CALB2 drives pancreatic cancer metastasis through inflammatory reprogramming of the tumor microenvironment

Abstract

Background: Early dissemination to distant organs accounts for the dismal prognosis of patients with pancreatic ductal adenocarcinoma (PDAC). Chronic, dysregulated, persistent and unresolved inflammation provides a preferred tumor microenvironment (TME) for tumorigenesis, development, and metastasis. A better understanding of the key regulators that maintain inflammatory TME and the development of predictive biomarkers to identify patients who are most likely to benefit from specific inflammatory-targeted therapies is crucial for advancing personalized cancer treatment.

Methods: This study identified cell-specific expression of CALB2 in human PDAC through single-cell RNA sequencing analysis and assessed its clinicopathological correlations in tissue microarray using multi-color immunofluorescence. Co-culture systems containing cancer-associated fibroblasts (CAFs) and patient-derived organoids (PDOs) in vitro and in vivo were employed to elucidate the effects of CALB2-activated CAFs on PDAC malignancy. Furthermore, CUT&RUN assays, luciferase reporter assays, RNA sequencing, and gain- or loss-of-function assays were used to unravel the molecular mechanisms of CALB2-mediated inflammatory reprogramming and metastasis. Additionally, immunocompetent KPC organoid allograft models were constructed to evaluate CALB2-induced immunosuppression and PDAC metastasis, as well as the efficacy of inflammation-targeted therapy.

Results: CALB2 was highly expressed both in CAFs and cancer cells and correlated with an unfavorable prognosis and immunosuppressive TME in PDAC patients. CALB2 collaborated with hypoxia to activate an inflammatory fibroblast phenotype, which promoted PDAC cell migration and PDO growth in vitro and in vivo. In turn, CALB2-activated CAFs upregulated CALB2 expression in cancer cells through IL6-STAT3 signaling-mediated direct transcription. In cancer cells, CALB2 further activated Ca²⁺-CXCL14 inflammatory axis to facilitate PDAC metastatic outgrowth and immunosuppression. Genetic or pharmaceutical inhibition of CXCL14 significantly suppressed CALB2-mediated metastatic colonization of PDAC cells in vivo and extended mouse survival.

Conclusions: These findings identify CALB2 as a key regulator of inflammatory reprogramming to promote PDAC metastatic progression. Combination therapy with αCXCL14 monoclonal antibody and gemcitabine emerges as a promising strategy to suppress distant metastasis and improve survival outcomes in PDAC with CALB2 overexpression.

Keywords: CALB2; CXCL14; Cancer-associated fibroblasts; Inflammatory reprogramming; Metastasis; Organoids; Pancreatic cancer.

Fig. 1

CALB2 is overexpressed both in CAFs and cancer cells and correlates with immunosuppressive TME. (A) UMAP plots of single-cell transcriptome data identified cell-specific expression of CALB2 in human PDAC tissues. (B) Heatmap illustrating the correlations of CALB2 expression with cell infiltration in the TME across multiple PDAC datasets. (C) Representative IHC staining of CALB2, FAP, and CK19 in human PDAC tissues. Black arrows indicate CALB2⁺CK19⁺FAP⁻ cancer cell and red arrows indicate CALB2⁺CK19^–FAP⁺ CAF. Scale bars, 200 μm (top), 50 μm (bottom). (D) Scatter plot demonstrating the correlation between the IHC mean density of CALB2 and FAP or CK19. (E) Tissue-based cyclic immunofluorescence for CALB2 (pink), FAP (orange), CK19 (green), in human PDAC tissues. Scale bars, 50 μm (top left), 20 μm (top right and bottom). (F) Comparison of the indicated cell types in matched cancer and adjacent tissues. (G) Comparison of the indicated cell types in tumors without or with metastasis. (H) Human PDAC tissues were classified into CALB2-High or CALB2-Low groups (left), cancer cell CALB2-High or CALB2-Low groups (middle), or CAFs CALB2-High or CALB2-Low groups (right), based on the cyclic immunofluorescence, followed by examining patient overall survival using Kaplan-Meier survival analysis. (I) Representative CALB2 and PD-L1 IHC staining in human PDAC tissues, and quantification of PD-L1 positive cells area per field (n = 36). Scale bars, 100 μm. (J) CALB2 IHC and Sirius Red staining in human PDAC tissues and quantification of collagen deposition using Sirius Red staining (n = 36). Scale bars, 50 μm. Error bars, mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant; by Pearson’s correlation test (D), paired t test (F), Student’s t test (G, I, and J) or log-rank test (H)

Fig. 2

CALB2 collaborates with hypoxia to activate an inflammatory fibroblast phenotype and enhances PDAC migration and growth. (A) Heatmap illustrating the gene set variation analyses (GSVA) score of the indicated CAF subpopulations using human scRNA-seq data. (B) Deciphering the differentiating signatures by trajectory inferences. (i-ii) Cell trajectory of PSCs, CALB2⁻ and CALB2⁺ CAF subpopulations. (iii) CALB2 expression in the process of CAF differentiation. Black arrows indicate the direction of pseudotime in trajectory plot. (C-D) PSCs were exposed to normoxia (20% O₂) or hypoxia (1% O₂) for 72 h, followed by RT-qPCR analysis (C) and western blotting (D). (E-G) PSCs were subjected to stable CALB2 overexpression (CALB2-OE), followed by examining cell growth using proliferation assay (E), the indicated proteins using western blotting (F), and IL6 production using ELISA (G). (H) Schematic diagram showing the establishment of the transwell co-culture system of organoid growth (left) or cell migration (right). (I) Transwell co-culture assays of growth of PDAC patient-derived organoids (PDOs) co-cultured with Control or CALB2⁺ CAFs for 5 days. (J) Transwell co-culture assays of migration of BxPC-3 and PANC-1 cells co-cultured with Control or CALB2⁺ CAFs for 24 h. (K-M) PDAC PDOs were orthotopically transplanted with Control or CALB2⁺ CAFs into the pancreas of NSG mice for 5 weeks (n = 5), followed by examining tumor growth (K), ex vivo pancreas imaging (L), representative H&E staining, and IHC of FAP and CALB2 (M). Scale bars, 500 μm (I), 100 μm (J), 1 cm (K), and 200 μm (M). Error bars, mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant; by Student’s t test

Fig. 3

CALB2 is upregulated in PDAC cells through IL6-STAT3 inflammatory signaling pathway. (A-B) PANC-1 cells were co-cultured with CALB2⁺ CAFs or control CAFs for 72 h, followed by western blotting (A) and RT-qPCR analysis (B). (C-D) PANC-1 cells were treated with 100 ng/mL rhIL6 over the time, followed by western blotting (C) and RT-qPCR analysis (D). (E) Measurement of IL6 concentration in the co-culture system containing CALB2⁺ CAFs or control CAFs using ELISA. (F) PANC-1 cells were pre-treated with 2.5 μg/mL Tocilizumab (IL6R neutralizing antibody) for 24 h, then cultured with CALB2⁺ CAFs or treated with 100 ng/mL rhIL6 for 48 h, followed by western blotting. (G-H) PANC-1 (G) or BxPC-3 (H) cells were treated with the indicated concentration of stattic (STAT3 inhibitor) and 100 ng/mL rhIL6 for 48 h, followed by western blotting. (I) Human PDAC organoids were dissociated into single cells and cultured with the following conditions for 5 days: control CAFs conditioned medium (CM) + DMSO (Control), CALB2⁺ CAFs CM + DMSO, and CALB2⁺ CAFs CM + 4 μΜ stattic, followed by microscopic imaging (top left), organoid tissue-based cyclic immunofluorescence to examine CALB2 expression (bottom left) and quantification of organoid growth (right). (J) Human PDAC organoids were dissociated into single cells and cultured with CALB2⁺ CAFs CM + 4 μM stattic or control DMSO for 5 days, and then were treated with control PBS or GEM for 48 h, followed by assaying organoid apoptosis and quantification. (K-L) CUT&RUN-qPCR assays showing binding of STAT3 to CALB2 promoter region was significantly enhanced by STAT3 overexpression in PANC-1 cells (K), while it was substantially impaired by STAT3 knockdown in CFPAC-1 cells (L). (M) Sequential deletions for evaluating the transcriptional activity of the CALB2 promoter in PANC-1 cells with or without STAT3 overexpression. (N) Relative luciferase activity of luciferase reporter plasmids bearing wild-type (WT) or mutant (MUT) CALB2 promoter in control or STAT3-overexpressing PANC-1 cells. Scale bars, 500 μm (I top), 100 μm (I bottom and J). Error bars, mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant; by one-way ANOVA (D, I, J, and L) or Student’s t test (B, E, K, M, and N)

Fig. 4

Downregulation of CALB2 inhibits epithelial-mesenchymal transition and metastatic outgrowth of PDAC. (A) Western blotting of CALB2 expression in human normal pancreatic ductal cells (HPNE), and human primary and metastatic PDAC cell lines. (B) The migration ability of control, or CALB2 KD or KO PDAC cells was determined by Transwell assays. (C-D) The influence of CALB2 knockdown (KD) or knockout (KO) on the in vitro proliferation (C) and sensitivity to gemcitabine (D) of PDAC cells was evaluated by CCK8 assays. (E) Significantly downregulated pathways (FDR < 0.05 and logFC > 0.5) identified by DAVID analysis of CALB2-KD AsPC-1 cells (n = 3) compared to control AsPC-1 cells (n = 3). (F) Western blot analysis of E-cadherin and N-cadherin in CALB2-KD AsPC-1 stable cell lines (left) or CALB2-KO CFPAC-1 stable cell lines (right). (G-H) IVIS living imaging (G), representative bright-field images of liver tissues and ex vivo liver imaging (H) after 5 weeks of splenic injection of CALB2-KO or control CFPAC-1 stable cell lines (n = 4 per group). (I-J) Relative expression of epithelial (I) and mesenchymal (J) genes in liver metastases from CALB2-KO (n = 3) and control (n = 4) tumors using RT-qPCR (2^−Δt). Scale bars, 200 μm (B), 1 cm (H). Error bars, mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant; by one-way ANOVA (B, C, and D) or Student’s t test (G, H, I, and J)

Fig. 5

CALB2 promotes PDAC metastasis through Ca²⁺-CXCL14 inflammatory axis. (A) Representative Ca²⁺ fluorescence images (left) and quantification of intensity (right) in CALB2 knockdown (KO) CFPAC-1 cells, or stable CALB2-overexpressing (CALB2-OE) BxPC-3 cells with or without 20 μM BAPTA treatment for 48 h. (B) Significantly upregulated pathways (FDR < 0.05 and logFC > 0.5) identified by DAVID analysis of stable CALB2-OE BxPC-3 cells (n = 3) compared to stable OE control (OE-NC) BxPC-3 cells (n = 3). (C) Volcano plots partially showing significantly upregulated genes encoding secreted protein (P < 0.05 and logFC > 1) in CALB2-OE BxPC-3 cells compared to control BxPC-3 cells. (D) CALB2-OE BxPC-3 cells were treated with or without 20 μM BAPTA for 48 h, followed by western blotting. (E) PANC-1 cells were transfected with CALB2 or control vector, followed by western blotting to identify candidate downstream molecules of CALB2. (F) CALB2-transfected PANC-1 cells were treated with increased concentrations of BAPTA for 48 h, followed by western blotting to identify candidate downstream molecules regulated by CALB2-Ca²⁺ axis. (G) Relative expression of CXCL14 in liver metastases from stable CALB2-KO (n = 3) and control (n = 4) CFPAC-1 cells using RT-qPCR (2^−Δt). (H) CALB2-OE BxPC-3 cells were subjected to stable CXCL14 KD (shCXCL14), followed by western blotting to confirm knockdown efficiency. (I) The effect of CXCL14 KD on the migration ability of CALB2-OE BxPC-3 cells was determined by Transwell assays. (J) IVIS living imaging and quantification of mice with splenic injection of shCXCL14 or control CALB2-OE stable BxPC-3 cells (n = 4 per group) at week 1 and week 6. (K) Representative ex vivo liver imaging and quantification, bright-field images of liver tissues, as well as H&E staining of metastatic nodules in mice from J. Scale bars, 200 μm (A and I), 1 cm (K top), 500 μm (K middle), 1000 μm (K bottom). Error bars, mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant; by one-way ANOVA (A, I, J, and K) or Student’s t test (G)

Fig. 6

CALB2 promotes immunosuppression and metastasis in KPC organoid allograft mice. (A) Schematic diagram depicting KPC organoid xenografts in the C57BL/6 mouse model. KPC organoids were stably transfected with Calb2-OE or control lentivirus and subsequently injected into the spleen of C57BL/6 mice to compare their ability to liver metastasis. (B) IVIS living imaging of the whole body (left) at week 1, 3, and 5, and quantification of the signals at week 5 (n = 5 each group; right). (C) Representative bright-field images and H&E staining images for metastatic lesions (left) and the quantification of metastatic nodules (right). Scale bars: 1 cm (left), 600 μm (middle), 200 μm (right). (D) Metastatic liver sections from C stained with IHC for CALB2 and CXCL14. Scale bars, 500 μm (left), 200 μm (right). (E) Representative microscopy images of bright field and green fluorescence of the indicated KPC organoids following single-cell passage for 5 days. Scale bars, 500 μm. (F) Significantly upregulated pathways (FDR < 0.05 and logFC > 1) identified by DAVID analysis of Calb2-OE liver metastasis (LM) KPC organoids (n = 3) compared to Calb2-OE KPC organoids (n = 3). (G) Volcano plots partially showing differential expression of Calb2, Cxcl14, and EMT-related genes in Calb2-OE-LM KPC organoids compared to Calb2-OE KPC organoids. Red, significant upregulated genes; blue, significantly downregulated genes; grey, non-significantly changed genes. Error bars, mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant; by Student’s t test (B and C)

Fig. 7

Combination of CXCL14 neutralizing antibody with gemcitabine effectively hinders CALB2-mediated metastasis and improves survival outcome. (A) Liver metastases from the Calb2-OE tumor were isolated to establish liver metastasis (LM) organoids. After one week of splenic injection of LM organoids into the C57BL/6 mice, isotype IgG, αCXCL14 (1 mg/kg, intravenously), gemcitabine (25 mg/kg, intraperitoneally) or combined chemotherapy (αCXCL14 + gemcitabine) were injected twice per week for 4 consecutive weeks (top). Animal survival was monitored up to 60 days after injection (bottom). (B) Representative IVIS bioluminescence images, bright-field images, and H&E staining for metastatic lesions after 5 weeks of splenic injection of Calb2-OE-LM KPC organoids. Scale bars: 1 cm (left), 2000 μm (middle), 500 μm (right). (C-D) Quantification of living imaging, ex vivo liver imaging (C) and metastatic nodules (D) from B (n = 6 per group). (E) Kaplan-Meier survival curves for mice with KPC liver metastasis-derived organoid xenografts treated with the indicated regimen (n = 10 per group). (F) Schematic diagram illustrating the inflammatory reprogramming mechanism by which CALB2 promotes liver metastasis of PDAC. Error bars, mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant; by one-way ANOVA (C-D) or log-rank test (E)