Journal: Cancer Research

Article Title: Adipocyte Precursor-Derived NRG1 Promotes Resistance to FGFR Inhibition in Urothelial Carcinoma

doi: 10.1158/0008-5472.CAN-23-1398

Figure Lengend Snippet: Adipocyte precursors promote resistance against erdafitinib in bladder cancer cell lines. A, Western blot analysis of FGFR3 in four bladder cancer cell lines: RT4, RT112, TSCCUP, and T24. β-Actin served as a loading control. B, Proliferation analysis of five bladder cancer cell lines RT4, RT112, TSCCUP, T24, and MB49 treated with vehicle (Veh) or erdafitinib (1, 10, 100, 1,000 nmol/L). Crystal violet staining was done on day 7. Three biological replicates were performed. Data are represented as mean ± SD. C, Western blot analysis of pAKT and pERK1/2 in RT4 and RT112 cells treated with DMSO (Veh) or erdafitinib (10 and 100 nmol/L) for 16 hours. β-Actin served as a loading control. D, Schematic diagram of the proliferation assay performed to investigate the effect of CM of different stromal cells on erdafitinib response. E, Proliferation analysis of RT4 and RT112 cells treated with 10 nmol/L erdafitinib in media control (ctrl) or CM of 3T3-L1 cells or hADSC. Crystal violet staining was performed on day 7. Data are normalized to cells treated with vehicle. Four biological replicates were performed. Data are represented as mean ± SD. F, Western blot analysis of pAKT and pERK1/2 in RT4 and RT112 cells treated with 10 nmol/L erdafitinib in media control or CM of 3T3-L1 cells. β-Actin served as a loading control. G, Proliferation analysis of RT4 cells and RT112 cells treated with 10 nmol/L erdafitinib in CM of 3T3-L1 cells or heat-inactivated (HI) CM of 3T3-L1. Data were normalized to cells treated with vehicle. Crystal violet staining was performed on day 7. Three biological replicates were performed. Data are represented as mean ± SD. Two-way ANOVA was used for statistical analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: MB49 cell line was purchased from Addexbio, RT112 cell line was purchased from DSMZ (German Collection of Microorganisms and Cell Cultures), and human adipose-derived stem cells (hADSC) were purchased from Lonza.

Techniques: Western Blot, Staining, Proliferation Assay

Journal: British Journal of Cancer

Article Title: Synthetic retinoid-mediated preconditioning of cancer-associated fibroblasts and macrophages improves cancer response to immune checkpoint blockade

doi: 10.1038/s41416-024-02734-3

Figure Lengend Snippet: a Tissue sections obtained from human ccRCC and UC cases who underwent ICB therapy were hematoxylin and eosin stained (top panels), and stained for Meflin mRNA by ISH (lower panels). Boxed areas are magnified in the middle panels. Dotted lines indicate the boundaries between the tumor and the stroma. Arrowheads indicate Meflin + CAFs. b Patients with ccRCC (top) or UC (bottom) treated with ICB therapy were stratified by the numbers of Meflin + CAFs in the stromal tissue. c Objective response rate (ORR) of Meflin-high and -low ccRCC and UC cases who received ICB therapy. d PFS (top) and OS (bottom) of Meflin-high and -low ccRCC or UC patients treated with ICB therapy. e , f C57BL/6 wild-type and Meflin KO female mice were subcutaneously transplanted with MB49 cells (1 × 10 6 cells/mouse) on Day 1, followed by intraperitoneal injection of anti-PD-L1 (αPD-L1) or isotype control IgG every three days from Day 5 to Day 14 ( e ). Tumor volume ( f ). The statistical methods used are a two-tailed unpaired t-test ( c , f ) and log-rank (Mantel–Cox) test ( d ). CAFs cancer-associated fibroblasts, ccRCC clear cell renal cell carcinoma, CR complete response, ICB immune checkpoint blockade, Ig immunoglobulin, ISH in situ hybridization, KO knockout, N.S. not significant, OS overall survival, PD progressive disease, PD-L1 programmed cell death ligand 1, PFS progression-free survival, PR partial response, SD stable disease, UC: urothelial carcinoma.

Article Snippet: The mouse UC cell line MB49 was purchased from Merck Inc (cat. no. SCC148).

Techniques: Staining, Injection, Control, Two Tailed Test, In Situ Hybridization, Knock-Out

Journal: British Journal of Cancer

Article Title: Synthetic retinoid-mediated preconditioning of cancer-associated fibroblasts and macrophages improves cancer response to immune checkpoint blockade

doi: 10.1038/s41416-024-02734-3

Figure Lengend Snippet: a C57BL/6 wild-type female mice were subcutaneously transplanted with mT5 cells (1 × 10 6 cells/mouse), followed by oral Am80 administration at the indicated periods and intraperitoneal injection of anti-PD-L1 antibodies. a, b, and c indicate the periods for oral Am80 administration. b Time courses of the volume of tumors developed in mice treated by the indicated regimens (left) and their body weights (right). c C57BL/6 wild-type or Meflin KO female mice were subcutaneously transplanted with MB49 cells (1 × 10 6 cells/mouse), followed by oral administration of DMSO or Am80 prior to intraperitoneal injection of anti-PD-L1 antibodies. d , e Tissue sections obtained from the MB49 tumors on Day 12 were stained for the Islr gene by ISH ( d ), followed by quantification of Islr + cells ( e ). Arrowheads in ( d ) indicate Islr + cells. f Time courses of the volumes of tumors of the indicated groups. Differences between groups were analyzed using 1-way ANOVA with the Tukey test ( b ), and Student t-test ( e , f ). HPF high-power field (×40 objective lens), ICB immune checkpoint blockade, ISH in situ hybridization, KO knockout, NS not significant, PDAC pancreatic ductal adenocarcinoma, PD-L1 programmed cell death ligand 1, UC urothelial carcinoma.

Article Snippet: The mouse UC cell line MB49 was purchased from Merck Inc (cat. no. SCC148).

Techniques: Injection, Staining, In Situ Hybridization, Knock-Out

Journal: British Journal of Cancer

Article Title: Synthetic retinoid-mediated preconditioning of cancer-associated fibroblasts and macrophages improves cancer response to immune checkpoint blockade

doi: 10.1038/s41416-024-02734-3

Figure Lengend Snippet: a C57BL/6 wild-type or Meflin KO female mice were subcutaneously transplanted with MB49 cells (1 × 10 6 cells/mouse), followed by oral administration of DMSO or Am80 and IHC for several immune cell markers. Four random HPFs from the intratumoral area for each tissue section were evaluated for quantification. b – e Cells positive for the indicated immune cell markers in each HPF were counted, followed by quantification. Twelve HPFs were evaluated for each group. The statistical methods used were 1-way ANOVA with the Tukey test ( b – e ). HPF high-power field (×40 objective lens), IHC immunohistochemistry, KO knockout, TIME tumor immune microenvironment.

Article Snippet: The mouse UC cell line MB49 was purchased from Merck Inc (cat. no. SCC148).

Techniques: Immunohistochemistry, Knock-Out

Journal: British Journal of Cancer

Article Title: Synthetic retinoid-mediated preconditioning of cancer-associated fibroblasts and macrophages improves cancer response to immune checkpoint blockade

doi: 10.1038/s41416-024-02734-3

Figure Lengend Snippet: a C57BL/6 wild-type or Meflin KO female mice were orally administered DMSO or Am80 every day from Day 1 to Day 7. The mice were subjected to intraperitoneal injection of 2 ml TGC (3%) on Day 4, followed by isolation of peritoneal macrophages on Day 7, and quantitative PCR (qPCR) for macrophage markers. b Expression levels of Nos2 and Mrc1 relative to Actb in the indicated macrophages as examined by qPCR. c Conditioned media (CM) from primary cultured mouse MSCs treated with either DMSO or Am80 (1 µM) for 48 h were added to control RAW264.7 cells or RAW264.7 cells that had been differentiated into M2-type macrophages using recombinant IL-4 (40 ng/ml, 48 h), followed by culture for 24 h and qPCR. d Total RNA extracted from MSCs treated with DMSO or Am80 (1 µM) for 48 h was examined for the expressions of Acta2 (left) and Islr (right) relative to Actb by qPCR. e Expression levels of Nos2 and Mrc1 relative to Actb in the indicated RAW264.7 cells as examined by qPCR. f , g Peritoneal macrophages (1 × 10 6 cells/mouse), isolated from C57BL/6 wild-type or Meflin KO female mice that were administered DMSO or Am80 after intraperitoneal injection of 2 ml 3% TGC, were subcutaneously co-transplanted with MB49 cells (1 × 10 6 cells/mouse) into wild-type mice on Day 1, followed by anti-PD-L1 therapy from Day 5 to Day 11 ( f ), and measurement of the volume of the developed tumors ( g ). Statistical differences were assessed with 1-way ANOVA with the Tukey test ( b ), Student t -test ( d , g ), and 1-way ANOVA with the Dunnett test ( e ). KO knockout, MSCs mesenchymal stem cells, NS not significant, qPCR quantitative polymerase chain reaction, PD-L1 programmed cell death ligand 1, TGC thioglycollate.

Article Snippet: The mouse UC cell line MB49 was purchased from Merck Inc (cat. no. SCC148).

Techniques: Injection, Isolation, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Control, Recombinant, Knock-Out

Journal: British Journal of Cancer

Article Title: Synthetic retinoid-mediated preconditioning of cancer-associated fibroblasts and macrophages improves cancer response to immune checkpoint blockade

doi: 10.1038/s41416-024-02734-3

Figure Lengend Snippet: a Publicly available datasets of single-cell RNA transcriptomic analysis of human bladder UC (left) and PDAC (right) were analyzed for the expression of ISLR and RARRES2 , which encode Meflin and chemerin, respectively. Shown are uniform manifold approximation and projection visualization of transcriptomes of all cells depicted by BBrowser. Arrows denote the fibroblast clusters that co-express ISLR and RARRES2 . b , c Selective expression of Rarres2 in Meflin + CAFs. Tumor sections from the MB49 model were double stained for Islr and Rarres2 by ISH ( b ). Boxed areas (a, b) are magnified in adjacent panels. Magenta and cyan arrowheads denote Islr and Rarres2 signals, respectively. The numbers of cells single- or double-positive for Islr and Rarres2 were counted and quantified ( c ). d Number of Rarres2 + cells detected in tissue sections prepared from MB49 tumors developed in wild-type and Meflin KO mice that were administered DMSO or Am80 (the experiment shown in Fig. ) were counted, followed by quantification. e , f C57BL/6 wild-type or Meflin KO female mice were subcutaneously transplanted with MB49 cells (1 × 10 6 cells/mouse), followed by intratumoral injection of PBS or recombinant chemerin-9 (0.2 mg/kg) during the indicated periods, and intraperitoneal injection of anti-PD-L1 antibody ( e ). Tumor volumes of the developed tumors were measured over time ( f ). g Schematic diagram showing a working hypothesis for the mechanism of Am80-mediated tumor sensitization to ICB therapy. The mechanisms by which Meflin induces the expression of chemerin and how it is involved in the induction of M1-like macrophages remain unaddressed in the present study. Statistically significance was assessed using 1-way ANOVA with the Tukey test ( d , f ). HPF high-power field (×40 objective lens), ICB immune checkpoint blockade, ISH in situ hybridization, KO knockout, PDAC pancreatic ductal adenocarcinoma, PD-L1 programmed cell death ligand 1, UC urothelial carcinoma.

Article Snippet: The mouse UC cell line MB49 was purchased from Merck Inc (cat. no. SCC148).

Techniques: Expressing, Staining, Injection, Recombinant, In Situ Hybridization, Knock-Out

Journal: International Journal of Molecular Sciences

Article Title: Combination of Cisplatin and Irradiation Induces Immunogenic Cell Death and Potentiates Postirradiation Anti–PD-1 Treatment Efficacy in Urothelial Carcinoma

doi: 10.3390/ijms22020535

Figure Lengend Snippet: Concurrent anti–PD-1 treatment facilitated abscopal effects induced by a single dose of irradiation in MB49 tumor-bearing mice. ( A ) Scheme for tumor inoculation and treatments. Six- to seven-week-old mice are inoculated subcutaneously with MB49 cells in the left hindlimb and in the right flank, and seven days later, receive irradiation with a single fraction of 10 Gy to the left hindlimb and anti–PD-1 treatment (or its isotype controls). ( B ) MB49 tumor growth curves of irradiated tumors in the left hindlimb (left) and nonirradiated tumors in the right flank (right). ( C ) Survival curves of mice. Log-rank test is used to compare survival curves. All data are shown as the mean ± standard error of the mean (SEM). Asterisks indicate p -values comparing two groups, as indicated in the figure. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: MB49, a mouse bladder UC cell line, was purchased from Merck KGaA (Darmstadt, Germany).

Techniques: Irradiation

Journal: International Journal of Molecular Sciences

Article Title: Combination of Cisplatin and Irradiation Induces Immunogenic Cell Death and Potentiates Postirradiation Anti–PD-1 Treatment Efficacy in Urothelial Carcinoma

doi: 10.3390/ijms22020535

Figure Lengend Snippet: The combination of cisplatin and irradiation potentiated the efficacy of postirradiation anti–PD-1 treatment in MB49 tumor-bearing mice. ( A ) Scheme for tumor inoculation, treatments, and T cell analysis. For the CRT model (top), six- to seven-week-old mice are injected with MB49 cells in the left hindlimb, and seven days later, receive cisplatin at 3 mg/kg and irradiation with a single fraction of 10 Gy to the left hindlimb. The mice are injected with MB49 cells in the right flank 14 days after irradiation, and seven days later, receive anti–PD-1 treatment (or its isotype controls). For the Non-CRT model (bottom), only a right flank tumor is established in the mice, and cisplatin and irradiation are not given. ( B ) MB49 tumor growth curves of irradiated tumors in the left hindlimb (left) and nonirradiated tumors in the right flank (right). ( C ) Survival curves of mice. The log-rank test is used to compare survival curves. ( D – G ) Proportions of CD45 + cells in the live cells ( D ), CD3 + cells in the CD45 + subpopulation ( E ), CD8 + CD4 − cells in the CD3 + subpopulation ( F ), and IFNγ + cells in the CD8 + CD4 − subpopulation ( G ) in single-cell suspensions of the irradiated and nonirradiated tumors on day seven in mice treated with CRT/postirradiation anti–PD-1 treatment compared to those of the nonirradiated tumors on day seven in mice treated with anti–PD-1 treatment alone ( n = 5/group). All data are shown as the mean ± standard error of the mean (SEM). Asterisks indicate p -values comparing two groups, as indicated in the figure. NS, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: MB49, a mouse bladder UC cell line, was purchased from Merck KGaA (Darmstadt, Germany).

Techniques: Irradiation, Cell Analysis, Injection

Journal: International Journal of Molecular Sciences

Article Title: Combination of Cisplatin and Irradiation Induces Immunogenic Cell Death and Potentiates Postirradiation Anti–PD-1 Treatment Efficacy in Urothelial Carcinoma

doi: 10.3390/ijms22020535

Figure Lengend Snippet: The combination of cisplatin and irradiation increases HMGB1 protein secretion and cell surface expression of calreticulin protein in MB49 cells. ( A ) MB49 cells are treated with cisplatin at 0.6 mg/L and/or irradiated with a single fraction of 10 Gy. Medium is collected two and five days after treatment, and HMGB1 protein expression levels in each medium are examined in duplicate by ELISA ( n = 3/group). ( B ) MB49 cells are treated with cisplatin at 0.6 mg/L and/or irradiated with a single fraction of 10 Gy. Cells are collected, and cell surface calreticulin protein expression levels are examined by flow cytometry ( n = 3/group). ( C , D ) Seven days after inoculation MB49 cells in the left hindlimb, mice are treated with cisplatin at 3 mg/kg and/or irradiation with a single fraction of 10 Gy. Tumor sections before and after CRT ( n = 3/group) are stained for HMGB1 ( C ) and calreticulin ( D ). Left, representative images; right, comparison of the mean (± standard error of the mean [SEM]) number of HMGB1- or calreticulin-positive cells counted in five fields of view (FOV). All data are shown as the mean ± SEM. Asterisks indicate p values comparing two groups, as indicated in the figure. NS, not significant; * p < 0.05; ** p < 0.01.

Article Snippet: MB49, a mouse bladder UC cell line, was purchased from Merck KGaA (Darmstadt, Germany).

Techniques: Irradiation, Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining, Comparison