Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: PD-L1 expression in GBM and PD-1 recruitment to the CARIS with GBM. A, Constitutive (UPN01) and inducible (UPN06) surface expression of PD-L1 in primary GBM cells after 24–48 hours of IFN-γ exposure. Representative results from two samples shown. UPN, unique patient number. B, PD-L1 median fluorescent intensity (MFI) on primary GBM ( n = 14) before and at 48 hours of IFN-γ (10 ng/mL) exposure; ***, P < 0.001, Wilcoxon signed-rank test. C, Fold-change in PD-L1 MFI in GBM cells from baseline, at 24, 48, and 72 hours. Each color represents a single patient donor, with some measured at multiple time points. Data are shown as individual values with the mean ± SD. D, PD-L1 expression in WT LN229-GBM cells and LN229 with PD-L1 deletion (LN229-PD-L1 KO) at baseline and at 24 hours of exposure to IFN-γ (10 ng/mL) or CAR28ζ T cells. E, MFI of PD-1 in the immune synapse between CARζ cells and WT LN229-GBM cells at 15, 30, and 60 minutes (****, P < 0.0001; ns, P > 0.5, Kruskal–Wallis and Wilcox pairwise); ≥20,000 events were captured, and 300–1,000 CAR + conjugates were examined for PD-1 recruitment to CARIS. The white box represents the IQR with horizontal lines at 25%, 50%, and 75%. F, Representative image capture showing the CARIS with tumor cell (WT LN229-GBM) and other parameters evaluated. Gating strategy is shown in the Supplementary material. G, Spearman correlation between PD-1 intensity and CAR intensity in the immune synapse at 15, 30, and 60 minutes. H, Spearman correlation between PD-1 intensity and actin intensity in the CARIS at 15, 30, and 60 minutes. ns, not significant.

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Expressing

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: Design and functional screening of PD-1 TR and CPR28 molecules. A, Schematic representation of the bicistronic vectors encoding for the HER2-CAR with truncated PD-1 (PD-1 TR ) or CPR28. The CPR28 dimer included a membrane proximal extracellular cysteine residue (C141) required for CD28 homodimerization, as indicated. B, Flow cytometry analysis showing coexpression of HER2-CAR and PD-1 CPR on T cells. CAR28ζ and NT T cells from the same donor were used as controls for assessment of CPR expression determined by surface PD-1 detection. C, Long-term cytolytic function of CAR28ζ/PD-1 TR , compared with CAR28ζ cells, against U373-GBM cells at effector to target ratios of 1:5 and 1:10 assessed using a cell-impedance based assay (xCELLigence). D, Comparison of the cytolytic ability of patient-derived CAR28ζ cells ( n = 3) coexpressing CPR28 dimer or CPR28 monomer against autologous HER2 + GBM cells at an effector to target ratio of 1:10 by assessment of tumor cell viability in an xCELLigence assay. In C and D , error bars represent the mean ± SD at each time point. ****, P < 0.0001, two-way ANOVA with the Tukey multiple comparisons test. E, IL-2 and ( F ) IFN-γ release by CAR28ζ, CAR28ζ/PD-1 TR , and CAR28ζ/CPR28 cells (100,000 T cells/well) upon stimulation with Fc-conjugated HER2 (0–2 μg/mL) and PD-L1 (0–5 μg/mL) proteins. Median values from a representative donor shown. G, CAR28ζ/CPR28 cell–induced lysis of Raji cells modified to express HER2 or HER2 and PD-L1 across different T-cell to tumor cell ratios but not the WT Raji cells (HER2 − /PD-L1 − ) or those expressing PD-L1 alone in a 51 Cr-release assay. T cells with CPR28 alone had no cytotoxic effect against HER2 + or PD-L1 + Raji cells, like NT T cells from the same donor. A, Created in BioRender. Navai, S. (2024) BioRender.com/l86h941 .

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Functional Assay, Membrane, Residue, Flow Cytometry, Expressing, Comparison, Derivative Assay, Lysis, Modification, Release Assay

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: Effect of decoupling signal 2 from the CAR on T-cell function. A, Illustration depicting the bipartite T-cell activation through signal 1 delivery from CAR engaging the HER2 antigen and signal 2 from binding of CPR with PD-L1 (or PD-L2). B, Percent increase in IFN-γ production by CAR28ζ/CPR28 and CARζ/CPR28 compared with CAR28ζ cells (50,000 T cells/well) at 24 hours of coculture with autologous GBM cells ( n = 5 patients). Effector (100,000 T cells) to target ratio of 1:1. **, P < 0.01; ****, P < 0.0001, one-way ANOVA with the Tukey multiple comparisons test. C, Cytolytic function of CAR28ζ/CPR28 and CARζ/CPR28 cells assessed using 4-hour 51 Cr-release assay at baseline and at 7 days of persistent T-cell stimulation through the CAR. NT T cells had poor viability after prolonged stimulation without added homeostatic cytokines and were not evaluable. ****, P < 0.0001, two-way ANOVA with the Tukey multiple comparisons test. D, Percent of total T cells expressing HER2-CAR on cell surface, and percent of CAR + T cells detected with PD-1 (surrogate marker for CPR), respectively, amongst different CPR/CART groups ( n = 4 patients) using flow cytometry. E, Median fluorescent intensity (MFI) of HER2-CAR detected in transduced T cells shown in D . In D and E , only statistically significant differences are shown, *, P < 0.05; **, P < 0.01; ****, P < 0.0001, one-way ANOVA with the Tukey multiple comparisons test. F, Multiplex analysis for proinflammatory cytokines (IL-2, MIP-1α, TNF-α, and GM-CSF) in autologous T-cell and GBM coculture ( n = 4 patients) supernatants at 24 hours. UPN, unique patient number. *, P < 0.05; **, P < 0.01; ****, P < 0.0001, two-way ANOVA with the Tukey multiple comparisons test. G, Assessment of long-term cytolytic function of CPR/CART against autologous GBM cells ( n = 3 patients) using cell impedance–based xCELLigence assay. Statistical differences (denoted by the color key) shown are in comparison to control treatment CAR28ζ cells overtime. ****, P < 0.0001, two-way ANOVA with the Tukey multiple comparisons test. In G, black arrow indicates addition of T cells. A, Created in BioRender. Navai, S. (2019) BioRender.com/p40z816 .

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Cell Function Assay, Activation Assay, Binding Assay, Release Assay, Cell Stimulation, Expressing, Marker, Flow Cytometry, Multiplex Assay, Comparison, Control

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: Phenotypic and functional profile of CART receiving bipartite activation signals through CPR41BB costimulation. A, In vivo functional screening of CARζ and CAR28ζ cells coexpressing CPR28 or CPR41BB against orthotopic xenografts of HER2 + PD-L1 + U373-GBM in SCID mice ( n = 5 per group). B, Fold change in tumor burden after treatment relative to the tumor volume before intratumoral injection of T cells (day 0), quantified by serial BLI. *, P < 0.05; **, P < 0.01, two-way ANOVA with the Tukey multiple comparisons test. Data are shown as the mean ± SD. C, CD8 + : CD4 + ratio in CAR-expressing T cells among CPR/CART from patients with GBM ( n = 4) as assessed by flow cytometry. Box plots show minimum to maximum with individual values. ns, P > 0.5, one-way ANOVA with the Tukey multiple comparisons test. D, Pie graph demonstrating immunophenotype distribution of CARζ/CPR41BB cells ( n = 4 patients) at baseline. Percentages shown represent the mean value. Immunophenotype is defined as follows: naïve, CCR7 + /CD45RA + ; central memory, CCR7 + /CD45RA − ; effector memory, CCR7 − /CD45RA − ; and terminal effector, CCR7 − /CD45RA + . E, The percentage CAR + CD8 + central memory (CCR7 + /CD45RA − ) T cells did not significantly differ amongst different CPR/CART groups ( n = 4 patients). The mean ± SEM is shown. ns, P > 0.5, one-way ANOVA with the Dunnett multiple comparisons test. F, Pie graph demonstrating immunophenotype distribution of CARζ/CPR41BB cells ( n = 4 patients) after 7 days of continued stimulation by repeat cocultures with autologous GBM cells. Percentages shown represent the mean value. On day 7 of repeated stimulation, CARζ/CPR41BB cells ( n = 4 patients) demonstrated ( G ) a significantly higher proportion of CAR + /CD8 + cells with the central memory phenotype compared with other CPR/CART groups and CAR28ζ, and ( H ) a significantly lower proportion of CAR + /CD8 + cells with the effector memory phenotype compared with CAR28ζ/CPR28 cells. The mean ± SEM is shown. *, P < 0.05; **, P < 0.01, one-way ANOVA with the Dunnett multiple comparisons test. I, Log-change in the median fluorescent intensity (MFI) of T-cell surface PD-1, LAG3, and TIM3 at 7 days of repeat coculture with autologous GBM cells ( n = 4 patients) from baseline. Box plots show minimum to maximum with individual values. *, P < 0.05; **, P < 0.01, two-way ANOVA with the Tukey multiple comparisons test. ns, not significant.

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Functional Assay, Activation Assay, In Vivo, Injection, Expressing, Flow Cytometry

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: Dynamics of CARζ/CPR41BB T-cell activation and CARIS formation in comparison with CAR41BBζ cells. A, CARζ/CPR41BB cells ( n = 3 donors) demonstrated significantly lower IL-2 and IFN-γ release compared with CAR41BBζ cells at 24 hours of coculture with LN229-GBM cells, with CAR28ζ cells consistently showing the higher Th1 cytokine production. Data are shown as the mean ± SD. **, P < 0.000; ****, P < 0.0001; two-way ANOVA with the Tukey multiple comparisons test. B, Western blot analysis for CAR-phosphoCD3ζ (pCD3) in T cells in a resting state (maintained in culture with IL-7/IL-15). The pCD3 to CD3 ratio is normalized to CARζ in each donor. *, P < 0.05; one-way ANOVA with the Tukey multiple comparisons test. C, Representative image capture showing the CARIS with tumor cell (LN229-GBM) and different CARζ/CPR41BB immune synapse parameters evaluated. Gating strategy is shown in the Supplementary Material. Spearman correlation ( D ) between the intensity of CAR and CPR at the CARIS and ( E ) between the intensity of actin and CPR at the CARIS, both assessed by imaging flow cytometry at 15, 30, and 60 minutes. F, CARζ/CPR41BB and CAR41BBζ cells show significantly higher percent (%) of F-actin at the CARIS compared with CARζ cells. ns, P > 0.5; **, P < 0.01; ***, P < 0.0001, two-way ANOVA with the Tukey post hoc test. Data are shown as the mean with 95% confidence interval (CI). G, CARζ/CPR41BB cells show significantly higher CPR intensity in the CARIS with WT LN229 GBM cells at 15, 30, and 60 minutes compared with conjugates with LN229-PD-L1 KO (Kruskal–Wallis and Wilcox pairwise). CPR intensity increased over time in both conditions. ns, not significant.

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Activation Assay, Comparison, Western Blot, Imaging, Flow Cytometry

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: Metabolomic parameters of CARζ/CPR41BB cells in comparison with CAR41BBζ cells. A, OCR measurements of resting (cultured in media containing in IL-7 and IL-15) CARζ/CPR41BB and CAR41BBζ cells ( n = 3 donors; 200,000 T cells per well) under basal metabolic conditions and after the addition of mitochondrial inhibitors. B, Comparison of maximal respiration between CARζ/CPR41BB cells and CAR41BBζ cells at baseline (day 0). P = 0.059, Student two-tailed t test. C, Basal OCR, maximal respiration, and SRC between CAR41BBζ and CARζ/CPR41BB cells after 7 days of stimulation with Fc-conjugated HER2 and PD-L1 proteins. ns, P > 0.05, Student two-tailed t test. D, The ECAR in resting CARζ/CPR41BB and CAR41BBζ cells. E, The ECAR of CARζ/CPR41BB and CAR41BBζ at baseline and at 7 days of continued stimulation with plate-bound HER2 and PD-L1 proteins. ns, P > 0.05; **, P < 0.01, Student two-tailed t test. F, Basal OCR to ECAR ratio in CARζ/CPR41BB compared with CAR41BBζ cells at rest and at 7 days of continued stimulation with HER2 and PD-L1 proteins. ns, P > 0.05; *, P < 0.05, Student two-tailed t test. G, The OCR in CD8 + CARζ/CPR41BB and CAR41BBζ cells ( n = 3 donors; 150,000 T cells per well) following 48-hour coculture with LN229-GBM cells (effector to tumor ratio 1:2). H, The SRC of CD8 + CARζ/CPR41BB and CAR41BBζ cells after 48 hours of coculture with LN229 GBM cells. ns, P > 0.05, Student two-tailed t test. The OCR and ECAR measurements are shown as the mean ± SEM. Data shown denote the IQR in the violin plot and the mean ± SEM in the bar graphs. ns, not significant.

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Comparison, Cell Culture, Two Tailed Test

Journal: Journal of translational medicine

Article Title: PD-L1 and IFN-γ modulate Non-Small Cell Lung Cancer (NSCLC) cell plasticity associated to immune checkpoint inhibitor (ICI)-mediated hyperprogressive disease (HPD).

doi: 10.1186/s12967-024-06023-8

Figure Lengend Snippet: Fig. 1 Clinical features of patient’s tumor before and after ICI treatment. Time points: Tdx, time of the diagnosis; Tb, time of baseline; Thy, time of hyperprogression. A Hematoxylin and eosin staining (H&E) and CD44 and PD-L1 immunohistochemical staining of tumor samples. Tdx: pleural effusion; Tb: pleural biopsy; Thy: subcutaneous lesion. Left: H&E, black line from top to bottom: 313.30 µm, 204.97 µm and 92.60 µm; middle: membrane CD44 expression on neoplastic cells, black line from top to bottom: 313.30 µm, 120.01 µm, 80.01 µm; right: PD-L1 expression on tumor cells by double stain for PDL1 and CD68 (PD-L1: brown, CD68: red), black line from top to bottom: 139.24 µm, 300 µm and 61.73 µm. B Imaging findings at the time of diagnosis (Tdx) (i.–iv.) and of progression to immunotherapy (Thy) (v.–viii.). Small right paratracheal lymph node (i.) progressed on immunotherapy (v., red arrow). Left pleural effusion (ii.) and left pleurodesis signs associated with the appearance of subcutaneous metastatic site (vi., red arrow). Left pleural effusion (iii.) and left pleurodesis signs associated with the appearance of a metastatic site at the left thoracic wall (vii., red arrow). Small right paraortic lymph node (iv.) progressed on immunotherapy (viii., red arrow). ICI: immune checkpoint inhibitors

Article Snippet: The silencing of PD-L1 in NSCLC-B cells was performed by using PD-L1 (CD274) Human Gene non-homology mediated CRISPR knock-out kit (KN413071; Origene, Rockville, MD, USA), according to the manufacturer’s instructions.

Techniques: Biomarker Discovery, Staining, Immunohistochemical staining, Membrane, Expressing, Imaging

Journal: Journal of translational medicine

Article Title: PD-L1 and IFN-γ modulate Non-Small Cell Lung Cancer (NSCLC) cell plasticity associated to immune checkpoint inhibitor (ICI)-mediated hyperprogressive disease (HPD).

doi: 10.1186/s12967-024-06023-8

Figure Lengend Snippet: Fig. 3 Comparison of immune-related genes and proteins between NSCLC-B and NSCLC-H cell lines. A Normalized expression (RLOG) of PD-L1 (CD274) gene, as identified by RNAseq analysis (Log2FC: −0.589, p-adj: 0.0003), and validation by flow cytometry (NSCLC-B, grey: unstained control, black: stained sample; NSCLC-H, orange: unstained control, red: stained sample). B GSEA analysis of curated list related to inflammatory response. C Heatmap showing the Log2FC of a subgroup of enriched score genes associated to GSEA analysis and reported in Additional file 5: Figure S1C, which are involved in immune-related cellular responses. Log2FC of differentially expressed genes in NSCLC-H compared to NSCLC-B is reported in the box and p-adj is reported on the right. D Western blot analysis for proteins involved in response to inflammatory stimuli and cell proliferation (n = 3, except for p-JAK2/t-JAK2 and p-IRF3/t-IRF3, in which n = 2). *p < 0.05, **p < 0.01, ***p < 0.001 by One Sample t test (each group vs theoretical mean of 100). Each bar represents mean with SEM. SEM: standard error of the mean

Article Snippet: The silencing of PD-L1 in NSCLC-B cells was performed by using PD-L1 (CD274) Human Gene non-homology mediated CRISPR knock-out kit (KN413071; Origene, Rockville, MD, USA), according to the manufacturer’s instructions.

Techniques: Comparison, Expressing, Biomarker Discovery, Flow Cytometry, Control, Staining, Western Blot

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a Schematic illustration of US-triggered deep-tissue activation of SPINs to release immunomodulators. b Schematic illustration of sonodynamic activation of SPINs to debulk tumor, enhance tumor immunogenicity, and release immunomodulators in situ as well as synergetic action of IDO inhibition and PD-L1 blocking on enhancing antitumor immunity with alleviated irAEs relative to free-drug administration.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Activation Assay, In Situ, Inhibition, Blocking Assay

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a Chemical structures of amphiphilic semiconducting polymeric modulators and schematic illustration of their self-assembly and surface modification to form SPINs. b The molar ratios of each component in different SPINs. c Zeta potentials and hydrodynamic sizes of different SPINs in 1× PBS buffer (pH = 7.4) ( n = 4). d Photographs of erythrocytes after incubation with 1× PBS buffer (negative control), 1% Triton X-100 (positive control), and 1× PBS buffer containing SPINs at the concentration of 100 µg/mL for 2 h, followed by centrifugation. e Hemolysis percentages of erythrocytes after incubation with SPINs at different concentrations for 2 h ( n = 4). f Schematic illustration of US irradiation of SPIN D2 solutions covered with a pork tissue. g ESR spectra of 1 O 2 for SPIN D2 (20 µg/mL) after US irradiation (1.2 W/cm 2 , 3 min) without or with coverage of pork tissues at different thicknesses. h Release profiles of aPD-L1 and NLG919 from SPIN D2 (40 µg/mL) after US irradiation for different time ( n = 4). i PD-L1/PD-1 binding activity assay after treatment with free aPD-L1 or SPIN D2 (40 µg/mL) with or without US irradiation ( n = 4). SPIN D2 – US versus SPIN D2 + US: P < 0.0001. Statistical significance was calculated via a two-tailed Student’s t test. *** P < 0.001. In ( g – i ), the power intensity of US irradiation was 1.2 W/cm 2 (1.0 MHz, 50% duty cycle). Data are presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Modification, Incubation, Negative Control, Positive Control, Concentration Assay, Centrifugation, Irradiation, Binding Assay, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a Schedule for the establishment of primary and distant tumors, triple systemic injection of SPINs (0.2 mL, 0.6 mg/mL) via tail vein, US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min), and analysis of immune responses. b , c Relative tumor volumes of primary ( b ) and distant ( c ) tumors of Panc02 tumor-bearing C57BL/6 mice ( n = 6) after systemic injection of saline, free-drug mixture (on day 0, 3, and 6, 4 mg/kg body weight for NLG919 and aPD-L1), or SPIN D2 (0.2 mL, 0.6 mg/mL) with or without US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min). SPIN D2 + US versus drug + US: P < 0.0001 for primary tumors ( b ); SPIN D2 + US versus drug: P < 0.0001 for distant tumors ( c ). d Survival curves of Panc02 tumor-bearing C57BL/6 mice ( n = 10) receiving different treatments as indicated. e Schematic illustration of treatment of rechallenged tumor mouse models using SPINs. f Growths of rechallenged tumors in Panc02 tumor-bearing mice after injection of saline or SPIN D2 (0.2 mL, 0.6 mg/mL) with US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min) ( n = 5). Saline versus SPIN D2 + US: P < 0.0001. g The survival curves of Panc02 tumor-bearing C57BL/6 mice after different treatments followed by tumor rechallenge ( n = 10). h Flow cytometry analysis of populations of effector memory T cells in the spleen of Panc02 tumor-bearing C57BL/6 mice after different treatments followed by tumor rechallenge ( n = 4). Saline versus SPIN D2 + US: P = 0.0059. i Differentially expressed gene numbers in tumor tissues of mice after different treatments. j Relative expression of Carl , Hmgb1-ps1 , Hmgb1-ps2 , Cd80 , Cd86 , Cd40 , Pdcd1 , Cd3e , Cd8a , Ifng , Gzmb , Cxcl1 , Cxcl2 , Cxcl9 , Cxcl10 , Cxcl11 , Ccl4 , Ccl5 , Il1b , Il2 , Il6 , Il7 , Il15 , Ido1 , and Cd274 in tumors of Panc02 tumor-bearing mice after different treatments (the experiment was repeated independently five times with similar results). k Unsupervised hierarchical clustering of relative gene expression in tumors of Panc02 tumor-bearing C57BL/6 mice after different treatments ( n = 5). Data are presented as mean values ± SD. Statistical significance was calculated via two-tailed Student’s t test; ** P < 0.01, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Injection, Irradiation, Flow Cytometry, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a Schematic of sono-immunotherapy of subcutaneous pancreatic mouse tumors covered with 5-cm tissue. b , c Relative tumor volumes of primary ( b ) and distant ( c ) tumors of Panc02 tumor-bearing C57BL/6 mice ( n = 5) after systemic injection of saline, free-drug mixture (on day 0, 3, and 6, 4 mg/kg body weight for NLG919 and aPD-L1), SPIN 0 or SPIN D2 (0.2 mL, 0.6 mg/mL) with or without US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min). The primary tumors were covered with 5-cm tissue under US irradiation. SPIN D2 + US versus SPIN 0 + US: P < 0.0001 for primary tumors ( b ); SPIN D2 + US versus SPIN 0 + US: P < 0.0001 for distant tumors ( c ). d Survival curves of Panc02 tumor-bearing C57BL/6 mice ( n = 10) after different treatments for 60 days. e Schematic of US-mediated deep-tissue sonodynamic therapy of orthotopic pancreatic rabbit tumors. f Radiolabeling stability of 131 I-SPIN 0 after storage in saline or 50% serum at 37 °C for different time ( n = 3). g , h SPECT imaging ( g ) and signal intensity ( h ) of orthotopic pancreatic rabbit tumors after systemic injection of 131 I-SPIN 0 (1.0 mL, 1.5 mg/mL) for different time ( n = 4). The white dotted circle indicated tumors. i Computed tomography (CT) imaging of orthotopic pancreatic rabbit tumors after systemic injection of saline or SPIN 0 (1.0 mL, 1.5 mg/mL) with or without US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 30 min). The white dotted circle indicated tumors. j Tumor volume of orthotopic pancreatic rabbit tumors ( n = 3) after treatments as indicated for different days. Saline + US versus SPIN 0 + US: P = 0.0108. k H&E staining images of orthotopic pancreatic rabbit tumors after different treatments. The experiment was repeated independently three times with similar results. l Survival curves of orthotopic pancreatic tumor-bearing rabbits ( n = 4) after different treatments for 20 days. Data are presented as mean values ± SD. Statistical significance was calculated via two-tailed Student’s t test; * P < 0.05, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Injection, Irradiation, Radioactivity, Single Photon Emission Computed Tomography, Imaging, Computed Tomography, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a , b Flow cytometry analysis of percentages of CD3 + CD4 + Th cells ( a ), and CD3 + CD8 + CTLs ( b ) in blood of mice ( n = 4) at 30 day after systemic administrations of saline, SPIN 0 , SPIN D2 (0.2 mL, 1.2 mg/mL) or free-drug mixture (8 mg/kg body weight for NLG919 and aPD-L1) with or without US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min). Saline – US versus drug − US: P = 0.0023; saline − US versus drug + US: P = 0.0006; drug + US versus SPIN D2 + US: P = 0.0071 for CD3 + CD4 + Th cells ( a ); saline − US versus drug − US: P = 0.0004; saline − US versus drug + US: P = 0.0001; drug + US versus SPIN D2 + US: P = 0.0093 for CD3 + CD8 + CTLs ( b ). c , d Flow cytometry analysis of percentages of CD3 + CD4 + Th cells ( c ), and CD3 + CD8 + CTLs ( d ) in spleen of mice ( n = 4) after different treatments for 30 days. Saline − US versus drug − US: P = 0.0008; saline − US versus drug + US: P = 0.0005; drug + US versus SPIN D2 + US: P = 0.0015 for CD3 + CD4 + Th cells ( c ); saline − US versus drug − US: P = 0.0001; saline − US versus drug + US: P = 0.0002; drug + US versus SPIN D2 + US: P = 0.0049 for CD3 + CD8 + CTLs ( d ). e Representative H&E staining images of liver after 30 days of treatments in different groups (white arrows indicate the infiltrated lymphocytes). The experiments were repeated independently three times with similar results. f Heatmap to show relative fold of cytokine levels in serum of mice after different treatments for 30 days relative to those in saline control group. g , h Serum levels of ALT ( g ) and AST ( h ) in mice ( n = 5) after different treatments for 30 days. Saline − US versus drug − US: P = 0.0010; saline − US versus drug + US: P = 0.0020; drug + US versus SPIN D2 + US: P = 0.0054 for ALT ( g ); saline − US versus drug − US: P = 0.0001; saline − US versus drug + US: P < 0.0001; drug + US versus SPIN D2 + US: P = 0.0013 for AST ( h ). i Summary comparison of the antitumor immunity and irAEs between SPIN D2 -mediated sono-immunotherapy and free-drug treatment. Data are presented as mean values ± SD. Statistical significance was calculated via two-tailed Student’s t test; ** P < 0.01, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Flow Cytometry, Irradiation, Staining, Two Tailed Test