Cytotoxicity of fourth-generation anti-Trop2 CAR-T cells against breast cancer

The treatment of breast cancer (BC) remains a formidable challenge due to the emergence of drug resistance, necessitating the exploration of innovative strategies. Chimeric antigen receptor (CAR)-T cell therapy, a groundbreaking approach in hematologic malignancies, is actively under investigation for its potential application in solid tumors, including BC. Trophoblast cell surface antigen 2 (Trop2) has emerged as a promising immunotherapeutic target in various cancers and is notably overexpressed in BC. To enhance therapeutic efficacy in BC, a fourth-generation CAR (CAR4) construct was developed. This CAR4 design incorporates an anti-Trop2 single-chain variable fragment (scFv) fused with three costimulatory domains –CD28/4-1BB/CD27, and CD3ζ. Comparative analysis with the conventional second-generation CAR (CAR2; 28ζ) revealed that anti-Trop2 CAR4 T cells exhibited heightened cytotoxicity and interferon-gamma (IFN-γ) production against Trop2-expressing MCF-7 cells. Notably, anti-Trop2 CAR4-T cells demonstrated superior long-term cytotoxic functionality and proliferative capacity. Crucially, anti-Trop2 CAR4-T cells displayed specific cytotoxicity against Trop2-positive BC cells (MDA-MB-231, HCC70, and MCF-7) in both two-dimensional (2D) and three-dimensional (3D) culture systems. Following antigen-specific killing, these cells markedly secreted interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α), IFN-γ, and Granzyme B compared to non-transduced T cells. This study highlights the therapeutic potential of anti-Trop2 CAR4-T cells in adoptive T cell therapy for BC, offering significant promise for the advancement of BC treatment strategies

ChREBP Regulates Itself and Metabolic Genes Implicated in Lipid Accumulation in β-Cell Line.

Carbohydrate response element binding protein (ChREBP) is an important transcription factor that regulates a variety of glucose-responsive genes in hepatocytes. To date, only two natural isoforms, Chrebpα and Chrebpβ, have been identified. Although ChREBP is known to be expressed in pancreatic β cells, most of the glucose-responsive genes have never been verified as ChREBP targets in this organ. We aimed to explore the impact of ChREBP expression on regulating genes linked to accumulation of lipid droplets, a typical feature of β-cell glucotoxicity. We assessed gene expression in 832/13 cells overexpressing constitutively active ChREBP (caChREBP), truncated ChREBP with nearly identical amino acid sequence to Chrebpβ, or dominant negative ChREBP (dnChREBP). Among multiple ChREBP-controlled genes, ChREBP was sufficient and necessary for regulation of Eno1, Pklr, Mdh1, Me1, Pdha1, Acly, Acaca, Fasn, Elovl6, Gpd1, Cpt1a, Rgs16, Mid1ip1,Txnip, and Chrebpβ. Expression of Chrebpα and Srebp1c were not changed by caChREBP or dnChREBP. We identified functional ChREBP binding sequences that were located on the promoters of Chrebpβ and Rgs16. We also showed that Rgs16 overexpression lead to increased considerable amounts of lipids in 832/13 cells. This phenotype was accompanied by reduction of Cpt1a expression and slight induction of Fasn and Pklr gene in these cells. In summary, we conclude that Chrebpβ modulates its own expression, not that of Chrebpα; it also regulates the expression of several metabolic genes in β-cells without affecting SREBP-1c dependent regulation. We also demonstrate that Rgs16 is one of the ChREBP-controlled genes that potentiate accumulation of lipid droplets in β-cells

Exogenous ChREBP regulates <i>Chrebpβ</i> expression.

<p>(A) Amino acid alignment among rat ChREBPβ, mouse ChREBPβ, caChREBP, and dnChREBP. GRACE, glucose response activation conserved element; bHLH, basic Helix-Loop-Helix-Leucine. (B) Schematic diagram of tetracycline-inducible vectors for overexpression of <i>caChREBP</i>, <i>dnChREBP</i>, and <i>eYFPnuc</i>. LTR, long terminal repeat; TRE, tetracycline-responsive promoter element; UBC, human ubiquitin C promoter; <i>rtTA3</i>, reverse tetracycline-transactivator 3; IRES, internal ribosomal entry site; <i>Puro</i>^<i>R</i>, puromycin resistance gene; WPRE, Woodchuck hepatitis posttranscriptional regulatory element; SIN LTR, self-inactivating long terminal repeat. (C) caChREBP induces <i>Chrebpβ</i> expression. We incubated caChREBP cells for 48h in RPMI with 11 mmol/l D-glucose in the presence of doxycycline 1 μg/mL. We isolated the RNA and performed RT-qPCR using ChREBP isoform-specific primers. The histograms are the means of relative RNA levels normalized to <i>Eef1g</i> and <i>Rpl13a</i> and expressed as fold activation over the activity seen in eYFPnuc cells incubated in RPMI with 11 mmol/l D-glucose in the presence of doxycycline 1 μg/mL. *, p< 0.05 compared with eYFPnuc cells. (D) dnChREBP reduces the <i>Chrebpβ</i> expression. We preincubated dnChREBP cells for 24h in RPMI with 5.5 mmol/l D-glucose in the presence of doxycycline 1 μg/mL to minimize expression of endogenous nuclear ChREBP and let the induced dnChREBP occupy ChoREs and switched to in RPMI with 25 mmol/l D-glucose in the presence of doxycycline 1 μg/mL for 48h to induce strong expression and activity of endogenous nuclear ChREBP. We isolated the RNA and performed RT-qPCR using ChREBP isoform-specific primers. The histograms are the means of relative RNA levels normalized to <i>Rns18</i> and <i>Hprt1</i> and expressed as fold activation over the activity seen in eYFPnuc cells incubated under the same condition. *, p< 0.05 compared with eYFPnuc cells. (E) Alignment of ChoRE sequence presents in <i>Chrebpβ</i> promoter among rat (at the position -109 to -93), mouse, dog, horse, rhesus, and human. Sequence assemblies and coordinates are as follows: Rat: Mar.2012 Chr12(-): 26638089–26638073, Mouse: Jul.2007 Chr5(+): 135565651–135565667, Rhesus: Jan.2006 Chr3(-):51178915–51178899, Horse: Jan.2007 ChrUn(-):175282430–175282414, Dog: May 2005 Chr6(+): 9619423–9619439, Human: Mar.2006 Chr7(-):72700300–72700284. Color coding: light grey, identical residues; dark grey, unconserved residues. (F) Functional analysis of putative ChoRE sequences at the position -109 to -92 on <i>Chrebpβ</i>. We co-transfected luciferase reporter driven by two copies of rat <i>Chrebpβ</i> ChoRE upstream of minimal TATA promoter with caChREBP in 832/13 cells in RPMI with 5.5 mmol/l D-glucose. <i>Gaussia</i> luciferase activity was measured at 48h and normalized to <i>Cypridina</i> luciferase, and expressed as fold activation over the activity seen in cells transfected with two copies of ChoRE with minimal TATA promoter and empty vector. *, p< 0.05.</p

ChREBP regulates the expression of metabolic genes in 832/13 β-cells.

<p>(A-B) Expression of genes encoding enzymes involved in glycolysis, lipogenesis, triglyceride biosynthesis and lipid oxidation in caChREBP cells (A) and dnChREBP cells (B). *<i>p</i>< 0.05 compared with eYFPnuc cells. (C-D) Expression of metabolic genes, including <i>Rgs16</i> and <i>Mid1ip1</i>, <i>Srebp1c</i> and its target <i>Irs2</i>, and genes involved in cell proliferation, <i>Hbegf</i> and <i>Myc</i>, in caChREBP cells (C) and dnChREBP cells (D). *<i>p</i>< 0.05 compared with eYFPnuc cells.</p

Effect of caChREBP on <i>Gpd1</i> or <i>Rgs16</i> ChoRE-containing promoters.

<p>(A) Alignment of ChoRE sequence presented on <i>Gpd1</i> promoters among rat (at the position -1943 to -1927), mouse, cow, dog, rhesus, chimpanzee, and human. Sequence assemblies and coordinates are as follows: Rat: Mar.2012 ChrX(+): 115873245–11587361, Mouse: Dec.2011 Chr15(+): 99716109–99716125, Cow: Oct.2011 Chr5(-): 32732081–32732065, Dog: Sep.2011 Chr27(-): 4623314–4623298, Rhesus: Oct.2010 Chr11(+): 47359512–47359528, Chimpanzee: Feb.2011 Chr12(-): 39238594–39238578, and Human: Feb.2009 Chr12(+): 50496575–50496591. Color coding: light grey, identical residues; dark grey, unconserved residues. (B) caChREBP cannot activate <i>Gpd1</i> ChoRE-containing promoter. We co-transfected luciferase reporter driven by two copies of rat <i>Gpd1</i> ChoRE (position -1943 to -1927 relative to TSS) upstream of minimal TATA promoter with caChREBP in 832/13 cells in RPMI with 5.5 mmol/l D-glucose. Gaussia luciferase activity was measured at 48h and normalized to Cypridina luciferase, and expressed as fold activation over the activity seen in cells transfected with two copies of ChoRE with minimal TATA promoter and empty vector. *, p< 0.05. (C) Sequence of rat Rgs16 proximal promoter (-104 to +196, relative to TSS). Bold text, Start codon. Color coding: light grey, Exon 1. (D) Alignment of ChoRE sequence presented on <i>Rgs16</i> promoter among rat (at the position +37 to +53), mouse, and human. Sequence assemblies and coordinates are as follows: Rat: Mar.2012 Chr13(+): 76145198–76145214, Mouse: Dec.2011 Chr1(+): 153740341–153740357, and Human: Dec.2013 Chr1(-): 182604401–182604385. Color coding: light grey, identical residues; dark grey, unconserved residues. (E) The effect of high glucose on the activity of natural <i>Rgs16</i> promoter and mutated <i>Rgs16</i> promoter. We transfected luciferase reporter driven by natural <i>Rgs16</i> promoter (position -1519 to +159 relative to TSS) or ChoRE-deleted <i>Rgs16</i> promoter in 832/13 cells cultured in RPMI with 5.5 mmol/l or 25 mmol/l D-glucose. Gaussia luciferase activity was measured at 48h and normalized to Cypridina luciferase, and expressed as fold activation over the activity seen in cells transfected with natural <i>Rgs16</i> promoter and exposed to 5.5 mmol/l D-glucose. *, p< 0.05. (F-G) Glucose and caChREBP stimulates <i>Rgs16</i> ChoRE-containing promoter. We transfected luciferase reporter driven by two copies of rat <i>Rgs16</i> ChoRE (position +37 to +53 relative to TSS) upstream of minimal TATA promoter in 832/13 cells in RPMI with 5.5 or 25 mmol/l D-glucose (F) or co-transfected with caChREBP or empty vector in 832/13 cells in RPMI with 5.5 mmol/l D-glucose (G). Gaussia luciferase activity was measured at 48h and normalized to Cypridina luciferase, and expressed as fold activation over the activity seen in cells transfected with two copies of ChoRE with minimal TATA promoter and exposed to 5.5 mmol/l D-glucose (F) or co-transfected with empty vector (G). *, p< 0.05.</p

<i>Rgs16</i> enhances accumulation of neutral lipid in 832/13 β-cells.

<p>(A) Schematic diagram of tetracycline-inducible vectors for overexpression of <i>Rgs16</i>. LTR, long terminal repeat; TRE, tetracycline-responsive promoter element; UBC, human ubiquitin C promoter; <i>rtTA3</i>, reverse tetracycline-transactivator 3; IRES, internal ribosomal entry site; <i>Puro</i>^<i>R</i>, puromycin resistance gene; WPRE, Woodchuck hepatitis posttranscriptional regulatory element; SIN LTR, self-inactivating long terminal repeat. (B-C) Overexpression of <i>Rgs16</i> triggers lipid accumulation in 832/13 cells. We incubated eYFPnuc cells, Rgs16 cells, and caChREBP cells for 72h in RPMI with 11 mmol/l D-glucose in the presence of doxycycline 1 μg/mL. We stained these cells for neutral lipid by Oil red O (B). Histograms (C) represent the amount of stained intracellular lipid compared with eYFPnuc cells. *, p< 0.05 compared with eYFPnuc cells. (D-E) Effects of <i>Rgs16</i> overexpression on genes encoding metabolic enzymes (D) and related metabolic genes (E). We incubated Rgs16 cells for 72h in RPMI with 11 mmol/l D-glucose in the presence of doxycycline 1 μg/mL. We isolated the RNA and performed RT-qPCR using gene-specific primers. The histograms are the means of relative RNA levels normalized to <i>Eef1g</i> and <i>Hprt1</i> and expressed as fold activation over the activity seen in eYFPnuc cells preincubated with 11 mmol/l D-glucose in the presence of doxycycline 1 μg/mL. *, p< 0.05 compared with eYFPnuc cells.</p

Suppression of TGF-β and IL-10 receptors on self-differentiated dendritic cells by short-hairpin RNAs enhanced activation of effector T-cells against cholangiocarcinoma cells

Cholangiocarcinoma (CCA) is an aggressive tumor that is associated with high rates of recurrence and mortality. This is due, in part, to the fact that CCA cells and their microenvironment secrete immunosuppressive cytokines, transforming growth factor-β (TGF-β) and interleukin-10 (IL-10), that inhibit dendritic cell (DC) functions, which, in turn, results in the decreased anti-tumor activity of T-cells. We hypothesized that the TGF-β receptor and IL-10 blockade on dendritic cells would improve DC function, thereby allowing improved activation of T cells against CCA cells. To test our hypothesis, we generated self-differentiated DCs (SD-DCs) via transduction of human peripheral blood monocytes with lentivirus expressing IL-4 and GM-CSF. SD-DCs were transduced with a second lentivirus containing short-hairpin RNAs (shRNAs) to knock-down TGF-βRII and IL-10RA mRNAs. Immunoblot confirmed the reduced expression levels of TGF-β and IL-10 receptors in both SD-DCs that were transduced with a single and/or combination of lentiviruses containing shRNAs. SD-DCs were thereafter pulsed with tumor antigens extracted from CCA cell lines in an effort to activate DC function. MHC class II (HLA-DR) and co-stimulatory molecules (CD40 and CD86) on SD-DCs were upregulated to levels comparable to those on DCs generated by the conventional method. Suppression of TGF-β and IL-10 receptors on SD-DCs influenced the effector T-cells to produce IFN-γ, which enhanced their ability to kill CCA cells. The preparation of adoptive effector T-cells holds the potential of becoming a novel therapy for cellular immunotherapy in CCA

Gender, age, and tumor location distribution of cases used in <i>KRAS</i> and <i>BRAF</i> mutation analysis.

<p>Gender, age, and tumor location distribution of cases used in <i>KRAS</i> and <i>BRAF</i> mutation analysis.</p

Identification of <i>BRAF</i> mutations in ultra-short DNA.

<p>A and B. Detection of BRAF V600E1 mutation (A) and V600K mutation (B) in 45-, 35-, or 25-base synthesized oligonucleotides (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198795#pone.0198795.s001" target="_blank">S1 Table</a>), which mimic severely fragmented single-stranded DNA. C. Cross-reactivity with non-target mutations, i.e. BRAF V600A, V600D, V600G, V600M, and V600R, presented in 45-base oligonucleotide templates (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198795#pone.0198795.s001" target="_blank">S1 Table</a>). Allele-specific primers for BRAF V600E and V600K were combined in the same quantitative PCR reaction.</p

Development of ultra-short PCR assay to reveal <i>BRAF</i> V600 mutation status in Thai colorectal cancer tissues

<div><p>The protein kinase BRAF is one of the key players in regulating cellular responses to extracellular signals. Somatic mutations of the <i>BRAF</i> gene, causing constitutive activation of BRAF, have been found in various types of human cancers such as malignant melanoma, and colorectal cancer. BRAF V600E and V600K, most commonly observed mutations in these cancers, may predict response to targeted therapies. Many techniques suffer from a lack of diagnostic sensitivity in mutation analysis in clinical samples with a low cancer cell percentage or poor-quality fragmented DNA. Here we present allele-specific real-time PCR assay for amplifying 35- to 45-base target sequences in <i>BRAF</i> gene. Forward primer designed for BRAF V600E detection is capable of recognizing both types of BRAF V600E mutation, i.e. V600E1 (c.1799T>A) and V600E2 (c.1799_1800delTGinsAA), as well as complex tandem mutation caused by nucleotide changes in codons 600 and 601. We utilized this assay to analyze Thai formalin-fixed paraffin-embedded tissues. Forty-eight percent of 178 Thai colorectal cancer tissues has <i>KRAS</i> mutation detected by highly sensitive commercial assays. Although these DNA samples contain low overall yield of amplifiable DNA, our newly-developed assay successfully revealed BRAF V600 mutations in 6 of 93 formalin-fixed paraffin-embedded colorectal cancer tissues which <i>KRAS</i> mutation was not detected. Ultra-short PCR assay with forward mutation-specific primers is potentially useful to detect BRAF V600 mutations in highly fragmented DNA specimens from cancer patients.</p></div