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. 2025 Aug 22;9:296. doi: 10.1038/s41698-025-01046-w

Targeting cancer-associated fibroblast-driven LIF/LIFR axis improves the therapeutic efficacy of gemcitabine and nab-paclitaxel in pancreatic cancer

Rakesh Bhatia 1,2,#, Imran Khan 1,#, Xiaoqi Li 1, Shailendra Gautam 1,3,4, Parthasarathy Seshacharyulu 1, Zahraa Wajih Alsafwani 1, Namita Bhyravbhatla 1, Moorthy P Ponnusamy 1,5, Maneesh Jain 1,5, Mokenge Malafa 6, Santhamma Bindu 7, Ahmed Gulzar 7, Hareesh Nair 7,✉, Surinder K Batra 1,5,✉, Sushil Kumar 1,5,✉
PMCID: PMC12373761  PMID: 40846751

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is inherently therapy resistant due to cancer cell-stroma crosstalk across several signaling pathways. Among these, the LIF/LIFR axis has been implicated in cancer cell and cancer-associated fibroblast (CAF) crosstalk. We evaluated the efficacy of EC359, a competitive inhibitor of LIFR, in combination with gemcitabine. EC359 reduced tumor burden by 90% compared to controls and by 55% compared to gemcitabine alone in cancer cell and CAFs co-implannation model. The RNA-seq analysis revealed a significant alteration in extracellular matrix components, stemness, microtubule assembly, and immune response, suggesting simultaneous targeting of cancer cell-intrinsic and stroma-mediated mechanisms by EC359. In autochthonous murine model of PDAC, EC359 enhanced the therapeutic efficacy of gemcitabine and nab-paclitaxel, accompanied by an increase in dendritic cells but a reduction in T-regulatory cells. Thus, EC359 reduces PDAC cell stemness, stabilizes microtubule assembly, and reduces the immunosuppressive microenvironment to improve the efficacy of standard-of-care in PDAC.

Subject terms: Drug discovery, Oncology

Introduction

Despite the recent advances, pancreatic ductal adenocarcinoma (PDAC) remains the fourth leading cause of cancer-related deaths in the United States, with a 5-year survival rate of 13% due to late diagnosis and inherent drug resistance1. Histologically, 80-85% of PDAC is composed of heterogenous stroma, which forms an obstructive barrier around the tumor cells, limiting the availability of therapeutics to pancreatic cancer cells (PCCs)2. Cancer-associated fibroblasts (CAFs) are a major component of the tumor microenvironment (TME) that significantly contribute to PDAC pathobiology3. The PCCs promote protumor CAF phenotypes by secreting factors such as Sonic hedgehog (SHH), interleukin-6 (IL6), platelet-derived growth factor (PDGF), and transforming growth factor-β (TGFβ)4–8. Similarly, CAFs promote tumor progression, invasion, and metastasis by secreting cytokines and growth factors9,10. This crosstalk between CAFs and PCCs also promotes the pro-survival phenotype in cancer cells, leading to inherent drug resistance, whereas CAF activation increases the extent of obstructive stroma11,12. Therefore, selective targeting of tumor-promoting crosstalk between CAFs and PCCs will significantly improve the clinical outcomes of the standard of care therapies in PDAC.

Leukemia inhibitory factor (LIF), a pleiotropic member of the IL6 family of cytokines, is a critical mediator of PDAC progression13–19. Its binding to the LIF-receptor (LIFR) and glycoprotein 130 (gp130) heterodimeric receptor complex activates downstream signaling pathways, including the JAK/STAT, ERK/MAPK, and PI3K/AKT pathways20. Genetic deletion of LIFR in the KPf/fCL murine model (KrasLSL-G12D/+; Trp53flox/flox; Pdx1-cre; Rosa26LSL-Luc/LSL-Luc) significantly delayed PDAC progression and increased the survival of animals. Moreover, KPf/fCL mice treated with anti-LIF monoclonal antibodies in combination with gemcitabine (Gem) significantly improved their survival and reduced tumor weight13. Furthermore, an engineered high-affinity soluble human LIFR inhibited LIF-mediated signaling pathways and tumor growth in PDAC xenograft mouse models21. These findings indicate that the LIF/LIFR axis is a potential therapeutic target16. A humanized anti-LIF antibody is in phase I clinical trial (NCT03490669) for dose escalation and toxicity studies22.

The LIF/LIFR axis has been targeted so far using monoclonal antibodies or ligand decoys, which may have limited availability in highly desmoplastic PDAC tumors. Furthermore, LIFR is activated by multiple ligands besides LIF; therefore, developing a competitive inhibitor that restricts the binding of multiple ligands to LIFR would be a more effective approach23,24. Additionally, small molecule inhibitors, which exhibit high tumor and oral bioavailability, are expected to perform better clinically. The EC359 is a first-in-class competitive LIFR inhibitor that blocks the binding of multiple ligands, including cardiotrophin-1, ciliary neurotrophic factor, and oncostatin-M, to LIFR25. Studies have shown that EC359 blocks the LIF/LIFR axis and inhibits the progression of triple-negative breast (TNBC) and endometrial cancers25,26. Although the clinical relevance of targeting the LIF/LIFR axis in PDAC has been well established13,27, the efficacy of EC359 in PDAC tumors in combination with first-line therapy, Gem, and nab-paclitaxel remains unexplored. In this study, we report that EC359 efficiently inhibits the CAF-mediated activation of the LIF/LIFR axis in PCCs. Findings from autochthonous, orthotopic co-implantation murine models, and RNA-seq analyses indicate that EC359 inhibits STAT3 signaling and PCC proliferation. Furthermore, in combination with Gem and nab-paclitaxel, EC359 efficiently induces apoptosis and enhances microtubule stabilization in PCCs. Moreover, the combination treatment also increases dendritic cell population and reduces the regulatory T-cells in the systemic circulation.

Results

EC359 inhibits CAF-mediated STAT3 signaling in PCCs

We evaluated the expression of LIFR in human PDAC tumor tissues and the tumors derived from KPC murine model. Our results show a significant LIFR expression on PCCs compared to the normal pancreatic ducts (Fig. 1A, B, Supplementary Fig. 1A). We validated the expression by IF analysis, which also showed an elevated LIFR expression on human PDAC cell lines (Fig. 1C, Supplementary Fig. 1B–D). Screening of different patient-derived CAFs showed higher expression of LIF, especially in 10.03-P cells (Fig. 1D). The recombinant LIF (rLIF) treatment in human (CD18/HPAF and SW1990) and mouse (KCT3266 and KCT3248) PCCs increased STAT3 phosphorylation (pSTAT3, Y705) in a dose and time-dependent manner (Fig. 1E, F), which was inhibited by pre-treatment with EC359 (2.5 µM, Fig. 1G, H).

Fig. 1. EC359 inhibits CAF-PCC crosstalk.

Fig. 1

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A Representative IHC images of LIFR expression in PDAC patient samples and KPC tumors. B Quantification of IHC analysis in KPC tumors and normal mouse pancreas (n = 3/group) demonstrated higher expression of LIFR in the PDAC ducts compared to normal pancreatic ducts. C Immunofluorescence (IF) imaging demonstrated the co-localization of LIFR and cell surface marker E-cadherin in CD18/HPAF and T3M4 PDAC cell lines. D Relative expression of hLIF in different CAF cell lines represented as the inverse of ΔCT (1/ΔCT) evaluated by qRT-PCR analysis. E Immunoblot analysis of pSTAT3 (Y705) levels in human PDAC cell lines CD18/HPAF and SW1990, and F Murine PDAC cell lines, KCT3266 and KCT3248, in response to recombinant LIF (rLIF) stimulation at concentrations of 1 ng/ml for 15, 30, and 60 min and 5 ng/ml for 15 and 30 min. Immunoblot analysis to evaluate the inhibitory potential of EC359 (2.5 µM) on LIF-mediated increase in pSTAT3 levels in human PDAC cell lines CD18/HPAF, T3M4, and SW1990 (G), and murine PDAC cell lines KCT3266 and KCT3248 (H). I Immunoblot analysis showing the pSTAT3 levels in SW1990 PDAC cell line in the presence of CM from hCAF and inhibition of STAT3 activation upon EC359 treatment. The data is presented as mean ± SEM. **p < 0.01. Recombinant human LIF (rhLIF), recombinant mouse LIF (rmLIF).

Further, to characterize the paracrine activation of the LIF/LIFR axis in PCC by CAFs, we treated PDAC cell lines with CAF condition medium (CM) and EC359 (Fig. 1I). The increased phosphorylation of STAT3 by CAF CM in PCCs was reduced by EC359 treatment (Fig. 1I), suggesting that EC359 can inhibit STAT3-mediated crosstalk between PCCs and CAFs.

EC359 enhances the efficacy of Gem treatment in orthotopic PDAC tumors by reducing cancer cell stemness

Next, we orthopically co-implanted PCCs and CAFs (1:1 ratio) in athymic (CD18/HPAF:hCAFs) and C57BL/6 (KCT3266:mCAFs) mice. After the tumors were palpable, animals were treated with vehicle, EC359 (E), Gem (G), and E + G (Fig. 2A). The E + G combination significantly decreased the tumor growth (~55%) compared to individual treatments and vehicle (~90%) (Fig. 2B). The IHC analysis revealed reduced expression of LIFR in the combination group compared to the other treatment groups (Fig. 2C, Supplementary Fig. 2A), likely due to the internalization and degradation28,29 of LIFR after EC359 binding. The IF analysis of tumor tissues revealed a decrease in PCC proliferation and stemness, as indicated by reduced staining with Ki-67 and CD44 (Fig. 2D-F). Indeed, studies have demonstrated that CAFs increase the stemness of cancer cells30. Supporting the findings, the E + G treatment significantly decreased the growth of KPC tumor-derived organoids compared to the control and individual treatment groups (Fig. 2G). Further, EC359 sensitizes the Gem-resistant Colo357 cells to Gem treatment, as indicated by decreased cell viability (Fig. 2H, I). These findings collectively indicate that EC359 increases the efficacy of Gem in PDAC by preferentially decreasing PCC stemness, which is responsible for therapy resistance.

Fig. 2. EC359 enhances the efficacy of Gem treatment.

Fig. 2

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A Schematic representation of orthotopic co-implantation of KCT3266: ImPaSC (1:1 ratio) in C57BL/6 (n = 7/group) and CD18/HPAF: hCAFs (1:1 ratio) in athymic mice. The animals were randomly divided into four treatment groups: control, EC359 (E, 5 mg/kg), Gem (G, 25 mg/kg/week), and EC359 in combination with Gem (E + G, EC359 5 mg/kg every alternate day with biweekly Gem 25 mg/kg/week). B The significant reduction in the tumor weight (g) in C57BL/6 and athymic mice demonstrated an enhanced efficacy of E + G therapy compared to G and E alone. C The zoomed area of the tumor tissues from C57BL/6 animals showed reduced expression of LIFR (10X) in the E + G group compared to either treatment alone. D Representative IF images (40x) and the quantification (E, F) showed significantly reduced expression of CD44 and Ki-67 in the E + G-treated group compared to either treatment alone. G Quantitative evaluation of the growth of KPC organoid after 72 h of treatment with control, E, G, and E + G showed the most significant growth reduction in the E + G group. The impact of EC359 on parental (H) and Gem-resistant (I) Colo357 cell lines in the presence of rLIF, evaluated using the real-time live imaging system IncuCyte® sx5 (dot plot depicts the percent confluence of cells over 57 h), showed sensitization of Gem-resistant cells by EC359. The data is presented as mean ± SEM. *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001.

EC359, in combination with Gem, modulates cancer cell-intrinsic and TME-mediated mechanisms

We performed bulk RNA sequencing on treated orthotopic PDAC tumors in C57BL/6 mice to gain insight into the mechanism of EC359 action. Based on differentially expressed genes (DEGs) with log2 fold change (FC) between treatment and control groups (Fig. 3A, Supplementary Fig. 2B and C), the gene ontology (GO) analysis indicated that the E + G treatment modulated the molecular functions (MF) like protein folding and regulation of enzymatic (hydrolases), and GTPase activities (Fig. 3B). Similarly, the components of TME, like extracellular matrix (ECM) and cell-intrinsic pathways, like membrane domain (signaling), and microtubule cytoskeleton and organization (cytoskeletal modifications) were significantly altered (Fig. 3C).

Fig. 3. The RNA-seq analysis suggests that EC359 targets cell-intrinsic and TME-mediated mechanisms.

Fig. 3

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A The volcano plot depicting the differentially expressed genes (DEG) in tumors treated with G and E + G. There were thirty downregulated and seventeen upregulated genes in the E + G treatment group. B The significant molecular functions (MFs) identified through Gene Ontology (GO) enrichment analysis for tumors treated with E + G compared to G alone. C Similarly, the Gene Ontology (GO) analysis for the significant cellular components (CC) in tumors treated with E + G and G indicated an association with microtubule assembly, extracellular matrix (ECM), and collagen-containing processes. D In contrast, the significant biological processes (BP) included spindle assembly and organization, as well as protein folding. E The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes revealed significant enrichment of ECM receptor interaction pathways (heatmap illustrates the differentially expressed genes).

Among the biological processes (BPs), mitotic spindle assembly and organization, as well as protein folding, are modulated by E + G treatment compared to Gem alone (Fig. 3D). Corroborating the findings, the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis also revealed that E + G treatment positively correlated with ECM-receptor interaction (Fig. 3E, subset of 24 genes associated with ECM remodeling). In addition, the combination treatment enriched the gene signature associated with antigen processing and presentation, as well as T-cell receptor signaling pathways, indicating a plausible switch from an immunosuppressive to an immune-responsive tumor microenvironment (TME) (Supplementary Fig. 2D, E). The RNA-seq analysis also suggests a global impact on transcription, RNA splicing (Bdp1, Txnl4a), metabolism (Ces2, Timm22), and stress response (Hapa1a) in PDAC tumors following E + G treatment. Together, RNA-seq analysis suggests that EC359, combined with Gem, reduced the tumor burden by modulating cancer cell-intrinsic and TME-mediated mechanisms.

EC359 promotes microtubule stabilization and cell cycle arrest in combination with Gem and nab-paclitaxel

The gene set enrichment analysis (GSEA) in E + G treated tumors revealed a significant association with microtubule structure assembly (Fig. 4A and Supplementary Fig. 3A). Interestingly, the expression of microtubule-associated protein Tubulin Polymerization Promoting Protein Family Member 3 (Tppp3) was significantly correlated with Epithelial Cell Adhesion Molecule (Epcam, a marker of malignant cells) compared to CAF-associated Podoplanin (Pdpn), suggesting a major contribution by malignant cells (Supplementary Fig. 3B). This intrigued us to test whether EC359 can increase the efficacy of microtubule-targeting drugs, nab-paclitaxel. The Food and Drug Administration (FDA) approved nab-paclitaxel for metastatic PDAC in combination with Gem in 201331, which disrupted cell cycle progression by inducing microtubule stabilization32. In line, compared to nab-paclitaxel alone, the IF analysis for tubulin bundles per cell indicated a significant increase in the stabilization of the α-tubulin bundles in PCCs upon treatment with nab-paclitaxel in combination with EC359 (Fig. 4B, C). Based on these findings, our subsequent analysis showed that EC359 in combination with Gem and nab-paclitaxel [E + G + A (EC359 + Gem + nab-paclitaxel)] arrested PDAC cells in the G1 phase (Fig. 4D, E). Moreover, there was a significant increase in apoptotic cells in the combination treatment group compared to various monotherapy groups (Supplementary Fig. 3C–E), indicating that EC359 may enhance the efficacy of the first-line therapy (Gem and nab-paclitaxel) in PDAC. We also observed that EC359 increased the expression of nucleoside transporter 1 (ENT1) in PDAC cells, a primary transporter for Gem (Fig. 4F), suggesting that inhibition of LIF/LIFR axis can counter acquired resistnace to GEM.

Fig. 4. EC359 potentiates Gem and nab-paclitaxel treatment.

Fig. 4

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A The GSEA suggests an association between microtubule assembly and E + G treatment, which led us to evaluate the role of EC359 in tubulin organization. B Representative images of IF analysis for the expression and organization of α-tubulin in murine KCT3248 PCCs after treatment with control, EC359 (E), nab-paclitaxel (A), and EC359 with nab-paclitaxel (E + A). C The quantification using ImageJ of average α-tubulin bundles per cell showed higher microtubule stabilization by EC359 in combination with nab-paclitaxel. D Representative flow cytometry images and percent distribution (E) of KCT3248 cells in different phases of the cell cycle (G1, G2, and S) after treatment with control, E, G, A, E + G, and E + G + A for 24 h showed an accumulation of EC359-treated cells in the G1 phase. F Relative mRNA expression of ENT1 transporter gene in murine KCT3248 PDAC cells treated with control, recombinant LIF (rLIF), rLIF+E, and rLIF+E + G for 24 h. The data is presented as mean ± SEM. *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001.

EC359 improves the efficacy of Gem and nab-paclitaxel treatment in the autochthonous murine model of PDAC

Following the lead from in vitro experiments, we evaluated the efficacy of combination therapy in the autochthonous KPC murine model of PDAC. Twenty-five to thirty weeks old KPC mice were randomly divided into a vehicle (human albumin), Gem and nab-paclitaxel (G + A), EC359 with Gem, and nab-paclitaxel (E + G + A) treatment groups as shown in Fig. 5A. The efficacy of EC359 was evaluated at suboptimum doses of Gem and nab-paclitaxel, as this will help reduce the toxicity of the chemotherapeutics. There was a significant reduction in tumor weight in animals treated with EC359 with G + A compared to the vehicle control group (Fig. 5B). We did not observe a significant reduction in the spleen weight in the combination groups (Fig. 5C). Histological evaluation of the tumor tissues indicated a significant increase in the apoptotic cells in tumors treated with E + G + A (Fig. 5D, E). Further, the combination group showed decreased expression of CD44 (stemness marker) (Supplementary Fig. 4) in cancer cells and a stromal marker, fibronectin1 (FN1) (Fig. 5D, F), suggesting the simultaneous targeting of PCCs and stromal compartment.

Fig. 5. EC359 improves the efficacy of Gem and nab-paclitaxel therapy in the autochthonous murine model.

Fig. 5

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A Schematic representation of the metronomic (suboptimum doses) therapy regimen in the KPC mouse model. The 25-30week-old KPC mice (n = 5/group) with palpable tumors were randomly divided into control, G + A, and E + G + A groups and subjected to 11 days regimen, with EC359 (10 mg/kg) on an alternate day, nab-paclitaxel, and Gem (50 mg/kg) on the 1st, 5th, and 9th days (suboptimum doses of G and A). B The average tumor weights (g) of resected tumors show a significant reduction in tumor weights in animals on the E + G + A regimen compared with controls. C There was no difference in the average spleen weight (g) among the different treatment groups. D Representative images of H&E staining and IHC. E Respective quantification showed a significant increase in apoptotic cells in KPC tumors after treatment with the E + G + A regimen. F Quantification (H-score) showed a significant decrease in the expression of FN1, suggesting a reduction in the activation of stroma in the E + G + A group. The percentage distribution of immune cells in the blood and spleen of animals treated with control, G + A, and E + G + A showed a significant increase in circulating dendritic cells (G, CD80 + CD86+ cells) but a decrease in T-regulatory cells (H, CD4+ CD25+ cells) in the E + G + A group. Concurrently, there was a significant increase in CD8 + T-cells in the spleens of animals treated with the E + G + A regimen (I). J The percent population of CD11b+ cells (Ly6Chigh Ly6Glow) and (Ly6Clow Ly6Ghigh) myeloid-derived suppressor cells in blood and spleen. K Representative IHC images and quantification (Percentage of positive cells) showed a significant increase in CD86+ expressing cells in tumors upon treatment with E + G + A, validating the antigen processing pathway identified in the RNA-seq analysis. The percentage of positive cells was calculated using the ImageJ image color deconvolution plugin on three independent areas in each tumor tissue. The data is presented as mean ± SEM. *p < 0.05; **p < 0.01, ***p < 0.001.

Based on our RNA-seq analysis, which revealed gene signature associated with antigen processing and presentation and T-cell receptor signaling (Supplementary Fig. 2D, E). Following the defined gating strategy that is depicted in the Supplementary Fig. 5A, we observed an increase in the antigen-presenting dendritic cells systemically in E + G + A animals compared to the vehicle group (Fig. 5G). In contrast, the T-regulatory (T-reg) cells (CD4+ CD25+) were significantly reduced in the spleen and blood of the combination group compared to the G + A group (Fig. 5H). However, there was no significant change in the CD8+ CD25+ population in the spleen of the combination group (Fig. 5I) and B-cells or myeloid cells in the systemic circulation (Fig. 5J and Supplementary Fig. 5B). Interestingly, there was an increase in CD86+ DC in KPC tumors treated with E + G + A compared to the vehicle group (Fig. 5K). These findings support the RNA sequencing analysis, which suggests enhanced antigen presentation due to EC359. Thus, EC359 enhances the efficacy of Gem and nab-paclitaxel in PDAC by targeting PCC stemness, promoting apoptosis, and improving immune responsiveness in the TME.

Discussion

Systemic chemotherapy remains the mainstay of clinical management for patients with PDAC1. Therapeutic regimens like FOLFIRINOX (5-fluorouracil, leucovorin, irinotecan, and oxaliplatin), and Gem with nab-paclitaxel forged the forefront of clinical care for advanced PDAC33,34. However, most patients respond poorly or develop resistance to these regimens, indicating a need for targeted therapies that simultaneously sensitize PCCs and modulate the stromal environment. Our study showed that EC359 stalled the crosstalk between CAFs and PCCs by effectively inhibiting the LIF/LIFR axis. Recently, Shi et al. also demonstrated the paracrine activation of the LIF/LIFR axis in PDAC, highlighting the importance of the LIF/LIFR axis as a potential therapeutic target13. The serum LIF levels increased significantly in PDAC mice, along with the expression of LIFR at both transcription and protein levels in PCCs35. The EC359 binds specifically to the LIFR ligand-binding domain and inhibits the activation of downstream signaling pathways [25]. Several studies have documented the efficacy of EC359 in targeting renal, ovarian, endometrial, and breast cancer25,36–38. Our initial studies with CAFs and PCCs co-implantation showed activation of the LIF/LIFR axis in the TME; however, EC359 treatment significantly reduced tumor weights in both syngeneic and immune-compromised orthotopic mouse models.

The bulk RNA sequencing analysis of orthotopic tumors after EC359 and Gem treatment indicated a reduction in the activation status of CAFs, which are the primary source of ECM components, including FN-1, periostin, matrix metalloproteinase-9, collagen1A1, 1A2, and 5A139,40. Additionally, Thbs4, which was upregulated in our RNA-seq analysis, is related to stromal response and acts as a tumor suppressor in colorectal cancer41. We found that E + G treatment led to the enrichment of gene signature associated with microtubule assembly, suggesting that EC359 can potentiate nab-paclitaxel-mediated targeting of PDAC. Paclitaxel inhibits cancer cell growth by promoting microtubule stabilization42. Indeed, EC359 enhanced the nab-paclitaxel-induced microtubule stabilization in PDAC cells, resulting in increased apoptosis of PCC.

LIF/LIFR signaling also plays a crucial role in altering the TME by regulating the infiltration of immune cells, including regulatory T-cells, effector T-cells, macrophages, and immature myeloid cells43–45. LIF regulates the expression of CXCL9 in macrophages, which prevents the infiltration of CD8 + T cells into the tumor and impedes the effectiveness of the anti-PD1 regimen. Co-administration of LIF-neutralizing and anti-PD1 antibodies results in tumor shrinkage, promotes immunological memory, and enhances survival46. The administration of MSC-1, a humanized monoclonal antibody that specifically binds to LIF, demonstrated antitumor and pro-inflammatory effects in mouse models of syngeneic colon cancer47. During a phase-I clinical trial on patients with advanced solid tumors, the administration of MSC-1 increased the M1:M2 ratio and decreased STAT3 phosphorylation22. Our GSEA analysis revealed that EC359, combined with Gem, enriched the gene signature associated with T-cell receptor signaling and antigen presentation. Furthermore, we demonstrated that EC359, combined with Gem and nab-paclitaxel, significantly enhanced the infiltration of the CD86 + DC population into tumors, as well as the number of antigen-presenting cells in the systemic circulation. Thus, EC359, in combination with Gem and nab-paclitaxel, suppressed immunosuppressive T-cells and enhanced the antigen presentation pathways systemically and within the tumor microenvironment (TME). Although EC359 showed therapeutic efficacy in both orthotopic and KPC animal models, the relatively lower reduction in tumor weight in KPC animals may be attributed to various reasons, including differences in disease penetrance, tumor microenvironment, and therapy regimens. Overall, our study demonstrated the effectiveness of EC359 in combination with Gem and nab-paclitaxel in PDAC, which will be evaluated in a clinical trial in the future.

Methods

Cell Culture

The human (CD18/HPAF, Colo357, SW1990), and murine (KCT3266 and KCT3248 derived from KPC tumors) PDAC cell lines, and fibroblasts were cultured at 37 °C with 5% carbon dioxide in a humidified chamber in DMEM supplemented with 10% FBS and 1% Penicillin-Streptomycin. Immortalized mouse pancreatic stellate cells, ImPaSC were gifted by Dr. Raul Urrutia, and patient-derived CAFs (10.32, 10.03, 9.17, and 9.26) were generated in our lab. The Gem-resistant cells were established by incrementing the Gem concentration in a gradient for 1 month and maintained in DMEM supplemented with 10 mM Gem, 10% fetal bovine serum (FBS), and 1% Penicillin-Streptomycin.

Cell viability, apoptosis, and growth kinetics

The cytotoxic activity was evaluated using the MTT assay, as described previously48. Briefly, cells were seeded into 96-well plates at 1 × 103 cells/well and treated as indicated for 24 h. The MTT (5 mg/ml) reagent-derived intracellular formazan crystals were dissolved in DMSO (100 µl). The IC50 concentrations of EC359, Gem, and nab-paclitaxel were calculated using the GraphPad Prism 10.0 software. For apoptosis, 5 × 105 cells/well were seeded into six-well plates and treated as indicated for 24 h. Apoptosis in PDAC cells was measured using AnnexinV/PI double staining as previously described49. The growth of human PDAC cells, Colo357 and Gem-resistant Colo357 cells, were analyzed for three days using Incucyte® (Sartorius, USA), as described previously50. As previously described, murine pancreatic organoids were established using the tumor specimens from the autochthonous murine model, KPC (KrasG12D; TP53R172H; Pdx1-Cre) mice51. The growth kinetics of organoids upon treatment with control (PBS), EC359 (E), Gem (G), and EC359 with Gem (E + G) hours were analyzed in real-time for 72 h in Incucyte® (Sartorius, USA) based on the size of the selected organoids and the data is presented as the percentage change compared to time zero51.

Immunoblotting Analysis

The protein expression in PDAC cells was evaluated by western blot as previously described52. The proteins (20-40 µg) were resolved by SDS-PAGE gel electrophoresis and transferred to PVDF membranes. The PVDV membranes were incubated with primary antibodies (List of antibodies in Supplementary Table 1) overnight at 4°C and processed using PierceTM ECL western blotting substrate (Thermo ScientificTM) chemiluminescent reagent and visualized using X-ray film and/or iBrightTM FL1500 imaging system (Thermo ScientificTM).

Animal Studies

All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Nebraska Medical Center (UNMC). For the orthotopic model, PDAC cells were mixed with CAFs (viability>95%) in a 1:1 ratio and implanted into mice pancreas at 2.5 × 105 cells in 50 µl phosphate buffer saline (PBS) in 6–8-weeks-old C57BL/6 (murine cells), and nude (human cells) mice (n = 7/group for 80% power to detect α = 0.05) as described previously53,54. As the tumors were palpable (~10 days), the animals were randomly divided into four groups and treated with vehicle (PBS), EC359 (E, 5 mg/kg body weight), Gem (G, 25 mg/kg body weight, IP), and a combination of EC359 and Gem (E + G). After the therapy, animals were euthanized using carbon dioxide (CO2) anaphylaxis followed by cervical dislocation, and organs were harvested for further analysis. Autochthonous murine model, KPC (KrasG12D; TP53R172H; Pdx1-Cre) mice were bred and maintained as previously described55. The 25-30-weeks-old KPC mice were randomly divided and treated with vehicle (human albumin), Gem and nab-paclitaxel (G + A), and EC359 with Gem and nab-paclitaxel (E + G + A). The animals were subjected to an 11-day therapy regime with EC359 (10 mg/kg, subcutaneous, Evastra Inc. GMP grade), nab-paclitaxel (50 mg/kg, Intravenous, Celgene Inc.), and Gem (50 mg/kg, Intraperitoneal, Cat. No. G6423m Millipore Sigma) as indicated in Fig. 5A. After the therapy, animals were euthanized, and organs were harvested for further analysis.

Immunohistochemistry (IHC) and Immunofluorescence (IF) Analysis

The PDAC tissue sections from patients and murine models were analyzed using IHC as previously described52. Paraffin-embedded tissue sections (5 µM thickness) were baked overnight at 56 °C, deparaffinization, and rehydration with gradient ethanol (100, 90, 70, 50, and 20%). The endogenous peroxidases were quenched with 3% hydrogen peroxide for 1 h in the dark, followed by antigen retrieval in citrate buffer (pH 6.0). The tissues were blocked using normal horse serum (Vector Laboratories) for 1 h, followed by overnight incubation with the primary antibody (Supplementary Table 1) at 4 °C. Later, the slides were incubated with a secondary antibody (Vector Laboratories) for 1 h, processed with DAB substrate, and counterstained with hematoxylin. The slides were scored by the pathologist (Intensity 0-3; Percent positive cells 0-100%). For IF analysis, PDAC cells were cultured on coverslips. Tissue sections and PDAC cells were blocked using 10% normal Goat serum (Impress Reagent Kit, Vector Laboratories, CA, USA) for 1 h followed by incubation with primary antibody overnight at 4 °C. After washing with PBS, samples were incubated with fluorochrome-conjugated secondary antibodies for 1 h. All sections and cover slips were washed and mounted using DAPI Fluoromount-G (SouthernBiotech, AL, USA). The photomicrographs were captured on a Zeiss 710 confocal Laser scanning microscope.

Quantitative Real-Time PCR (qRT-PCR) and Digital Droplet PCR (ddPCR)

Total RNA from the cells was isolated using RNeasy Kit (Qiagen, MD, USA). Frozen tumor tissues were ground in liquid nitrogen, and the total RNA was isolated using the mirVanaTM miRNA isolation kit (Thermo ScientificTM) followed by cDNA synthesis using iScript cDNA synthesis kit (BioRad, USA). Gene expression was analyzed by quantitative real-time PCR (qRT-PCR) using a CFX Connect (Bio-Rad, USA) as described56. Furthermore, the copy number of genes was analyzed by ddPCR using the QX200 (Bio-Rad, USA), and the results were analyzed using the QX Manager as previously described57. The primer sequences are provided in Supplementary Table 2.

RNA sequencing

The RNA isolated from orthotopic tumors treated with vehicle, EC359, Gem, and EC359+Gem was analyzed by RNA sequencing (Novogene Co. Ltd., CA) using the Arraystar RNA Sequencing service, which includes library preparation, sequencing, quality control, and mapping (mapping statistic summary, transcript coverage). The differential gene expression analysis (differentially expressed gene/transcript analysis, hierarchical clustering, and plots of differentially expressed genes) and functional analysis of differentially expressed coding genes (GO, KEGG Pathway, GSEA) were also performed58. Next, we examined the correlation between Epcam or Pdpn with microtubule pathway genes using log2(FPKM + 1) values.

Immune phenotyping

Flow cytometry was used to analyze various immune cell subsets (antiCD3+-APC, antiCD4+-PerCPCy55, antiCD8+-PE, antiCD25+-A700, and antiCD19+-V450), dendritic cells (antiCD86+-PECy7 and antiCD80+-PE), and macrophage populations (antiCD11b+-A700, antiLy6C+-PER55, and antiLy6G+-FITC) from the blood and spleen samples (Supplementary Table 1). The blood and spleen samples were subjected to red blood cell (RBC) lysis, washed, and processed for analysis. The single-cell suspensions (0.2 × 106 cells) were washed and incubated with fluorochrome-conjugated primary antibodies for 1 h on ice. The cells were washed in flow buffer (PBS, pH 7.2 + 1% FBS), fixed with 4% paraformaldehyde, and analyzed using a BD LSRFortessa. The acquired data were analyzed using FlowJo.

Statistical analysis

All the statistical analysis was performed by GraphPad Prism (version 10.0). The data is presented as mean ± SEM for each group, and the significance between the two groups was evaluated by student t-test. One-way ANOVA and the Tukey comparison test calculated the significant difference between multiple groups. P ≤ 0.05 was considered statistically significant.

Supplementary information

Supplementary Information (2.9MB, pdf)

Acknowledgements

The authors are indebted to the National Institute of Health (NIH) funding support. The authors/work on this manuscript were supported, in parts, by grants from the NIH (R44 CA235991, R01 CA247471, RO1 CA210637, RO1 CA206444, RO1 CA183459, UO1 CA200466, PO1 CA217798, R01 CA290859, R01 CA195586). Mice schematic was generated using Biorender.

Author contributions

Conceptualization: S.K., S.K.B. Experiments and data collection: R.B., I.K., S.G., X.L., S.K., S.S., Z.A., M.P.P., M.J., M.M., S.K.B. Data Analysis and interpretation: I.K., R.B., X.L., S.K., H.N., S.K.B. Visualization and Bioinformatic Analysis: I.K., S.K., Z.A., M.M. Supervision: S.K., S.K.B. Writing-orginal draft: I.K. Writing-review & editing: S.K., S.K.B., H.N.

Data availability

All data supporting the study's findings are available from the corresponding authors upon reasonable request. The RNA sequencing data sets used in the study are deposited to the GEO repository under the accession code GSE300401.

Code availability

The differentially expressed genes (DEG) between the groups were analyzed via R Package ballgown (2.10.0).

Competing interests

S.K.B. is one of the founders of Sanguine Diagnostics and Therapeutics, Inc. H.N., S.B. and A.G. are employees of Evestra and holders of a patent on EC359. The other authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Rakesh Bhatia, Imran Khan.

Contributor Information

Hareesh Nair, Email: hnair@evestra.com.

Surinder K. Batra, Email: sbatra@unmc.edu

Sushil Kumar, Email: skumar@unmc.edu.

Supplementary information

The online version contains supplementary material available at 10.1038/s41698-025-01046-w.

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Associated Data

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

Supplementary Materials

Supplementary Information (2.9MB, pdf)

Data Availability Statement

All data supporting the study's findings are available from the corresponding authors upon reasonable request. The RNA sequencing data sets used in the study are deposited to the GEO repository under the accession code GSE300401.

The differentially expressed genes (DEG) between the groups were analyzed via R Package ballgown (2.10.0).

Articles from NPJ Precision Oncology are provided here courtesy of Nature Publishing Group

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