Journal: Cancer Cell International

Article Title: GPR34 inhibition reprograms tumor-associated macrophages and enhances the sensitivity of anti-PD-1 therapy in hepatocellular carcinoma

doi: 10.1186/s12935-025-04030-3

Figure Lengend Snippet: GPR34 is upregulated in the tissues of patients with HCC and is mainly expressed in intratumoral macrophages. ( A ) Heatmap displaying the expression patterns of selected genes constituting the model in TCGA-LIHC database. ( B ) Comparative analysis of GPR34 mRNA expression between HCC and paired adjacent non-tumor tissues in TCGA-LIHC database. ( C ) Comparative analysis of GPR34 mRNA expression in HCC and paired adjacent non-tumor tissues obtained from Zhongshan Hospital. ( D ) Immunohistochemistry (IHC) scores of GPR34 expression in HCC tissues versus adjacent peritumor tissues. ( E ) Representative immunofluorescence images showing co-localization of GPR34 (green) with the macrophage marker CD68 (red) in HCC tissues. ( F ) Violin plots illustrating GPR34 expression across different cell populations from GSE125449 . ( G ) Violin plots illustrating GPR34 expression across different cell populations rom GSE146115 . ( H ) Analysis of single-cell data from GSE125449 using the TISCH tool, with a t-SNE plot of all single cells colored by GPR34 expression level. ( I ) Analysis of single-cell data from GSE146115 using the TISCH tool, with a t-SNE plot of all single cells colored by GPR34 expression level

Article Snippet: In contrast to the control group, pharmacological inhibition of GPR34 using GPR34 receptor antagonist 2 (also named Compound D2, obtained from MedChemExpress) resulted in a significant increase in the expression of the M1 marker CD86 while decreasing the expression of the M2 marker CD206.

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Marker, Single Cell

Journal: Cancer Cell International

Article Title: GPR34 inhibition reprograms tumor-associated macrophages and enhances the sensitivity of anti-PD-1 therapy in hepatocellular carcinoma

doi: 10.1186/s12935-025-04030-3

Figure Lengend Snippet: Inhibition of GPR34 promotes macrophage towards M1 polarization. ( A ) Positive correlations between GPR34 expression and the levels of M2 markers (CD163, IL10, MRC1 and CLEC10A) in TCGA-LIHC. ( B ) Schematic representation of in vitro induction of TAM-like BMDMs. ( C ) Western blot analysis of GPR34 expression in untreated or TCM-educated BMDMs. β-Actin was used as the loading control. ( D-E ) The mRNA levels of GPR34 in the indicated groups. ( F-G ) Flow cytometry analysis of CD86 and CD206 on TCM-educated BMDMs treated with either 0.5 µM or 1.0 µM GPR34 receptor antagonist 2 (GPR34 inhibitor) or vehicle. ( H ) qRT-PCR analysis of the mRNA levels of immune-regulatory genes in the indicated groups

Article Snippet: In contrast to the control group, pharmacological inhibition of GPR34 using GPR34 receptor antagonist 2 (also named Compound D2, obtained from MedChemExpress) resulted in a significant increase in the expression of the M1 marker CD86 while decreasing the expression of the M2 marker CD206.

Techniques: Inhibition, Expressing, In Vitro, Western Blot, Control, Flow Cytometry, Quantitative RT-PCR

Journal: Cancer Cell International

Article Title: GPR34 inhibition reprograms tumor-associated macrophages and enhances the sensitivity of anti-PD-1 therapy in hepatocellular carcinoma

doi: 10.1186/s12935-025-04030-3

Figure Lengend Snippet: GPR34 modulates macrophage polarization through the PI3K/AKT pathway. ( A-B ) GSEA analysis demonstrates the downregulation of signature genes of PI3K/AKT pathways in GPR34 high macrophages versus GPR34 low macrophages. ( C ) Western blot analysis of PI3K/AKT pathways in TAMs treated with either GPR34 inhibitor (1 µM) or vehicle. ( D-E ) Flow cytometry analysis of CD86 and CD206 on TCM-educated BMDMs treated with either GPR34 inhibitor (1 µM), or SC79 (10 µM), SC79 (10 µM) + GPR34 inhibitor (1 µM). ( F-G ) Proportion of IFN-γ + CD8 + T cells and GZMB + CD8 + T cells after co-culturing with TCM-educated BMDMs treated with either GPR34 inhibitor (1 µM), SC79 (10 µM), or SC79 (10 µM) + GPR34 inhibitor (1 µM)

Article Snippet: In contrast to the control group, pharmacological inhibition of GPR34 using GPR34 receptor antagonist 2 (also named Compound D2, obtained from MedChemExpress) resulted in a significant increase in the expression of the M1 marker CD86 while decreasing the expression of the M2 marker CD206.

Techniques: Western Blot, Flow Cytometry

Journal: Cancer Cell International

Article Title: GPR34 inhibition reprograms tumor-associated macrophages and enhances the sensitivity of anti-PD-1 therapy in hepatocellular carcinoma

doi: 10.1186/s12935-025-04030-3

Figure Lengend Snippet: The anti-cancer effect of GPR34 inhibition in vivo depends on macrophages. ( A ) Schematic of the experimental design for treating C57BL/6 mice bearing HCC tumors with clodronate liposomes (Clodro) and a GPR34 inhibitor. ( B-E ) Analysis of orthotopic HCC tumors in mice treated with the GPR34 inhibitor, Clodro, GPR34 inhibitor combined with Clodro, or control. Representative bioluminescence images and corresponding statistical analyses depicting tumor weights are shown. ( F-H ) Tumor growth curves and tumor weights of Hepa1-6 subcutaneous tumors in C57BL/6 mice treated with GPR34 inhibitor, Clodro, GPR34 inhibitor combined with Clodro, or an isotype control

Article Snippet: In contrast to the control group, pharmacological inhibition of GPR34 using GPR34 receptor antagonist 2 (also named Compound D2, obtained from MedChemExpress) resulted in a significant increase in the expression of the M1 marker CD86 while decreasing the expression of the M2 marker CD206.

Techniques: Inhibition, In Vivo, Liposomes, Control

Journal: Cancer Cell International

Article Title: GPR34 inhibition reprograms tumor-associated macrophages and enhances the sensitivity of anti-PD-1 therapy in hepatocellular carcinoma

doi: 10.1186/s12935-025-04030-3

Figure Lengend Snippet: GPR34 inhibitor boosts the efficacy of anti-PD-1 antibody in HCC in vivo. ( A ) Schematic diagram of the treatment protocol for anti-PD-1 therapy and GPR34 inhibitor in C57BL/6 mice bearing HCC tumors. ( B-E ) Analysis of orthotopic HCC tumors in mice treated with GPR34 inhibitor alone, anti-PD-1 alone, combination therapy of GPR34 inhibitor with anti-PD-1, or an isotype control. ( F-G ) Tumor weights of Hepa1-6 subcutaneous tumors in C57BL/6 mice treated with either GPR34 inhibitor alone, anti-PD-1 alone, combination therapy of GPR34 inhibitor with anti-PD-1, or an isotype control. ( H-I ) Flow cytometry analysis of CD11b + F4/80 + CD86 + cells, CD11b + F4/80 + CD206 + cells, CD8 + T cells, IFN-γ + CD8 + T cells and PD-1 + CD8 + T cells in the indicated groups

Article Snippet: In contrast to the control group, pharmacological inhibition of GPR34 using GPR34 receptor antagonist 2 (also named Compound D2, obtained from MedChemExpress) resulted in a significant increase in the expression of the M1 marker CD86 while decreasing the expression of the M2 marker CD206.

Techniques: In Vivo, Control, Flow Cytometry

Journal: Cancer Cell International

Article Title: GPR34 inhibition reprograms tumor-associated macrophages and enhances the sensitivity of anti-PD-1 therapy in hepatocellular carcinoma

doi: 10.1186/s12935-025-04030-3

Figure Lengend Snippet: Macrophage GPR34 expression is markedly linked to the prognosis and immunotherapy response in patients with HCC. ( A ) Representative IHC images demonstrating both low and high levels of GPR34 and CD68 expression in HCC tissues. ( B-C ) OS curves for patients patients with stratified on the basis of low and high GPR34 or CD68 expression in the Zhongshan cohort ( n = 88). ( D ) OS curves for patients with HCC categorized by low and high co-expression levels of GPR34/CD68 in the Zhongshan cohort. ( E ) Magnetic Resonance Imaging (MRI) scans showcasing patients with HCC undergoing anti-PD-1 therapy with either progressive disease (PD) or partial response (PR). ( F ) Representative IF images illustrating the expression patterns of GPR34, CD68, and CD8 in tumor tissues from anti-PD-1-treated patients with HCC showing PD or PR. ( G-I ) Expression profiles of GPR34, CD68, and CD8 in patients with HCC receiving anti-PD-1 therapy

Article Snippet: In contrast to the control group, pharmacological inhibition of GPR34 using GPR34 receptor antagonist 2 (also named Compound D2, obtained from MedChemExpress) resulted in a significant increase in the expression of the M1 marker CD86 while decreasing the expression of the M2 marker CD206.

Techniques: Expressing, Magnetic Resonance Imaging

Journal: bioRxiv

Article Title: A CRISPRi/a platform in iPSC-derived microglia uncovers regulators of disease states

doi: 10.1101/2021.06.16.448639

Figure Lengend Snippet: a, Strategy for stable integration of six transcription factors integrated in AAVS1 and CLYBL loci by TALEN-mediated integration: The doxycycline-inducible reverse transcriptional activator (rtTA3G) is driven by the constitutive CAG promoter. Human MAFB, CEBPα and IRF8 are driven by the tet response element (TRE3G) in the CLYBL locus. Human PU.1, CEBP® and IRF5 are driven by TRE3G in the AAVS1 locus. All transcription factors are separated from each other via T2A ribosome skipping sequences. b, Overview of the differentiation process for generating iTF-Microglia. Top : timeline with media and cytokines, bottom : representative phase-contrast images of cells on the indicated days. Scale bar: 100 μm. c, Expression of six inducible transcription factors during iTF-Microglia differentiation. Transcript abundance (TPM) of MAFB, CEBPα, IRF8 cassette and the PU.1, CEBPβ, IRF5 cassette at Day 0, Day 9 and Day 15 of differentiation. n = 3 biological replicates. d, Representative immunofluorescence micrographs of iTF-Microglia on Day 8 of differentiation stained for microglia markers GPR34 and IBA1. Nuclei were labelled by Hoechst 33342. Scale bar: 100 μm. e, Expression of iPSC and microglia marker genes in iPSCs and derived iTFMicroglia on Day 9 and Day 15 of differentiation. The heatmap displays normalized and genecentered transcripts per million (TPM) counts for selected genes (rows) for 3 biological replicates of timepoints (columns).iTF-Microglia express microglia homeostatic markers and activation markers, while losing their expression of iPSC markers. Asterisks highlight microgliaselective markers.

Article Snippet: Primary antibodies used for immunofluorescence in this study were as follows: anti-mouse 1:150 GPR34 (R&D Systems; Cat. No. MAB4617), anti-rabbit 1:1000 IBA-1 (Wako; Cat. No. 019-19741), anti-rabbit 1:200 TFRC (abcam; Cat. No. ab84036), anti-rabbit 1:1000 synaptophysin (Synaptic Systems; Cat. No. 101 004).

Techniques: Expressing, Immunofluorescence, Staining, Marker, Derivative Assay, Activation Assay

Journal: The Journal of Experimental Medicine

Article Title: The lysophosphatidylserine receptor GPR174 constrains regulatory T cell development and function

doi: 10.1084/jem.20141827

Figure Lengend Snippet: Elevated expression of transcripts encoding GPR174 and other LysoPS receptors in T reg cells. (A) Diagram of the construct used to target the Gpr174 locus by homologous recombination. For more details, see Materials and methods. (B and C) Measurement of dTomato-GPR174 reporter allele expression in thymocytes (left) or splenocytes (right) by flow cytometry from 8-wk-old male Gpr174 −/Y mice. (B) Thymocyte populations are as follows: gray shaded, CD4 + CD8 + DP; blue dashed, CD8 + SP; purple dotted, CD25 − CD4 SP; and red, CD25 + CD4 SP T reg cells. Splenocyte populations are as follows: gray shaded, background (splenocytes from wild-type mice); blue dashed, naive CD8 + T cells; purple dotted, CD25 − naive CD4 + T cells; and red, CD25 + CD4 + T reg cells. (C) The mean fluorescence intensity (MFI) of dTomato-GPR174 is shown for the indicated cell populations. B cells were identified as B220 + IgD high splenocytes. Each dot represents a measurement from a separate mouse; n = 4. (D) Expression of dTomato-GPR174 was measured in naive CD4 + T cells cultured under Th0, Th1, or Th17 polarizing conditions for 5 d; representative flow cytometry data are shown. (E) The mRNA expression levels of the LysoPS receptors Gpr174 , Gpr34 , P2ry10 , and P2ry10-L were measured by RT-PCR in the indicated sorted thymocyte and splenocyte populations from 8-wk-old wild-type mice. Populations were gated as described in Materials and methods. Cells were sorted in triplicate, and each dot represents the relative expression in a separate sorted cell population from a distinct mouse; n = 3; error bars show SD. All data in B–E are representative of at least three independent assays.

Article Snippet: Gpr34 −/Y mice ( ) were provided by T. Schoeberg and A. Schulz (Institut für Biochemie, Universität Leipzig, Leipzig, Germany).

Techniques: Expressing, Construct, Homologous Recombination, Flow Cytometry, Fluorescence, Cell Culture, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular Biology Reports

Article Title: G-protein coupled receptor 34 regulates the proliferation and growth of LS174T cells through differential expression of PI3K subunits and PTEN

doi: 10.1007/s11033-021-07068-4

Figure Lengend Snippet: GPR34 expression confirmed by RT-PCR of 34 CRC patients and the Kaplan–Meier survival analysis of 208 CRC patients in TCGA datasets, and Knockdown of GPR34 inhibit the proliferation and colony formation of LS174T cells in vitro. a GPR34 expression in tumor samples and adjacent normal tissues by RT-PCR. b Kaplan–Meier survival curves of GPR34 related to overall survival (OS). GPR34 was positively correlated with OS. The red lines represent the subjects with low GPR34 low expression, and the purple lines represent the subjects with high expression. c Effect of GPR34 knockdown by GPR34-shRNA on protein levels. Image J software analysis. Data represent mean ± SD of triplicate experiments. ** P < 0.01. d Effect of GPR34 knockdown induced by GPR34-shRNA on mRNA levels. Total RNAs from the indicated cell lines were isolated and the cDNAs were synthesized. Real-time quantitative PCR was performed to determine GPR34 mRNA levels, which are expressed as the levels relative to that of β-actin. Data represent mean ± SD of triplicate experiments. *P < 0.05. e A soft agar assay showed that GPR34 knockdown impaired LS174T colony formation in vitro. The colony number is shown on the vertical axis as the mean ± SD of triplicate wells. Circles represent plates treated with LS174T-Vector cell or LS174-GPR34-ShRNA cell lines. **P < 0.01. f Inhibition of LS174T growth and proliferation by GPR34 knockdown. LS174T-vector cells and LS174T cells transduced with a GPR34 specific shRNA were grown in vitro for the indicated time. A cell counting kit-8 assay showed that GPR34 knockdown significantly impair the proliferative activities of LS174T cells in vitro. The cell viability (fold change) is shown on the vertical axis as the mean ± SD of triplicate wells (2-way ANOVA, **P < 0.01 LS174T-vector vs., LS174-GPR34-ShRNA, n = 3)

Article Snippet: We used the following antibodies: β-actin (1:2000 dilution, mouse, #HRP-60008, Proteintech), GPR34 (1:2000 dilution, mouse, #H00002857-B01P, Abnova), PI3K Ab Sampler Kit (1:1000 dilution, Rabbit, #9655, CST), P-Akt pathway Sampler Kit (1:1000 dilution, Rabbit, #9916, CST), p-ERK (1:2000 dilution, Rabbit, #4370, CST), ERK (1:1000 dilution, Rabbit, #4695,CST) and PTEN and PDK1 Antibody Sampler Kit (1:1000 dilution, Rabbit, #9652, CST).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Knockdown, In Vitro, shRNA, Software, Isolation, Synthesized, Real-time Polymerase Chain Reaction, Soft Agar Assay, Plasmid Preparation, Inhibition, Transduction, Cell Counting

Journal: Molecular Biology Reports

Article Title: G-protein coupled receptor 34 regulates the proliferation and growth of LS174T cells through differential expression of PI3K subunits and PTEN

doi: 10.1007/s11033-021-07068-4

Figure Lengend Snippet: Xenograft tumor growth is impaired in vivo by knockdown of GPR34 in LS174T cells. a Tumors established in null BALB/c mice by subcutaneous injection of 2 × 10 6 LS174T-GPR34-shRNA, LS174T-vector cells. Thirty days later, subcutaneous injection of LS174T cells resulted in tumor formation in all 15 mice (tumor formation rate 100%). b Tumors were excised and weighed. c The tumor/body ratio (weight) is shown on the vertical axis as the mean ± SD of 5 mice (*P < 0.05, LS174T-vector vs. LS174-GPR34-ShRNA, n = 5/group)

Article Snippet: We used the following antibodies: β-actin (1:2000 dilution, mouse, #HRP-60008, Proteintech), GPR34 (1:2000 dilution, mouse, #H00002857-B01P, Abnova), PI3K Ab Sampler Kit (1:1000 dilution, Rabbit, #9655, CST), P-Akt pathway Sampler Kit (1:1000 dilution, Rabbit, #9916, CST), p-ERK (1:2000 dilution, Rabbit, #4370, CST), ERK (1:1000 dilution, Rabbit, #4695,CST) and PTEN and PDK1 Antibody Sampler Kit (1:1000 dilution, Rabbit, #9652, CST).

Techniques: In Vivo, Knockdown, Injection, shRNA, Plasmid Preparation

Journal: Molecular Biology Reports

Article Title: G-protein coupled receptor 34 regulates the proliferation and growth of LS174T cells through differential expression of PI3K subunits and PTEN

doi: 10.1007/s11033-021-07068-4

Figure Lengend Snippet: The stimulatory effect of LysoPS, GPR34-Mab and PI3K inhibitor assay on the proliferation of LS174T cells. a Proliferation assay showing a significant stimulatory effect of lysoPS on LS174T-vector cells. Cancer cells were treated with various concentrations of lysoPS for 72 h. The cell viability (fold change) is shown on the vertical axis as the mean ± SD (1-way ANOVA, **P < 0.01 vs. control, n = 3). b Proliferation assay showing a significant stimulatory effect of lysoPS with 20 μM on LS174T-GPR34-ShRNA cells or LS174T-vector cells at 0 h, 24 h, 48 h, 72 h. The cell viability (fold change) is shown on the vertical axis as the mean ± SD (2-way ANOVA, **P < 0.01 vs. control, n = 3). c Frua-2-AM Calcium Assay in LS174T cell lines. LS174T cell lines was treated by lysoPS and PBS for 72 h, separately. The cell intensity was monitored within 30 min every 15 s. d Proliferation assay showing a significant inhibitory effect of GPR34-Mab blocking on LS174T-vector cells. Cancer cells were treated with 0.2 μg/mL of GPR34-Mab for 72 h and cell viability was analyzed. The cell viability (fold change) is shown on the vertical axis as the mean ± SD (**P < 0.01 vs. control, n = 3). e A66 (PI3KCA-specific inhibitor), TGX-221 (PI3KCB-specific inhibitor), and LY29004 (PI3K universal inhibitor) blocking assay. After treatment with A66 (100 nM), TGX-221 (100 nM), A66 + TGX-221 (each 100 nM), or LY29004 (3 μM), a cell viability assay using cck-8 was performed. Cell viability (fold change) of these cells is shown on the vertical axis as mean ± SD of triplicate wells. As indicated, all three inhibitors, A66, TGX-221, and LY290045, were found to inhibit the growth and proliferation of LS174T-vector to various degrees compared with DMSO treatment. The cell viability (fold change) is shown on the vertical axis as the mean ± SD (**P < 0.01 vs. DMSO, n = 3). f GPR34 controls the proliferation of LS174T cells through different pathways based on distinct PI3K subunits. PI3K/PTEN, GPR34/PI3K/ERK, GPR34/PI3K/PDK1/AKT and represent potential pathways which regulate the growth and proliferation of cancer cells. Basal expression of PI3KCB (p110β), and PI3K Class III, compared with LS174T-vector cells. PI3KCB was found to be downregulated in LS174T-GPR34-shRNA. Both phosphorylated and non-phosphorylated PTEN were constitutively (significantly) upregulated. Low levels of p-Src, pc-Raf, Ras and p-ERK expression were observed in LS174T-GPR34-shRNA cells, and lower expression levels of p-PDK1 and p-AKT(T308) were detected in LS174T-GPR34-shRNA cells

Article Snippet: We used the following antibodies: β-actin (1:2000 dilution, mouse, #HRP-60008, Proteintech), GPR34 (1:2000 dilution, mouse, #H00002857-B01P, Abnova), PI3K Ab Sampler Kit (1:1000 dilution, Rabbit, #9655, CST), P-Akt pathway Sampler Kit (1:1000 dilution, Rabbit, #9916, CST), p-ERK (1:2000 dilution, Rabbit, #4370, CST), ERK (1:1000 dilution, Rabbit, #4695,CST) and PTEN and PDK1 Antibody Sampler Kit (1:1000 dilution, Rabbit, #9652, CST).

Techniques: Proliferation Assay, Plasmid Preparation, Control, shRNA, Calcium Assay, Blocking Assay, Viability Assay, CCK-8 Assay, Expressing

Journal: Molecular Biology Reports

Article Title: G-protein coupled receptor 34 regulates the proliferation and growth of LS174T cells through differential expression of PI3K subunits and PTEN

doi: 10.1007/s11033-021-07068-4

Figure Lengend Snippet: GPR34 signal transduction. GPR34 is a G-protein coupled receptor that activates classical signaling cascades downstream this G-protein family. This includes activation of GPR34/PI3K/ERK and R34/PI3K/PDK1/AKT pathway, which is critical for cell proliferation

Article Snippet: We used the following antibodies: β-actin (1:2000 dilution, mouse, #HRP-60008, Proteintech), GPR34 (1:2000 dilution, mouse, #H00002857-B01P, Abnova), PI3K Ab Sampler Kit (1:1000 dilution, Rabbit, #9655, CST), P-Akt pathway Sampler Kit (1:1000 dilution, Rabbit, #9916, CST), p-ERK (1:2000 dilution, Rabbit, #4370, CST), ERK (1:1000 dilution, Rabbit, #4695,CST) and PTEN and PDK1 Antibody Sampler Kit (1:1000 dilution, Rabbit, #9652, CST).

Techniques: Transduction, Activation Assay