Novel Targeting Strategies to Reprogram Cancer-Associated Fibroblasts in the Tumor Microenvironment
Ahmed Mostafa is a PhD student in the department Advanced Organ bioengineering and Therapeutics. Promotors are prof.dr. J. Prakash and prof.dr. D. Stamatialis from the Faculty of Science & Technology.
The tumor microenvironment (TME) represents a complex and dynamic landscape pivotal in the progression, metastasis, and response to therapy of cancers. Within this microenvironment, the tumor stroma plays a critical role not only in supporting tumor cells but also in influencing their behavior and fate. The stroma, composed of a diverse array of non-malignant cells including cancer-associated fibroblasts (CAFs), immune cells, endothelial cells, and the extracellular matrix (ECM), is not a mere bystander but an active participant in tumor biology. A key player in the tumor stroma are CAFs, the most abundant cells in many tumor stromas, notably in pancreatic ductal adenocarcinoma (PDAC), are known to undergo a phenotypic transformation in response to signals from tumor cells, namely myofibroblastic CAFs (myCAFs) and inflammatory CAFs (iCAFs). Both phenotypes secrete growth factors, cytokines, and ECM proteins, contributing distinctly to the surrounding pro-tumorigenic microenvironment. Current strategies targeting CAFs highlight a multifaceted approach to cancer therapy, focusing on inhibiting CAF activity, reverting CAFs to a quiescent state, and disrupting ECM production and remodeling. Despite progress, challenges remain, including the heterogeneity of CAF, the need for effective, novel biomarkers, and hurdles of clinical translation. In this thesis, we focused on the inhibition of CAF-mediated pro-tumorigenic functions within the TME by developing CAF-specific targeted drug delivery systems and exploring new therapeutic agents. We identified the selective expression of integrin alpha 5 (ITGA5) and alpha 11 (ITGA11) on these CAF phenotypes and developed polymeric nanoparticle drug delivery systems based on our previously identified AV3 and AXI targeting peptides, respectively.
Initially, Chapter 1 details the thesis introduction, objectives and the chapters outline. In Chapter 2 we explore the complex role of fibroblasts in fibrosis and cancer. Fibroblasts, traditionally recognized for their function in maintaining tissue integrity and facilitating wound healing, are highlighted for their transformation into an aggressive phenotype under pathological conditions. This transformation contributes to the induction of organ fibrosis and supports tumor progression through the secretion of abundant ECM, growth factors, and cytokines. The chapter systematically dissects the biology of fibroblasts, emphasizing their diversity, heterogeneity, and the various targeting strategies employed to modulate their activity in disease states. We further critically analyze various therapeutic modalities aimed at fibroblasts, including nanoparticles and biological agents, proposing them as innovative approaches to counteract fibrosis and tumor progression.
In Chapter 3, we conduct a comprehensive review on PDAC models, providing an in-depth analysis of the current state of research and the challenges in developing effective therapies for PDAC. The chapter emphasizes the importance of understanding the complex TME of PDAC, which plays a significant role in tumor progression, metastasis, and resistance to treatment. The TME's complexity is highlighted as a major hurdle in translating promising laboratory findings into successful clinical therapies. Our review systematically explores various in vitro and in vivo models that researchers have developed to study PDAC, each with its own set of advantages and limitations. It points out that while in vitro models, including 2D cultures, spheroids, organoids, and more advanced 3D and chip-based models, offer controlled environments to study specific aspects of PDAC biology and drug responses, they often lack the complete representation of the in vivo TME. These models, however, are crucial for high-throughput screening of therapeutics and understanding specific mechanisms of action in a controlled setting. In vivo models, particularly genetically engineered mouse models (GEMMs) and patient-derived xenografts (PDXs), are recognized for their ability to more accurately mimic the human PDAC TME, including its dense stroma and immune landscape.
Later, Chapter 4 introduces a novel approach for targeting solid tumors, particularly focusing on overcoming therapeutic resistance associated with the TME. It leverages a dual delivery system comprising electrospun nanofibers loaded with a p21-activated kinase 1 (PAK1) inhibitor, FRAX597, and the chemotherapeutic agent paclitaxel (PTX). The research underscores the pivotal role of CAFs in promoting tumor progression, metastasis, and chemoresistance through ECM remodeling and crosstalk with cancer cells, advocating for targeted disruption of these interactions as a therapeutic strategy. The chapter outlines the fabrication of polycaprolactone-based nanofibers using electrospinning, achieving sustained release of FRAX597 and PTX over 16 days. In vitro models, including stroma-rich 3D heterospheroids, were employed to evaluate the therapeutic efficacy of this dual delivery system. The findings reveal that nanofibers containing both FRAX597 and PTX significantly inhibit tumor growth and viability in these models by more than 90%, compared to nanofibers loaded with either drug alone. This effect is attributed to the reduction of key stromal markers within the TME, such as collagen 1 and α-smooth muscle actin (α-SMA), indicating effective stromal targeting and enhanced chemotherapeutic efficacy.
In Chapter 5, we investigate the significance of ITGA5 in different CAF subtypes as an additional therapeutic target, and the development of a targeted therapeutic strategy using an ITGA5-targeting peptide, AV3. Our findings demonstrate that ITGA5, although overexpressed on both subtypes, is significantly overexpressed in myCAFs compared to iCAFs, both in vivo in a murine KPC PDAC model and in vitro in lab-activated CAF populations. This differential expression of ITGA5 in CAF subtypes underscores its potential as a drug delivery target. In the challenge of developing targeted therapy, AV3-functionalized polymeric nanoparticles were developed. These nanoparticles showed a preferential uptake by both myCAFs and iCAFs in vitro and enhanced tumor accumulation in vivo. Flow cytometry analysis confirmed the preferential targeting of myCAFs and iCAFs by AV3 nanoparticles in the PDAC model, with minimal macrophage accumulation. This preferential targeting is crucial for developing effective therapies that can navigate the complex TME.
In Chapter 6, we extensively investigate the impact of YL109, an aryl hydrocarbon receptor (AhR) agonist, on the TME of PDAC, particularly focusing on its interaction with CAFs. CAFs, including myCAFs and iCAFs, play a crucial role in tumor progression and response to therapy. The study explores the effects of YL109 on these CAFs, using various in vitro and in vivo assays, and assesses its potential as a targeted therapy when encapsulated in AV3 nanoparticles. Key findings include YL109's ability to inhibit myCAF and iCAF activities, affecting pathways like Wnt/β-catenin, and its impact on tumor cell migration and invasion through the suppression of myCAF-mediated paracrine activation of epithelial-mesenchymal transition (EMT). Furthermore, YL109 was found to reduce iCAF-mediated M2 macrophage polarization, indicating its broader influence on the immune landscape within the TME. When combined with chemotherapy, YL109 enhanced the efficacy of standard treatments and sensitized 3D tumor spheroids to chemotherapy. In vivo, YL109 encapsulated in AV3 nanoparticles significantly reduced tumor growth and improved the survival rate in a KPC PDAC model. The study confirms the role of AV3 nanoparticles in delivering YL109 directly to the tumor site, thus enhancing its effectiveness.
In Chapter 7, our study presents an innovative approach to targeting the desmoplastic TME in PDAC, focusing on CAFs, particularly the myCAF subtype. The identification of ITGA11 as a receptor selectively expressed on myCAFs in PDAC tissues underscores its potential as a therapeutic drug delivery target. The development of the ITGA11-binding peptide (AXI) and its functionalization onto polymeric nanoparticles for targeted drug delivery showcases a promising strategy for PDAC therapy. The utilization of the KPC PDAC-bearing murine model to demonstrate the in vivo efficacy of AXI-functionalized nanoparticles (AXI-NPs) in selectively accumulating within myCAFs in the tumor stroma further validates this targeted approach. This specificity could potentially lead to improved therapeutic outcomes by directly affecting the TME components that support tumor growth and resistance to treatment. By demonstrating enhanced accumulation of AXI-NPs in ITGA11-expressing PSCs and myCAFs in vitro and confirming preferential tumor accumulation and myCAF selectivity in vivo through biodistribution studies and flow cytometry analysis, the study provides a solid foundation for the development of targeted therapies aimed at modulating the TME in PDAC.
To investigate the applicability of AXI nanoparticles reported in the previous chapter, we further extended into targeting the TME in PDAC, in Chapter 8, focusing on the inhibition of oncogenic lipid synthesis by myCAFs through the use of ITGA11-targeted nanomedicine loaded with the lipase inhibitor URB602. We address the critical role of the MAGL-LPAR pathway in myCAF-mediated tumor progression and therapy resistance. By employing a variety of experimental techniques, including immunocytochemistry, gene expression analysis, and in vivo murine models, the study presents a multifaceted approach to disrupting myCAF functions and their contribution to PDAC growth and metastasis. Our findings reveal that URB602 effectively downregulates key myCAF markers and genes, impacting their migratory capacity and disrupting LPAR-mediated oncogenic lipid signaling pathways. Furthermore, the encapsulation of URB602 within AXI integrin 11-targeted nanoparticles enhances its efficacy in targeting myCAFs, providing a novel approach to selectively modulate the TME in PDAC. The in vivo studies underscore the enhanced therapeutic potential of combining URB602 with gemcitabine, showing significant reduction in tumor growth and myCAF status in the PDAC model.
In Chapter 9, we present a comprehensive study on the role of iCAFs within the TME and explores the therapeutic potential of FGFR4 inhibition by AZD4547 in modulating iCAF activity. iCAFs are characterized by their secretion of inflammatory cytokines, contributing to TME immunosuppression and metastasis. Key findings include the demonstration that AZD4547 effectively inhibits iCAF activity and their paracrine effects on tumor cells, leading to reduced EMT and decreased metastatic capabilities. Importantly, AZD4547 treatment attenuates iCAF-derived immunosuppressive effects on macrophages, shifting the immune landscape towards a less pro-tumorigenic state. In 3D tumor spheroids and a KPC murine model of PDAC, AZD4547 significantly reduced tumor growth, indicating its potential in targeting the TME to impede cancer progression.
In Chapter 10, we investigated the therapeutic potential of PT100, a dual inhibitor of dipeptidyl peptidase 4 (DPP4) and fibroblast activation protein (FAP), targeting CAFs in the TME of PDAC. Investigating the effects on myCAFs and iCAFs in vitro and in a KPC PDAC murine model, our research underscores PT100's dual inhibitory capacity on CAFs' fibrotic and inflammatory activities. PT100 diminished myCAF-induced contractility and iCAF-mediated cytokine secretion, leading to reduced EMT progression, tumor cell migration, and altered macrophage polarization. In vivo, PT100 significantly reduced myCAF and iCAF populations, M2-like macrophages, and suppressed collagen type 1 and CXCL-12 expression, alongside intratumoral hypoxia and angiogenesis. Our findings highlight PT100's broad inhibitory effects on both myCAF and iCAF activities, underscoring its potential in modulating the TME to attenuate cancer progression. Finally, Chapter 11 summarizes the thesis key results and discusses CAF targeting challenges and shares future perspectives insights on overcoming translational challenges.
Overall, this thesis has made several contributions to the field of the tumor microenvironment. First, several pathways to modulate CAFs including PAK1, AhR, FGFR4/STAT3, MAGL/LPAR and β-Catenin/Wnt are investigated. Many of these pathways have not been investigated earlier in literature in context of CAFs subtypes. Second, different targeting tools were developed to deliver therapeutics to the tumor stroma via local route of administration using nanofibers and via systemic route using nanoparticles.
