Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Enhanced dendritic cell maturation by TNF-alpha or cytidine-phosphate-guanosine DNA drives T cell activation in vitro and therapeutic anti-tumor immune responses in vivo.

doi: 10.4049/jimmunol.165.11.6278

Figure Lengend Snippet: FIGURE 5. Tumor specifity of vaccination with DC after coculture with tumor cells. DC were cocultured with irradiated Colon-26 cells (a and d) or Renca cells (b and c) for 4 days in the presence of 200 U/ml GM-CSF and 20 ng/ml IL-4 and additionally stimulated for 48 h with 6 mg/ml ODN-1826. Renca cells (5 3 105) were injected 7 days after vaccination with ODN-1826-stimulated DC cocultured with Colon-26 cells F, (a, p 5 0.290) or in mice vaccinated with ODN-1826-stimulated DC cocultured with Renca cells (b, f, p , 0.001). Tumor growth after injection of 5 3 105 Renca cells in unvaccinated mice (control group ¿) was rapid, leading to death at day 23. In addition, 5 3 105 Colon-26 cells were injected 7 days after vaccination with ODN-1826-stimulated DC cocultured with Renca cells (c, l, p 5 0.250) or ODN-1826-stimulated DC cocultured with Colon-26 cells (d, Œ, p , 0.001). One representative experiment of two (n 5 60) is shown. Each curve represents one mouse except for the top curve (¿), which is the mean 6 SEM of untreated control mice (n 5 5).

Article Snippet: The murine, BALB/c-derived Colon-26 carcinoma line as well as the murine Renca cells (Cell Lines Service, Heidelberg, Germany) were maintained in DMEM supplemented with 10% FCS, 1% L-glutamine, and antibiotics.

Techniques: Irradiation, Injection, Control

Journal: Frontiers in Oncology

Article Title: Mutational Analysis of PBRM1 and Significance of PBRM1 Mutation in Anti-PD-1 Immunotherapy of Clear Cell Renal Cell Carcinoma

doi: 10.3389/fonc.2021.712765

Figure Lengend Snippet: The expression level of PBRM1 varies in different tumors and tumor stages. (A) The expression status of the PBRM1 gene in diverse cancer types were analyzed through the TIMER dataset. *P < 0.05; **P < 0.01; ***P < 0.001. (B) Based on the CPTAC dataset, the expression level of PBRM1 total protein between normal tissues and primary tumor tissues of breast invasive carcinoma (BRCA), colon adenocarcinoma (COAD), lung adenocarcinoma (LUAD), and kidney renal clear cell carcinoma (KIRC, ccRCC) were analyzed. (C) Based on the TCGA data, the expression levels of the PBRM1 gene were analyzed by the main pathological stages (stage I, stage II, stage III, and stage IV) of BRCA, COAD, LUAD, ccRCC. Log2 (TPM+1) was applied for log-scale. (D) PBRM1 protein expression levels in Stage I and Stage IV of our ccRCC samples. The expression levels were detected using immunohistochemistry and analyzed by unpaired t-test.

Article Snippet: Mouse ccRCC cell line Renca was purchased from ATCC and maintained in RPMI-1640 containing 10% fetal bovine serum, non-essential amino acids (NEAA), and sodium pyruvate according to the ATCC instruction at 37°C with 5% CO 2 .

Techniques: Expressing, Immunohistochemistry

Journal: Frontiers in Oncology

Article Title: Mutational Analysis of PBRM1 and Significance of PBRM1 Mutation in Anti-PD-1 Immunotherapy of Clear Cell Renal Cell Carcinoma

doi: 10.3389/fonc.2021.712765

Figure Lengend Snippet: Mutation frequency of PBRM1 in tumors reproduced from the Cancer Genome Atlas (TCGA) database. (A) Alternation frequency of PBRM1 in various tumors are shown. (B) Mutation types of PBRM1 in ccRCC are shown. (C) 3D structure of PBRM1 and the mutation cite is shown. (D) Protein-protein interaction network of PBRM1 are shown. The top 10 experimentally determined genes ranked by degree are shown.

Article Snippet: Mouse ccRCC cell line Renca was purchased from ATCC and maintained in RPMI-1640 containing 10% fetal bovine serum, non-essential amino acids (NEAA), and sodium pyruvate according to the ATCC instruction at 37°C with 5% CO 2 .

Techniques: Mutagenesis

Journal: Frontiers in Oncology

Article Title: Mutational Analysis of PBRM1 and Significance of PBRM1 Mutation in Anti-PD-1 Immunotherapy of Clear Cell Renal Cell Carcinoma

doi: 10.3389/fonc.2021.712765

Figure Lengend Snippet: The mutation of PBRM1 affects the survival rates of patients. Overall survival (A) and disease-free survival (B) of PBRM1 high and PBRM1 low patients in different tumors. GEPIA2 tool was used to analyze the TCGA dataset. BRCA, breast invasive carcinoma; COAD, colon adenocarcinoma; LUAD, lung adenocarcinoma; KIRC, kidney renal clear cell carcinoma (ccRCC). (C) Overall survival of KIRC (ccRCC) patients based on the PBRM1 expression level and tumor grade. Kaplan-Meier curves with positive results are given.

Article Snippet: Mouse ccRCC cell line Renca was purchased from ATCC and maintained in RPMI-1640 containing 10% fetal bovine serum, non-essential amino acids (NEAA), and sodium pyruvate according to the ATCC instruction at 37°C with 5% CO 2 .

Techniques: Mutagenesis, Expressing

Journal: Frontiers in Oncology

Article Title: Mutational Analysis of PBRM1 and Significance of PBRM1 Mutation in Anti-PD-1 Immunotherapy of Clear Cell Renal Cell Carcinoma

doi: 10.3389/fonc.2021.712765

Figure Lengend Snippet: Low expression of PBRM1 correlates with decreased CD4 cells in human ccRCC. (A) The correlation of PBRM1 expression with CD4, CD8 within tumors from 533 ccRCC patients were analyzed using the TIMER database. (B) Opal Multiplex Immunohistochemistry detection of PBRM1, CD4, CD8 in human ccRCC samples.180 ccRCC samples were tested and classified as High (>80%), Medium (50-60%), Low (<20%) according to the expression level of PBRM1 in the tumor tissues. (C) Represents the numbers of CD4, CD8 cells in (B) . PBRM1 High, PBRM1 Low groups were calculated by unpaired t-test, **P < 0.01; ***P < 0.001. Data indicates Mean+SD.

Article Snippet: Mouse ccRCC cell line Renca was purchased from ATCC and maintained in RPMI-1640 containing 10% fetal bovine serum, non-essential amino acids (NEAA), and sodium pyruvate according to the ATCC instruction at 37°C with 5% CO 2 .

Techniques: Expressing, Multiplex Assay, Immunohistochemistry

Journal: Frontiers in Oncology

Article Title: Mutational Analysis of PBRM1 and Significance of PBRM1 Mutation in Anti-PD-1 Immunotherapy of Clear Cell Renal Cell Carcinoma

doi: 10.3389/fonc.2021.712765

Figure Lengend Snippet: Low expression of PBRM1 causes reduction of tumor-infiltrating CD4 cells in ccRCC mouse model. (A) PBRM1 protein expression level in PBRM1 high and PBRM1 low Renca cells were detected by western blot. PBRM1 low Renca cells were obtained using shRNA to PBRM1. β-actin was used as an internal control. (B) Growth curves of PBRM1 High and PBRM1 Low tumors in ccRCC mouse model. (C) Flow cytometry image of tumor-infiltrating T cells. TIL cells were isolated from tumors and gated CD45 + CD3 + cells. Percentage (D) and absolute numbers (E) of CD4 and CD8 cells in tumors of PBRM1 High and PBRM1 Low mice model. CD4 andCD8 cells gated in CD45 + CD3 + cells. Data represents 3 independent experiments at least 6 mice per group. *P < 0.05; ns, no significance.

Article Snippet: Mouse ccRCC cell line Renca was purchased from ATCC and maintained in RPMI-1640 containing 10% fetal bovine serum, non-essential amino acids (NEAA), and sodium pyruvate according to the ATCC instruction at 37°C with 5% CO 2 .

Techniques: Expressing, Western Blot, shRNA, Control, Flow Cytometry, Isolation

Journal: Frontiers in Oncology

Article Title: Mutational Analysis of PBRM1 and Significance of PBRM1 Mutation in Anti-PD-1 Immunotherapy of Clear Cell Renal Cell Carcinoma

doi: 10.3389/fonc.2021.712765

Figure Lengend Snippet: Anti-PD-1 immunotherapy increased CD4 T cell infiltration in ccRCC mouse model. (A) Treatment scheme of anti-PD-1 immunotherapy in ccRCC mouse model. Anti-PD-1 antibody and control antibody were administrated at day 6, day 9, and day 12 after tumor inoculation. (B) Growth curves of PBRM1 High and PBRM1 Low tumors treated with anti-PD-1 antibody or control isotype antibody in ccRCC mouse model. Test was stopped when mice died or tumors reached 1000mm 3 in size. (C) Survival rates of mice in different groups. Data were analyzed with GraphPad Prism 8.0.2. Absolute cell numbers (D) and percentages (E) of CD4 and CD8 T cells in tumors of different groups were shown. All live mice were euthanized at day39 and tumor infiltrating T cells were detected by Flow Cytometry. Data are presented as mean ± SD with at least 6 mice for each group. *p < 0.05; ns, no significance.

Article Snippet: Mouse ccRCC cell line Renca was purchased from ATCC and maintained in RPMI-1640 containing 10% fetal bovine serum, non-essential amino acids (NEAA), and sodium pyruvate according to the ATCC instruction at 37°C with 5% CO 2 .

Techniques: Control, Flow Cytometry

Journal: Frontiers in Oncology

Article Title: Alphataxin, a Small-Molecule Drug That Elevates Tumor-Infiltrating CD4 + T Cells, in Combination With Anti-PD-1 Therapy, Suppresses Murine Renal Cancer and Metastasis

doi: 10.3389/fonc.2021.739080

Figure Lengend Snippet: Tumor Growth Curves of Subcutaneously Implanted Tumor Cells. Renca cells (2 x 10 5 ) were subcutaneously implanted into 8 female BALB/c mice per group and allowed to grow for 2 weeks before the mice remained untreated (●, n = 7) or were treated with an anti-PD-1 antibody ( , 7 mg/kg, n = 6) via IP injection twice per week or with Alphataxin via daily oral gavage at a dose of 2.5 mg/kg (●), 5 mg/kg ( ), or 10 mg/kg ( ) (n = 8/arm). In combination treatment, mice were treated with anti-PD-1 (7mg/kg) via IP injection twice per week and with Alphataxin via daily oral gavage at a dose of 2.5 mg/kg ( ), 5 mg/kg ( ), or 10 mg/kg ( ) (n = 8/arm). Tumor volume was calculated periodically using calipers. Alphataxin (10 mg/kg) significantly suppressed tumor growth as compared with Alphataxin (5 mg/kg) or Alphataxin (2.5 mg/kg) (P < 0.001, n = 8/arm). Data were normalized to baseline tumor volume and are presented as the means and standard deviations.

Article Snippet: For a study conducted by AJES Life Sciences (IACUC# 764665-4), orthotopic implantation was achieved by injecting 5 x 10 2 Renca cells in 100 μl of HBSS mixed 1:1 with Matrigel (BD Biosciences) into the left kidney of 8-wk-old female BALB/c mice, and treatment was initiated 7 days after implantation.

Techniques: Injection

Journal: Frontiers in Oncology

Article Title: Alphataxin, a Small-Molecule Drug That Elevates Tumor-Infiltrating CD4 + T Cells, in Combination With Anti-PD-1 Therapy, Suppresses Murine Renal Cancer and Metastasis

doi: 10.3389/fonc.2021.739080

Figure Lengend Snippet: Long-term Remission Achieved by Treatment with Alphataxin and an Anti-PD-1 Antibody. Mice were orthotopically implanted with 5x 10 3 Renca-GL cells, and treatment was initiated the following day. (A) Kidneys were excised at the time of euthanization and stained with H&E. Images were captured at identical magnification, and representative images at 1X are shown. (B) Longitudinal dorsal images of the orthotopic kidney tumor growth of mice. A representative animal that showed similar bioluminescence with at least 2 other animals within the same treatment arm after 25 days of treatment was selected from each treatment arm. The mice in the combination treatment arm were bifurcated into 2 groups, those with tumors (“Combination +”, 4 th row, n = 4) and those without tumors (“Combination −”, 5 th row, n = 2). The luminescence color scale represents photons/sec/cm 2 /steradian x 10 9 with minimum 4.48e6 and maximum 19.4e9. (C) As compared with untreated mice and monotherapy, after 25 days of treatment the tumor volumes (BLI) were significantly smaller in mice receiving combination treatment with tumors ( P = 0.05, n = 4) and without tumors ( P = 0.02, n = 2). After 25 days of treatment, there were no significant differences between untreated and Alphataxin-treated mice ( P = 0.24, n = 6/arm) or between untreated and anti-PD-1-treated mice ( P = 0.06, n = 6) although the difference between untreated and anti-PD-1-treated mice have practical statistical importance. Means and standard deviations are depicted. (D) Median longitudinal tumor growth demonstrates that as compared with untreated animals ( ), Alphataxin ( ) and anti-PD-1( ) individually impeded tumor growth and in combination + animals ( ), substantially impeded tumor growth while in combination – animals ( ), tumor growth regressed. Asterisks designate statistically significant differences (*P < 0.05).

Article Snippet: For a study conducted by AJES Life Sciences (IACUC# 764665-4), orthotopic implantation was achieved by injecting 5 x 10 2 Renca cells in 100 μl of HBSS mixed 1:1 with Matrigel (BD Biosciences) into the left kidney of 8-wk-old female BALB/c mice, and treatment was initiated 7 days after implantation.

Techniques: Staining

Journal: Frontiers in Oncology

Article Title: Alphataxin, a Small-Molecule Drug That Elevates Tumor-Infiltrating CD4 + T Cells, in Combination With Anti-PD-1 Therapy, Suppresses Murine Renal Cancer and Metastasis

doi: 10.3389/fonc.2021.739080

Figure Lengend Snippet: Increases in the Tumor-infiltrating CD4/CD8 Ratio Achieved by Treatment with Alphataxin or an Anti-PD-1 Antibody. Excised kidneys from mice in each treatment arm of (5 x 10 2 orthotopically implanted Renca cells and 5 x 10 3 orthotopically implanted Renca-GL cells) were immunohistochemically stained to determine the numbers of CD4 + and CD8 + TILs which were counted in all the tumor fields of the stained slides. Because the tumors varied in size, yielding large variation in the number of TILs, the CD4/CD8 ratio is depicted to produce more accurate comparisons. (A) In the Renca cell orthotopic study, after 21 days of treatment, the tumor-infiltrating CD4/CD8 ratio in Alphataxin-treated animals was significantly greater than that in untreated and anti-PD-1 antibody-treated mice ( P = 0.04). (B) In the Renca-GL study, tumor-infiltrating CD4/CD8 ratio in Alphataxin-treated mice was significantly greater than that in untreated ( P < 0.001, n = 3) and anti-PD-1-treated mice ( P < 0.001, n = 3). In anti-PD-1 treatment, CD4/CD8 ratio was significantly greater than in untreated animals ( P = 0.02, n = 3). Combination treatment is not depicted due to the lack of suitable kidney sectioning. Means and standard deviations are depicted. The CD4/CD8 ratios were not normally distributed and were compared by the Mann-Whitney rank- sum test. (C) In the Renca-GL study, there were more CD4 + TILs (green bars) in the tumors of Alphataxin-treated mice than in those of anti-PD-1 antibody-treated mice ( P = 0.086, n = 3), which demonstrates practical statistical importance. The numbers of CD8 + TILs (blue bars) were not different among the treatment arms ( P = 0.94), n =3). Note that the scales for the CD4/CD8 ratio and TIL counts are not equivalent in different graphs. TILs were normally distributed and were tested by ANOVA. Asterisks designate statistically significant differences (*P < 0.05, ***P < 0.001).

Article Snippet: For a study conducted by AJES Life Sciences (IACUC# 764665-4), orthotopic implantation was achieved by injecting 5 x 10 2 Renca cells in 100 μl of HBSS mixed 1:1 with Matrigel (BD Biosciences) into the left kidney of 8-wk-old female BALB/c mice, and treatment was initiated 7 days after implantation.

Techniques: Staining, MANN-WHITNEY

Journal: Journal for Immunotherapy of Cancer

Article Title: Immunization with short peptide particles reveals a functional CD8 + T-cell neoepitope in a murine renal carcinoma model

doi: 10.1136/jitc-2021-003101

Figure Lengend Snippet: Multivalent immunization reveals Nes2LR as a functional neoepitope. (A) Approach used for in vivo screening of RENCA neoepitopes. (B) Binding synthetic neoepitope candidate pool to CPQ or 2HPQ liposomes. (C) BALB/c mice were immunized with CPQ or 2HPQ liposomes admixed with all 20 predicted neoepitope candidates (50 ng per peptide) on days 0 and 7, then challenged with RENCA cells subcutaneously on day 14, and tumor growth was monitored. (D) Eighteen days after tumor inoculation, spleens were collected from the group immunized with CPQ, and the neoepitope pool, and splenocytes were restimulated with individual neoepitope candidates and assessed for intracellular IFN-γ staining. (E) Mice were immunized with CPQ admixed with individual neoepitope candidates (0.5 µg peptide per mouse) on days 0 and 7, then challenged with RENCA cells on day 14. Tumor sizes on day 21 are shown. (F) Splenocytes from immunized mice were cultured with Ag and interleukin-2 for 5 days before serving as effector cells to lyse tumor target cells at indicated effector to target (E:T) cell ratio. (G) Flowchart of the neoepitope identification and processing during the screening. Mice were vaccinated with CPQ with the Nes2LR neoepitope along with another 19 or 59 unrelated short peptides on days 0 and 7, then challenged with RENCA cells on day 14 with a total Ag dose of 3 µg peptide and assessed for (H) percentage of Nes2LR-tetramer + cells in the CD8 + T-cell population, (I) tumor growth and (J) percentage of mice bearing tumors not exceeding 1 cm. Error bars show mean±SD for n=3 or four replicates (B, F, respectively), or n=5 mice (C, D) or n=3 mice (E) or n=10 mice (H–J). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, analyzed by the (B, C) two-tailed unpaired Student’s t-test or (D, F) one-way analysis of variance with Dunnett’s multiple comparisons post-test or (J) log-rank test. IFN-γ, interferon gamma; Nes2LR, nesprin-2 L4492R; RENCA, renal adenocarcinoma.

Article Snippet: Short RENCA neoepitope peptides were synthesized by GenScript.

Techniques: Functional Assay, In Vivo, Binding Assay, Liposomes, Staining, Cell Culture, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: Immunization with short peptide particles reveals a functional CD8 + T-cell neoepitope in a murine renal carcinoma model

doi: 10.1136/jitc-2021-003101

Figure Lengend Snippet: Antitumor effects of Nes2LR neoepitope immunization when converted into particle format by admixture with CPQ liposomes. (A) Cryo-TEM images of CPQ liposomes with or without Nes2LR peptide bound. (B) Binding percentage of Nes2LR peptide to CPQ and 2HPQ liposomes. BALB/c mice were vaccinated on days 0 and 7, and then blood was collected for flow cytometry analysis on day14; mice were challenged with RENCA tumor cells subcutaneously on day 14. (C) Tumor volume of mice. (D) Mice with tumor sizes smaller than 1 cm. (E) Percentage of Nes2LR-tet + cells in the CD8 + cell population in blood. (F) Tumor growth of mice. (G) Tumor volume of mice on day 26. (H) Percentage of mice with tumor sizes smaller than 1 cm. Error bars show mean±SD for n=5 mice. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, analyzed by (B) two-tailed unpaired Student’s t-test or (E, G) one-way analysis of variance with Bonferroni multiple comparisons post-test or (H) log-rank test. Nes2LR, nesprin-2 L4492R; RENCA, renal adenocarcinoma.

Article Snippet: Short RENCA neoepitope peptides were synthesized by GenScript.

Techniques: Liposomes, Binding Assay, Flow Cytometry, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: Immunization with short peptide particles reveals a functional CD8 + T-cell neoepitope in a murine renal carcinoma model

doi: 10.1136/jitc-2021-003101

Figure Lengend Snippet: CPQ/Nes2LR as a therapeutic vaccine delayed ectopic and orthotopic primary RENCA tumor growth and lung metastasis. BALB/c mice were inoculated with RENCA cells subcutaneously on day 0 and then primed on day 3 or 6 and boosted 7 days later; the dose of Ag was 0.5 µg per mouse. Tumor growth (A) and survival (B) of untreated mice or mice vaccinated with CPQ/Nes2LR and 2HPQ/Nes2LR. Error bars show mean±SD for n=5 mice per group. BALB/c mice were injected with RENCA cells intravenously on day 0, then the vaccine with an Ag dose of 0.5 µg per mouse was applied on days 3 and 10. Mice were sacrificed on day 23, and lungs were excised and stained with Bouin’s solution. (C) Image of lungs from mice that received the indicated vaccine. (D) Lung nodule counts. (E) Lung weights. Error bars show mean±SD for n=5 mice per group. BALB/c mice were inoculated with mouse RENCA LUC+ tumors orthotopically on day 0 and CPQ/Nes2LR vaccine with 2 µg Ag on days 2 and 9. BL quantification (F), survival (G), and corresponding BL imaging (H) of untreated mice or mice vaccinated with CPQ/Nes2LR. Overall survival based on Kaplan-Meier analysis. Error bars show mean±SD for n=8 mice per group. **P<0.01, ***P<0.001, ****P<0.0001, analyzed by long rank test (B, G) or one-way analysis of variance with Dunnett’s multiple comparisons post-test (D, E) and two-tailed unpaired Student’s t-test on day 39 (F). BL, bioluminescence; Nes2LR, Nesprin-2 L4492R; RENCA, renal adenocarcinoma.

Article Snippet: Short RENCA neoepitope peptides were synthesized by GenScript.

Techniques: Injection, Staining, Imaging, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: Immunization with short peptide particles reveals a functional CD8 + T-cell neoepitope in a murine renal carcinoma model

doi: 10.1136/jitc-2021-003101

Figure Lengend Snippet: Synergistic effects of CPQ/Nes2LR vaccine and immune checkpoint blockade combination therapy. BALB/c mice were inoculated with tumor cells subcutaneously on day 0 and untreated (Ctrl) or treated with CPQ/Nes2LR on days 10 and 17, treated with ICB (anti-CTLA-4 and anti-PD-1 antibodies on days 12, 14, 19, and 21) or treated with the combination. (A) Tumor growth of mice. (B) Mice with tumor sizes smaller than 1 cm. Error bars show mean±SD for n=5 mice per group. BALB/c mice were inoculated with RENCA tumor cells subcutaneously on day 0 and then untreated (Ctrl) or vaccinated with CPQ/Nes2LR, 2HPQ/Nes2LR on days 10 and 17. Mice were sacrificed on day 22; spleens and tumors were collected for analysis. (C) Percentage of CD8 + T cells in the live cells. (D) Percentage of Nes2LR tet + cells in the CD8 + T-cell population. Percentage of PD-1 + (E), TIM-3 + PD-1 + , (F) and LAG-3 + PD-1 + (G) among Nes2LR tet + CD8 + T-cell population. (H) Percentage of IFN-γ producing cells in the CD8 + T-cell population after 10 µg/mL Ag stimulation in vitro. Error bars show mean±SD for n=5 mice per group. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, analyzed by (A) one-way ANOVA with Dunnett’s multiple comparisons post-test or long rank test (B) or two-way ANOVA (C–H) with Tukey multiple comparisons post-test. The asterisk in A shows a comparison to untreated group on day 29. ANOVA, analysis of variance; CTLA-4, cytotoxic T-lymphocyte-associated protein 4; Ctrl, control; ICB, immune checkpoint blockade; IFN-γ, interferon gamma; Nes2LR, Nesprin-2 L4492R; ns, not significant; PD-1, programmed cell death protein 1; RENCA, renal adenocarcinoma.

Article Snippet: Short RENCA neoepitope peptides were synthesized by GenScript.

Techniques: In Vitro, Comparison, Control

Journal: Journal of Nanobiotechnology

Article Title: A multifunctional PEGylated liposomal-encapsulated sunitinib enhancing autophagy, immunomodulation, and safety in renal cell carcinoma

doi: 10.1186/s12951-024-02664-5

Figure Lengend Snippet: The biodistribution pattern of lipo-sunitinib in RCC models. A Lipo-sunitinib was labeled with the lipophilic tracers DiI or DiR to assess biodistribution and observation in vitro and in vivo. B A498 and RENCA cells were subjected to IF to evaluate the observation of DiI-labeled lipo-sunitinib. DAPI and DiI were used to represent nuclei and observed lipo-sunitinib, respectively. C, D Lipo-sut DiR and free DiR were intravenously injected into RCC orthotopic models, and the distribution of DiR signals was tracked over a time period of 0–48 h. E The primary organs were harvested for ex vivo detection of DiR signals to analyze the biodistribution of lipo-sut-DiR and free DiR groups (T=tumor, K=kidney, B=brain, H/LG=heart/lungs, L=livers, S=spleen). F The DiR signals from RCC kidneys were compared with those from normal kidneys. G RCC orthotopic tumors were harvested from DiI-labeled lipo-sunitinib, and DiI fluorescence signals were detected with excitation and emission wavelengths of 555 and 584 nm, respectively (T = tumor tissue, N = normal tissue). Scale bar, 100 μm

Article Snippet: RENCA cells and A498 cells were purchased from Bioresource Collection and Research Center (BCRC, Hsinchu City, Taiwan) and maintained in Roswell Park Memorial Institute (RPMI) 1640 and Eagle’s Minimum Essential (MEM) medium contenting 10% fetal bovine serum (FBS) and 1 × penicillin/streptomycin (PS), then incubated with 5% CO 2 and 95% air mixture at 37 °C humanity incubator.

Techniques: Labeling, In Vitro, In Vivo, Injection, Ex Vivo, Fluorescence

Journal: Journal of Nanobiotechnology

Article Title: A multifunctional PEGylated liposomal-encapsulated sunitinib enhancing autophagy, immunomodulation, and safety in renal cell carcinoma

doi: 10.1186/s12951-024-02664-5

Figure Lengend Snippet: Lipo-sunitinib strongly upregulates cytotoxicity through autophagy induction in RCC. A , B MTT assays were conducted to compare the cytotoxicity of sunitinib and lipo-sunitinib in RENCA and A498 cell lines. C RENCA cells treated with lipo-sunitinib underwent RNA-seq analysis, revealing significantly upregulated pathways compared to the non-treated group. D RNA-seq data for lipo-sunitinib were subjected to GSEA analysis with a focus on macrophagy and autophagosome pathways. E IF analysis of RENCA and A498 cells included detection of LC3B and p62 expression using GFP and RFP channels, with DAPI staining representing nuclei. F In addition, LC3B protein expression in RENCA and A498 cells were assessed using western blotting assays. G The subcutaneous RCC xenograft models were constructed using BALB/c mice with RENCA cells subcutaneously injection. Vehicle and sunitinib groups were administrated 0.1% DMSO and 30 mg/kg sunitinib through daily gavage, respectively, and the H tumor volume of RCC was measured twice daily. Mice are sacrificed on day 14, and all tumors were isolated from mice for I weighting and J imaging. K Body weights of mice were also measured twice daily after treatment started. L Several organs of the subcutaneous RCC xenograft model (liver, heart, and kidneys) were subjected to H&E staining. Scale bar, 50 μm

Article Snippet: RENCA cells and A498 cells were purchased from Bioresource Collection and Research Center (BCRC, Hsinchu City, Taiwan) and maintained in Roswell Park Memorial Institute (RPMI) 1640 and Eagle’s Minimum Essential (MEM) medium contenting 10% fetal bovine serum (FBS) and 1 × penicillin/streptomycin (PS), then incubated with 5% CO 2 and 95% air mixture at 37 °C humanity incubator.

Techniques: RNA Sequencing, Expressing, Staining, Western Blot, Construct, Injection, Isolation, Imaging