Blockade of histamine receptor H1 augments immune checkpoint therapy by enhancing MHC-I expression in pancreatic cancer cells

Abstract

Background: Although immune checkpoint blockade (ICB) therapy has proven to be extremely effective at managing certain cancers, its efficacy in treating pancreatic ductal adenocarcinoma (PDAC) has been limited. Therefore, enhancing the effect of ICB could improve the prognosis of PDAC. In this study, we focused on the histamine receptor H1 (HRH1) and investigated its impact on ICB therapy for PDAC.

Methods: We assessed HRH1 expression in pancreatic cancer cell (PCC) specimens from PDAC patients through public data analysis and immunohistochemical (IHC) staining. The impact of HRH1 in PCCs was evaluated using HRH1 antagonists and small hairpin RNA (shRNA). Techniques including Western blot, flow cytometry, quantitative reverse transcription polymerase chain reaction (RT-PCR), and microarray analyses were performed to identify the relationships between HRH1 and major histocompatibility complex class I (MHC-I) expression in cancer cells. We combined HRH1 antagonism or knockdown with anti-programmed death receptor 1 (αPD-1) therapy in orthotopic models, employing IHC, immunofluorescence, and hematoxylin and eosin staining for assessment.

Results: HRH1 expression in cancer cells was negatively correlated with HLA-ABC expression, CD8⁺ T cells, and cytotoxic CD8⁺ T cells. Our findings indicate that HRH1 blockade upregulates MHC-I expression in PCCs via cholesterol biosynthesis signaling. In the orthotopic model, the combined inhibition of HRH1 and αPD-1 blockade enhanced cytotoxic CD8⁺ T cell penetration and efficacy, overcoming resistance to ICB therapy.

Conclusions: HRH1 plays an immunosuppressive role in cancer cells. Consequently, HRH1 intervention may be a promising method to amplify the responsiveness of PDAC to immunotherapy.

Keywords: Histamine receptor H1; Major histocompatibility complex class I; Pancreatic cancer cell.

Fig. 1

HRH1 expression in PDAC correlates with low HLA-ABC expression, CD8 T cell infiltration, and poor survival in human PDAC. A HRH1 mRNA expression in normal pancreatic tissue, adjacent non-tumor tissue, and PDAC tissue in public data. B Representative images of immunohistochemical (IHC) for HRH1 in normal pancreas and PDAC. The blue arrow indicates a normal duct. The red arrow indicates PDAC cells. Quantification of HRH1 expression intensity in normal ducts and paired tumor cells, n = 20. C Kaplan–Meier analysis of overall survival according to HRH1 expression by R2: Genomics Analysis and Visualization Platform. D The correlation plot of the mRNA relationships between HRH1 and CD3D⁺/CD45⁺ and CD8A⁺/CD45⁺, respectively (public data: CRA001160). E The correlation plot of the mRNA relationships between HRH1 and T cell exhaustion scores using GEPIA 2.0. F Representative images of IHC for HRH1, HLA-ABC, CD8, and GZMB in human PDAC tissues. The red arrow indicates GZMB. G The relationship between HRH1 protein levels and CD8, HLA-ABC, or GZMB protein levels in human PDAC tissues, n = 43. Scale bar = 100 µm (B, F). Median (A, B); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. HRH1, histamine receptor H1; PDAC, Pancreatic ductal adenocarcinoma; GZMB, Granzyme B

Fig. 2

HRH1 antagonists enhance aPD-1 therapeutic efficacy via MHC-I upregulation. A Schema of the treatment plan for mouse cancer cell lines and the mouse CAF1 co-orthotopic transplantation model. Seven days after transplantation, treatment was started. B-G Results of the treatment in each group, (B, E, F) tumor picture, weight, and volume, (C) detection of tumor and metastasis, (D, G) representative image of IHC. Scale bar = 100 µm. (H) Kaplan–Meier survival curve for the co-orthotopic transplantation model. Median (B, E, F); error bars, mean ± SD (D, G); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant. αPD-1, anti-programmed cell death protein 1; CAF1, mouse cancer-associated fibroblast 1; IHC, immunohistochemical; SD, standard deviation

Fig. 3

HRH1 inhibition of human cancer cells enhances MHC-I expression in human cancer cells. The expression of HLA-ABC, B2M, and HLA-related pathway proteins was determined using western blotting and FCM. A Various types of human cancer cell lines were treated with HRH1 antagonists for 48 h. B and C HLA-ABC or B2M expression of MIA PaCa-2 or KP-2 treated with HRH1 antagonists (20 µM) for 48 h or shHRH1 (sh1). D Protein expression of MIA PaCa-2 or KP-2 treated with HRH1 antagonists (20 µM) for 48 h or KP-2 shNC and shHRH1 (sh1). E Protein expression of MIA PaCa-2 treated with Az (20 µM) or combined with histamine (10 µM) for 48 h, (F) KP-2 treated with Az (20 µM) or combined with histamine (5 µM) for 24 h. Error bars, mean ± SD (C, F); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. MHC, major histocompatibility complex; HLA, Human Leukocyte Antigen; HLA-ABC, HLA Class 1 ABC; B2M, Beta-2 microglobulin

Fig. 4

Cholesterol biosynthesis pathway is related to MHC-I expression and simvastatin-reduced azelastine or HRH1 knockdown-related MHC-I upregulation. A Demographics of microarray analysis of MIA PaCa-2 or KP-2 treated with Az (20 µM) or H₂O for 48 h (upper), and the Metascape analysis pathway-enriched upregulated genes (lower). B RT-PCR for cholesterol biosynthesis-related gene expression in MIA PaCa-2 or KP-2 treated with Az (20 µM) for 48 h or KP-2 shHRH1 (sh1), n ≥ 3 per group. C The correlation plot shows cholesterol biosynthesis-related genes between T cell exhaustion scores by GEPIA 2.0. D MSMO1 or DHCR7 protein expression in whole lysis of siDHCR7 or siMSMO1, or Az (20 µM) or combination. E MFI of HLA-ABC expression using FCM for Az (20 µM) or combination, n = 3 per group. F CANX and CALR protein expression in KP-2 treated by Az (20 µM) or combined with simvastatin (10 µM) for 48 h. G MFI of HLA-ABC and B2M using FCM for MIA PaCa-2 or KP-2 treated alone or in combination, n ≥ 3 per group. Error bars, mean ± SD (B, E, G); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. MHC-I, major histocompatibility complex class I; RT-PCR, quantitative reverse transcription polymerase chain reaction; MFI, median fluorescence intensity; FCM, Flow cytometry

Fig. 5

Azelastine induces quiescent-like fibroblasts to reverse the antigen presentation of cancer cells. A Schema of SUIT-2 and CAF1 subcutaneous or orthotopic models for 4 weeks of Az treatment, (B and C) tumor picture, volume and/or weight, (D) representative image of H&E, sirius red, IHC of α-SMA and PCNA. E α-SMA and IL-6 protein expression in whole lysis of human CAF1, CAF2, and CAF3. F Representative image of CAF1 stained with BODIPY. G The effect of treatment on migration and invasion of CAF1. H Representative image of the IHC for HLA-ABC. I Schema of MIA PaCa-2 with CAFs indirect co-culture. J MFI of HLA-ABC or B2M of MIA PaCa-2 detected by FCM, n = 3 per group. Scale bar = 100 µm (D, F, H). Median (C); error bars, mean ± SD (B, D, F, G, J); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. CAF1, human cancer-associated fibroblast 1; Az, Azelastine; H&E, hematoxylin and eosin; α-SMA, α-smooth muscle actin; PCNA, Proliferating cell nuclear antigen; IL-6, Interleukin 6

Fig. 6

Cancer cell-specific HRH1 depletion enhances αPD-1 therapeutic efficacy in mice. A HRH1 protein expression in mouse whole lysis of cancer cell lines (KPC-1 to KPC-7) and M2 macrophages. B MHC-I protein expression in whole lysis of KPC-2 and KPC-3 treated with Az or H₂O for 48 h. C MFI of MHC-I using FCM for KPC-1, KPC-2, and KPC-3 treated with Az (40 µM) or H₂O for 48 h, n = 3 per group. D MHC-I protein expression in KPC-1 shHRH1 (sh1) and shNC. E MFI of MHC-I using FCM for KPC-1 shHRH1 (sh1, sh2) and shNC, n ≥ 3 per group. F–H The orthotopic co-transplanted syngeneic tumors (KPC-1 shNC, shHRH1 (sh1 or sh2), and mouse CAF1) for treatment of 2 weeks, (F) tumor picture, volume, and weight, (G) H&E of KPC-1 (H) representative image of IHC. Scale bar = 20 µm Median (F); error bars, mean ± SD (C, E, H); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001