Breaking the Gemcitabine Resistance Barrier in Extrahepatic Cholangiocarcinoma | Absin Powers Top-Tier Research: Targeting the CAF-HGF-c-MET Axis to Restore Therapeutic Efficacy

Extrahepatic cholangiocarcinoma (eCCA) is characterized by high malignancy, insidious onset, and propensity for metastasis. First-line chemotherapy with gemcitabine frequently becomes ineffective due to acquired drug resistance. How do cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) drive tumor progression and therapeutic resistance? A landmark study published in Acta Pharmaceutica Sinica B provides compelling evidence, with the Absin Organoid ATP Viability Assay Kit supporting pivotal drug sensitivity experiments throughout, offering precise translational evidence for clinical combination therapy strategies.

I. Core Research Strategy: From Microenvironment to Therapeutic Targets, Closing the Loop on Drug Resistance

The research team established an integrated eCCA preclinical platform encompassing primary cell lines, patient-derived organoids (PDOs), and patient-derived xenografts (PDX), systematically addressing three fundamental scientific questions:

Key Models and Technical Approaches

II. Landmark Findings: The CAF-HGF-c-MET Axis as the Core Mechanism of eCCA Drug Resistance

1. CAFs Drive Comprehensive Malignant Progression of eCCA (Original Fig. 2)

Figure 2 Effect of CAFs on eCCA progression. (A) Immunofluorescence staining of CAFs/NFs showed positive expression of α-SMA, Vimentin and FAP. Scale bar: 200 μm. (B) Effect of CAF-CM/NF-CM on proliferation of TFK-1 and CBC3T-1 cells. Negative control (NC) (n = 3). (C, D) Effect of CAF-CM/NF-CM on migration and invasion of TFK-1 and CBC3T-1 cells (n = 3). Scale bar: 200 μm. (E, F) Representative fluorescent images of PDOs monocultures or co-cultures with CAFs/NFs and quantitative analysis of total organoid area (n = 3). PDOs (green), CAFs/NFs (red). Scale bar: 200 μm. (G) Representative tumor images of cell-derived xenografts: cancer cells, cancer cells + NFs and cancer cells + CAFs. (H, I) Mean tumor volume and tumor weight (n = 8). (J) Representative images of H&E and IHC staining of α-SMA, CK7 and Ki67 (n = 8). Data are shown as mean ± SD. ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

2. HGF is the Critical Secreted Effector of CAF-Mediated Tumorigenesis (Original Fig. 3)

Figure 3 HGF is a key cytokine in CAF-CM affecting eCCA progression. (A, B) Cytokine expression of CAF-CM and NF-CM was detected by cytokine array. HGF, CCL5, Angiogenin and TSP-1 expression was significantly elevated. (C) Levels of HGF in conditioned media from CAFs and NFs derived from six patients with eCCA, measured by ELISA (n = 6). (D) Viability of eCCA cells under different treatment conditions-normal control (NC), CAF-CM, CAF-CM with HGF-neutralizing antibody (CAF-CM + HGF Ab), and recombinant HGF (HGF)—measured at 0, 24, 48, 72, and 96 h (n = 3). (E, F) Representative images of Transwell assay of eCCA cells treated with CAF-CM or CAF-CM + HGF Ab or HGF for 48 h (n = 3). Scale bar: 200 μm. (G, H) Schematic and ELISA-based quantitative analysis of HGF secretion in different co-culture models (n = 3): (i) PDO monoculture; (ii) indirect co-culture in which CAFs are seeded in the matrix surrounding PDOcontaining Matrigel; (iii) indirect co-culture using a Transwell insert (TI) to allow paracrine interaction without physical contact; (iv) direct co-culture in which PDOs and CAFs are co-embedded in Matrigel to allow cell—cell contact. (I) Representative fluorescence images of organoids in monoculture or direct co-culture with CAFs under different conditions and quantitative analysis of total organoid area (n = 3). Scale bar:200 μm. Data are shown as mean ± SD. ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

3. HGF/c-MET/PI3K/AKT Signaling Mediates Gemcitabine Resistance (Original Fig. 4/5)

Figure 4 Inhibition of HGF/c-MET signaling pathway suppresses CAFs-induced eCCA progression. (A) Representative IHC images of c-MET expression in eCCA (n = 27) and para-tumor tissues (n = 9). (B) IHC staining intensity of c-MET in eCCA tissues (n = 27) and para-tumor tissues (n = 9) of TMAs. (C) Representative images of multiplex immunofluorescence staining (α-SMA, green; p-c-MET, yellow; CK19, red) in eCCA and para-tumor tissue. (D) Percentage of p-c-MET +CK19 +cells to CK19 +cells in eCCA tissues and para-tumor tissues. (E) Mean fluorescence intensity of α-SMA +in eCCA and para-tumor tissues. (F) Western blot analysis of p-c-MET, total c-MET, p-PI3K, total PI3K, p-AKT, and total AKT in TFK-1 and CBC3T-1 cells treated with recombinant HGF at different time points (15, 30, 60, 120 min). (G, H) Western blotting of the c-MET/PI3K/AKT signaling pathway in TFK-1 and CBC3T-1 cells under the following conditions: normal control (NC), CAFCM, CAF-CM supplemented with an HGF-neutralizing antibody (CAF-CM + HGF Ab), or recombinant HGF (n = 3). (I, J) Western blot analysis of c-MET/PI3K/AKT pathway activity in eCCA cells treated with CAF-CM, with or without the c-MET inhibitors JNJ-38877605 or crizotinib (n = 3). Data are shown as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 5 Targeting the HGF/c-MET signaling pathway enhances gemcitabine treatment sensitivity in vitro. (A) Cell viability of TFK-1 and CBC3T-1 cells treated with gemcitabine or gemcitabine combined with HGF (n = 3). (B) Representative images of the sensitivity of gemcitabine to PDOs monoculture systems or direct co-culture systems with CAFs (n = 3). PDOs (green), CAFs (red). The percentage of total area of the PDOs was quantified and the viability was determined. Scale bar: 500 μm. (C) Schematic diagram for the construction of TFK-1 gemcitabineresistant cells (TFK-1R). (D) Western blot analysis of c-MET protein levels in parental TFK-1 cells and gemcitabine-resistant TFK-1 cells (TFK-1R). (E) Cell viability of 100 μmol/L gemcitabine-treated TFK-1 and TFK-1R cells (n = 3). (F) Cell viability of TFK-1R cells treated with 100 μmol/L gemcitabine or in combination with c-MET inhibitor under conditions of added HGF (n = 3). (G) Dose—response curves of gemcitabine alone or in combination with c-MET inhibitors for treatment of TFK-1 and CBC3T-1 cells under conditions of added HGF (n = 3). (H) Western blot analysis of c-MET protein levels in PDOs established from 10 cases of eCCA. (I, J) Representative images and dose—response curves of gemcitabine alone or in combination with c-MET inhibitor in a direct co-culture system of PDOs and CAFs. Patient 8 (P8) (n = 3). Scale bar: 500 μm. Data are shown as mean ± SD. ns, no significance; **P < 0.01; ***P < 0.001.

4. Combination Therapy Demonstrates Significant Efficacy in Organoid and PDX Models (Original Fig. 6/7)

Figure 6 The combination of gemcitabine and c-MET inhibitors reduced cell-derived xenotransplantation growth. (A) Schematic diagram of he process of co-injection of cancer cells and CAFs into mice subcutaneously. (B—D) Representative tumor images of cell-derived xenografts treated with gemcitabine, c-MET inhibitors, or gemcitabine combined with c-MET inhibitor. Tumor volume and mass were calculated (n = 5).(E) H&E and IHC staining were used to assess the expression of Ki67, p-c-MET, p-PI3K and α-SMA in subcutaneous tumor tissues of mice. i.p., intraperitoneal injection; i.g., intragastric. Scale bar, 100 μm. Data are shown as mean ± SD. ns, no significance; *P < 0.05; **P < 0.01;****P < 0.0001.

Figure 7 The combination of gemcitabine and c-MET inhibitors reduced PDX tumor growth. (A) Schematic diagram of the PDX model construction process used for screening drugs. Passage 0 (P0), passage 4 (P4), passage 5 (P5). (B) Representative tumor images of PDX treated with gemcitabine alone or gemcitabine in combination with c-MET inhibitors. (C, D) Tumor volume and mass statistics (n = 8). (E) H&E and IHC staining were used to assess the expression of Ki67, p-c-MET, p-PI3K and α-SMA in PDX tissues. Scale bar, 100 μm. Data are expressed as mean ± SD. *P < 0.05; **P < 0.01; ****P < 0.0001.

III. Absin Core Product Support: Organoid ATP Viability Assay Kit

Figure 5 Targeting the HGF/c-MET signaling pathway enhances gemcitabine treatment sensitivity in vitro. (B) Representative images of the sensitivity of gemcitabine to PDOs monoculture systems or direct co-culture systems with CAFs (n = 3). PDOs (green), CAFs (red). The percentage of total area of the PDOs was quantified and the viability was determined. Scale bar: 500 μm.(I, J) Representative images and dose—response curves of gemcitabine alone or in combination with c-MET inhibitor in a direct co-culture system of PDOs and CAFs. Patient 8 (P8) (n = 3).Scale bar: 500 μm. Data are shown as mean ± SD. ns, no significance; **P < 0.01; ***P < 0.001.

IV. Research Significance and Clinical Translation

This study provides the first demonstration in the eCCA subtype that:

Absin delivers high-performance reagents that empower tumor microenvironment and drug resistance mechanism research, providing robust support for clinical translation in cholangiocarcinoma and other malignancies.

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