?Color cancer (Crc) is the third most common cancer in the world, with an average 5-year overall survival rate (OS) of 60%. Primary Crc can be cured by surgery; however, during the metastatic stage, OOS                                                                                                                                                                                                                                  

?At diagnosis, 20%–30% of patients suffer from synchronous liver metastasis (LM), and 50%–75% of all CRC patients will experience liver metastasis, which is the main cause of death. To improve OS, we need to further understand mcrc and identify other therapeutic targets. Although preclinical studies focus on primary tumors (Ptu) and metastasis processes, the clinical challenge lies in the optimal treatment of metastatic patients. In addition, there is no commonly used immunoactive animal model to reproduce Crc and metastasis growth, making us challenging to understand metastaticity.

?Tumor cells grow in complex microenvironment, involving extracellular and intracellular signals that drive tumor growth, invasion and metastasis. Changes in tissue mechanical properties can both precede disease development and promote disease development. Tumor "hardness" is related to the prognosis of several tumor types. Previous studies have shown that disease progression in several solid tumors, including breast cancer and pancreatic ductal adenocarcinoma, is related to their elastic modulus.

?Tissue stiffness is the degree to which tissue resists deformation, which is described by the relationship between stress and strain. At the tissue level, stiffness is controlled by the cytoskeleton and extracellular matrix (Ecm). Fibrous collagen is the most abundant scaffolding protein and has a significant impact on tissue stiffness. Abnormal collagen rich in collagen i (col-i) remodeling has been identified as the main cause of tissue sclerosis during cancer progression. As ec

The main source of m    , cancer-associated fibroblasts (CAF) further change the tumor mechanical environment by expressing lysyl oxidase (LOX). Lysyl oxidase is an amine oxidase that can initiate the covalent intra- and intermolecular cross-linking process of collagen. In experimental models, inhibiting stromal sclerosis through                                                                                                                                                                                                            

?Tumor microenvironment also plays a key role in providing blood supply to tumors. The use of monoclonal antibodies, vegf traps or multi-target tyrosine kinase inhibitors targeting vascular endothelial growth factor (vegf) signaling pathways to inhibit angiogenesis. Currently, insufficient efficacy and drug resistance limit the benefits of anti-angiogenic therapies. In addition, endothelial cells (ec) regulate their barrier capacity and growth according to the mechanical environment. Specifically, hard matrix promotes ec   

Proliferation and may lead to defects in vascular integrity, which suggests that the mechanical environment may affect the behavior of ec       in tumors, thereby affecting the efficacy of anti-vegf. This study aims to clarify whether metastatic angiogenesis is affected by the mechanical microenvironment, which can be regulated by metastasis-related fibroblast (MAF) activation in mcrc, and whether this activation can be changed to improve the efficacy of anti-angiogenic therapy.

1. Maf in crc highly activated

? Significantly higher expression of asma, p-mlc2 and col-i        remodeling characteristics of maf                                                                                                         

?Next, primary fibroblasts were isolated from ptu(caf) and lm(maf) of the patients. Maf    showed a significant upregulation of asma                                                                                                                                                                                                                                    �

2. Advanced Crc Patients’ ECM Reconstructive and Sclerotic Characteristics

?To identify gene expression characteristics related to liver metastasis, we performed RNA sequencing (RNA-seq) of patients with Crc                                                                                                                                                                                                                                              �

?We identified 3,721 differentially expressed genes. Go identified myofibroblasts/ECM remodeling characteristics upregulated in MaF. Kegg identified ECM-receptor interactions and Fa-character characteristics and Pid identified integrin signals (Figure h). In addition, gene set enrichment analysis (GSA) revealed strong enrichment of actin-mediated cell contraction, Fa-character, myogenesis and angiogenesis characteristics of differentially expressed genes (Figure i-m).

3. Mafs increase the hardness of the microenvironment and support angiogenesis

?We performed gel remodeling analysis and observed that maf showed a more dense and complex f-actin network (Figure    a), and the gel was contracted to a greater extent than caf (Figure b). In fresh and cryopreserved tissues, we observed a significant increase in matrix hardness at lm (Figure c-d). Matrix hardness was associated with the expression of col-i, asma and p-mlc2 in samples from the same patient (Figure e-g).

?Matrix sclerosis regulates ec       Proliferation, angiogenesis, vascular growth and branching. To study this link, we evaluated the vasculature in lm     and observed the correlation between matrix hardness and vascular area of ​​cd31 (Figure h). When cultured in the gel, fibroblasts were allowed to remodel the matrix, and the degree of germination induction of ec           The degree of germination induces ec    (Figure i-k). Therefore, maf    supports angiogenesis of cytokines accompanied by local     Em                                                                                                                                                         �

4. Renin-angiotensin system inhibits target fibroblasts

?Analysis of freshly isolated maf and hepatogenic fibroblasts qpcr showed that all key components of the ras system (the expression of angiotensin [agt], renin [ren], angiotensin convertase [ace] and at-1[agt1] was significantly increased). In addition, the levels of ras and agt1 were significantly higher than those of cafs (

Figure l-o). To characterize the effect of ras targeting on maf function, we conducted a gel contraction test after treatment with losartan (an At-1 blocker) or captopril (an ACE inhibitor). Both significantly reduced both at low concentrations (1 mm of losartan and 5 mm of captopril) and at high concentrations (10 mm of losartan and 50 mm of captopril).

5, ras    Inhibiting and reducing the hardness of the transfer matrix and reshaping the microenvironment

?Tissue stiffness of patients treated with anti-ras drugs was significantly reduced compared with those of the hypertension-free group and non-ras-treatment group 2 (Figures a-b). Further evaluation (by staining of col-1, asma and pmmlc2 in the same patient group) could explain the difference in metastatic stiffness by downregulating maf-activation. Although hypertension was associated with an increase in pmmlc2 staining, we did not observe the effects on asma and col-i (Figure c-h). In all groups,

Anti-Ras treatment showed a significant reduction in maf activation and ecm deposition (Figure c-h). The effect of anti-Ras drugs was independent of specific RAS inhibitory treatment. A positive correlation between metastatic stiffness (different conditions ± hypertension ± anti-Ras drugs) and col-i, asma and p-mlc2 expression was observed (Figure i-k), indicating that maf activation levels contributed to tissue stiffness at LM. In summary, patients receiving anti-Ras treatment showed low myofibroblasts/Ecm      characteristics, which explained the decrease in metastatic sclerosis.

6, at1r   Signal transduction mediated through rhoa         Maf     

?Next, the authors want to determine how ras                                                                                                                                                                                                                                                       �

Reduced activity     rhoa                                                                                                                                                                                                                                                       

7, ras   Inhibition increases the anti-angiogenic effect of bevacizumab

?Matrix hardness was not affected by bevacizumab alone (Figure a-b). In the bev- group, anti-ras treatment resulted in a decrease in tissue hardness compared with non-ras-treated hypertensive patients and patients without hypertension (Figure a,c). Similarly, the same reduction in matrix hardness was observed in the bev group (all p<0.001) in the bev group (Figure a,d). In addition, the reduction in hardness in the anti-ras treatment group was not related to specific treatments. Bevacizumab alone did not affect co

l-i,asma    and        p-mlc2. To evaluate the combined effect of antiangiogenic therapy (bevacizumab treatment) and ras inhibition on the vasculature, we measured the vascular density in a large group of      Crc     LM                                                                                                                                                                                                         �

8. Reduced angiogenesis inhibition by ras and ecology

?    ec    Proliferation increases with increasing hardness, vegf        The proliferation further induced, and at low hardness, vegf     the effect of the highest (Figure a-c). In addition, we analyzed the presence or absence of vegf                                                                                                                                                                                                              �

?To understand how the combination of anti-ras and bevacizumab affects ec in metastases, we quantified their effects on ec proliferation. Bevacizumab alone resulted in ec proliferation reduction 56.1%±11.0%. Since metastatic stiffness is associated with ec proliferation within metastases independent of treatment, we evaluated whether ras                                                                                                                                                                                                              �

9, ras       Anti-angiogenic inhibition improves vascular integrity

? Interestingly, both vegf and matrix sclerosis effectively lead to vascular permeability. To further characterize the effect of anti-ras drugs on vascular integrity, we analyzed their effects on ec-connection stability by immunostaining the tight junction protein zo-1 (closed small band 1). Although bevacizumab treatment was suggested to lead to the elimination of immature blood vessels, we observed that bevacizumab was not changed on zo-1 tight junctions (Figure a-b). Consistent with previous findings in other disease models, that increasing matrix stiffness enhances endothelial permeability, we also observed

There is a negative correlation between the coverage and tissue hardness of crc     lm                                                                                                                                                                                                                                                  

?      klf2                                                                                                                                                                                                                                                       

10. Endothelial cells yap/taz are the center regulated by lm       Matrix hardness and vegf

?Then we analyzed the activation status of ec                                                                                                                                                                                                                                                         �

Egf                                                                                                                                                                                                                                                              

?Research advantages:

1 The tumor is affected by the mechanical properties of its microenvironment. Using patient samples and atomic force microscopy, we found that the tissue hardness of liver metastases is higher than that of primary colorectal tumors. Highly activated metastases-associated fibroblasts increase the hardness of tissues, thereby enhancing the resistance to angiogenesis and anti-angiogenesis treatment.

2 Drugs targeting the renin-angiotensin system are usually used to treat hypertension and can inhibit fibroblast contraction and extracellular matrix deposition, thereby reducing liver metastasis and increasing the anti-angiogenic effect of bevacizumab.

3 Patients treated with bevacizumab have prolonged survival time when they are treated with renin-angiotensin inhibitors, which highlights the importance of the mechanical microenvironment that regulates the treatment regimen.

?Research limitations:

1. For patients with colorectal and lung cancer treated with bevacizumab, bevacizumab treatment itself can induce hypertension. In this study, no distinction was made between hypertension not related to bevacizumab and hypertension caused by bevacizumab.
Chapter completed!