Lecture 6

University of Nottingham Lecture Notes

Fibroblasts – Hijacking Wound Healing Mechanisms

Presented by: Prof. Jacqui Shields, Translational Medical Sciences

Today's Learning Objectives and Outcomes

  • Understanding the normal functions of fibroblasts

  • Identifying sources of fibroblasts in cancers

  • Exploring fibroblast populations present in tumors

  • Discussing the influences of fibroblasts on cancer cell behavior

  • Introducing the potential for therapeutically targeting fibroblasts

The Tumor Microenvironment (TME)

  • The TME consists of various components:

    • Blood vessels

    • Lymphatic vessels

    • Immune cells

    • Fibroblasts

    • Adipocytes (fat cells)

    • Extracellular matrix (ECM)

  • In some cancers, fibroblasts may constitute up to 90% of tumor mass

Fibroblasts – A Brief History

  • 1858: First description by Rudolf Virchow as "Spin-delzellen des Bindegewebes" meaning "spindle-shaped cells of the connective tissue"

  • 1895: The term “fibroblast” suggested to describe cells producing new connective tissue during healing (Ziegler)

  • 1963: Establishment of 3T3 fibroblast cell line from mouse embryos allowing identification of various properties:

    • Fibroblast growth factors (FGFs)

    • Differentiation into bone, cartilage, and adipose cells (Junker, 2010)

    • Role in ECM production and remodeling

  • 2018: Single-cell RNA sequencing validates cellular heterogeneity

The Irritation Theory

  • Proposed by Virchow in 1858 suggesting that cancer arises from chronic irritation.

    • Mechanisms include:

    • Mechanical irritation

    • Chemical affects

    • Thermal irritation

  • Involvement of inflammatory cells is crucial, linking inflammation and cancer

  • Burrows (1924) emphasized the relationship between epithelial and connective tissues, indicating that cancer may result from an imbalance in these interactions.

The Wound that Doesn’t Heal

  • Dvorak (1986): Introduced the concept that tumors behave like wounds that do not complete the healing process, perpetuating chronic inflammation and abnormal tissue remodeling.

Normal Function of Fibroblasts

  • Functions include:

    • ECM synthesis and remodeling

    • Angiogenesis (formation of blood vessels)

    • Production of growth factors and cytokines (e.g., VEGF, TGFβ, HGF, EGF, IGF, NGF)

    • Cell proliferation and epithelial-mesenchymal transition (EMT)

    • Fibroblasts play critical roles in maintaining tissue integrity across various body tissues.

  • They are classified as non-immune, non-epithelial cells primarily derived from the primitive mesenchyme.

Normal Wound Healing Process

  1. Hemostasis

    • Blood clot formation involves fibrin, fibrinogen, and platelets.

  2. Inflammatory Phase

    • Fibroblasts sense cell loss at the wound site and release chemotactic signals to recruit immune cells.

    • Immune cells eliminate infection and debris.

  3. Proliferative Phase

    • Fibroblasts produce matrix components (collagen, fibronectin, GAGs) that form granulation tissue and promote wound contraction.

    • Epithelial cells migrate to create a new surface.

  4. Remodeling Phase

    • Fibroblasts guide macrophages to enhance phagocytic activity and tissue remodeling processes, ultimately restoring strength and flexibility.

    • Fibroblasts may either become inactive or undergo apoptosis, marking healing completion.

Fibroblast Activation

  • Fibroblasts activate during wounds by transforming into myofibroblasts, influenced by:

    • Soluble factors (e.g., TGFβ)

    • Mechanical factors (e.g., stiffness in the ECM)

  • Major features of activation:

    • Increased migratory and proliferative capabilities

    • Two-step activation process:

    1. Proto-myofibroblast Stage:

      • Characterized by increased secretory and contractile capacities due to stress fiber formation and modified cell surface fibronectin expression (mediated via YAP-TAZ signaling pathways).

    2. Myofibroblast Stage:

      • Expression of alpha-smooth muscle actin (α-SMA) and collagens (I and III), heavily influenced by the local TGF-beta environment (Tomasek et al., 2002).

Fibroblasts in Cancer

Chronic Activation and Tumor Evolution

  • Cancer evolution involves:

    • Tumor initiation occurs with an insult detected leading to epithelial hyperproliferation.

    • Development of a stress-shielded environment conducive to fibroblast recruitment.

    • Quiescent fibroblasts transition to cancer-associated fibroblasts (CAFs), which contribute to ECM remodeling and tumor progression.

  • Key transitional phases:

    • Carcinoma in situ: Activation of fibroblasts possibly driven by epithelial transformations or external insults. CAFs exhibit similarities in gene expression to those in invasive breast cancer.

    • Invasive Carcinoma: CAFs facilitate the breach of the basement membrane through matrix metalloproteinases (MMPs) and enhance communication with tumor epithelial cells.

    • Senescent CAFs: These cells may lose proliferative capacity but secrete pro-inflammatory mediators, perpetuating tumorigenesis.

Fibroblast Populations in Cancer - Origins

  • Major Sources of CAFs:

    • Resting, quiescent tissue resident fibroblasts transform into the CAF phenotype.

  • Studies reveal diverse origins, including:

    • Embryonic fibroblasts during EMT

    • Mesenchymal stem cells (MSCs) recruited from the bone marrow.

    • Differentiation of adipocytes into fibroblasts mediated by tumor-derived factors such as Wnt3a.

Fibroblast Populations in Cancer - Activation

  • Activation of fibroblasts is influenced by numerous signals from the tumor microenvironment, including:

    • Growth factors: TGFβ, PDGF, FGF, EGF, and cytokines (IL-1β, IL-6, LIF)

    • Reacting to mechanical environmental factors such as stiffness and oxidative stress

  • The process leads to activation pathways involving SMAD, NF-kB, JAK-STAT, and MAPK signalling cascades.

fibroblast Populations in Cancer - Heterogeneity

  • CAFs exhibit considerable heterogeneity:

    • They consist of various subpopulations that can influence tumor progression differently.

    • Studies have shown that depleting specific CAF populations (e.g., αSMA+ vs. FAP+) led to different impacts on tumor growth.

  • This emphasizes the necessity of characterizing fibroblast populations accurately to draw meaningful biological conclusions in cancer research.

Influence of Fibroblasts on Cancer Cell Behavior

  • CAFs significantly support tumor cell proliferation directly by releasing:

    • Cytokines, growth factors, and exosomes (which modulate PI3K/mTOR pathways)

  • They also affect the microenvironment to promote proliferation via:

    • Enhancing ECM stiffness
      ot altered metabolism
      offsetting normal nutrient and oxygen distribution.

CAFs as Major Drivers of Invasion

  • CAFs facilitate:

    • Tumor cell invasion and metastasis through multiple mechanisms including:

    • Promoting survival and metastasis by remodeling the ECM

    • Activating other stromal cellular components

    • Establishing a pre-metastatic niche.

CAF-Secreted Factors and Migration

  • Conditioned medium from CAFs has been shown to induce a migratory phenotype in cancer cells, markedly through:

    • Epithelial-to-mesenchymal transition (EMT)

    • Providing mechanical forces to align ECM components conducive to collective cancer cell migration.

CAFs and Tumor Cell Invasion via ROCK Pathways

  • CAF-supported invasive assays highlight:

    • The role of podoplanin-expressing CAFs working through Rho-associated kinases (ROCK) in cancer cell migration.

    • Inhibition of ROCK signaling has demonstrated reduced tumor invasion, while the tumor model response varies based on specific integrins and E-cadherin expressions of the cancer cells.

CAFs Impact on Tumor Cell Metabolism

  • Reverse Warburg Effect:

    • Cancer cells induce a state of aerobic glycolysis in CAFs, resulting in the secretion of lactate and pyruvate, enhancing energy production in cancer cells for ATP synthesis (Pavlides et al., 2009).

Beyond the Cancer Cell

  • CAFs perform numerous supportive roles via angiogenesis, releasing:

    • Vascular endothelial growth factor (VEGF), CXCL12, placental growth factor (PGF), and hepatocyte growth factor (HGF).

  • These factors play pivotal roles in recruiting endothelial progenitor cells to the tumor site.

CAFs and Tumor Immunity

  • CAFs hinder tumor immunity through:

    • Recruitment of immune cells, excluding them, directly and indirectly modulating immune responses.

  • Strategies targeting fibroblast-centric immune modulation are explored in advanced studies.

Why Do We Care?

  • Dense breast tissue is an independent risk factor for breast cancer due to high fibrous tissue content, increasing disease susceptibility by four to six times.

  • CAFs can lead to therapy resistance by modifying responses to chemotherapeutic agents, including increased survival pathways driven by HGF and IL-6.

Therapeutic Targeting of Fibroblasts

  • Targeting CAFs is a promising approach to interrupt their pro-tumor activities, potentially through:

    • Interfering with activation or function

    • Reverting back to a normal quiescent state

    • Depletion of CAF populations to reduce tumorigenicity.

Summary

  • The role of the stroma in modulating tumor immunity

  • Strategies for therapeutic interventions targeting CAFs and their byproducts

  • Essential themes include the hijacking of fibroblasts in cancer comparable to normal physiological wound healing processes, phenotypic and functional heterogeneity of CAFs, and their multi-faceted support of tumor cell activities.