Empowering pancreatic tumor homing with augmented anti-tumor potency of CXCR2-tethered CAR-NK cells

Abstract

Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells are a promising immunotherapy for solid cancers; however, their effectiveness against pancreatic cancer is limited by the immunosuppressive tumor microenvironment. In particular, low NK cell infiltration poses a major obstacle that reduces cytotoxicity. The current study aimed to enhance the tumor-homing capacity of CAR-NK cells by targeting the chemokine-chemokine receptor axis between NK and pancreatic cancer cells. To this end, data from a chemokine array and The Cancer Genome Atlas pan-cancer cohort were analyzed. Pancreatic cancer cells were found to secrete high levels of ligands for C-X-C motif receptor 1 (CXCR1) and CXCR2. Subsequently, we generated anti-mesothelin CAR-NK cells incorporating CXCR1 or CXCR2 and evaluated their tumor-killing abilities in 2D cancer cell co-culture and 3D tumor-mimetic organoid models. CAR-NK cells engineered with CXCR2 demonstrated enhanced tumor killing and strong infiltration of tumor sites. Collectively, these findings highlight the potential of CXCR2-augmented CAR-NK cells as a clinically relevant modality for effective pancreatic cancer treatment. By improving their infiltration and tumor-killing capabilities, these CXCR2-augmented CAR-NK cells have the potential to overcome the challenges posed by the immunosuppressive tumor microenvironment, providing improved therapeutic outcomes.

Keywords: CAR-NK; MT: Regular Issue; NK infiltration; chemokine receptors; chemotaxis; pancreatic cancer; tumor microenvironment.

Graphical abstract

Figure 1

Profiling of chemokine ligands secreted from pancreatic cancer cells (A and B) A human chemokine array was performed using culture supernatant from human pancreatic cancer cells, including Capan-2, AsPC-1, and PANC-1. Human primary pancreatic stellate cells (HPaSteC) were used as a negative control. (B) The expression of secreted chemokine ligands from each cell line was quantified and normalized to standard positive spots by measuring their pixel intensity using the ChemiDoc software (n = 2). (C) Relative mRNA expression of CXCR1 or CXCR2 ligands was determined using total cell extracts for each cell line. Quantitative data were obtained from three independent experiments per group (n = 3). p values were determined using a one-way ANOVA followed by a multiple comparison test; ∗∗∗∗p < 0.0001, ns: not significant. (D) CXCL score distribution in The Cancer Genome Atlas pan-cancer cohort. Whisker-box plots were used to represent the distribution of the CXCL score, including those for CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8, across different cancer types. The red color represents pancreatic adenocarcinoma. Abbreviations for cancer types are described in the materials and methods section. (E) Kaplan-Meier overall survival analysis was performed on patients with pancreatic cancer (n = 176) using high vs. low CXCL scores, with a cutoff of 5.336. p values were calculated using a standard log rank test.

Figure 2

Generation and characterization of CXCR1/2-tethered human primary natural killer (pNK) cells (A and B) Flow cytometry analysis of the endogenous surface expression of chemokine receptor CXCR1–3 in pNK cells. Resting (A) and ex vivo expanded (B) pNK cells were obtained from peripheral blood mononuclear cells (PBMCs) of two different healthy donors. (C) Lentiviral constructs of CXCR1 and CXCR2 were used for overexpression. (D) Expanded pNK cells were transduced with lentivirus carrying the CXCR1 or CXCR2 constructs to generate CXCR1-overexpressing (CXCR1-pNK) or CXCR2-overexpressing (CXCR2-pNK) pNK cells, respectively. The transduction efficacy was analyzed using flow cytometry. (E) Flow cytometry analysis of the expression of activating receptors on engineered pNK cells. Quantitative data were obtained from three independent experiments per group (n = 3). p values were determined using one-way ANOVA followed by a multiple comparison test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns: not significant. (F) Evaluation of cancer-killing efficacy of engineered pNK cells toward pancreatic cancer cells at indicated effector-to-target (E:T) ratios. Cancer cell viability was assessed by measuring the luciferase activity.

Figure 3

Enhanced migration of human primary natural killer (pNK) cells overexpressing CXCR1 or CXCR2 toward pancreatic cancer cells (A) Schematic illustration of the Transwell-mediated chemotaxis assay designed to evaluate the migration of CXCR1-and CXCR2-overexpressing pNK cells. (B) For visualization of each cancer cell type, Vybrant DiO fluorescence dye was used to stain Capan-2, AsPC-1, and PANC-1 cells. Vybrant DiD-labeled pNK cells were subsequently added to the Transwell insert. Fluorescence images were captured from the bottom of the lower well 30 min after seeding pNK. The numbers of migrating pNK cells in the lower well were quantified via live cell imaging analysis. p-values were determined using a student`s t-test; ∗∗p < 0.01, ∗∗∗p < 0.001. (C) Migration of CXCR1- and CXCR2-augmented pNK cells following pre-treatment of cancer cells with navarixin, a selective CXCR1/2 antagonist, for 24 h. Quantitative analyses were conducted with two-way ANOVA followed by a multiple comparison test; ∗∗∗p < 0.001, ns: not significant. Data are represented as means ± standard error of the mean (SEM).

Figure 4

Biodistribution of human primary natural killer (pNK) cells augmented with CXCR1 or CXCR2 in Capan-2-bearing xenograft mice (A) Experimental design scheme of the in vivo biodistribution analysis of engineered pNK cells. Vybrant DiD-labeled pNK cells were intravenously injected into Capan-2-bearing subcutaneous xenograft mice. (B) Visualization of the targeting of DiD-labeled pNK cells to Capan-2 tumor sites at the indicated time points using the IVIS system. Arrows indicate the subcutaneously injected tumors. (C) Visualization of infiltrated pNK cells in excised tumors 48 h after pNK cell injection, with ex vivo fluorescence imaging using the IVIS system. Quantification of infiltrated pNK cells. Data represent the means ± standard error of the mean (SEM). ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns: not significant; a one-way ANOVA with a multiple comparison test was used. (D) Tumor sections were stained with DAPI for counterstaining. Arrows indicate DiD-labeled pNK cells. (E) Proportion of live infiltrating pNK cells from the total tumor tissues of two individual mice, assessed by flow cytometry. CD56 and DiD-positive NK cells were quantified 48 h post-NK cell injection.

Figure 5

Amplified anti-cancer activity of CXCR2-augmenting anti-MSLN CAR-NK toward mesothelin (MSLN)-positive cancer with enhanced infiltration (A and B) The generation of a second-generation CAR gene containing SS scFv, which targets MSLN, augmented with CXCR1 or CXCR2 in human primary natural killer (pNK) cells. (B) Flow cytometry analysis of transduction efficiency of engineered CAR-NK. (C) Flow cytometry analysis of the surface expression of MSLN on pancreatic cancer cells, including Capan-2 and PANC-1. Capan-2 shows strong expression of MSLN, whereas PANC-1 shows weak expression. (D) In vitro cancer cell-killing efficacy of CAR-NK combined with CXCR1 or CXCR2. Engineered CAR-NK cells were co-incubated with luciferase-overexpressing cancer cells of each type at the indicated E:T ratios for 24 h, and then luminescence signals from live cancer cells were quantified. p values were determined using a two-way ANOVA followed by a multiple comparison test for data from triplicate of each group; ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (E–G) Chemotaxis assay of CXCR1-or CXCR2-tethered CAR-NK cells toward Capan-2 cells. (E) Schematic representation of the chemotaxis assay. Each NK cell was stained with Hoechst, whereas Capan-2 cells stably expressed GFP. (F) Quantification of infiltrated CAR-NK cells, measured every 4 h up to 12 h after adding NK cells in the Transwell, as analyzed using a live cell imaging system. p-values were determined using a two-way ANOVA followed by a multiple comparison test for data from triplicate of each group; ∗∗p < 0.01, ∗∗∗p < 0.001. (G) Representative images showing the chemoattractive movement of pNK cells toward GFP-expressing Capan-2 at the indicated time points.

Figure 6

Human primary natural killer (pNK) cell-mediated killing of patient-derived organoids (A) Schematic illustration of pNK-organoid co-culture. The 3D organoids and pNK cells were co-cultured using a micro-pillar system, and the viability of dTomato-overexpressing organoids was assessed. (B) Representative brightfield images of patient-derived pancreatic cancer organoid lines, SNU-2571-CO. (C) Human chemokine array analysis of total lysates from SNU-2571-CO organoids. (D) Relative mRNA expression of CXCR1 or CXCR2 ligands from total organoid extracts. p values were determined using a Student’s t test; ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns: not significant. (E and F) pNK-mediated killing of patient-derived organoids. (E) Representative fluorescence images of dTomato-expressing SNU-2571-CO organoids co-cultured with each group of pNK cells at the indicated effector (NK cells) vs. target (organoids) ratios. (F) Relative viability of organoids analyzed using mean fluorescence intensity (MFI) and normalized to the MFI of the organoid-only group. The graph represents the mean ± SEM of data from quadruplets of each group. p values were determined using a two-way ANOVA followed by a multiple comparison test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (G) Representative images showing pNK cells infiltrated into organoids at the indicated E:T ratios; brightfield (BF) images of the cancer organoids (orange) and infiltrated NK cells (green).

Figure 7

Working mechanism of anti-MSLN chimeric antigen receptor (CAR)-engineered natural killer (CAR-NK) cells augmented with CXCR2 Pancreatic cancers exhibit strong secretion of CXCR1 and CXCR2 ligands, such as CXCL1, CXCL2, CXCL3, CXCL5, CXCL7, and CXCL8. Anti-MSLN CAR-pNK cells were genetically augmented with CXCR1 or CXCR2. CXCR2-tethered CAR-NK cells exhibit synergistic anti-tumor activity against MSLN-positive pancreatic cancer cells, accompanied by enhanced NK cell infiltration within in vitro 2D co-cultures and 3D tumor microenvironment (TME)-mimicking patient-derived model.