Journal: JCI Insight

Article Title: Differential CXCR4 expression on hematopoietic progenitor cells versus stem cells directs homing and engraftment

doi: 10.1172/jci.insight.151847

Figure Lengend Snippet: ( A – D ) Irradiated NSG mice were transplanted with MPB CD34 + HSPC that were transduced with a GFP-encoding LV vector (gene transfer 63%–66%) either i.v. or i.b.m. at the indicated cell doses. Human cell engraftment was analyzed by determining the percentage of human CD45 + (hCD45 + ) cells and GM hCD45 + (hCD45 + GFP + ) cells in the BM obtained via BM aspirate at 12 weeks to assess short-term engraftment ( A and B ) and from pooled BM from femurs, tibias, and iliac crests at 24 weeks to determine long-term human engraftment ( C and D ). Bars represent median engraftment ± 95% CI. Each symbol represents an individual mouse; n = 18–20 mice per cell dose per transplant method. Mice were transplanted using 4 unique MPB donors in 4 experiments; statistical analysis was performed using 1-way ANOVA comparing i.v. and i.b.m. groups at each cell dose with correction for multiple comparisons. ( E – H ) Lineage output of the human xenograft. BM was stained with antibodies specific for T cell lineage (hCD3), B cell lineage (hCD19), myeloid lineage (hCD33), and HSPC (hCD34) to determine the lineage output of the transplanted total HSPC ( E and G ) and GM HSPC ( F and H ) at 12 weeks and 24 weeks after transplant. Statistical analysis was performed using 2-way ANOVA. * P < 0.05, ** P < 0.01.

Article Snippet: CD34 + CD38 – cells were isolated from G-CSF MPB by magnetic selection with human CD34 + CD38 – Isolation Kit (Miltenyi Biotec, 130-114-822) and cryopreserved.

Techniques: Irradiation, Transduction, Plasmid Preparation, Staining

Journal: JCI Insight

Article Title: Differential CXCR4 expression on hematopoietic progenitor cells versus stem cells directs homing and engraftment

doi: 10.1172/jci.insight.151847

Figure Lengend Snippet: ( A ) MPB CD34 + HSPC were injected i.v., i.b.m. using a standard syringe, or slow i.b.m. using a Hamilton syringe over 1 minute, and homing of CD34 + cells to the BM was analyzed 20–22 hours later. To determine if mechanical pressure in the injected femur alters homing, irradiated CD34 – cells were injected i.b.m., and CD34 + cells were injected i.v. into the same animal. Homing was determined in the injected femurs (IF) and non-IF with i.b.m. injections, and in both femurs (BF) combined with i.v. injection. Symbols represent individual animals; n = 3 mice per group; statistical analysis was performed using 2-way ANOVA. ( B and C ) For i.b.m. injections, BM from the IF and non-IF was analyzed for expression of CXCL12 and VCAM1 on mouse stromal cells (hCD45 – PKH-26 – , mouse lineage – mCD45 – mCD51 + cells). n = 5 mice per group; data were normalized to i.v. injected group; statistical analysis was performed by Mann-Whitney U test. ( D and E ) CXCR4 and VLA4 homing receptor expression of CD34 + HSPC, CD34 + CD38 + HPC, and CD34 + CD38 – CD90 + HSC-enriched populations. Data were normalized to CXCR4 and VLA4 expression on CD34 + HSPC. Symbols represent unique MPB donors; n = 5 per group; statistical analysis was performed using ANOVA. ( F ) Adhesion of CD34 + HSPC, CD34 + CD38 + HPC, and CD34 + CD38 – CD90 + HSC to CXCL12. Symbols represent unique MPB donors; n = 5 per group; statistical analysis was performed using ANOVA. ( G ) Binding of CD34 + HSPC, CD34 + CD38 + HPC, and CD34 + CD38 – CD90 + HSC to the VLA4 affinity detection ligand LDV-FITC. Data were normalized to LDV-FITC expression in CD34 + HSPC. Symbols represent unique MPB donors; n = 3 per group; statistical analysis was performed using 1-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001. In B – E and G , data were normalized to the indicated group, to account for variability in MFI between different experiments/donors.

Article Snippet: CD34 + CD38 – cells were isolated from G-CSF MPB by magnetic selection with human CD34 + CD38 – Isolation Kit (Miltenyi Biotec, 130-114-822) and cryopreserved.

Techniques: Injection, Irradiation, Expressing, MANN-WHITNEY, Binding Assay

Journal: JCI Insight

Article Title: Differential CXCR4 expression on hematopoietic progenitor cells versus stem cells directs homing and engraftment

doi: 10.1172/jci.insight.151847

Figure Lengend Snippet: ( A – C ) High CXCR4 expression on HPC mediates their preferential homing locally with i.b.m. transplant; CD34 + HSPC were transduced with a BFP LV vector, and the CD34 + CD38 + BFP + HPC were sorted by flow cytometry 72 hours after gene transfer. HPC were blocked with AMD3100 (CXCR4 antagonist) or BIO5192 (VLA4 antagonist) before i.v. or i.b.m. delivery ( A ). Homing of hCD34 + cells was analyzed in the injected femur (IF) and non-IF for i.b.m. injected mice and in both femurs combined in i.v. injected mice. Data were normalized to i.v. injected control CD34 + CD38 + cells, and the fold increase is indicated within the bar ( B and C ). n = 5–6 mice per group; statistical analysis was performed by 1-way ANOVA. ( D – F ) Induction of high CXCR4 expression on CD34 + CD38 – HSC enriched population via gene transfer confers them with a homing advantage despite higher abundance of CD34 + CD38 + HPC. CD34 + HSPC were transduced with a BFP LV vector or a BFP-CXCR4 LV vector and sorted for transduced CD34 + CD38 – BFP + cells and untransduced CD34 + CD38 + BFP – HPC at 72 hours. Transduced CD34 + CD38 – cells were mixed with untransduced CD34 + CD38 + cells and transplanted i.v. or i.b.m., and homing of CD34 + CD38 – BFP + cells into BM was analyzed at 20–22 hours ( D ). CXCR4 expression on injected cells at time of transplant is shown and the MFI on injected cells at time of transplant was as follows: CD34 + CD38 – control, 16,930; CD34 + CD38 + control, 35,098; and CD34 + CD38 – CXCR4 transduced, 37,453. ( E ). Homing in BM is shown ( F ). n = 3–10 mice per experimental arm; statistical analysis was performed using 1-way ANOVA. * P < 0.05.

Article Snippet: CD34 + CD38 – cells were isolated from G-CSF MPB by magnetic selection with human CD34 + CD38 – Isolation Kit (Miltenyi Biotec, 130-114-822) and cryopreserved.

Techniques: Expressing, Transduction, Plasmid Preparation, Flow Cytometry, Injection, Control

Journal: JCI Insight

Article Title: Differential CXCR4 expression on hematopoietic progenitor cells versus stem cells directs homing and engraftment

doi: 10.1172/jci.insight.151847

Figure Lengend Snippet: CD34 + CD38 – HSC-enriched cells were obtained using immunomagnetic sorting, transduced with a GFP LV vector (gene transfer efficiency was 69%), and transplanted into NSG mice i.v. or i.b.m. in 2 limiting dilution doses. ( A and B ) Long-term (24 week) engraftment of total (hCD45 + ) and GM human (hCD45 + GFP + ) cells is shown. Symbols represent individual animals; n = 3–4 mice per treatment condition; statistical analysis was performed by comparing the modes of delivery comparing the different cell doses using 1-way ANOVA. * P < 0.05. ( C and D ) Lineage output at 24 weeks shows that the long-term human graft was multilineage, composed of B, T, and myeloid cells and CD34 + HSPC. Statistical analysis was performed using 2-way ANOVA.

Article Snippet: CD34 + CD38 – cells were isolated from G-CSF MPB by magnetic selection with human CD34 + CD38 – Isolation Kit (Miltenyi Biotec, 130-114-822) and cryopreserved.

Techniques: Transduction, Plasmid Preparation

Journal: JCI Insight

Article Title: Differential CXCR4 expression on hematopoietic progenitor cells versus stem cells directs homing and engraftment

doi: 10.1172/jci.insight.151847

Figure Lengend Snippet: ( A ) Construct design. The aa sequence of VPR and its mutant version generated is shown. VPR, a small HIV-1 accessory protein, is carried in viral particles bound to gag via residues in its central region that fold into 3 α-helices. The C-terminal domain has 6 arginine residues that potentiate nuclear localization, G 2 M arrest, and apoptosis. S79 phosphorylation is important for cell cycle arrest. We truncated VPR at the 78 aa and mutated VPR R77Q . W54 and Q65 interacts with DNA damage response (DDR) proteins via UNG2 and DCAF and were mutated to VPR W54R and VPR Q65R . The triple mutated and truncated VPR (VPR MT ) would lack pathogenicity (residues known to be associated with VPR toxicity are highlighted in red) but allow binding to gag. VPR MT was fused to CXCR4 cDNA via the HIV-1 protease cleavage site (PCS) to generate VPR MT -CXCR4. ( B ) K562 cells were transduced with LV CXCR4 vector-like particles (VLP; empty vector particles lacking the vector genome) at increasing particle concentration and analyzed for CXCR4 expression using flow cytometry. Mean fluorescence intensity (MFI) of CXCR4 is listed against volume of VLP added. ( C and D ) A GFP-encoding LV was either packaged using standard packaging plasmids (LV, black) or packaged with VPR MT -CXCR4 plasmid in addition in order to package the CXCR protein attached to the LV capsid (LV CXCR4 , red). CXCR4 expression on MPB CD34 + cells transduced with LV CXCR4 vector compared with cells transduced with the control LV vector 24 hours following gene transfer is shown. n = 4; statistical analysis was performed by Mann-Whitney U test. ( E ) The time course of CXCR4 expression in LV CXCR4 HSPC, normalized to that of control LV HSPC, is shown, with expression peaking at 24 hours that returns to baseline by 72 hours. Symbols represent individual MPB donors; n = 3.

Article Snippet: CD34 + CD38 – cells were isolated from G-CSF MPB by magnetic selection with human CD34 + CD38 – Isolation Kit (Miltenyi Biotec, 130-114-822) and cryopreserved.

Techniques: Construct, Sequencing, Mutagenesis, Generated, Phospho-proteomics, Binding Assay, Transduction, Plasmid Preparation, Concentration Assay, Expressing, Flow Cytometry, Fluorescence, Control, MANN-WHITNEY

Journal: JCI Insight

Article Title: Differential CXCR4 expression on hematopoietic progenitor cells versus stem cells directs homing and engraftment

doi: 10.1172/jci.insight.151847

Figure Lengend Snippet: Experimental schema for assessment of homing and engraftment of CD34 + CD38 – cells transduced with GFP LV or GFP LV CXCR4 that were transplanted into NSG mice either via i.v. or i.b.m. delivery. ( A ) The number of animals used for the homing experiment and engraftment experiments is indicated under each experimental arm. ( B ) Homing of CD34 + CD38 – cells in the BM. Data were normalized to i.v. injected control CD34 + CD38 – cells, and the fold increase is indicated within the bar. Symbols represent individual mice; statistical analysis was performed using ANOVA. ( C and D ) Long-term engraftment of CD34 + CD38 – cells was assessed by determining the percentage of human CD45 + GFP – cells ( C ) and human CD45 + GFP + ( D ) cells 24 weeks following transplant. Data were normalized to i.v. transplanted GFP LV transduced CD34 + CD38 – cells, and the fold increase is indicated within the bar. Gene transfer was 60% with GFP LV and 52% with GFP LV CXCR4 . Symbols represent individual mice; statistical analysis was performed by 1-way ANOVA. * P < 0.05, ** P < 0.01.

Article Snippet: CD34 + CD38 – cells were isolated from G-CSF MPB by magnetic selection with human CD34 + CD38 – Isolation Kit (Miltenyi Biotec, 130-114-822) and cryopreserved.

Techniques: Transduction, Injection, Control

Journal: Journal of Cancer

Article Title: Integrative Analysis Identified CD38 As a Key Node That Correlates Highly with Immunophenotype, Chemoradiotherapy Resistance, And Prognosis of Head and Neck Cancer

doi: 10.7150/jca.59730

Figure Lengend Snippet: CD38 is closely related to HNSC immune microenvironment.

Article Snippet: Then, 50 μg lysate was electrophoresed on 10% separation gel and transferred to a polyvinylidene fluoride (PVDF) membrane (HyClone Laboratories, Logan, UT, USA), which was blocked with 5% non-fat milk diluted in phosphate-buffered saline (PBS)-Tween20 for approximately 2 h. Primary antibody solution was then added and incubated at 4 °C for 12 h. The primary antibodies used were: Anti-phospho-phosphatidylinositol 3-kinase (PI3K) p85 alpha (Tyr607), anti-phospho-PI3 Kinase p85 beta (Ser605), anti‐phospho‐PI3K p85 (Tyr458), anti-CD38, anti-phosphatase and tensin homolog deleted on chromosome ten (PTEN), anti-NF-κb, anti-mTOR, anti-p53, and anti-PD-L1 (at dilution ratios of 1:500-1:2000 using 5% Skim milk, Affinity Biosciences (Cincinnati OH, USA) supplied the antibodies for PI3K, CD38, PTEN, and Cell Signaling Technology (Danvers, MA, USA) supplied the PD-L1 antibodies.

Techniques:

Journal: Journal of Cancer

Article Title: Integrative Analysis Identified CD38 As a Key Node That Correlates Highly with Immunophenotype, Chemoradiotherapy Resistance, And Prognosis of Head and Neck Cancer

doi: 10.7150/jca.59730

Figure Lengend Snippet: CD38 and the number of immune cells in the HNSC microenvironment correlate positively. The TIMER database was used to analyze the correlation between the expression of CD38 in HNSC and the expression of biomarkers in immune infiltrating cells. (A-K) Scatter plots representing correlation between CD38 expression and the biomarkers of (A) B cells (CD19 and CD79A); (B) Monocytes (CD86); (C) CD8+ T cells (CD8A and CD8B); (D) Neutrophils (CCR7 and ITGAM); (E) Tregs (CCR8 and STAT5B); (F) TAMs (CD68 and IL10); (G) Exhausted T cells (CTLA4, HAVCR2 and PDCD1); (H) M1 Macrophages (IRF5); (I) M2 Macrophages (CD163, MS4A4A and VSIG4); and (J) Th1 cells (IFNG, STAT1, and TBX21)) in HNSC samples. (K) Th2 cells (STAT6).

Article Snippet: Then, 50 μg lysate was electrophoresed on 10% separation gel and transferred to a polyvinylidene fluoride (PVDF) membrane (HyClone Laboratories, Logan, UT, USA), which was blocked with 5% non-fat milk diluted in phosphate-buffered saline (PBS)-Tween20 for approximately 2 h. Primary antibody solution was then added and incubated at 4 °C for 12 h. The primary antibodies used were: Anti-phospho-phosphatidylinositol 3-kinase (PI3K) p85 alpha (Tyr607), anti-phospho-PI3 Kinase p85 beta (Ser605), anti‐phospho‐PI3K p85 (Tyr458), anti-CD38, anti-phosphatase and tensin homolog deleted on chromosome ten (PTEN), anti-NF-κb, anti-mTOR, anti-p53, and anti-PD-L1 (at dilution ratios of 1:500-1:2000 using 5% Skim milk, Affinity Biosciences (Cincinnati OH, USA) supplied the antibodies for PI3K, CD38, PTEN, and Cell Signaling Technology (Danvers, MA, USA) supplied the PD-L1 antibodies.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: Integrative Analysis Identified CD38 As a Key Node That Correlates Highly with Immunophenotype, Chemoradiotherapy Resistance, And Prognosis of Head and Neck Cancer

doi: 10.7150/jca.59730

Figure Lengend Snippet: CD38 mRNA level of in head and neck squamous cell carcinoma . Overexpression gene rank, fold change, and related p -values are shown, based on Oncomine 4.5 analysis. (A-D) Box plot showing CD38 mRNA expression in Ginos Head-Neck, Pyeon Multi-cancer, Toruner Head-Neck, and Cromer Head-Neck datasets, respectively.

Article Snippet: Then, 50 μg lysate was electrophoresed on 10% separation gel and transferred to a polyvinylidene fluoride (PVDF) membrane (HyClone Laboratories, Logan, UT, USA), which was blocked with 5% non-fat milk diluted in phosphate-buffered saline (PBS)-Tween20 for approximately 2 h. Primary antibody solution was then added and incubated at 4 °C for 12 h. The primary antibodies used were: Anti-phospho-phosphatidylinositol 3-kinase (PI3K) p85 alpha (Tyr607), anti-phospho-PI3 Kinase p85 beta (Ser605), anti‐phospho‐PI3K p85 (Tyr458), anti-CD38, anti-phosphatase and tensin homolog deleted on chromosome ten (PTEN), anti-NF-κb, anti-mTOR, anti-p53, and anti-PD-L1 (at dilution ratios of 1:500-1:2000 using 5% Skim milk, Affinity Biosciences (Cincinnati OH, USA) supplied the antibodies for PI3K, CD38, PTEN, and Cell Signaling Technology (Danvers, MA, USA) supplied the PD-L1 antibodies.

Techniques: Over Expression, Expressing

Journal: Journal of Cancer

Article Title: Integrative Analysis Identified CD38 As a Key Node That Correlates Highly with Immunophenotype, Chemoradiotherapy Resistance, And Prognosis of Head and Neck Cancer

doi: 10.7150/jca.59730

Figure Lengend Snippet: Relationship between CD38 expression and clinical parameters in healthy subjects and patients with HNSC. (A) Box diagram showing the relative transcript levels of CD38 in HNSC and healthy samples. (B) Relative CD38 transcription level in healthy individuals or in patient with HNSC in disease stage 1, 2, 3, or 4 shown as a box plot. (C) Relative CD38 transcription level in healthy individuals of any race or in patients with HNSC of Caucasian, African-American, or Asian ethnicity, shown using a box plot. (D) Relative C D38 transcription level in healthy individuals of either gender or in female or male patients with HNSC, shown as a box plot. (E) Relative CD38 transcription level in healthy individuals with any age or in patients with HNSC at ages of 21-40, 41-60, 61-80, or 81-100 years, shown as a box plot. (F) Relative CD38 transcription level in healthy individuals or in patients with HNSC with tumor grade 1, 2, 3, or 4, as shown in a box plot. Data are the mean ± SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Then, 50 μg lysate was electrophoresed on 10% separation gel and transferred to a polyvinylidene fluoride (PVDF) membrane (HyClone Laboratories, Logan, UT, USA), which was blocked with 5% non-fat milk diluted in phosphate-buffered saline (PBS)-Tween20 for approximately 2 h. Primary antibody solution was then added and incubated at 4 °C for 12 h. The primary antibodies used were: Anti-phospho-phosphatidylinositol 3-kinase (PI3K) p85 alpha (Tyr607), anti-phospho-PI3 Kinase p85 beta (Ser605), anti‐phospho‐PI3K p85 (Tyr458), anti-CD38, anti-phosphatase and tensin homolog deleted on chromosome ten (PTEN), anti-NF-κb, anti-mTOR, anti-p53, and anti-PD-L1 (at dilution ratios of 1:500-1:2000 using 5% Skim milk, Affinity Biosciences (Cincinnati OH, USA) supplied the antibodies for PI3K, CD38, PTEN, and Cell Signaling Technology (Danvers, MA, USA) supplied the PD-L1 antibodies.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: Integrative Analysis Identified CD38 As a Key Node That Correlates Highly with Immunophenotype, Chemoradiotherapy Resistance, And Prognosis of Head and Neck Cancer

doi: 10.7150/jca.59730

Figure Lengend Snippet: Relationship between prognosis and CD38 expression in patients with HNSC. (A) The relationship between the expression of CD38 and OS (overall survival) in patients with HNSC. (B) Relationship between the tumor grade and CD38 in different clinical tumors of HNSC and the OS in patients with HNSC.

Article Snippet: Then, 50 μg lysate was electrophoresed on 10% separation gel and transferred to a polyvinylidene fluoride (PVDF) membrane (HyClone Laboratories, Logan, UT, USA), which was blocked with 5% non-fat milk diluted in phosphate-buffered saline (PBS)-Tween20 for approximately 2 h. Primary antibody solution was then added and incubated at 4 °C for 12 h. The primary antibodies used were: Anti-phospho-phosphatidylinositol 3-kinase (PI3K) p85 alpha (Tyr607), anti-phospho-PI3 Kinase p85 beta (Ser605), anti‐phospho‐PI3K p85 (Tyr458), anti-CD38, anti-phosphatase and tensin homolog deleted on chromosome ten (PTEN), anti-NF-κb, anti-mTOR, anti-p53, and anti-PD-L1 (at dilution ratios of 1:500-1:2000 using 5% Skim milk, Affinity Biosciences (Cincinnati OH, USA) supplied the antibodies for PI3K, CD38, PTEN, and Cell Signaling Technology (Danvers, MA, USA) supplied the PD-L1 antibodies.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: Integrative Analysis Identified CD38 As a Key Node That Correlates Highly with Immunophenotype, Chemoradiotherapy Resistance, And Prognosis of Head and Neck Cancer

doi: 10.7150/jca.59730

Figure Lengend Snippet: Activation of the PI3K pathway induced by overexpression of CD38. Western blotting showed that the level of PD-L1 (A) and PI3K phosphorylated at Tyr607 and Ser605 (C,D) and were increased, while the level of PTEN (B) was decreased after overexpression of CD38.

Article Snippet: Then, 50 μg lysate was electrophoresed on 10% separation gel and transferred to a polyvinylidene fluoride (PVDF) membrane (HyClone Laboratories, Logan, UT, USA), which was blocked with 5% non-fat milk diluted in phosphate-buffered saline (PBS)-Tween20 for approximately 2 h. Primary antibody solution was then added and incubated at 4 °C for 12 h. The primary antibodies used were: Anti-phospho-phosphatidylinositol 3-kinase (PI3K) p85 alpha (Tyr607), anti-phospho-PI3 Kinase p85 beta (Ser605), anti‐phospho‐PI3K p85 (Tyr458), anti-CD38, anti-phosphatase and tensin homolog deleted on chromosome ten (PTEN), anti-NF-κb, anti-mTOR, anti-p53, and anti-PD-L1 (at dilution ratios of 1:500-1:2000 using 5% Skim milk, Affinity Biosciences (Cincinnati OH, USA) supplied the antibodies for PI3K, CD38, PTEN, and Cell Signaling Technology (Danvers, MA, USA) supplied the PD-L1 antibodies.

Techniques: Activation Assay, Over Expression, Western Blot

Journal: Journal of Cancer

Article Title: Integrative Analysis Identified CD38 As a Key Node That Correlates Highly with Immunophenotype, Chemoradiotherapy Resistance, And Prognosis of Head and Neck Cancer

doi: 10.7150/jca.59730

Figure Lengend Snippet: Expression of CD38 in nasopharyngeal carcinoma cell lines. (A) Western blotting analysis showing the levels of CD38 in nasopharyngeal carcinoma epithelial cells (NP69) and different nasopharyngeal carcinoma cell lines (CNE2, HNE2, 6-10B, HONE1, HK1, and C666-1). (B) Detection of CD38 levels in radiotherapy-tolerant cell lines HNE2-IR and CNE2-IR. (C) HNE2 and CNE2 cells were both transfected with the CD38 expression vector and control vector, CD38 levels were detected using immunoblotting. (D,E) Clone formation experiment to verify the successful construction of HNE2-IR and CNE2-IR. The experiments were performed in triplicate and repeated at least two times. Student's t test was used for statistical analysis. * P < 0.01; ** P < 0.001.

Article Snippet: Then, 50 μg lysate was electrophoresed on 10% separation gel and transferred to a polyvinylidene fluoride (PVDF) membrane (HyClone Laboratories, Logan, UT, USA), which was blocked with 5% non-fat milk diluted in phosphate-buffered saline (PBS)-Tween20 for approximately 2 h. Primary antibody solution was then added and incubated at 4 °C for 12 h. The primary antibodies used were: Anti-phospho-phosphatidylinositol 3-kinase (PI3K) p85 alpha (Tyr607), anti-phospho-PI3 Kinase p85 beta (Ser605), anti‐phospho‐PI3K p85 (Tyr458), anti-CD38, anti-phosphatase and tensin homolog deleted on chromosome ten (PTEN), anti-NF-κb, anti-mTOR, anti-p53, and anti-PD-L1 (at dilution ratios of 1:500-1:2000 using 5% Skim milk, Affinity Biosciences (Cincinnati OH, USA) supplied the antibodies for PI3K, CD38, PTEN, and Cell Signaling Technology (Danvers, MA, USA) supplied the PD-L1 antibodies.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Control

Journal: Journal of Cancer

Article Title: Integrative Analysis Identified CD38 As a Key Node That Correlates Highly with Immunophenotype, Chemoradiotherapy Resistance, And Prognosis of Head and Neck Cancer

doi: 10.7150/jca.59730

Figure Lengend Snippet: Resistance in CD38-high cells to radiation treatment at 4 Gy and 6 Gy. (A,B) The effects of CD38 overexpression on colony formation in HNE2 and CNE2 cells under radiation dose of 0, 2, 4, and 6 Gy. The experiments were performed in triplicate and repeated at least three times. Student's t test was used for statistical analysis. **, P < 0.01; ***, P < 0.001.

Article Snippet: Then, 50 μg lysate was electrophoresed on 10% separation gel and transferred to a polyvinylidene fluoride (PVDF) membrane (HyClone Laboratories, Logan, UT, USA), which was blocked with 5% non-fat milk diluted in phosphate-buffered saline (PBS)-Tween20 for approximately 2 h. Primary antibody solution was then added and incubated at 4 °C for 12 h. The primary antibodies used were: Anti-phospho-phosphatidylinositol 3-kinase (PI3K) p85 alpha (Tyr607), anti-phospho-PI3 Kinase p85 beta (Ser605), anti‐phospho‐PI3K p85 (Tyr458), anti-CD38, anti-phosphatase and tensin homolog deleted on chromosome ten (PTEN), anti-NF-κb, anti-mTOR, anti-p53, and anti-PD-L1 (at dilution ratios of 1:500-1:2000 using 5% Skim milk, Affinity Biosciences (Cincinnati OH, USA) supplied the antibodies for PI3K, CD38, PTEN, and Cell Signaling Technology (Danvers, MA, USA) supplied the PD-L1 antibodies.

Techniques: Over Expression

Journal: Cancers

Article Title: Neoantigen Dendritic Cell Vaccination Combined with Anti-CD38 and CpG Elicits Anti-Tumor Immunity against the Immune Checkpoint Therapy-Resistant Murine Lung Cancer Cell Line LLC1

doi: 10.3390/cancers13215508

Figure Lengend Snippet: The adenosine pathway in the LLC1 tumor microenvironment. ( A ) RNA was extracted from ASB-XIV and LLC1 tumor cells and subjected to RNA-Seq analysis. The mRNA expressions of immunosuppressive molecules in these two cell lines were shown. ( B ) The expressions of CD38, CD39, and CD73 on LLC1 were examined by flow cytometry in vitro. ( C ) C57BL/6 mice ( n = 5) received a subcutaneous injection of 5 × 10 5 LLC1. The combination of 2 mg/Kg A2A and 1 mg/Kg A2B adenosine receptor antagonists (SCH 58261 and PSB 1115) was administered on days 7, 9, 11, 13, 15, 17, and 19. ( D ) LLC1 tumor growth was compared in mice ( n = 8) that received 30μg CpG monotherapy, L82 LP-pulsed DC vaccination (1 × 10 6 ), and CpG, or the triple combination of DC vaccination, CpG, and A2R antagonists. DC vaccines were prophylactically administered twice at a 2-week interval.

Article Snippet: Monoclonal antibodies (mAbs) specific for PD-1 (RMP1-14), CTLA-4 (9H10), CD38 (NIMR5), and CD16/32 (2.4G2) were purchased from BioXcell (Lebanon, NH, USA).

Techniques: RNA Sequencing Assay, Flow Cytometry, In Vitro, Injection

Journal: Cancers

Article Title: Neoantigen Dendritic Cell Vaccination Combined with Anti-CD38 and CpG Elicits Anti-Tumor Immunity against the Immune Checkpoint Therapy-Resistant Murine Lung Cancer Cell Line LLC1

doi: 10.3390/cancers13215508

Figure Lengend Snippet: The triple combination of DC vaccination, CpG and anti-CD38 mAb treatment inhibited LLC1 tumor growth. ( A ) C57BL/6 mice were divided into five groups (five mice per group): (1) 30μg CpG treatment; (2) CpG plus 250μg anti-CD38 mAb; (3) 1×10 6 L82 LP-pulsed DC vaccination with CpG; (4) the triple combination of L82 LP-pulsed DC vaccination, CpG, and anti-CD38 mAb; and (5) the quadruple combination of DC, CpG, anti-CD38 mAb, and 200μg of anti-PD-1 mAb. LLC1 cells (5 × 10 5 ) were subcutaneously inoculated and tumor growth was monitored. ( B ) On day 14, tumor-infiltrating cells were extracted from these mice ( n = 5) and subjected to flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Monoclonal antibodies (mAbs) specific for PD-1 (RMP1-14), CTLA-4 (9H10), CD38 (NIMR5), and CD16/32 (2.4G2) were purchased from BioXcell (Lebanon, NH, USA).

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: In vivo metabolomics identifies CD38 as an emergent vulnerability in LKB1 -mutant lung cancer

doi: 10.1101/2023.04.18.537350

Figure Lengend Snippet: LKB1 deficient NSCLC show shifts in NAD + homeostasis and upregulation of the CD38 NADase. A , Metabolites that show significant changes in KL mouse lung tumors compared to KP tumors. Values are shown as z score of fold-change. B , C, Flow cytometry analysis showing quantification of CD38 expression in neoplastic (EPCAM+) cells in Kras / Lkb1 (KL) and Kras / Trp53 (KP) tumors. B , (left panel) percent positive tumor cells and (right panel) median fluorescent intensity (MFI). Student’s t test, two-tailed. ** P <0.01, **** P <0.0001. C , Representative FACS blots of CD38 expression. D , CD38 mRNA levels in human KP and KL lung cancer patient specimens, from TCGA dataset. Unpaired t test with Welch’s correction, two-tailed. ** P <0.01. E,F, Lung cancer patient specimens with LKB1 wt and mutant status were stained with anti-CD38 antibodies. E , Representative IHC images. Scale bar, 100 μm. F , Quantification of CD38 IHC score. Student’s unpaired t test, two tailed. *** P <0.001.

Article Snippet: In the present study, 17 human lung ADC samples were performed IHC staining with an anti-CD38 antibody (Cell Signaling Technology, Cat #51000).

Techniques: Flow Cytometry, Expressing, Two Tailed Test, Mutagenesis, Staining

Journal: bioRxiv

Article Title: In vivo metabolomics identifies CD38 as an emergent vulnerability in LKB1 -mutant lung cancer

doi: 10.1101/2023.04.18.537350

Figure Lengend Snippet: LKB1 is a negative regulator of CD38 expression in lung cancer cells. A , B , A series of KRAS mutant NSCLC cell lines with or without LKB1 mutation were analyzed for CD38 expression via FACS. A , Representative FACS plots. B , Quantification. n=3 biological replicates for each cell line. C , Western blot analysis of CD38 protein levels in KRAS mutant lung cancer cell lines with or without LKB1 mutation. D , E, LKB1 mutant (Lx337) and LKB1 wildtype (Lx210) lung cancer PDXs were tested for CD38 expression by FACS analysis. D , Representative FACS plots. E , Quantification of percent CD38+ tumor cells in the PDXs. n=4 tumors per group. Student’s t test, two tailed. * P <0.05. F, G, A549 NSCLC cells were transduced with empty vector (pBABE) or ectopically expressing LKB1 wildtype (LKB1) or kinase-dead mutant (LKB1-KD). F , Cells were tested for CD38 expression by FACS analysis. n=3 each group. Each dot represents one independent experiment. One-way ANOVA Dunnett’s multiple comparisons test compared with pBABE group. *** P <0.001. G , Western blot analysis of CD38 protein levels.

Article Snippet: In the present study, 17 human lung ADC samples were performed IHC staining with an anti-CD38 antibody (Cell Signaling Technology, Cat #51000).

Techniques: Expressing, Mutagenesis, Western Blot, Two Tailed Test, Transduction, Plasmid Preparation

Journal: bioRxiv

Article Title: In vivo metabolomics identifies CD38 as an emergent vulnerability in LKB1 -mutant lung cancer

doi: 10.1101/2023.04.18.537350

Figure Lengend Snippet: LKB1-SIK signaling suppresses CD38 transcriptionally. A , Relative phosphorylation levels of SIK3 T221 in human lungs cancer specimens with the indicated LKB1 genotypes (from the CPTAC dataset). Student’s unpaired t test, two tails. * P <0.05. B, FACS analysis showing CD38 expression on KP lung tumors with sgRNA mediated targeting of Lkb1 or Sik family members versus control. * P <0.05, *** P <0.001, **** P <0.0001. C , Representative FACS analysis of CD38 levels in KP lung cancer cells shown in ( B ). D , Western blot of CD38 levels in KP lines without Lkb1 or Sik family kinases expression, or KL lines without Lkb1 expression. E , Relative CD38 mRNA levels in the lines shown in panel B . One-way ANOVA, Dunnett’s multiple comparisons test compared with KP Cas9. ** P <0.01, **** P <0.0001. F , Representative MRI scan of lung tumor nodules (circled) with the indicated genotypes. H, heart. G , Quantification of tumor volume changes 6 weeks after tumor implantation. Unpaired student’s t test in comparison with KP sgCtrl group. * P <0.05. H , Metabolite changes with statistical significance comparing mouse KP lung tumor nodules with or without Sik1/2/3 loss. I , CREB reporter assay in the indicated cells. n=3 each cell line. Ordinary one-way ANOVA Dunnett’s multiple comparison test, each group compared with the KP Cas9 group. n=3 each group. * P <0.05, **** P <0.0001. J , EP300 ChIP-seq track of CD38 promoter using the A549 cell line from the ENCODE database.

Article Snippet: In the present study, 17 human lung ADC samples were performed IHC staining with an anti-CD38 antibody (Cell Signaling Technology, Cat #51000).

Techniques: Phospho-proteomics, Expressing, Control, Western Blot, Tumor Implantation, Comparison, Reporter Assay, ChIP-sequencing

Journal: bioRxiv

Article Title: In vivo metabolomics identifies CD38 as an emergent vulnerability in LKB1 -mutant lung cancer

doi: 10.1101/2023.04.18.537350

Figure Lengend Snippet: Daratumumab induces killing of CD38 positive lung cancer cells through antibody-dependent cellular cytotoxicity (ADCC). A-B, Cytotoxicity assay in LKB1 mutant A549 ( A ), HCC44 ( B ) or LKB1 wild type H358 ( C ) target cells using calcein-acetoxymethyl (AM) release in the presence of NK cell line NK-92. Lytic activity was measured 4 hours after co-culturing (E:T=100:1) in the presence of Daratumumab (20 μg/ml). Student’s t test, two-tailed analysis was performed to compare with no NK cell control as indicated. D , Indicated cells were cultured with NK-92 cells (E:T=100:1) in the presence of increasing concentrations of daratumumab. The percentage of cell lysis for each cell line was measured 4 hrs after co-culturing. Paired student’s t test for LKB1 mutant cells (A549 or H1944) compared with LKB1 wild type control H358. E-F, Mouse A549 ( E ) or HCC44 ( F ) xenograft tumor growth in response to daratumumab treatment. For each mouse, 3x10 6 A549 cells ( E ) or 5x10 5 HCC44 ( F ) were implanted into nu/nu mice subcutaneously. Daratumumab or hIgG1 (12.5mg/kg, once per week) was dosed when tumor size reached100 mm 3 . Multiple unpaired student’s t test was performed for each of the time points indicated. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: In the present study, 17 human lung ADC samples were performed IHC staining with an anti-CD38 antibody (Cell Signaling Technology, Cat #51000).

Techniques: Cytotoxicity Assay, Mutagenesis, Activity Assay, Two Tailed Test, Control, Cell Culture, Lysis

Journal: bioRxiv

Article Title: Non-invasive imaging of cell-based therapies using acoustic reporter genes

doi: 10.1101/2024.11.01.621111

Figure Lengend Snippet: a , Schematic of lentiviral vectors designed to report T cell activity state by expressing GVs downstream of the NFAT promoter. T cell activation is chemically induced by incubating cells with PMA and ionomycin, which trigger the NFAT promoter to express rtTA, and in the presence of doxycycline, activate transcription of GV genes. b , Representative BURST images (top) and signal quantification (bottom) of Jurkat cells transduced with the vectors in panel ( a ) with or without chemical activation, normalized to wild-type (WT) cells (not shown). c , Percentage of Jurkat cells in panel ( b ) expressing both fluorescent reporters (GFP and BFP), with and without activation. d , Imaging T cell activation in response to CD19-CD3 BTE-mediated engagement with CD19+ Raji cells, using cells engineered with the vectors shown in ( a ). e , Representative BURST images (top) and signal quantification (bottom) of Jurkat cells engineered to express GVs upon receptor-mediated activation in the presence of Dox. f , Percentage of cells from panel ( e ) expressing both fluorescent reporters (GFP and BFP), with and without receptor engagement. Statistical comparisons were performed using Fisher’s LSD test, with each condition compared to resting state T cells (without PMA/ionomycin or BTE stimulation). P-value for ( e ): 0.0173. All other p-values < 0.0001. Significance levels: *p<0 . 05, **p<0 . 01, ***p<0 . 001, ****p<0 . 0001 . Error bars represent mean ± s.e.m. of N = 3 biological replicates. Each data point represents the arithmetic mean of N = 2 technical replicates. Scale bars for all ultrasound images represent 1 mm.

Article Snippet: To induce receptor-mediated T cell cytotoxicity, CD19-CD3 BTE (1 ng/mL, BPS Bioscience) and IL-2 (100 U/mL) were added.

Techniques: Activity Assay, Expressing, Activation Assay, Transduction, Imaging

Journal: bioRxiv

Article Title: Non-invasive imaging of cell-based therapies using acoustic reporter genes

doi: 10.1101/2024.11.01.621111

Figure Lengend Snippet: a , Workflow for engineering T cells isolated from human PBMCs to express doxycycline-inducible GVs and imaging BTE-mediated homing of cytotoxic T cells into target tumors. Top: T cells are transduced with the 3-vector lentiviral system, and GFP expressing cells are sorted to select for transduction of the assembly factor vectors. The cells are then expanded in vitro for downstream characterization and in vivo injections. Bottom: T cells are systemically administered to immunocompromised mice bearing subcutaneous CD19+ Raji cell tumors, accompanied by intraperitoneal (IP) injections of BTE every 48 hours and IP injections of doxycycline for 72 hours prior to ultrasound imaging. b , BURST SBR (left) and representative images (right) of GV-expressing and WT T cells. Paired one-tailed t test, p = 0.0115. Error bars represent mean ± s.e.m. of N = 5 PBMC donors; each data point is the arithmetic mean of N = 3 technical replicates. c , Percentage of live Raji cells in a cytotoxicity assay, where T cells are co-cultured with CD19+ Raji cells at effector-to-target (E:T) ratios of 1:1 and 4:1. Welch’s two-tailed t-test, N = 6 biological replicates (2 PBMC donors and 3 replicates per donor). d , GFP and BFP fluorescence measurements from ( c ), normalized to the maximum mean fluorescence intensity (MFI) of each reporter. e , BURST images overlaid on anatomical B-mode grayscale images of representative Raji tumors in mice injected with GV-expressing T cells, either Dox-induced or uninduced, and mice injected with WT T cells. Red lines indicate tumor boundary used for signal quantification. f , Quantification of BURST signal inside tumors. Welch’s two-tailed t test, Dox+ vs. Dox-, p = 0.0067; Dox+ vs. WT, p = 0.0063. N = 10 mice for Dox+, N = 6 for Dox-, N = 6 for WT. g , Histology of an example tumor infiltrated by GV-expressing T cells and its corresponding BURST image. White: Raji-Antares cells; Magenta: CD8+ cytotoxic T cells; Green: GFP+ T cells; Blue: BFP+ T cells. h , Schematic of T cell distribution in the Dox-induced tumor from ( g ). i , Correlation of the BURST signal within tumors with the absolute numbers of GFP+ and BFP+ T cells in corresponding histological sections. Pearson correlation: r = 0.89, p = 0.0072, N = 7 mice. One mouse (gray data point) was excluded due to a significant number of T cells located outside the imaging plane whose GV expression was not captured by BURST (see ). All scale bars represent 1 mm.

Article Snippet: To induce receptor-mediated T cell cytotoxicity, CD19-CD3 BTE (1 ng/mL, BPS Bioscience) and IL-2 (100 U/mL) were added.

Techniques: Isolation, Imaging, Transduction, Plasmid Preparation, Expressing, In Vitro, In Vivo, One-tailed Test, Cytotoxicity Assay, Cell Culture, Two Tailed Test, Fluorescence, Injection