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. 2025 Jul 19. Online ahead of print. doi: 10.1039/d5md00488h

Development of a CXCR4 antagonistic peptide, P12, to suppress pancreatic cancer progress via enhancing T cell responses and sensitizing cells to gemcitabine

Xin Huang a,†, Hang Wu a,†, Ke Zhu a,†, Xuanxin Liu a, Dapeng Li a, Yuanhao Liu b, Tao Wang a, Tao Wen a, Xiaocui Fang c, Jian Liu a, Yanlian Yang c, Jie Meng a,✉, Chen Wang c,✉, Haiyan Xu a,‡,✉
PMCID: PMC12320945  PMID: 40766864

Abstract

The C–X–C motif chemokine receptor 4 (CXCR4) is overexpressed by pancreatic cancer cells. This work developed a CXCR4 antagonistic peptide P12, which was identified by pancreatic-cell-based selection from among the de novo designed peptides and was able to specifically bind to the pancreatic cancer cells as well as fibroblasts and macrophages in vitro and in vivo. CXCL12-mediated migration of tumor cells and adhesion to stromal cells were effectively inhibited by P12, and the phosphorylation of Erk and P38 was down-regulated. P12 increased the sensitivity of the tumor cells and fibroblasts to gemcitabine (GEM). The combination of P12 with GEM (P12+GEM) increased the infiltration of CD8+ T cells and reduced fibroblasts in the tumor microenvironment, as well as increasing the toxicity of the lymphocytes to the tumor cells with upregulated blood levels of INF-γ and TNF-α. Collectively, P12+GEM decreased the tumor weight and prolonged the survival of tumor-bearing mice significantly. In conclusion, P12 is a potent and selective CXCR4 antagonist that effectively enhances anti-tumor immune responses and overcomes the gemcitabine resistance of pancreatic cancer.

CXCR4 antagonistic peptide, P12, prolongs the survival of pancreatic tumor bearing mice via inhibiting the phosphorylation of Erk and p38, sensitizing the tumor cells to gemcitabine, and enhancing the anti-tumor immune responses.graphic file with name d5md00488h-ga.jpg

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer with a 5 year overall survival rate that remains lower than 11%1 and that exhibits poor responses to chemotherapeutics and immune therapies.2 The relevant mechanisms of resistance to chemotherapies and immune therapies may be associated with multiple interactions between the pancreatic cancer cells and the large amounts of stromal cells in the tumor microenvironment.3 It has been proposed that the C–X–C motif chemokine receptor 4 (CXCR4) and its ligand CXCL12 perform critical roles in mediating pancreatic cancer–stroma crosstalk.4,5 Stroma are prevalent in the tumor microenvironment of PDAC, and fibrotic stroma are primarily composed of fibroblasts and collagen.6 At the same time, CXCR4 is overexpressed in PDAC cells, and the stromal fibroblasts are also CXCR4 positive and secrete CXCL12 to activate the tumor cells to proliferate, metastasize, and develop drug resistance.7,8 Furthermore, clinical data also indicate that PDAC patients with high CXCR4 expression levels have worse outcomes.9,10

Gemcitabine (GEM) is a standard first-line drug for the treatment of metastatic or advanced PDAC;11 however, it only achieves a slight improvement in PDAC survival, and the combination of GEM with other cytotoxic drugs showed limited improvements of the prognosis of PDAC.12 The GEM chemo-resistance of PDAC has been suggested to be partly mediated by the CXCL12/CXCR4 axis through intracellular downstream activation of Erk1/2 signaling pathways.13 Moreover, the abnormally high expression of CXCR4 of PDAC and the interactions between the tumor cells and stroma have been demonstrated to block the infiltration of T cells and induce T cell apoptosis.14,15 Therefore, as CXCR4 is responsible for tumor proliferation and metastasis, as well as a crucial mediator of the tumor microenvironment, it has been considered an important therapeutic target.

Efforts to develop CXCR4 antagonists have demonstrated their therapeutic effects on pancreatic cancer in combination with different drugs, showing the promising potential of this strategy. For example, the small molecule antagonist AMD3100 was demonstrated to increase pancreatic tumor cell death with lymphocyte expansion when combined with the checkpoint inhibitors CTLA-4 and PD-L1 (ref. 16) and PD-1.17 In a clinical study, the CXCR4 antagonistic peptide BL8040 in combination with pembrolizumab and a regime of chemotherapeutics resulted in positive outcomes in a phase IIa clinical trial of metastatic PDAC.18,19

In this context, we report a novel CXCR4 antagonistic peptide, P12, which was identified via pancreatic-cell-based selection from among the de novo designed peptides. We showed that P12 was able to specifically bind to CXCR4-positive pancreatic cancer cells as well as fibroblasts and macrophages, significantly inhibiting the CXCR4/CXCL12 axis to decrease migration of the tumor cells, reduce the adhesion of the tumor cells to the macrophages and fibroblasts, and down-regulate CXCR4-mediated phosphorylation of ERK and P38. Importantly, P12 effectively ameliorated the immune suppression of the tumor microenvironment and increased the GEM sensitivity, which collectively resulted in the prolonged survival of the orthotopic pancreatic cancer mice.

Results

P12 is identified to have high affinity to CXCR4 of pancreatic cancer cells

First, the linear peptide P12 was identified as an antagonist of CXCR4 via pancreatic cancer-cell-based selection using our de novo designed peptides. The design concept is based on the previous analysis of amino acid interaction patterns, which details the amino acid–amino acid interaction patterns.20–23 This concept provides the general principles for constructing the group of candidate peptides that were subjected to screening. to obtain the CXCR4-targeted antagonistic peptide, we designed a CXCR4-targeted peptide library by integrating sequence- and structural-based characteristics. Using a cellular fluorescence imaging screening approach, CXCR4-targeted antagonistic peptide P12 was identified. As shown in Fig. 1A, the sequence of P12 is QGCRRRNTVDDWISRRRAL with a molecular weight of 2202.45 Dalton. A scramble sequence of P12 (P12-scramble) was synthesized (QDGCRTRDRNWSRIRVRAL) as well. Theoretical predictions for the binding of P12 and P12-scramble to CXCR4 were conducted using AlphaFold3 (Fig. 1B and C), and showed that P12 was able to bind to the ECL2 of CXCR4, while P12-scramble did not show this binding capacity (Fig. 1D and E). Detailed information of regarding the protein–protein interactions between P12 or P12-scramble and CXCR4 is provided in Tables S1 and S2. Additionally, Fourier transform infrared (FTIR) spectroscopy was used to investigate the conformations of P12 and P12-scramble according to a previous publication.24 Second-derivative FTIR spectra showed that P12 adopted predominantly α-helix (the amide I band at 1650 cm−1), β-sheet (1627 cm−1), and β-turn (1674 cm−1) structures. These accounted for 44.5%, 20.3%, and 35.2% of the amide I peak area, respectively. In contrast, the peak areas for P12-scramble corresponded to 36.0% α-helix, 30.1% β-sheet, and 33.9% β-turn (Fig. 1F and G).

Fig. 1. Molecular information of P12 and P12-scramble and their interaction with CXCR4 predicted by AlphaFold3. A. Molecular structural formula of P12 and P12-scramble. B and C. Binding patterns of P12 to CXCR4 and magnification of the binding pattern of P12 to ECL2 of CXCR4, respectively. D and E. Binding pattern of P12-scramble to CXCR4 and magnification of the binding pattern of P12-scramble to ECL2 of CXCR4, respectively. F and G. Second-derivative FTIR spectra of P12 and P12-scramble, in which the solid and dashed curves represent experimental and fitted data, respectively. β-Sheet, green; α-helix, red; β-turn, blue.

Fig. 1

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The affinity of P12 toward CXCR4 of pancreatic cancer tumor cells was examined using confocal microscopy and flow cytometry assays. The confocal microscopy observations clearly showed that FITC-P12 (green fluorescence) bound to the CXCR4 (red fluorescence) of PANC-1 cells (Fig. 2A). Results from flow cytometry were consistent with the confocal observations, and also confirmed the affinities of P12 to CCC-ESF-1 and THP-1 cells in a concentration-dependent manner (Fig. 2B). Similarly, P12 showed good affinity to mouse-derived cells including pan02, NIH/3T3, and RAW264.7 cells (Fig. 3). Moreover, a diagnosed human pancreatic ductal cancer tissue sample with typical ductal neoplasia (Fig. S1) was positively stained with CXCR4 (red fluorescence) and FITC-P12, and most of the green fluorescence was co-located with the red fluorescence, as evidenced by the yellow color when the images were merged (Fig. S2). The affinity Kd of P12 was also estimated to be 0.869 ± 0.096 μM by analyzing the mean fluorescence intensity of flow cytometry (Fig. S3) according to previous publications,20–23 while P12-scramble showed low binding capacity (Fig. S4). These results indicated that P12 was able to recognize and bind to CXCR4 of the pancreatic cancer cells as well as that of the macrophages and fibroblasts, and clearly revealed that the binding properties were highly sequence-dependent.

Fig. 2. Affinity of P12 to CXCR4 of human-derived ductal pancreatic cancer cells, fibroblasts and macrophages. A. Confocal microscopy images of P12 binding to PANC-1. Red: CXCR4 (Alexa Fluor 647), green: FITC-P12, blue: nuclei (DAPI). Results from flowcytometry for P12 binding to PANC-1 (B), CCC-ESF-1 (C) and THP-1 (D) (n = 3, *p < 0.05, **p < 0.01).

Fig. 2

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Fig. 3. Affinity of P12 to CXCR4 of mouse-derived ductal pancreatic cancer cells, fibroblasts and macrophages. A. Confocal microscopy images of P12 binding to pan02. Red: CXCR4 (Alexa Fluor 647), green: FITC-P12, blue: nuclei (DAPI). Results from flowcytometry for P12 binding to pan02 (B), NIH/3T3 (C) and RAW264.7 (D) (n = 3, *p < 0.05, **p < 0.01).

Fig. 3

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P12 inhibited CXCR4/CXCL12 axis-mediated cell signaling in the pancreatic tumor cells

Next, we investigated whether P12 could inhibit the interaction between CXCR4 and CXCL12, since stromal cells in the tumor microenvironment such as fibroblasts and macrophages largely secrete CXCL12 to activate the tumor cells, which increases the adhesion between the cancer cells and stromal cells as well as enhancing the migration of the cancer cells. Western blot (WB) results showed that supplementation of CXCL12 effectively activated the CXCR4 downstream signaling proteins of PANC-1 (Fig. 4A) and pan02 cells (Fig. 4B), as evidenced by the up-regulated phosphorylation of Erk and P38. Notably, when pretreated with P12, both pancreatic cancer cells showed down-regulation of the levels of p-Erk and p-P38, which clearly indicated that P12 was able to effectively inhibit the activation of intracellular signaling pathways mediated by the CXCR4/CXCL12 axis.

Fig. 4. P12 inhibited CXCL12-mediated phosphorylation of CXCR4 downstream signaling proteins in the pancreatic cancer cells. Representative WB images and quantifications of p-Erk and p-P38 for PANC-1 cells (A–C) and pan02 cells (D–F) (n = 3, *p < 0.05, **p < 0.01).

Fig. 4

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The interference of P12 with the CXCL12-mediated migration of pancreatic cancer cells was measured using a transwell assay. It was shown that more PANC-1 (Fig. 5A) or pan02 cells (Fig. 5B) penetrated the membrane of the upper well when CXCL12 was added to the bottom well compared to the control. However, when both cells were incubated with P12 at 10 μM for 1 h prior to being seeded, the numbers of penetrating cells were greatly reduced, as evidenced by the fading crystal violet color and the quantification of the spectral absorbance at 570 nm (Fig. 5C and D). In addition, the inhibitory effect was also examined in two other pancreatic cell lines using the spectrum of crystal violet; the results showed that the CXCL12 mediated-migration of AsPC-1 and BXPC-3 cells was significantly inhibited by the pre-incubation of P12 (Fig. 5E and F).

Fig. 5. P12 inhibited the CXCL12-mediated migration of pancreatic cancer cells. A and B. Representative images of the transwell assay for PANC-1 and pan02, respectively. Quantification of PANC-1 (C), pan02 (D), AsPC-1 (E), and BXPC-3 cell (F) migration measured by the spectral absorbance of crystal violet (n = 3, *p < 0.05, **p < 0.01 vs. CXCL12).

Fig. 5

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The inhibitory effect of P12 on the adhesions between the cancer cells and macrophages or fibroblasts was examined, because one of the key functions of the CXCR4/CXCL12 axis is to mediate the adhesions. It was shown that in the control group, a high density of THP-1 (Fig. 6A), CCC-ESF-1 (Fig. 6B), RAW264.7 (Fig. 6C) and NIH/3T3 (Fig. 6D) cells labeled with green fluorescence adhered to the PANC-1 cell or pan02 cell layers, while the supplementation of P12 resulted in a reduction in adhered cells. As a quantitative example, supplementation of P12 at 1 μM and 10 μM resulted in about a 25% and 50% decrease in the THP-1 cell adhesion. Quantification of the adhesion for the other kinds of cells also displayed similar trends.

Fig. 6. The inhibitory effect of P12 on the adhesions between pancreatic cancer cells and fibroblasts or macrophages. A and B: Representative images of THP-1 and CCC-ESF-1 adhered on PANC-1 cells. C and D. Representative images of RAW264.7 and NIH/3T3 adhering on pan02 cells. Green: calcein AM. E: Quantifications of the adhesion for the four cells supplemented with P12 at different concentrations (n = 3, *p < 0.05, **p < 0.01).

Fig. 6

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Pancreatic cancer cells can secrete CXCL12 to undergo self-activation of their CXCR4,25,26 which is also one of the key factors inducing drug resistance.27 Therefore, we treated both the mouse-derived (pan02 and NIH/3T3) and human-derived (PANC-1 and CCC-ESF) pancreatic cells and fibroblasts cells with GEM at different concentrations after prior incubation with P12 at 5 μM for 2 h. It was shown that with the aid of P12, GEM induced stronger cytotoxicity on the both the pan02 (Fig. 7A)/PANC-1 (Fig. 7B) and the NIH/3T3 (Fig. 7C)/CCC-ESF-1 (Fig. 7D) cells than it did alone, which clearly indicated that P12 was able to promote the sensitivity of the cells to GEM. It could be noticed that the sensitizing effects were stronger on the murine-origin cells than the human ones. We also investigated whether P12 was able to sensitize the pancreatic tumor cells to other chemotherapeutics. Results showed that no significant sensitizing effect was detected with irinotecan, which is of the one chemotherapeutics in FOLFIRINOX (Fig. S5). To understand the underlying mechanisms, we examined the impacts of GEM and irinotecan on the expression of CXCR4 in the tumor cells. The results showed that the GEM incubation caused the CXCR4 level of pan02 cells to increase significantly, and P12 pretreatment reduced the degree of the CXCR4 up-regulation; however, P12 did not reduce the increase in CXCR4 level caused by irinotecan (Fig. S6).

Fig. 7. P12 sensitized pancreatic cells and fibroblasts to GEM. The viability of pan02 (A), NIH/3T3 (B), PANC-1 (C), and CCC-ESF-1 (D). The cells were exposed to GEM at different concentrations after prior incubation with P12 at 5 μM for 2 h (n = 3, *p < 0.05, **p < 0.01).

Fig. 7

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Therapeutic effect of P12 on pancreatic cancer in vivo

The therapeutic effects of P12 were examined using an orthotopic pancreatic cancer mouse model. The administration regimes are schematically presented in Fig. 8A. For the control group (Ctrl), 5% glucose was given once a day; for the P12 group (P12) and combination P12/GEM group (P12+GEM), 100 mg kg−1 of P12 was given once a day for four days in one week. GEM (10 mg kg−1) was given twice a week for the GEM-only group and the P12+GEM group. It was shown that P12 or GEM alone did not prolong the survival period of the pancreatic cancer mice, while the combination of P12 and GEM significantly prolonged the survival, demonstrating a synergistic effect (Fig. 8B). Morphological observations showed the presence of many deep-stained pancreatic cancer cells forming typical ductal structures (blue star) in the Ctrl group, while in the GEM group, the amount of ductal pancreatic cancer structures decreased and a necrotic and fibrotic area could be observed in the center of the tumor tissue. For the P12 group, there were pancreatic ducal structures comparable with those in the GEM group; however, lymphocytes (green arrow) with round dark-stained nuclei were seen surrounding the ductal structures (Fig. 8C), providing a remarkable sign showing that the immunological environment of the tumor was changed by P12. More importantly, the combination P12/GEM group exhibited the morphological features of GEM, which induces a reduction in ductal carcinoma structures, and P12, which drives lymphocyte infiltration. At the same time, both the size and weight of the tumor mass for mice receiving P12+GEM was reduced significantly compared with that of the Ctrl group (Fig. 8D and E).

Fig. 8. Treatment outcomes of P12 in combination with GEM. A. Administration regimens. B. Survival of pancreatic cancer mice that received different treatments (n = 7, **p < 0.01 vs. Ctrl). C. Representative images of H&E staining of the pancreatic tumor tissue of mice in the different groups. Ductal pancreatic tumor structures are indicated by the blue stars, and the lymphocyte cells are indicated by green arrows. D. Photos of pancreatic tumors from the different groups. E. Tumor weights for the different groups (*p < 0.05, n = 4).

Fig. 8

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Immunofluorescence staining of CD8 and SMA antibodies indicated that the combination treatment increased CD8+ T cells in the tumor tissue to the highest level. Although P12 alone was able to increase the amount of CD8+ T cells, GEM alone did not cause the CD8+ T cells to increase (Fig. 9A). At the same time, fibroblasts in the tumor microenvironment were reduced significantly as well for mice receiving P12 and P12+GEM, as evidenced by the decreased levels of the SMA (green fluorescence) (Fig. 9B), which was consistent with the results shown in Fig. 7B and D. These results represented an important sign that P12 and P12+GEM were potent at overcoming the immune-suppressive tumor microenvironment.

Fig. 9. P12 affected the immune suppressive tumor microenvironment of pancreatic cancer. A. Representative fluorescence images of CD8+ T cells in the tumor tissue. Green: CD8 (FITC), blue: nuclei (DAPI). B. Representative images of SMA expression in the tumor tissue. Green: SMA (FITC), blue: nuclei (DAPI).

Fig. 9

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Next, the lymphocyte toxicity assay was conducted following the protocol illustrated in Fig. 10A. It was shown that the lymphocytes separated from the spleens of the mice from the P12 or P12+GEM groups killed more pan02 cells than those from the spleens of GEM group and Ctrl group (Fig. 10B), with higher ratios of CD8/CD4 (Fig. 10C). At the same time, the concentrations of TNF-α and IFN-γ in the blood of the mice were significantly increased in P12 group and P12+GEM group (Fig. 10D and E). These results highlighted the role of P12 in the activation of killing T cells, indicating that P12 was able to exert effective anti-tumor immunological effects through interference with CXCR4/CXCL12.

Fig. 10. P12 induced anti-tumor immunological effects in vivo. A. Schematic graph of the lymphocyte toxicity assay. B. Survival of cells after splenic lymphocytes killing. The relative viability of pan02 cells incubated with the spleen lymphocytes of Ctrl mice was set as 100%. C. The CD8+/CD4+ ratio of the splenocytes. D and E: Concentration of IFN-γ and TNF-α in the blood for each group (n = 4, *p < 0.05, **p < 0.01).

Fig. 10

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Bio-distribution of P12

The bio-distribution of FITC-P12 in the orthotopic pancreatic cancer mice was investigated using fluorescent intensity measurements of the blood, pancreas, liver and kidney, as well as using ex vivo fluorescence imaging. As shown, the intraperitoneally injected P12 was rapidly detected in the circulating blood and reached the peak concentration at 0.5 h, then decreased rapidly with time (Fig. 11A). From the results in Fig. 11A, the half-life time of P12 in the blood was calculated to be 0.96 h, which could reflect the metabolic stability of P12. It can be seen that P12 largely accumulated in the pancreas and reached peak concentration at 0.5–1.0 h post injection, followed by a gradually decrease to 6 h. Compared with the pancreatic accumulation, the accumulation of P12 in the liver decreased faster and was undetectable 2 h after injection, reaching a peak at 0.5 h (Fig. 11C). It should be noted that the amount of P12 accumulated in the pancreatic tissue was greater compared to that in the liver and kidney, which provided indirect evidence of the specific affinity of P12 to CXCR4. The quantification of the fluorescence intensity for the organs also showed the preferential accumulation of P12 in the pancreas cancer (Fig. 11B).

Fig. 11. Biodistribution of P12 in mice bearing orthotopic pancreatic cancer. A. P12 in the serum at different time points post FITC-P12 injection. B. Ex vivo fluorescent imaging of the pancreas, liver, and kidney with the injection of FITC-P12. C. Quantification of the fluorescence intensity in the three organs (n = 3).

Fig. 11

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Discussion

CXCR4 is highly expressed in more than 23 kinds of cancer cells including PDAC.28 The CXCR4/CXCL12 axis mediates multiple signal transduction pathways and a variety of cellular functions such as cell migration, proliferation, invasion, and survival. In the past decade, CXCR4 antagonistic peptides have become attractive therapeutic options for cancer treatment due to their high specificity and favorable safety profiles. In addition to BL-8040, which has demonstrated improved efficacy against pancreatic cancer in combination with pembrolizumab and the NAPOLI-1 regimen in metastatic PDAC in a IIa clinical trial as well as being found to be safe and well tolerated in a multicenter, single-arm, phase II study,18,19 LY2510924 has demonstrated its safety profile to be acceptable in the advanced pancreatic cancer population.29 Several derivatives of the endogenous peptide inhibitor of CXCR4 (EPI-X4) were reported to have therapeutic effects in a pancreatic cancer animal model.30 Nevertheless, so far, few are clinically available for pancreatic cancer treatment, highlighting the unmet clinical demands and developing space.

In this work, a chemically synthesized CXCR4 antagonistic peptide, P12, was identified from among our de novo designed peptides using pancreatic-cancer-cell-based selection, and its antagonistic function was verified in an orthotopic pancreatic cancer mouse model, which confirmed that P12 was potent against CXCR4 of the pancreatic cells. We have previously developed another CXCR4 antagonistic peptide, E5, for the treatment of acute myeloid leukemia,31,32 while P12 is a novel sequence displaying much stronger antagonistic effects on pancreatic cancer cells than E5, because P12 was screened using pancreatic cell lines. This observation may be plausibly considered as a manifestation of epitope heterogeneity, which has been documented in a number of previous reports.33,34 Such heterogeneity could conceivably lead to varied interactions between ligands and tumor antigens in different organs, such as that illustrated in the current study for different peptide antagonists targeting CXCR4 for the treatment of pancreatic cancer and acute myeloid leukemia. Extended and systematic mechanistic studies on such heterogeneity effects are highly important and should be pursued in future efforts.

Gemcitabine is a first-line chemotherapeutic in the treatment of pancreatic cancer; however, drug resistance readily occurs after the beginning of clinical treatment.35,36 CXCR4/CXCL12 can mediate the resistance of pancreatic cancer to gemcitabine by stimulating the intrinsic cell survival signaling pathway and restricting GEM-induced toxicity.37 In this study, we observed that the survival signaling effector Erk of the pancreatic cancer cells PANC-1 or pan02 was activated in response to CXCL12 (Fig. 3), and P12 was able to significantly inhibit this activation, and thus to increase the cytotoxicity of GEM in vitro. In addition, it was detected that GEM upregulated CXCR4 significantly for the tumor cells, but P12 pretreatment was able to reduce the degree of the CXCR4 up-regulation effectively, which indicated that P12 sensitized GEM via CXCR4 antagonism of the tumor cells. Several investigations supported our observations, showing that the CXCR4/CXCL12 axis is involved in GEM resistance.38–40 Consistently, the combination of P12 and GEM prolonged the survival of tumor-bearing mice significantly as well as reducing the tumor burden, which underscored the potential contribution of P12 to the long-standing hurdles in PDAC therapy. Unlike in the case of GEM, the tumor cells were not sensitized to irinotecan by P12, although irinotecan made the CXCR4 level increase as well. These results strongly suggested that the sensitizing effect of P12 was chemotherapeutic-dependent, implying the importance of choosing and optimizing chemotherapeutics in combination with CXCR4 antagonists. The underlying mechanisms merit further investigation.

It should be noted that P12 also elicited anticancer immune responses effectively, as evidenced by the infiltration of CD8+ T cells in the tumor tissues, enhanced killing effects of spleen lymphocytes, and the upregulated expression of anti-tumor cytokines. The significantly increased anticancer immune responses could be attributed to the combination effect. As it is known that CXCR4 is overexpressed in PDAC cells and tumor-associated suppressor cells, its ligand CXCL12 creates a protective niche that promotes tumor growth and blocks T-cell infiltration.7,8,41 CXCR4 antagonists, for example, BL-8040 and AMD3100, have been proven to enhance T cell infiltration. The injection of BL-8040 increased the tumor infiltration of CD8+ effector T cells, sensitized the drug response and improved anti-tumor efficacy.18 Intratumoral AMD3100 administration was also reported to induce immune response.42 Our results further confirmed that P12 was able to enhance the immune activity of T cells against tumor cells by disrupting the CXCL12/CXCR4 axis, alleviating the axis-mediated immune exclusion for PDAC.

Importantly, P12 displayed excellent pancreatic cancer targeting capacity in vivo. As shown, P12 was able to remain in the pancreas with cancer for about 6 h, which was quite different from the situation in the liver and kidney. The quick clearance of P12 in the liver and kidney indicated that P12 was mainly metabolized and excreted from these two organs. This performance enabled P12 to accumulate and carry out its an antagonistic function inside the tumor tissue, which inhibited the activity of not only the tumor cells but also of the fibroblasts, and thus increased the GEM sensitivity and enhanced the immunological responses against the cancer cells.

Conclusions

In conclusion, the chemically synthesized CXCR4 antagonistic peptide P12 was developed, which inhibited the CXCL12-mediated migration and adhesion of pancreatic cancer cells and down-regulated the phosphorylation of Erk and P38. P12 significantly improved the therapeutic efficiency of GEM, as evidenced by the prolonged survival, decreased tumor weights and CD8+ T cell infiltration via disruption of the CXCR4/CXCL12 axis.

Methods

Peptides and gemcitabine

The peptide P12 (QGCRRRNTVDDWISRRAL), FITC labeled P12 (FITC-P12) and P12-scramble (QDGCRTRDRNWSRIRVRAL) were purchased from Anhui Guoping Pharmaceutical Co., Ltd. Gemcitabine (GEM) was purchased from Bide Pharmatech Co., Ltd.

Cells and animals

Cell lines were purchased from the Cell Resource Center, Peking Union Medical College (which is part of the National Science and Technology Infrastructure, the National Biomedical Cell-Line Resource, NSTI-BMCR. http://cellresource.cn), including human pancreatic cancer cells PANC-1, AsPC-1 and BXPC-3, mouse pancreatic cancer cells pan02, human monocytes THP-1, mouse macrophage cells RAW264.7, human fibroblast cells CCC-ESF-1, and mouse fibroblast cells NIH/3T3. The THP-1 cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS, Gibco), and the others were maintained in DMEM high glucose medium containing 10% FBS at 37 °C with 5% CO2. Specific pathogen-free C57BL/6 mice (female, 6–8 weeks) were purchased and bred in the Center for Experimental Animal Research of Institute of Basic Medical Sciences.

FTIR spectrometry for peptide conformation analysis

Peptide solutions of 100 μL of 100 μM were deposited onto a CaF2 window and dried. FTIR spectra were collected using a PerkinElmer Spectrum One FTIR Spectrometer (Waltham, MA, USA) over a wavenumber range of 4000 cm−1 to 450 cm−1 at a resolution of 2 cm−1. The deconvoluted spectra underwent second-order derivation, analyzed using PeakFit software (v4.12; Jandel Scientific, San Jose, CA, USA). Second-derivative profiles were calculated using Origin software (v9.1; OriginLab, Northampton, MA, USA).

Peptide binding assays in vitro

Confocal microscopy assay: 2 × 105 PANC-1 and pan02 cells were seeded and cultured overnight on glass slides in 24-well plates. Next, the cells were incubated with 4% formaldehyde for 15 minutes, followed by incubation with FITC-P12 for 1 h and Alexa Fluor® 647 anti-CXCR4 (Abcam) for 30 min. The cell nuclei were counterstained with DAPI. For the Ctrl group, the cells were treated following the above procedures, except that the FITC-P12 was replaced by the same volume of phosphate buffer saline (PBS, pH = 7.4).

The human pancreatic ducal cancer patient was diagnosed by a professional pathologist of the Department of Pathology, Peking Union Medical College Hospital. The paraffin-embedded tumor tissues were cut into 5 μm slides, de-waxed, and then stained with hematoxylin–eosin (H&E) following the conventional procedures. The slides were incubated with FITC-P12 for 1 h, then co-stained with Alexa Fluor® 647 anti-CXCR4 (Abcam) for 30 min.

The fluorescence-labeled cells and tissues were subjected to confocal microcopy (FV1000, Olympus) for observation.

Flow cytometry assay: 2 × 105 PANC-1, THP-1, CCC-ESF-1, pan02, RAW264.7, and NIH/3T3 cells were incubated with biotin-P12 at different concentrations for 1 h, followed by incubation with FITC-streptavidin (Biolegend) for 30 minutes at 4 °C. After that, the cells were subjected to flow cytometry (Accuri™ C6, BD Biosciences). The MFI for each cell line was recorded and analyzed. For P12-scramble, the PANC-1 cells were incubated with P12-scramble at different concentrations for 1 h. To estimate the cell-based Kd of P12, the MFI obtained from the flow cytometry measurement was analyzed according to previous publications.20–23

Transwell assay

First, 2 × 105 PANC-1 or pan02 cells were suspended in serum-free medium and seeded in the upper chamber of 24-well Millicell hanging cell culture inserts (8 μm, Millipore) following incubation in DMEM high glucose medium with or without P12 at a concentration of 1 μM or 10 μM at 37 °C for 1 h. DMEM high glucose medium containing 5% FBS was added to the lower chamber for all groups. For the CXCL12 control and P12 groups, CXCL12 (Peprotech) was added to the lower chamber at 100 ng mL−1. After 24 h of incubation, the cells in the upper layer of the insert were removed. The inserts were soaked in 4% formaldehyde for 15 minutes, followed by staining with 0.1% crystal violet. The inserts were observed under a microscope (EVOS M7000, Thermo Fisher). The crystal violet was dissolved, and the absorbance of the solution at 570 nm was detected using a microplate reader (Varioskan Lux, Thermo Fisher). The relative migration of each group was calculated by normalization with the CXCL12-induced migration. For the migration of AsPC-1 and BxPC-3 cells, the same protocol was applied, except the CXCL12 concentration in the lower chamber was changed to 200 ng mL−1.

Cell adhesion assay

2 × 104 PANC-1 or pan02 cells were seeded in 96-well culture plates to form a monolayer. P12 was added to the cells at 0, 1 or 10 μM and incubated for 1 h. Next, THP-1 or RAW264.7, CCC-ESF-1 or NIH/3T3 cells (2 × 104), all labeled with Calcein AM (2 μM, Invitrogen), were gently added to the pancreatic cancer cell monolayers after the medium containing P12 was removed. After 15 min of incubation at 37 °C, the wells were gently washed twice to remove non-adherent cells and subjected to microscopy. The fluorescent intensity at 515 nm was measured. The relative cell adhesion was calculated by normalizing the fluorescence intensity of each group to that of control.

Western blot assay

4 × 105 PANC-1 or pan02 cells were seeded in 6-well plates and incubated with P12 for 1 h at 37 °C, followed by incubation with CXCL12 (100 ng mL−1) for 15 min at 37 °C. After being washed with cold PBS, the cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors, followed by electrophoretic separation and membrane transfer. Next, the membrane was incubated overnight at 4 °C with primary antibodies for phosphorylated-Erk (p-Erk), Erk, phosphorylated-P38 (p-P38), P38, and β-actin (CST). The immunocomplex on the membrane was visualized using a gel imaging system (Tanon). The blot density of the bands was quantified using Image J software.

Cell viability

A total of 5 × 103 cells per well PANC-1, pan02, CCC-ESF-1, and NIH/3T3 were incubated with GEM at different concentrations following incubation with P12 at 5 μM for 2 h and measured using a cell count kit (CCK-8, Dojindo). The PANC-1 cells were incubated with GEM at 1, 5, or 10 μM for 72 h. The CCC-ESF-1 cells were incubated with GEM at 1, 5, 10, 20, or 50 μM for 72 h. The pan02 cells were incubated with GEM at 0.05, 0.1, 0.5 or 1 μM for 24 h. The NIH/3T3 cells were incubated with GEM at 0.01, 0.05, 0.1, 0.5 or 1 μM for 24 h. After that, the cells were rinsed twice with PBS and incubated with a medium containing 10 μL CCK-8 reagents for 2 h. Additionally, 5 × 103 per well pan02 cells were incubated with P12 at 5 μM for 2 h, followed by incubation with irinotecan (Aladdin) at 1, 5, or 10 μM for 24 h. The absorbance of the medium was measured at 450 nm using a microplate reader (Varioskan Lux, Thermo Fisher). The relative cell viability was calculated using the protocol provided by the manufacturer, in which the viability of cells cultured in complete medium was set as 100%.

Administration regimens in the orthotopic pancreatic cancer murine model

The orthotopic pancreatic cancer murine model was established using the following protocol. The pan02 cells were collected and concentrated to prepare a cell suspension of 2 × 107 cell per mL. The cells were mixed with an equal volume of ice-cold Matrigel™ matrix solution (BD Biosciences). Female C57BL/6 mice were anesthetized, and their pancreases were gently exposed. The pan02 cells of the 50 μL matrix solution were injected into the pancreases, which were reset after the injection. The abdomen and skin were closed by layer-by-layer stitching with 4–0 suture.

On the 14th day post-tumor cell inoculation, the pancreatic-tumor-bearing mice were randomly divided into 4 groups (n = 7) and received intraperitoneal injections. For the control group (Ctrl), 5% glucose was given once a day for 4 days in a week; for the P12 (P12) and combined P12/GEM (P12+GEM) group, 100 mg kg−1 of P12 was given once a day for 4 days a week. GEM (10 mg kg−1) was given twice a week for the GEM-only and P12+GEM groups. The survival of the mice administered different treatments was monitored with two-week treatments. For the therapeutic effect assay, a one-week treatment was applied, and the mice were sacrificed on the third day after the last injection. The tumor, spleen and peripheral blood of the mice were collected. The tumor mass was analyzed by H&E and immuno-fluorescence staining that was conducted using anti-CD8 and anti-SMA antibody (Servicebio).

Immunological cytotoxic assay

The pan02 cells (target cells) were seeded at 1 × 104 per well on 96-well plates. The spleen lymphocytes (effecter cells, 1 × 105 per well) were separated by Ficoll solution (Sigma) from the mice of each group with one-week treatment, counted, and added to the pan02 cells. The ratio of lymphocytes to tumor cells was 10 : 1. After 4 h of incubation, the lactate dehydrogenase (LDH) reagent (Dojindo) was added according to manufacturer instructions, and the optical densities of the supernatants were measured at 490 nm. The rates of pan02 cells killed by the lymphocytes for each group were calculated according to the formula provided by the manufacturer. The spleen lymphocytes were incubated with anti-CD4 (FITC, Biolegend) and anti-CD8 (APC, Biolegend) for 1 h at 4 °C and then analyzed using flow cytometry (Attune NxT, Thermo Fisher).

ELISA assays for IFN-γ and TNF-α

The serum was collected from the coagulated peripheral blood of each group by centrifugation. Interferon gamma (IFN-γ) and tumor necrosis factor-a (TNF-α) were quantified using the corresponding ELISA assay kit (Neobioscience) according to the manufacturer protocol.

Pharmacokinetics and bio-distribution of P12 in cancer mice

FITC-P12 (10 mg kg−1) was intraperitoneally injected into the orthotopic pancreatic cancer mice 14 days after the cancer cells were inoculated. For the control group, 5% glucose was injected (n = 3). Peripheral blood samples were collected from the mouse orbits at 0.5, 1, 2, 3, 4, 6, 12, and 24 h after the injection (n = 3 for each time point). The coagulated blood was centrifuged at 4 °C at 3000 rpm for 10 minutes to collect the supernatant and transferred to a 96-well black plate. The fluorescence intensity was measured at Ex488 nm/Em525 nm (Varioskan Lux, Thermo Fisher). The pancreas, liver, and kidneys of the mice were collected, and the fluorescence intensity was measured and calculated using an IVIS imaging system (Xenogen, Caliper Life Science). The half-life time of P12 in the blood was calculated using GraphPad Prism version 9.0 (GraphPad Software Inc., San Diego, CA, USA).

Ethics statement

All animal experiments were approved by and performed in accordance with the guidelines of the Animal Care and Use Committee of IBMS/PUMC (ACUC-A02-2023-023). The histochemical and immunofluorescence staining involving human pancreatic cancer tissue samples were reviewed and approved by the Research Ethics Committee of Peking Union Medical College Hospital (I-24YSB0803). The patients/participants provided their written informed consent to participate in this study.

Statistics

The statistical analyses were performed using GraphPad Prism version 9.5.1 (GraphPad Software Inc., San Diego, CA). One-way analysis of variance (ANOVA) was utilized for examining variations among three or more groups, followed by LSD post hoc test to determine differences between specific group pairs. Multi-t-test was applied to compare the two values between the P12 and no P12 group. The survival curve comparison was analyzed using a log-rank (Mantel–Cox) test. A P value of <0.05 was considered statistically significant.

Author contributions

Conceptualization, C. W., H. X.; investigation, X. H., H. W., K. Z., X. L., D. L., T. W., T. Wen, X. F., J. L., Y. Y., J. M.; writing—original draft, J. M., H. X.; writing—review & editing, J. M., C. W., H. X.; funding acquisition, T. W., C. W., H. X.; resources, Y. L.; supervision, C. W., H. X.

Conflicts of interest

The authors declare no conflicts of interest.

Supplementary Material

MD-OLF-D5MD00488H-s001

Acknowledgments

This work was funded by the National Key Research and Development Program of China (2022YFA1205803), CAMS Innovation Fund for Medical Science (CIFMS 2021-I2M-1-026), Strategic Priority Research Program of Chinese Academy of Sciences (XDB36000000) and Beijing Natural Science Foundation grant (7244372). The authors would like to thank Prof. Chenxuan Wang and Ms Jin Cheng for their kind help as well as technical support in the measurement of FTIR spectroscopy analysis.

Data availability

Document S1. Fig. S1 and S2. See DOI: https://doi.org/10.1039/D5MD00488H.

Original western blot images have been deposited at Mendeley at [https://doi.org/10.17632/sctxx6w7bg.1] and are publicly available as of the date of publication. Microscopy data reported in this paper will be shared by the lead contact upon request.

Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Haiyan Xu: xuhy@pumc.edu.cn.

Notes and references

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

MD-OLF-D5MD00488H-s001
MD-OLF-D5MD00488H-s001.pdf (935KB, pdf)

Data Availability Statement

Document S1. Fig. S1 and S2. See DOI: https://doi.org/10.1039/D5MD00488H.

Original western blot images have been deposited at Mendeley at [https://doi.org/10.17632/sctxx6w7bg.1] and are publicly available as of the date of publication. Microscopy data reported in this paper will be shared by the lead contact upon request.

Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Haiyan Xu: xuhy@pumc.edu.cn.

Articles from RSC Medicinal Chemistry are provided here courtesy of Royal Society of Chemistry

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