Paricalcitol and hydroxychloroquine modulates extracellular matrix and enhance chemotherapy efficacy in pancreatic cancerDownload PDF Download PDF ArticleOpen accessPublished: 27 September 2025Dhana Sekhar Reddy Bandi1,Sujith Sarvesh1,Jeremy Foote2,Doug Welsch1,3,Changde Cheng1,3,4,5,Mehmet Akce1,Ganji Purnachandra Nagaraju ORCID: orcid.org/0000-0002-4989-52341 &…Bassel F. El-Rayes ORCID: orcid.org/0000-0003-0405-55031 Cancer Gene Therapy (2025)Cite this articleSubjectsPancreatic cancerTargeted gene repairAbstractPancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor prognosis and limited therapeutic options. In a previous publication, our group defined some of the mechanisms that vitamin D analogue paricalcitol (P) and hydroxychloroquine (H) potentiated the effects of gemcitabine-based chemotherapy in PDAC. Based on this, we hypothesized that PH may potentiate 5-fluorouracil (5FU) and Oxaliplatin-based chemotherapy, and this may involve a novel mechanism of extracellular matrix (ECM) modulation. The combination of PH with 5FU+Oxaliplatin significantly increased the cell death, apoptosis, and S-phase cell cycle arrest as compared to untreated or 5FU + Oxaliplatin-treated MIA PaCa-2, HPAC and KPC cell lines. In vivo, the combination therapy inhibited PDAC growth and altered the immune landscape by activating T and NK cells. Proteomic analysis revealed significant reduction in ECM proteins, specifically integrin beta-4 (ITGB4). Confirmation of the role of ITGB4 was performed through genetic knockdown of ITGB4, which led ECM inhibition. In conclusion, the combination of PH significantly enhances the efficacy of Oxaliplatin and 5FU. We identified a new mechanism of action of PH through inhibiting ITGB4, leading to ECM modulation. These results suggest that the combination of PH with cytotoxic chemotherapy should be tested in PDAC clinical trials.IntroductionPancreatic ductal adenocarcinoma (PDAC) is one of the most deadly malignancies [1]. It is estimated that by 2030, PDAC will become second leading cause of cancer-related deaths in the U.S. The 5-year survival rate for PDAC is poor (~11%) due to its tendency for early metastasis and resistance to systemic treatments, including cytotoxic and immune-based therapies [2, 3]. The resistance to systemic therapies is partly due to the presence of collagen-rich dense extracellular matrix (ECM) [4].The PDAC ECM consists of several components, such as collagens, glycoproteins, fibronectins, proteoglycans, hyaluronic acid, and laminins. Cancer-associated fibroblasts (CAF) play a pivotal role in the development and maintenance of the ECM. PDAC cells interact with ECM components through integrin’s and receptor tyrosine kinases to form focal adhesion complexes, which help in growth, metastasis, and drug resistance [5, 6]. ECM affects the behavior of immune and angiogenic cells in the tumor microenvironment (TME), promoting a tumorigenic effects [7]. The compact and heterogeneous physical network of ECM components inhibit drug delivery, further contributing to resistance and poor prognosis [8]. Therefore, the ECM has pleotropic effects in PDAC, facilitating cancer cell survival, metastasis, and chemo-resistance. Modulating the ECM represents a rational approach to inhibit PDAC progression and promote the effects of systemic therapies.Previous reports have indicated that vitamin D analogs, such as paricalcitol (P) and hydroxychloroquine (H) have effects on CAF and immune TME. Vitamin D analogs has been shown to suppress activated CAF in PDAC and acts as master transcriptional regulator of pancreatic stellate cells (PSCs) to reprise in the quiescent state resulting in stromal remodeling [9, 10]. It has also been shown to suppress the expression of several ECM proteins, such as smooth muscle alpha-actin (α-SMA) in breast cancer [11,12,13,14]. H selectively reduced the deposition of ECM in hepatic stellate cells by inhibiting α-SMA and collagen I [15]. H alleviates renal interstitial fibrosis by inhibiting PI3K/Akt signaling and attenuates ECM and EMT by suppressing NF-κB signaling [16]. H has also been shown in clinical trials to modulate the immune microenvironment in PDAC [17]. The effects of the combination of H and P with 5FU-based chemotherapy on modulation of ECM and immune microenvironment in PDAC has not been previously studied.Clinical trial targeting ECM components, including matrix metalloproteinase or hyaluronidase in PDAC have resulted in limited success [18]. The heterogeneity of CAF’s and redundant ECM composition and structure limits the benefit of these therapies, which are very specifically designed against one component or pathway in the ECM. PDAC cells frequently develop resistance mechanisms, such as alterations in integrin expression and ECM remodeling, which reduce the effectiveness of such treatments. In our prior study, we reported the addition of HP to gemcitabine resulted in immune modulation and effects on autophagy [19]. These effects were observed in preclinical models and clinical samples from paired biopsies obtained from patients on a trial [19]. While gemcitabine-based therapy is a standard chemotherapy for PDAC, accumulating evidences suggests that 5-FU-based regimens may have shown superior efficacy [20]. Encouraged by the observed effects of PH with gemcitabine, we planned to assess this combination with 5FU-based therapy. In addition, our objective was to evaluate if the mechanisms involved in potentiation of 5FU-based therapy by H and P were mediated through mechanisms involving modulation of immune cells and ECM proteins in the TME.ResultsParicalcitol and hydroxychloroquine enhances the growth inhibitory effects of 5FU and Oxaliplatin in both murine and human PDAC cellsThe effects of PH in combination with 5FU and Oxaliplatin (oxali) on murine and human PDAC cells were evaluated by dosing the cells with varying concentrations of the drugs and then quantifying the survival rates by cell viability using MTT assay. Human PDAC cell lines MIA PaCa-2 and HPAC, and murine KPC PDAC cells were cultured in the presence of PH, 5FU+Oxali either alone or in combination for 72 h (Fig. 1A–C). The combination treatment was extremely efficient in urging cytotoxicity in all the cell lines. The combination index (CI) values, calculated with PRISM software for dose-effect analysis, designated the presence of a robust synergism between 5FU + Oxali with PH, exclusively at lower and intermediary drug doses compared to chemotherapy alone. We then asked whether this potential combination has effect on long-term survival of PDAC cells using clonogenic assays. Treatment of PH with 5FU (10 µM) and Oxali (20 µM) inhibited colony-forming ability of PDAC cells (Fig. 1D, E). In addition to this, we have also checked if PH sensitizes the combination of 5FU, Oxaliplatin, and Irinotecan in PDAC. To this end, we have performed MTT assay in MIA PaCa-2, HPAC, and murine KPC cells and found that the combination treatment significantly suppressed cell growth compared to chemotherapy alone (Supplementary Fig. 1A–C). This increased suppression suggests that PH may acts as chemosensitzer. Furthermore, clonogenic assay revealed that the combination of 5FU, Oxaliplatin, and Irinotecan resulted in marked reduction in colony formation compared to any other treatment group, indicating substantial impairment in the long-term proliferative capacity (Supplementary Fig. 1D, E).Fig. 1: PH potentiates the growth inhibition of 5FU and Oxaliplatin in PDAC cells.A–C The indicated cancer cell lines were treated with various concentration of 5FU (15 μM) and Oxaliplatin (25 μM), paricalcitol (350 nM), and hydroxychloroquine (25 μM) for 3 days and subjected to MTT assays. Relative percentage cell viability was plotted with respect to DMSO treated cells. D The indicated cancer cell lines were treated with DMSO, PH, 5FU+oxali, and combination (PH + 5FU+Oxali) for 2–4 weeks, and long-term cell survival was measured using clonogenic assays. Representative images are shown. E Colony counts from the clonogenic assay data shown in (D). Data represent the mean ± standard error of three biological replicates. Statistical significance was assessed using two-way ANOVA. ns not significant, *p