniques: Expressing, Transduction, Staining, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: Simultaneous CRISPR/Cas9-induced double-strand breaks are lethal in models of pancreatic cancer

doi: 10.1172/JCI190121

Figure Lengend Snippet: ( A ) SVs were categorized by whether the breakpoints resulted from noninduced DSBs (0-target SV), from 1 site that was CRISPR/Cas9 targeted (1-target SV), or from both sites being targeted (2-target SV). ( B ) Quantification of SVs through WGS analyses of Panc10.05 surviving/resistant colonies after treatment with multitarget sgRNAs. N = 2 except for 164R(14) ( N = 1); mean ± SEM. ( C ) Number of translocations detected in each Panc10.05 surviving colony. N = 2 except for 164R(14) ( N = 1); mean ± SEM. ( D ) Sequences at breakpoint junctions were analyzed to identify indels and microhomology sequences (mh). Shown are the percentages of breakpoint types in each surviving colony. N = 2 except for 164R(14) ( N = 1); mean only. ( E ) Example of a 0-target deletion from a 715F(5) sgRNA–resistant colony. The red dotted lines indicate the 3 bp homology region on both upstream and downstream sequences.

Article Snippet: With the exception of Panc10.05, which is an ATCC line, all other cell lines and their derivatives (including Panc10.05-derived lines) are available through Material Transfer Agreements.

Techniques: CRISPR

Journal: The Journal of Clinical Investigation

Article Title: Simultaneous CRISPR/Cas9-induced double-strand breaks are lethal in models of pancreatic cancer

doi: 10.1172/JCI190121

Figure Lengend Snippet: ( A ) Mutation frequency of sgRNA target sites in each CRISPR/Cas9 surviving colony used for xenograft experiment. ( B – D ) Tumor growth experiment of CRISPR/Cas9 surviving colonies in subcutaneous xenograft models. In addition to a nontransduced cell line (NTC), surviving colonies from clonogenicity experiment transduced with NT sgRNA (NT colony #1 and NT colony #2) and multitarget sgRNAs 531F(2), 715F(5), and 551R(8) were injected into nude mice for tumor growth. ( B ) Tumor volume measurements postxenograft. Dunnett’s test between NTC and NT #1: P = 0.050, NT #2: P = 0.145, 531F(2): P = 0.349, 715F(5): P = 0.0002, 551R(8): P = 0.498 on week 5. N = 10 ( N = 8 for NTC on day 30 and 33 due to early death); mean ± SEM. ( C ) Tumor weight measurements. Dunnett’s test between NTC and NT #1: P = 0.341, NT #2: P = 0.437, 531F(2): P = 0.457, 715F(5): P = 0.041, and 551R(8): P = 0.531. N = 10; mean ± SEM. ( D ) Body weight of mice 5 weeks postxenograft. Dunn-Šidák test between NTC and the other treatment groups showed no significant differences. N = 5 except for NTC ( N = 4 due to early death); mean ± SEM. ( E ) Cell survival of Panc10.05 551R(8)–resistant colony that was retransduced with nontargeting sgRNA (NT2) or multitargeting sgRNAs — 551R(8), 230F(12), and 164R(14) — as detected by alamar blue cell viability assay and normalized to NT2. N = 3; mean ± SEM. ( F ) Cell survival of TS0111- and Panc10.05 715F(5)–resistant colony that was retransduced with nontargeting sgRNA (NT2) or multitargeting sgRNAs — 715F(5), 230F(12), and 164R(14) — as detected by alamar blue cell viability assay and normalized to NT2. N = 3; mean ± SEM.

Article Snippet: With the exception of Panc10.05, which is an ATCC line, all other cell lines and their derivatives (including Panc10.05-derived lines) are available through Material Transfer Agreements.

Techniques: Mutagenesis, CRISPR, Transduction, Injection, Viability Assay