Research progress and targeting strategies of tumor-associated extracellular matrix

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Research progress and targeting strategies of tumor-associated extracellular matrix

Research progress and targeting strategies of tumor-associated extracellular matrix.

Foreword

The development of cancer immunotherapy, especially immune checkpoint blockade therapy, has led to major breakthroughs in cancer treatment.

However, less than one-third of cancer patients are able to achieve significant and durable treatment effects with cancer immunotherapy.

Over the past few decades, we have learned that the tumor microenvironment ( TME ) of chronic inflammation plays a major role in tumor immunosuppression.

As the core member of TME, tumor-associated extracellular matrix ( ECM ) has become a research hotspot in recent years.

A growing body of research suggests that tumor-associated ECM is one of the major barriers to more successful cancer immunotherapy cases.

The ECM is an acellular three-dimensional macromolecular network composed of collagen, proteoglycans ( PGs )/glycosaminoglycans ( GAGs ), elastin, fibronectin ( FN ), laminin, and several other glycoproteins.

Both in normal tissue and in tumors, stromal components and cell adhesion receptors bind to each other to form a complex network in which a variety of cells reside.

For many years, the ECM has been considered an inert cytoskeleton that only provides structure to cells.

However, in the past two decades, more functions affecting the biochemical and biophysical processes of cells have been discovered, and the ECM is regarded as a repository and binding site for bioactive molecules.

Cell surface receptors transmit signals from the ECM to cells to regulate multiple cellular functions such as survival, growth, migration, differentiation, and immunity, which are essential for maintaining normal homeostasis.

Numerous studies have shown that tumor-associated ECM is involved in promoting tumor cell growth, invasion, metastasis, and angiogenesis, as well as resisting cell death and drug proliferation.

Therefore, an in-depth understanding of the relationship between ECM and tumor immune response will help to develop the potential of targeting tumor-associated ECM to improve cancer immunotherapy.

Extracellular matrix and tumor

The extracellular matrix is a complex network of extracellularly secreted macromolecules, such as collagens, enzymes, and glycoproteins , whose primary functions are involved in the structural scaffolding and biochemical support of cells and tissues.

In general, the ECM can be divided into basement membrane ( BM ) and interstitial matrix ( IM ), which support epithelial/endothelial cells, respectively, as well as the underlying stromal compartment and pericellular membrane.

The degradation of the surrounding ECM is an important component of invasive cancer growth, and more importantly, the degradation of the ECM is accompanied by the deposition of different tumor-specific ECMs, resulting in increased density and stiffness.

The basement membrane, composed of collagen, laminin, PGs, and FN, sits at the interface between parenchyma and connective tissue, providing an anchoring lamella for parenchyma cells to hold them together and prevent them from tearing.

In epithelial cancers, the BM acts as a structural barrier to cancer cell invasion, infiltration, and extravasation.

Changes in the BM are frequently observed during the progression of cancer, often cancer cells invade the BM by producing ECM remodeling enzymes such as matrix metalloproteinases , exploiting the natural pores in the BM, or forcibly passing through these pores.

Under physiological conditions, the IM is a loose ECM composed of collagens I and III, elastin fibers, and glycoproteins that penetrate deep into the BM. Fibroblasts, resident immune cells, vasculature, and lymphatic vessels are embedded. However, in some tumors, collagen fibers in the IM are thicker, more organized, and denser due to increased deposition of collagen fibers.

As the cancer progresses, the stromal collagen fibers become more and more neat, especially at the tumor margin, thereby promoting the invasion of cancer cells.

Furthermore, the lysyl oxidase ( LOX ) family catalyzes the formation of collagen cross-links.

In tumors, increased LOX expression leads to excessive cross-linking of collagen, and at the same time, increased collagen deposition leads to increased stiffness, which induces solid stress in the tumor.

In addition to collagen alterations, there is pathological expression of various glycoproteins in the ECM, all of which form a niche that promotes cell migration, adhesion, and metastasis.

The main components of the extracellular matrix

Collagen

Collagen, one of the major components of the ECM, is involved in the structural formation of cancer fibrosis and solid cancers along with matrix glycoproteins such as FN, laminin, elastin, and versican.

Collagen biosynthesis is consistently regulated in multiple ways by fibroblasts, cancer cells, and other stromal cells such as macrophages .

Other substances in the ECM, such as FN, hyaluronic acid ( HA ), laminin, and matrix metalloproteinases ( MMPs ), via integrins, discoid domain receptors ( DDR ), tyrosine kinase receptors, and some signaling The pathway interacts with collagen, thereby affecting the behavior and activity of cancer cells.

Changes in stromal components within cancer ultimately form a mutual feedback loop that affects cancer prognosis, recurrence, and treatment resistance.

Integrins are major receptors for collagen, are widely expressed and promote cell migration, and may be a key pathway for tumor angiogenesis, chemoresistance, and metastasis.

Integrin α11β1 is a stromal cell-specific fibrillar collagen receptor that is overexpressed in cancer-associated fibroblasts ( CAFs ). Collagen crosslinking correlates with stromal α11 expression, and loss of tumor stromal α11 expression causes collagen reorganization and reduced stiffness.

Forced expression of β1 integrin significantly stimulates Src and extracellular signal-regulated kinase ( ERK ) phosphorylation, increases cell stiffness and accelerates cell motility, resulting in low cancer cell elasticity and high metastatic capacity. Integrins also control local activation of the ECM and cell surface TGF-β.

In addition, non-integrin collagen receptors DDRs ( DDR1 and DDR2 ) are expressed on the surface of tumor cells and belong to the receptor tyrosine kinase ( RTK ) family.

DDR exhibits delayed and sustained activation upon interaction with collagen, and DDR collagen signaling plays an important role in cancer proliferation and progression.

In addition to the above-mentioned role of collagen, the relationship between collagen and tumor-associated macrophages ( TAM ) in anti-tumor immunity should not be underestimated.

TAMs are considered to be a major limiting factor in the efficacy of cancer immunotherapy, whereas high-density collagen can guide macrophages to acquire an immunosuppressive phenotype.

Proteoglycan

Proteoglycans, as components of the ECM, play a key role in providing intrinsic signals required to coordinate key events in cancer immune regulation.

PGs are closely related to cancer-related inflammatory processes and regulate key events in innate and adaptive immunity, respectively.

Versican is a member of the hyalectan family of large chondroitin sulfate PGs ( CSPGs) that have been shown to be overexpressed in many cancers.

VCAN releases inflammatory cells from the circulation by binding to TSG-6 and IαI.

In addition, VCAN interacts with inflammatory cells through HA indirectly or directly through receptors such as CD44, PSGL-1, and TLR, and activates signaling pathways to promote the synthesis and secretion of inflammatory cytokines, such as TNF-α, IL-6 and NFκB.

Biglycan ( BGN ), a member of small leucine-rich PGs ( SLRPGs ), is overexpressed and secreted in a variety of cancers and has been implicated in the regulation of immune responses.

However, its oncogenic or tumor-suppressive potential is unclear.

In addition to VCAN and BGN, heparan sulfate proteoglycan ( HSPG ) also has multifunctional roles in inflammation, such as regulating multiple steps of the leukocyte recruitment cascade, activating lymphocytes, and inducing immature dendritic cells in mice ( DC ) phenotypic maturation.

Glycosaminoglycan

Hyaluronic acid, a simple linear non-sulfated GAG composed of repeating units of N-acetylglucosamine ( GlcNAc ) and glucuronic acid ( GlcUA ), accumulates in a variety of human solid tumors.

The biological activity of HA depends on its molecular weight and the receptors it interacts with, including CD44, lymphatic endothelial receptor ( LYVE-1 ) and HA endocytic receptor ( HARE ), maintain homeostasis and inhibit cell proliferation in normal tissues and migration.

High molecular weight HA can be cleaved by hyaluronidase and free radicals into low molecular weight ( LMW ) polymers of 7 to 200 kDa, and these LMW-HAs promote inflammation, immune cell recruitment, and epithelial cell migration.

The components mentioned above are just a few of the thousands of ECM components.

They not only perform their respective functions, but also interact and participate in the dynamic changes of the ECM and immune responses.

The mechanism of action of extracellular matrix in regulating tumor immunity

A growing body of research suggests that ECM remodeling plays an important role in the inflammatory and immune milieu that shape tumors.

ECM cytoskeleton remodeling, structural plasticity, and mechanical forces are key factors in immune synaptic trafficking, activation, and formation.

Research progress and targeting strategies of tumor-associated extracellular matrix

Rigid extracellular matrix inhibits cancer cell death and reduces antigen release

Research progress and targeting strategies of tumor-associated extracellular matrix

The tumor extracellular matrix is approximately 1.5 times stiffer than the surrounding normal tissue, and by exerting physical forces on the hardened ECM on the host tissue, tumors can enhance cellular ECM adhesion and break cell-to-cell contacts, leading to their growth and survival.

Collagen cross-linking is induced in rigid ECM, which enhances phosphatidylinositol 3-kinase ( PI3K ) activity, thereby improving cancer cell viability.

Cancer cell survival is also affected by the release of MMPs from cells that degrade multiple components of the ECM and interact with integrins to promote the activation of intracellular signaling STAT3.

In addition, the ECM is also indirectly involved in the activation of the ERK pathway, which contributes to the proliferation of cancer cells.

In addition to promoting the survival of cancer cells, the stiffness of the ECM is also an obstacle to the efficient uptake or delivery of drugs to intratumoral areas.

The enhanced survival potential of tumor cells also reduces cell death and the release of cancer cell antigens, as the first and key step in initiating anti-cancer immunity, the reduction of cancer cell antigen release will weaken cancer immunity.

Extracellular matrix interferes with tumor antigen presentation

The functional basis of the ICIs response is tumor immunogenicity, which is mainly determined by tumor antigenicity and antigen presentation efficiency.

APCs, including macrophages, DCs, and B cells, are responsible for presenting antigens and triggering immunity through different mechanisms.

DCs are sentinel APCs of the immune system, however, only mature DCs are able to induce antitumor immunity, whereas antigens presented by immature DCs may lead to immune tolerance and fail to induce T cell responses.

Notably, the ultimate fate of DC function is determined by signals from the microenvironment, and ECM components may induce a DC phenotype with low immunogenicity.

Studies of mouse myeloid DCs interacting with laminin have shown that mouse ovarian tumors produce a variety of laminins, and that DCs cultured on these laminins upregulate AKT and MEK signaling pathways and reduce immunity, leading to tumor growth.

In addition to HS, HA can also regulate DC maturation in a TLR4-dependent manner.

It was found that exposure of DCs to HA fragments increased the expression of activation markers such as MHC II, CD80, CD86, and CD40, and promoted DC activation.

In addition, HA can utilize VACN to form a temporary matrix. The HA-VACN interaction is important for the recruitment of inflammatory cells.

Extracellular matrix influences initiation and activation of effector T cells

In general, naive T cells are located within lymph nodes ( LNs ), encounter and become activated with antigen-loaded DCs.

In LNs, various stromal cell subsets form dense 3D cellular networks, which provide an opportunity for naive T cells to interact with antigens presented by DCs to initiate immune responses.

In LNs, immunity or tolerance induction can affect the expression of laminin α4 and α5 in all stromal cells ( SCs ).

Laminin α5 is upregulated in immune and inflammatory responses; conversely, α4 is increased in tolerance induction. Functionally, laminin 411 and laminin 511 act as co-suppressive and co-stimulatory ligands for CD4+ T cells, respectively, and laminin 411 inhibits CD4+ T cell activation and Th1, Th2, and Th17 polarization, but Promotes induction of Treg polarization.

Laminin 511 is recognized by CD4+ T cells via α6 integrin and α-dystroglycan to inhibit T cell activation, proliferation and differentiation.

Extracellular matrix regulates T cell migration

Effector T cells from LNs to tumor sites are critical for the density and diversity of tumor-infiltrating T cells, which are closely related to the prognosis and efficacy of cancer immunotherapy.

The T cell trafficking process is highly dynamic and controlled by a complex set of mechanisms involving complex interactions between T cells and endothelial cells ( ECs ).

The trafficking of T cells is also highly dependent on the microenvironment. T cells utilize porous three-dimensional ECM as a scaffold for integrin-dependent and receptor-independent amoeba motility.

Laminin can act as a ligand to bind to immune cell membrane receptors ( mainly integrins ) and initiate integrin-mediated signaling.

The rigid ECM may act as a physical barrier for T cells to infiltrate the tumor and influence the preferential localization of T cells.

For example, in pancreatic ductal adenocarcinoma ( PDAC ) and lung cancer models, stromal density and structure induce T cell localization and migration into the tumor stroma rather than tumor cell nests.

In addition to rigidity, certain ECM components may also play a role in regulating T cell motility.

Dense collagen-rich ECM has direct and indirect effects on T cell infiltration and function.

In the ECM rich in collagen fibers, CD8+ T cells move faster and more persistently.

Extracellular matrix interferes with T cell recognition and killing of cancer cells

In various cancers, collagen fibers are thicker and more tightly packed around cancer cell nests than the tumor stroma.

Stiff ECM acts as a steric barrier around tumor cells, limiting the accessibility of CD8+ T cells and thus interfering with recognition.

Spatial analysis of cancers suggests that cancers with excess ECM deposition are resistant to immune checkpoint inhibition.

Collagen density reduces proliferation and tumoricidal activity of tumor-infiltrating T cells.

Whole transcriptome analysis of 3D cultured T cells revealed that high-density matrix induced downregulation of a marker of cytotoxic activity ( CD101 ) and upregulation of a marker of Treg ( CIP2A ).

Furthermore, PD-L1 expression in tumor cells plays an important role in evading the “kill” step.

Rigid substrates enhance PD-L1 expression in lung cancer cells through an actin-dependent mechanism, suggesting that rigidity as a tumor environment upregulates PD-L1 expression and leads to immune system escape and tumor growth.

Targeting strategies for tumor-associated extracellular matrix

Tumor-associated ECM can be therapeutically targeted in a variety of ways, including targeting ECM molecules, ECM remodeling enzymes, altering the structure or physical properties of the matrix, or modulating fibroblast function. Currently, there are several joint studies with ICI in clinical stage.

Research progress and targeting strategies of tumor-associated extracellular matrix

Direct targeting

Research progress and targeting strategies of tumor-associated extracellular matrix

Several studies suggest that modulators of PG activity may be a new approach in the field of cancer immunotherapy.

As shown in preclinical studies, the VCAN fragment versikine, a damage-associated molecular pattern, contributes to immune sensing in myeloma and enhances T cell activation for immunotherapy.

Non-glycinated endoglycan polypeptide ( ESM-1 ) inhibits tumor growth by increasing leukocyte infiltration and enhancing innate immune response in vivo.

In addition, placental growth factor-2 ( PIGF-2 ) heparin-binding domain ( HBD )-conjugated immune checkpoint inhibitors exhibit extremely high affinity for a variety of ECM proteins.

Peritumoral injection of PIGF-2-anti-PD-L1 can increase the retention rate in tumor tissue.

In addition to high efficacy, it also reduced systemic toxicity in the B16F10 melanoma model.

The accumulation of excess HA can lead to increased interstitial pressure and impair perfusion and chemotherapy to the tumor.

In preclinical studies, pegylated recombinant human hyaluronidase alpha ( PEGFH20 ) has been shown to successfully degrade HA in tumors and remodel tumor stroma, thereby improving perfusion and drug delivery.

Thus, the recent phase II HALO-202 clinical trial ( NCT 01839487 ) demonstrated that treatment with PEGFH20 plus gemcitabine and Nab-paclitaxel significantly improved PFS in previously untreated patients with metastatic PDAC.

However, the phase III trial showed that addition of PEGFH20 to gemcitabine and Nab-paclitaxel increased ORR but not OS, which does not support further studies of PEGFH20 in metastatic PDAC.

Removal of HA by hyaluronidase can not only improve the efficacy of chemotherapy, but also improve the success rate of immunotherapy, which has been demonstrated in animal models.

Furthermore, hyaluronidase ( HAase ) increases the permeability of tumor tissue by breaking down HA in the tumor ECM, thereby enhancing nanovaccine-induced tumor infiltration of tumor-specific T cells.

In the presence of hyaluronidase, both nanovaccine and therapeutic monoclonal antibody delivery were enhanced.

Indirect targeting

Collagen is the most abundant component of the ECM and is mainly secreted by fibroblasts.

Fibroblast depletion studies have focused on targeting fibroblast-activating protein ( FAP )-positive fibroblast populations. In a mouse melanoma model, depletion of FAP-expressing CAFs induces a decrease in immunosuppressive myeloid cells.

However, a phase II trial of FAP inhibition with the small molecule inhibitor Talabostat failed to demonstrate clinical efficacy in colorectal cancer.

In addition to removing CAFs, other approaches have focused on targeting downstream cellular responses to affect the ECM.

Currently, research hotspots are mainly on matrix-binding proteins, integrins and their downstream signaling mechanisms, such as targeting ECM-regulated signaling pathways using small-molecule kinase inhibitors, such as plaque adhesion kinase ( FAK ) and Rho-associated protein kinase ( ROCK ) .

Summary

Accumulating evidence suggests that the extracellular matrix plays an extremely important role in tumor immunity, and targeting tumor-associated ECM has the potential to improve tumor immunotherapy.

However, the compositional and structural complexity of the ECM and the significant intratumoral heterogeneity are not fully understood, which may limit the application of ECM-targeted therapy.

Fortunately, advances in technologies such as multiplex immunohistochemistry, tissue decellularization techniques, single-cell sequencing, and mass spectrometry are working to address these issues.

In the future, strategies to modulate tumor-associated ECM are expected to generate new approaches to further optimize the therapeutic strategy of tumor immunotherapy and prolong the overall survival of cancer patients.

references:

1. Tumor-Associated Extracellular Matrix: Howto Be a Potential Aide to Anti-tumor Immunotherapy? Front Cell Dev Biol. 2021;9: 739161.

Research progress and targeting strategies of tumor-associated extracellular matrix

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Research progress and targeting strategies of tumor-associated extracellular matrix

Research progress and targeting strategies of tumor-associated extracellular matrix

Foreword

Extracellular matrix and tumor