Journal: Bioactive Materials

Article Title: Feeder-free differentiation of human iPSCs into natural killer cells with cytotoxic potential against malignant brain rhabdoid tumor cells

doi: 10.1016/j.bioactmat.2024.02.031

Figure Lengend Snippet: Activation markers of CD56 bright CD16 bright iPSC-NK cells. (A) The Immunocytochemistry data showed positive expression of the activation markers such as SH2D1A, NKG2D, CD107a, and inhibitory markers, KIR2DL and CD94 upon coculture with (i) CHLA-02-ATRT or (ii) CHLA-05-ATRT. (B) (i) The percentage expression of the activation markers, NKG2D and NKp46, in iPSC-NK cells before and after coculture was performed using flow cytometry. (ii) The relative gene expression of NK cell markers, including NKG2D, CD107a, NKp46, CD16a and CD56 after IL-15 treatment was also measured. KIR2DL1, killer cell immunoglobulin-like receptor 2DL1; SH2D1A, SH2 domain-containing protein 1A; NKG2D, NKG2-D type II integral membrane protein. The scale bar in (A) represents 100 μm, which corresponds to a 20x magnification. The statistically significant data are represented with “*”, “**”, and “***” for p- values <0.05, <0.01, and <0.001, respectively.

Article Snippet: In this study, two distinct types of ATRT cell lines, namely CHLA-05-ATRT (ATCC® CRL-3037TM, ATCC) and CHLA-02-ATRT (ATCC® CRL-3020TM, ATCC), were used to test the cytotoxic effect of iPSC-NK cells.

Techniques: Activation Assay, Immunocytochemistry, Expressing, Flow Cytometry, Gene Expression, Membrane

Journal: Bioactive Materials

Article Title: Feeder-free differentiation of human iPSCs into natural killer cells with cytotoxic potential against malignant brain rhabdoid tumor cells

doi: 10.1016/j.bioactmat.2024.02.031

Figure Lengend Snippet: Degranulation marker expression and cytokine production in CD56 bright CD16 bright iPSC-NK cells after exposure to ATRT cells. (A) (i) Phase–contrast images showing coculture of iPSC-NK cells with CHLA-02-ATRT and CHLA-05-ATRT. (ii) Immunocytochemistry of degranulation marker CD107a in iPSC-NK cells after coculture with ATRT cells. (iii) Relative degranulation measurement in iPSC-NK cells before and after coculture with ATRT. “**”, p- value<0.01. (B) Cytokines production by human iPSC-NK cells. (i) Immunofluorescent images show positive TNF-alpha and IL-6 expression. (ii) iPSC-NK cells expressing IFN-gamma after exposure to CHLA-05-ATRT are quantified via flow cytometry. (iii) Cytokines released by iPSC-NK cells after exposure to interleukins, CHLA-05-ATRT, and CHLA-02-ATRT were quantified through ELISA assay. “*”, p -value<0.05. ns, non-significant difference. IL-6: Interleukin 6, TNF-a: Tumor necrosis factor alpha, IFN-gamma: Interferon-gamma. iNK:CD56 bright CD16 bright iPSC-derived NK cells, ILs: IL2, IL12, IL15, IL18, and IL21. Scale bar, 100 μm. The magnification is 20x for ICC images and 10x for phase-contrast images.

Article Snippet: In this study, two distinct types of ATRT cell lines, namely CHLA-05-ATRT (ATCC® CRL-3037TM, ATCC) and CHLA-02-ATRT (ATCC® CRL-3020TM, ATCC), were used to test the cytotoxic effect of iPSC-NK cells.

Techniques: Marker, Expressing, Immunocytochemistry, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Bioactive Materials

Article Title: Feeder-free differentiation of human iPSCs into natural killer cells with cytotoxic potential against malignant brain rhabdoid tumor cells

doi: 10.1016/j.bioactmat.2024.02.031

Figure Lengend Snippet: The cytotoxicity analysis of interleukins (ILs)-exposed iPSC-NK cells against ATRT cells. (A) Flow cytometry-based Calcein-AM assay. The quadrant plot displays the percentage expression of the live cell marker (Calcein-AM) on the x-axis and the dead cell marker (ETHD-1) on the y-axis for CHLA-02-ATRT or CHLA-05-ATRT after coculture with iPSC-NK cells. (B) The table depicts the percentage expression of live and dead cells as measured in (A). The live/dead ratio is observed to be lower in the coculture of both CHLA-02-ATRT and CHLA-05-ATRT with interleukin-exposed iPSC-NK cells compared to unexposed iPSC-NK cells. ILs: IL2, IL12, IL15, IL18, and IL21.

Article Snippet: In this study, two distinct types of ATRT cell lines, namely CHLA-05-ATRT (ATCC® CRL-3037TM, ATCC) and CHLA-02-ATRT (ATCC® CRL-3020TM, ATCC), were used to test the cytotoxic effect of iPSC-NK cells.

Techniques: Flow Cytometry, Calcein AM Assay, Expressing, Marker

Journal: Bioactive Materials

Article Title: Feeder-free differentiation of human iPSCs into natural killer cells with cytotoxic potential against malignant brain rhabdoid tumor cells

doi: 10.1016/j.bioactmat.2024.02.031

Figure Lengend Snippet: Decrease in the viability of cancer cells upon exposure to CD56 bright CD16 bright iPSC-NK cells. (A) Ratio-dependent decrease in CHLA-02-ATRT and CHLA-05-ATRT cell viability upon coculture with CD56 bright CD16 bright iPSC-NK cells; (B) Ratio-dependent decrease in the viability of other types of tumor cells upon coculture with CD56 bright CD16 bright iPSC-NK cells, including SF8628, a diffuse intrinsic pontine glioma cell line, G401, a kidney rhabdoid tumor cell line, and A549, a lung carcinoma cell line. iNK, CD56 bright CD16 bright iPSC-NK. Statistical significance is denoted by *, **, and *** for p -values less than 0.05, 0.01, and 0.001, respectively.

Article Snippet: In this study, two distinct types of ATRT cell lines, namely CHLA-05-ATRT (ATCC® CRL-3037TM, ATCC) and CHLA-02-ATRT (ATCC® CRL-3020TM, ATCC), were used to test the cytotoxic effect of iPSC-NK cells.

Techniques:

Journal: Bioactive Materials

Article Title: Feeder-free differentiation of human iPSCs into natural killer cells with cytotoxic potential against malignant brain rhabdoid tumor cells

doi: 10.1016/j.bioactmat.2024.02.031

Figure Lengend Snippet: Further maturation of iPSC-NK cells into CD56 -ve CD16 bright phenotype. (A) (i) Phase-contrast imaging on Day 65 and Day 70. (ii) Immunocytochemistry (ICC) images of CD56 and CD16. (iii) Flow cytometry quantification shows the highly positive expression of CD16 and low expression for the CD56 marker. (B) Immunocytochemistry detects the expression of CD56 and CD16 in iPSC-NK cells after activation using CHLA-02-ATRT, IL-15, and a combination of interleukins (IL2, IL12, IL15, IL18, and IL21). (C) The percentage expression of CD16 and CD56 under each condition remains relatively stable. Scale bar: 100 μm.

Article Snippet: In this study, two distinct types of ATRT cell lines, namely CHLA-05-ATRT (ATCC® CRL-3037TM, ATCC) and CHLA-02-ATRT (ATCC® CRL-3020TM, ATCC), were used to test the cytotoxic effect of iPSC-NK cells.

Techniques: Imaging, Immunocytochemistry, Flow Cytometry, Expressing, Marker, Activation Assay

Journal: Bioactive Materials

Article Title: Feeder-free differentiation of human iPSCs into natural killer cells with cytotoxic potential against malignant brain rhabdoid tumor cells

doi: 10.1016/j.bioactmat.2024.02.031

Figure Lengend Snippet: Characterization of the activation markers in CD56 -ve CD16 bright iPSC-NK cells and their cytotoxicity toward ATRT cells. (A–B) Immunocytochemistry and flow cytometry data indicate the absence of expression for NKG2D and NKp46 markers. At this stage, NKG2D in iPSC-NK cells was not upregulated after coculture with CHLA-02-ATRT. (C) Effects of ILs-exposed iPSC-NK cells versus ILs-unexposed iPSC-NK cells on cell viability of CHLA-02-ATRT and CHLA-05-ATRT cells have no statistically significant decrease in cell viability compared to the untreated group, E: T = 0:1. ns, non-significant difference. The data were verified by two independent experiments, and each experiment has triplicates (n = 3).

Article Snippet: In this study, two distinct types of ATRT cell lines, namely CHLA-05-ATRT (ATCC® CRL-3037TM, ATCC) and CHLA-02-ATRT (ATCC® CRL-3020TM, ATCC), were used to test the cytotoxic effect of iPSC-NK cells.

Techniques: Activation Assay, Immunocytochemistry, Flow Cytometry, Expressing

Journal: Cancer Science

Article Title: Hypoxia‐related carbonic anhydrase 9 induces serpinB9 expression in cancer cells and apoptosis in T cells via acidosis

doi: 10.1111/cas.16133

Figure Lengend Snippet: Increased expression of the serpinB9 gene in RENCA/hCA9 cells. (A) Gene array analysis was conducted using total RNAs extracted from RENCA and RENCA/hCA9 cells. Red and blue lines indicate fourfold upregulation and downregulation in RENCA/hCA9, respectively. (B) Gene Ontology enrichment analysis of genes with more than fourfold upregulated expression was performed using Metascape. (C) The correlation between CA9 and s erpinB9 gene expression in 43 human kidney cancer cell lines from the Cancer Cell Line Encyclopedia data (version 23Q2) was assessed, with results shown as a scatter plot. TPM, transcripts per million; ρ , Spearman's rank correlation coefficient.

Article Snippet: Three distinct SerpinB9 siRNAs and three distinct mouse CA9 siRNAs, along with a control siRNA, were purchased from ORIGENE.

Techniques: Expressing

Journal: Cancer Science

Article Title: Hypoxia‐related carbonic anhydrase 9 induces serpinB9 expression in cancer cells and apoptosis in T cells via acidosis

doi: 10.1111/cas.16133

Figure Lengend Snippet: SerpinB9 contributes to immune resistance to cytotoxic T lymphocyte (CTL)‐mediated cytotoxicity. (A) Expression of serpinB9 in RENCA and RENCA/hCA9 tissues is displayed. Scale bar, 100 μm. (B) Both RENCA and RENCA/hCA9 cells were cultured under normoxic or hypoxic conditions or treated with deferoxamine (DFO) (100 μg/mL) for 3 days. The harvested cells were tested for protein expression of serpinB9. (C) RENCA cells were transfected with a control or one of three different mouse CA9 siRNAs and then cultured under hypoxic conditions (1% O 2 ). Quantitative RT‐PCR was performed 2 days later. (D) RENCA cells were transfected with control or mouse CA9 siRNA (C) and cultured under normoxic (20% O 2 ) or hypoxic (1% O 2 ) conditions. After 2 days, the serpinB9 expression levels were determined. (E) The sensitivity of AH1 peptide‐pulsed RENCA and RENCA/hCA9 cells to AH1 peptide‐specific CTLs was determined using a 5‐h 51 Cr release test. (F) Two days post siRNA transfection, immunoblots were performed to assess expression of serpinB9. Three separate serpinB9 siRNAs were transfected. Cont, control. (G) Two days after transfection with serpinB9(C) siRNA, RENCA/hCA9 cells were pulsed with the AH1 peptide and used as targets for AH1 peptide‐specific CTLs. * p < 0.05, ** p < 0.01 (Student's t ‐test); 5‐h 51 Cr release test was conducted. E/T, effector/target ratio.

Article Snippet: Three distinct SerpinB9 siRNAs and three distinct mouse CA9 siRNAs, along with a control siRNA, were purchased from ORIGENE.

Techniques: Expressing, Cell Culture, Transfection, Control, Quantitative RT-PCR, Western Blot

Journal: Cancer Science

Article Title: Hypoxia‐related carbonic anhydrase 9 induces serpinB9 expression in cancer cells and apoptosis in T cells via acidosis

doi: 10.1111/cas.16133

Figure Lengend Snippet: CA9 increases serpinB9 expression and induces T cell apoptosis through acidification. (A) RENCA cells were transfected with human or mouse CA9 genes. On days 2, 4, and 6, cells were harvested to examine the expression of CA9 and serpinB9. (B) Quantitative RT‐PCR was used to determine the levels of mRNAs transcribed from hCA9 , mCA9 , and serpinB9 . (C) The hCA9 and serpinB9 protein levels in RENCA, RENCA/hCA9, and the new hCA9‐expressing RENCA cell lines (N1, N2, and N3) were determined. (D) RENCA and RENCA/hCA9 cells were cultured in SB‐free or SB‐containing medium with CO 2 for 5 days. Cell counts and pH levels of the culture medium are displayed. Data from three wells are presented. * p < 0.05, ** p < 0.01 (Student's t ‐test). (E) RENCA and CT26 cells were cultured under two distinct pH conditions (6.5 and 7.4) using sodium bicarbonate‐free medium in a CO 2 incubator for 5 days. Subsequently, cells were harvested to check serpinB9 expression. (F) T cells extracted from naïve BALB/c spleen cells, RENCA, and RENCA/hCA9 cells were cultured under three different pH conditions (6.5, 7.0, and 7.4) in a sodium bicarbonate‐free medium in a CO 2 incubator for 3 days. T cells, after staining with PE‐conjugated anti‐CD4 mAb and APC‐conjugated anti‐CD8 mAb, were then stained with annexin V‐FITC and examined via flow cytometry. Cancer cells, post staining with annexin V‐FITC and PI, were analyzed by flow cytometry. (G) T cells purified from naïve BALB/c spleen cells, RENCA, and RENCA/hCA9 cells were cocultured in a SB‐free or SB‐containing medium in a CO 2 incubator for 2 and 4 days. Post staining with PE‐conjugated anti‐CD4 mAb and APC‐conjugated anti‐CD8 mAb, they were further stained with annexin V‐FITC and analyzed by flow cytometry. Data are presented as means ± SD from three samples. * p < 0.05, ** p < 0.01 (Student's t ‐test). SB, sodium bicarbonate.

Article Snippet: Three distinct SerpinB9 siRNAs and three distinct mouse CA9 siRNAs, along with a control siRNA, were purchased from ORIGENE.

Techniques: Expressing, Transfection, Quantitative RT-PCR, Cell Culture, Staining, Flow Cytometry, Purification

Journal: Cancer Science

Article Title: Hypoxia‐related carbonic anhydrase 9 induces serpinB9 expression in cancer cells and apoptosis in T cells via acidosis

doi: 10.1111/cas.16133

Figure Lengend Snippet: Prognosis among renal cell carcinoma (RCC) patients with high serpinB9 expression. (A) The Kaplan–Meier plotter univariate analysis illustrates the overall survival duration based on CA9 mRNA and serpinB9 mRNA expression in kidney renal clear cell carcinoma patients. (B) Kaplan–Meier plotter univariate analysis represents the overall survival time relative to the ratio of serpinB9 mRNA/ CA9 mRNA expression in kidney renal clear cell carcinoma patients. (C) The Kaplan–Meier univariate plot reveals the overall survival duration according to the levels of CA9 mRNA and serpinB9 mRNA expression in patients with renal papillary cell carcinomas.

Article Snippet: Three distinct SerpinB9 siRNAs and three distinct mouse CA9 siRNAs, along with a control siRNA, were purchased from ORIGENE.

Techniques: Expressing