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

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.

Fig. 1. EC359 inhibits CAF-PCC crosstalk.

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).

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

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.

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

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).

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

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.

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

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 (Ly6C^high Ly6G^low) and (Ly6C^low Ly6G^high) 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.