Research progress and targeting strategies of tumor-associated extracellular matrix

preface

The development of cancer immunotherapy, especially immune checkpoint blockade therapy, has made major breakthroughs

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

ECM is a non-cellular three-dimensional macromolecular network consisting of collagen, proteoglycans (PGs)/glycosaminoglycans (GAGs), elastin, fibronectin (FN), laminin, and several other glycoproteins

For many years, ECM was considered an inert cell scaffold that provided only structure

Numerous studies have shown that tumor-associated ECMs are involved in promoting the growth, invasion, metastasis, and angiogenesis of tumor cells, as well as resisting cell death and drug proliferation

Extracellular matrix and tumors

The extracellular matrix is a complex network of extracellularly secreted macromolecules such as collagen, enzymes, and glycoproteins whose primary functions involve structural scaffolding and biochemical support

The basement membrane consists of collagen, laminins, PGs, and FNs, located at the interface between parenchymal tissue and connective tissue, providing an anchored flake layer for parenchymal cells to hold them together and prevent them from tearing

Under physiological conditions, IM is a loose ECM consisting of collagen I and III, elastin fiber and glycoprotein, deep into BM

In addition, the lysysyl oxidase (LOX) family catalyzes the formation

The main component of the extracellular matrix

collagen

Collagen is one of the main components of ECM, and together with matrix glycoproteins such as FN, laminin, elastin and versican, it is involved in the structural formation

Integrins are the main receptors of collagen, widely expressed and promote cell migration, and may be a key pathway for tumor angiogenesis, chemotherapy resistance, and metastasis

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

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

Proteoglycans

Proteoglycans, as components of ECM, play a key role

Versican, a member of the hyalectan family of macrochondroitin sulfate PGs (CSPG), has been shown to be overexpressed in many cancers

Biglycan (BGN) is a member of small leucine-rich PGs (SLRPG) that are overexpressed and secreted in a variety of cancers, linked to

In addition to VCAN and BGN, heparan sulfate protein polysaccharides (HSPG) also play a multifunctional role in inflammation, such as regulating multiple steps of the leukocyte recruitment cascade, activating lymphocytes, and inducing phenotype maturation
of immature dendritic cells (DCs) in mice.

Glycosaminoglycans

Hyaluronic acid is a simple linear non-sulfated GAG consisting of repeating units of N-acetyl glucosamine (GlcNAc) and glucuronic acid (GlcUA) that accumulate in a variety of human solid tumors
.
The biological activity of HA depends on its molecular weight and the receptors that interact with it, including CD44, lymphoendothelial receptors (LYVE-1) and HA endocytosis receptors (HARE), which maintain internal environment stability in normal tissues and inhibit cell proliferation and migration
.
High molecular weight HA can be lysed into low molecular weight (LMW) polymers from 7 to 200 kDa by hyaluronidase and free radicals, which promote inflammation, immune cell recruitment, and epithelial cell migration
.

The components mentioned above are only a small fraction of the thousands of
ECM components.
They not only perform their respective functions, but also interact, participating in dynamic changes
in ECM and immune responses.

The extracellular matrix modulates the mechanism of action of tumor immunity

A growing body of research suggests that the remodeling of ECM plays an important role
in the inflammatory and immune environments in which tumors form.
The remodeling, structural plasticity and mechanical force of the ECM cytoskeleton are key factors in the transport, activation and formation of immune synapses
.

The rigid extracellular matrix inhibits cancer cell death and reduces the release of antigens

The tumor's extracellular matrix is about 1.
5 times harder than the surrounding normal tissue, and by applying physical force to the ECM that hardens on the host tissue, the tumor can enhance cell ECM adhesion and break the cell-to-cell contact, leading to its growth and survival
.
Collagen crosslinking is induced in rigid ECM, which enhances phosphatidylinositol 3-kinase (PI3K) activity, thereby improving the viability
of cancer cells.
The survival of cancer cells is also affected by the release of MMPs by cells, which degrade multiple components of ECM and interact with integrins, thereby promoting the activation
of intracellular signal STAT3.
In addition, ECM is 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 hardness of ECM is also an obstacle
to the effective uptake of drugs or their delivery to the area within the tumor.
The enhancement of the survival potential of tumor cells also reduces cell death and the release of cancer cell antigens, as the first and critical step in initiating anti-cancer immunity, the reduction of cancer cell antigen release will weaken cancer immunity
.

The extracellular matrix interferes with tumor antigen presentation

The functional basis of the ICIs response is the immunogenicity of the tumor, which is mainly determined
by the tumor antigen and antigen presentation efficiency.
APCs, including macrophages, DC, and B cells, are responsible for presenting antigens and triggering immunity
through different mechanisms.
DC is the outpost of the immune system, however, only mature DC cells are able to induce anti-tumor immunity, while antigens presented by immature DC cells may lead to immune tolerance and cannot induce T cell responses
.

It is important to note that the ultimate fate of DC function is determined by signals from the microenvironment, while ECM components may induce DC phenotypes
with low immunogenicity.
Studies of the interaction of medullary DC with laminin in mice have shown that mouse ovarian tumors produce multiple laminins, and the DC cultured on these laminins upregulates the AKT and MEK signaling pathways and reduces immune capacity, leading to tumor growth
.

In addition to HS, HA can also regulate the maturity
of DC in a TLR4-dependent manner.
It was found that DC exposure 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 use VACN to form a temporary matrix
.
HA-VACN interactions are important
for the recruitment of inflammatory cells.

The extracellular matrix affects the initiation and activation of effector T cells

In general, the original T cells are located within the lymph nodes (LNs), meet and are activated
by the antigen-loaded DC.
In LNs, various subpopulations of stromal cells form dense 3D cell networks, which provides an opportunity for naïve T cells to interact with antigens presented by DC, triggering an immune response
.

In LNs, immunity or tolerance induction can affect the expression
of lamins α4 and α5 in all stromal cells (SC).
In immune and inflammatory reactions, laminin alpha5 is upregulated; In contrast, α4 increased
in tolerance induction.
Functionally, laminin 411 and laminin 511 act as co-inhibition and co-stimulation ligands of CD4+ T cells, respectively, and laminin 411 inhibits activation of CD4+ T cells and polarization of Th1, Th2 and Th17, but promotes the induction
of Treg polarization.
Laminin 511 is recognized by CD4+ T cells by α6 integrin and α-dystroglycan to inhibit T cell activation, proliferation, and differentiation
.

The extracellular matrix regulates the migration of T cells

Effector T cells from LNs to tumor sites are critical for the density and diversity of tumor-infiltrating T cells, which is closely related
to the prognosis and efficacy of cancer immunotherapy.
T cell transport processes are highly dynamic and controlled by a complex set of mechanisms involving complex interactions
between T cells and endothelial cells (ECs).

The transport of T cells is also highly dependent on the microenvironment
.
T cells utilize porous three-dimensional ECM as scaffolds
for integrin-dependent and receptor-independent amoebic exercises.
Laminin can act as ligands to bind to immune cell membrane receptors (primarily integrins) and initiate integrin-mediated signaling
.

Rigid ECM may act as a physical barrier for T cells to infiltrate tumors and affect the preferential localization
of T cells.
For example, in pancreatic ductal adenocarcinoma (PDAC) and lung cancer models, matrix density and structure induce T cell localization and migration to the tumor stromal rather than the tumor cell nest
.

In addition to rigidity, certain ECM components can also play a role
in regulating the movement of T cells.
Dense collagen-rich ECM has a direct and indirect effect on the infiltration and function of
T cells.
In collagen-rich ECM, CD8+ T cells move faster and last longer
.

The extracellular matrix interferes with T cell recognition and killing of cancer cells

In various cancers, collagen fibers are thicker and tighter wrapped around
the nest of cancer cells than the tumor stromal.
Stiff ECM acts as a spatial barrier around tumor cells, limiting the accessibility of CD8+ T cells and thus interfering with recognition
.

Spatial analysis of cancers showed that cancers with excessive ECM deposition were resistant
to immune checkpoint suppression.
Collagen density reduces the proliferation and tumoricidal activity
of tumor invasive T cells.
Full transcriptome analysis of 3D cultured T cells showed downregulation of the high-density matrix-induced cytotoxic activity marker (CD101) and upregulation
of the Treg marker (CIP2A).

In addition, the expression of PD-L1 in tumor cells plays an important role
in evading the "kill" step.
Rigid substrates enhance PD-L1 expression in lung cancer cells through actin-dependent mechanisms, suggesting that rigidity as a tumor environment upregulates PD-L1 expression and leads to immune system escape and tumor growth
.

Targeted strategies for tumor-associated extracellular matrices

Tumor-associated ECMs 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 regulating fibroblast function
.
Currently, several joint studies with ICI are in the clinical stage
.

Direct targeting

Some studies suggest that PG activity modulators may be a new approach
in the field of cancer immunotherapy.
As preclinical studies have shown, VCAN fragments of damage-related molecular patterns versikine contribute to immune sensing in myeloma and enhance T cell activation immunotherapy
.
Non-glycinated endoglycan polypeptides (ESM-1) inhibit tumor growth
by increasing leukocyte infiltration in vivo and enhancing the innate immune response.
In addition, placental growth factor-2 (PIGF-2) heparin-binding domain (HBD) conjugated immune checkpoint inhibitors exhibit extremely high affinity
for multiple ECM proteins.
Pertuscular injection of PIGF-2-anti-PD-L1 improves retention rates
within tumor tissue.
In addition to its high efficiency, it reduces the systemic toxicity
of the B16F10 melanoma model.

The accumulation of excess HA can lead to an increase in interstitial pressure and impair perfusion and chemotherapy
of the tumor.
In preclinical studies, polyethylene glycolated recombinant human hyaluronidase α (PEGFH20) has been shown to successfully degrade HA in tumors and remodel the tumor matrix, thereby improving perfusion and drug delivery
.
Thus, the recent Phase II HALO-202 clinical trial (NCT 01839487) showed that treatment with PEGFH20 plus gemcitabine and Nab paclitaxel significantly increased PFS in patients with previously untreated metastatic PDAC
.
However, Phase III trials showed that the addition of PEGFH20 to gemcitabine and Nab paclitaxel increased ORR but did not improve OS, which did not support further studies
of PEFH20 in metastatic PDAC.

Hyaluronidase removal of HA not only improves the effectiveness of chemotherapy, but also increases the success rate of immunotherapy, as animal models have demonstrated
.
In addition, hyaluronidase (HAase) increases the permeability of tumor tissue by breaking down HA in tumor ECM, thereby enhancing tumor infiltration of tumor-specific T cells induced by
nano-vaccines.
In the presence of hyaluronidase, both the delivery of nanovaccines and therapeutic monoclonal antibodies is enhanced
.

Indirect targeting

Collagen is the most abundant component of ECM and is secreted primarily by fibroblasts
.
Research to remove fibroblasts has focused on targeting fibroblast populations
that are positive for fibroblast activating protein (FAP).
In mouse melanoma models, removal of FAP-expressing CAF induces immunosuppressive myeloid cytopenia
.
However, phase II trials using the small molecule inhibitor Talabostat to inhibit FAP failed to demonstrate clinical efficacy
in colorectal cancer.

In addition to removing CAF, other approaches focus on targeting downstream cellular responses to influence ECM
.
At present, the research hotspots are mainly matrix binding proteins, integrins and their downstream signaling mechanisms, such as the use of small molecule kinase inhibitors to target ECM-regulated signaling pathways, such as adhesion plaque kinase (FAK) and Rho-associated protein kinase (ROCK
).

brief summary

There is growing evidence that the extracellular matrix plays an extremely important role in tumor immunity, and targeting tumor-associated ECMs has the potential
to improve tumor immunotherapy.
However, the compositional and structural complexity of ECM and the significant intratumoral heterogeneity are not fully understood, which may limit the application
of targeted therapies for ECM.
Fortunately, advances in technologies such as multiplex immunohistochemistry, tissue decellularization, single-cell sequencing, and mass spectrometry are working to solve these problems
.
In the future, strategies to regulate tumor-associated ECMs are expected to yield new approaches to further optimize treatment strategies for tumor immunotherapy and prolong the overall survival of
cancer patients.

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