Underlying mechanisms and drug intervention strategies for the tumour microenvironment

Generally, the TME is mainly composed of immune cells, vascular cells, fibroblasts, etc. Recruiting the above cells to the primary cancer site would construct a special TME and provide soluble paracrine signals to promote cancer progression (Fig. 1).

Before cancer cell metastasis, primary focal cancer cells can secrete a variety of cytokines in remote organs, affecting and changing the formation of the organ metastasis microenvironment (pre-metastatic niche) (Fig. 1). It is worth noting that all events that adjust the formation of the niche before metastasis are deemed to occur before the cancer cells reach the niche. Cancer cells prepare and form pre-metastatic niches in distal organs, which refer to the arrangement of pre-metastatic signals, including exosomes, growth factors, cytokines, etc., to regulate their position before cancer metastasis.

Cancer stem cells and cancer cells exudate from the primary focus, infiltrate into the extracellular matrix, and promote angiogenesis or cell infiltration into the circulatory system, thus evading the host’s defence mechanism through chemotaxis, causing it to migrate to specific vascular sites, adhere, and then exudate blood vessels and return to a specific environment until metastatic foci are formed (Fig. 1). Metastatic cancer cells usually reside in distal tissues and organs in a dormant state. The mechanism of controlling metastatic dormancy includes the regulation of the expression of genes in disseminated cancer cells (DTCs), including genetic and/or epigenetic control, as well as the regulation mechanism of the TME [66]. For example, the niche after liver metastasis, which develops after cancer cells enter the liver, can be divided into four key stages (i) microvascular, (ii) preangiogenesis, (iii) angiogenesis and (iv) growth stages [67].

The TME is also a protective barrier for cancers. The interaction between cancer cells and the TME enables some cancer cells to escape apoptosis and develop drug resistance. Microenvironment-mediated drug resistance may be caused by the adhesion of cancer cells to interstitial fibroblasts or extracellular matrix components, or by soluble factors secreted by stromal cells, or mediated by the immune response [77]. For example, stromal cells from lymph nodes promote resistance to 5-fluorouracil and oxaliplatin through SDF1/CXCR4-dependent mechanisms [78]. Studies on melanoma have also confirmed that CAFs promote the metastasis and drug resistance of melanoma cells by increasing the expression of MMP1 and MMP2 [79]. Gemcitabine and 5-fluorouracil can activate MDSCs, and induce the production of IL-1β, which induces the Th17 response and weakens the anticancer effect [80]. Therefore, taking the TME as the target may destroy the interaction between the TME and cancer cells and make follow-up treatment more effective.

The liquid TME is mainly formed by the interaction between haematopoietic cancer cells and stromal cells, such as B-cell lymphoma, including follicular lymphoma (FL), mantle cell lymphoma (MCL), chronic lymphocytic leukaemia (CLL), classical Hodgkin’s lymphoma (CHL) and mucosa-associated lymphoid tissue lymphoma (MALT), providing striking examples of a pivotal interaction of haematopoietic cancer cells with stromal cells [81]. The important components of its microenvironment are monocyte-derived milk cells (NLCs), mesenchymal stromal cells, T cells and NK cells, which communicate with cancer cells through a complex network of adhesion molecules, chemokine receptors, cancer necrosis factor (TNF) family members and soluble factors [82]. Unlike solid cancers, some of these cell types and precursors were already present in secondary lymphoid organs before the onset of lymphoma [83]. For example, the specialized fibroblast reticular cells (FRCs) that make up the trunk of SLOs are essential for organ development and the division of T and B cell regions and participate in the acquired immune response [84]. There is a great difference between liquid cancers and solid cancers, and the mechanisms of their microenvironment and components are very different. For example, there is a detailed understanding of TAMs in solid cancers, but the mechanism of TAMs in liquid cancers is not well understood [85]. Through the study of the solid TME, we can provide some new ideas for the study of the liquid TME.

Chemotherapeutic drugs, also known as cytotoxic drugs, usually have anti-cancer effects by acting on key cellular biological events necessary for cancer cell survival and proliferation. Their effects on various components in the TME have become a research point (Fig. 2).

Chemotherapy can activate the local immune state, which regulates the anti-cancer T cell response by enhancing the effector T cell response, disrupting the immunosuppressive pathway and increasing cancer antigenicity [86‐88]. By comparing the therapeutic effects of 27 patients with osteosarcoma before and after treatment, neoadjuvant chemotherapy was related to an the increase in the density of CD8⁺ T cells, CD3⁺ T cells, Ki67⁺CD8⁺ T cells and PD-L1⁺ immune cells, and the myeloid suppressor cells of HLA-DR-CD33⁺ decreased significantly after treatment [89]. Oxaliplatin (OXP) alone can eliminate immunosuppressive cells, suppress cancer growth and induce an anti-cancer immunostimulatory microenvironment. For example, for the model of the abdominal metastasis of colon cancer, the administration of OXP can increase cancer infiltration and activation of CD8⁺ T cells, reduce cancer CD11b⁺F4/80^high macrophages, and reduce spleen MDSCs, thus affecting the cancer immune microenvironment [125].

Through preclinical studies and phase I clinical trials of four patients with advanced solid cancers (thyroid cancer, colon cancer, pancreatic cancer and melanoma), Winterhoff et al. found that PG545 suppresses growth factor-mediated cell invasion; reduces the phosphorylation of AKT, EGFR and ERK induced by HB-EGF; and significantly abates the cancer burden, which is strengthened when combined with carboplatin in the SKOV-3 model or paclitaxel in the A2780 model [90]. In a mouse model of pancreatic ductal adenocarcinoma (PDAC), gemcitabine (GEM) or paclitaxel (PTX) could inhibit EMT, to reduce the frequency of CTCs and the logarithm of CTCs in circulating cancer cells, thus reducing cancer metastasis [91].

There is no doubt about the status of chemotherapy as the earliest treatment for cancer. In the model of brain metastasis of breast cancer, fludarabine is highly selective for cells with low expression of X-inactivated specific transcript (XIST), which significantly inhibits the growth of brain cancer cells, delays the occurrence of brain metastasis, and has no obvious toxicity [92]. Chemotherapy has its own advantages, but patients have poor tolerance and strong drug resistance. It may affect the TME to promote the spread of cancer cells to secondary sites [93]. Therefore, how to reduce the side effects of chemotherapy in microenvironment treatment in clinical practice may be of great concern.

The TME is complex and diverse, in which various components and characteristics are closely related to cancer occurrence and development, and thus, targeted regulation of components or signalling pathways in the TME has become the key to suppressing cancer proliferation and invasion (Fig. 3). For instance, Shao et al. confirmed that rapamycin-mediated autophagy can result in a reduction in the expression of Bcl-2 and survivin and an increase in the expression of Smac in TAMs. The upregulation of TAM autophagy inhibits the propagation of colon cancer cells, induces apoptosis, and changes the expression of radiosensitivity-related proteins [94].

The same drug may have an effect on different targets in the microenvironment of different types of cancers. For example, in a mouse model of human hepatocellular carcinoma xenotransplantation, apatinib can effectively reduce cancer angiogenesis, inhibit cancer growth and prolong animal survival [95]. In an osteosarcoma model, apatinib suppresses the invasion and migration of cancer cells and the expression of PD-L1 by targeting STAT3 to inhibit the EMT. Interestingly, it can also block the PI3K/AKT and VEGFR2/RAF/MEK/ERK signalling pathways in cholangiocarcinoma cells, thus affecting VEGF-mediated cell proliferation and invasion [126]. This suggests that the existing targeted drugs may have greater potential and are worthy of our study.

Different targets in the microenvironment of the same cancer type are often affected by different drugs to inhibit cancer proliferation and metastasis. In experimental mouse models of breast cancer, WRG-28 inhibits cancer invasion and migration by targeting DDR2, reduces the supporting effect of the matrix on cancer, and thus inhibits the colonization of metastatic breast cancer cells in the lungs [127]. Lee et al. screened 51 drugs that are in clinical trials or approved by the FDA and found that bortezomib (BTZ) and phenobarbital (PST) can reduce the survival rate of CAFs by inducing caspase-3-mediated apoptosis and inhibit the proliferation of cancer cells in a breast cancer mouse transplantation model [96]. In addition, Uriesalgo et al. showed that targeting apelin with apelin inhibitors can inhibit angiogenesis and growth in breast and lung cancer models without increasing TME hypoxia, improve vascular function, and reduce the infiltration of polymorphonuclear myeloid derived suppressor cells [97]. Therefore, the synergistic effect of different targeted drugs on the TME can be considered for the prevention and treatment of cancer.

Therapeutic and targeted delivery at the cancer site can be achieved by modifying exosomes with corresponding targeting ligands, for example, in mouse models of breast and ovarian cancer, DOX enhances its targeting through exosomes and effectively inhibits cancer progression [128]. The discovery of an antibody-functionalized exosome-targeting delivery system may be a new human cancer drug delivery system. In vivo experiments have shown that the A33 antibody-functionalized exocrine targeted delivery of doxorubicin can inhibit the growth of colorectal cancers, prolong the survival time of mice and reduce cardiotoxicity [129].

Similarly, for cisplatin-resistant non-small-cell lung cancer, dasatinib inhibited the self-renewal ability of H460R and A549R cells and reduced the M2 polarization of TAMS [130]. Takigawa et al. suggested that regorafenib affects the interaction between MSCs and cancer cells by targeting the TME, thus inhibiting the proliferation and migration of colon cancers in mice [98]. In a randomized, placebo-controlled phase II clinical trial, tasquiimod, a new oral targeted therapy for the TME, increased progression-free survival (PFS) in prostate cancer patients with (MCRPC) metastatic castration resistance [131]. Targeted therapy is a part of precision therapy, but for some cancers, its curative effect is not obvious, and there are some limitations and side effects. Therefore, while exploring potential targets and targeted drugs, it may be more important to apply the existing research results to the clinical setting and investigate how to better cooperate with other treatment methods to enhance the therapeutic effect.

Cancer immunotherapy is a type of immune function that stimulates or removes immunosuppression as a cancer treatment strategy to effectively inhibit cancer development. It produces immune memory, effectively restrains the resistance of malignant cancer to prevent the proliferation of cancer recurrence, restarts and maintains cancer-immune circulation, and restores the body’s normal anticancer immune responses. The goal of cancer immunotherapy is to initiate a self-sustaining cancer immune cycle that can self-amplify and spread while minimizing the self-inflammation associated with treatment [132] (Fig. 4).

Immunotherapy has a unique mechanism of action on stromal cells. Clinically, cytotoxic T lymphocyte associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) are two checkpoints that can be successfully targeted [99]. Zhang et al. developed a new generation of anti-CTLA-4 antibodies. Preclinical studies have proven that they selectively consume regulatory T cells in the TME to enhance antibody-dependent cell-mediated cytotoxicity/phagocytosis (ADCC/ADCP) and reduce immunotherapy-related adverse events (IRAEs) [100]. The preservation of CTLA-4 checkpoints may be more effective in removing Tregs from the TME, thus improving the efficacy [101].

On the other hand, anti-PD-L1/PD-1 antibodies can restore T cell immunity by interfering with the PD-L1/PD-1 pathway, which leads to lasting remission in some cancer patients [102]. Tauriello et al. found the inhibition of liver metastasis of colon cancer by transforming growth factor-β in mice, thus releasing cytotoxic T cells to respond to cancer cells to prevent metastasis, and thus, the use of TGF-β inhibitors to achieve immuno-osmosis is sufficient to increase the sensitivity to anti-PD-1/PD-L1 checkpoint therapy [103]. It is not surprising that the combination of TGF-β inhibitors and anti-PD-L1 antibodies can reduce the TGF-β signal in stromal cells, promote T cell infiltration to the cancer centre, and stimulate anti-cancer immunity and cancer regression [133]. In the preclinical model of prostate cancer, the inhibition of CXCR4 by plerixafor can reduce invasiveness in vitro, which in turn reduces cancer spread and related angiogenesis [134].

In addition, Georgoudaki et al. found that in breast and colon cancer as well as melanoma models, immunotherapy with “macrophage receptor with collagen structure” (MARCO) can prevent cancer proliferation and migration and improve the immunogenicity of the TME. E-programming of macrophages in the TME with monoclonal antibodies is a feasible method for cancer immunotherapy [135]. Marsh et al. found that embelin can inhibit the growth of pancreatic cancer in KrasG12D mice by increasing the infiltration of Th1 cells, NK cells, CTLs, γδT cells and NKT cells, and reducing the infiltration of Th17, PMN-MDSC, and IL-8- and IL-6-positive immune cells, thus regulating the cancer-immune microenvironment [136]. Jeffery et al. proved for the first time that anti-c-FMS antibody affects the establishment of breast cancer cells in bone through a mouse model of breast cancer [137].

Immunotherapy is changing the treatment of solid cancers, and current clinical work is focused on developing immunotherapy combinations to transform non-responders into responders, deepen their responses, and overcome their drug resistance. Therefore, the identification of immune markers can predict the response potential of immunotherapy and determine the best combination of immunotherapy for specific patients to carry out effective immunotherapy for cancer patients [138]. For example, human epidermal growth factor receptor-2⁺ (HER-2⁺) breast cancer and TNBC are more likely to have interstitial infiltrating immune cells (TILs), than luminal breast cancer, and there is a linear relationship between the TILs content and clinical results. The possibility of expressing programmed death ligand-1 (PD-L1) in the TME is also higher than that in luminal breast cancer [139‐141].

Progression-free survival and overall survival were longer in stage III NSCLC patients undergoing chemoradiotherapy when the density of CD8+ cancer-infiltrating lymphocytes in the pre-treated biopsy specimens was higher than that in patients with low CD8+ cancer infiltrating lymphocyte density [142]. This is through the immune mechanism to improve disease control, and thus, the baseline cancer-infiltrating lymphocyte status can be used as a predictive biomarker of checkpoint inhibitory immunotherapy and as a prognostic biomarker. There may also be a variety of immunosuppressive mechanisms in the TME, including CTLA-4, PD-L2 and interleukins [143]. Strategies that combine multiple approaches to detect the immune status of the TME may be more effective in treating immune checkpoint inhibitors [144].

As a new treatment, immunotherapy is not aimed at tissues and cancer cells but at the body’s own immune system to change the immune state of the TME and prevent and treat cancer progression. However, although the therapeutic effect is good, the proportion of the effective population is low. Therefore, the establishment of predictive biomarkers of checkpoint immunotherapy is very important to maximize the effectiveness of treatment. For patients for whom immunotherapy is ineffective at the current checkpoint, unnecessary toxicity can be avoided in time, and alternative treatment strategies can be adopted. Although immunotherapy research is in a bottleneck at present, it has broad application prospects in the clinical setting.

The use of nanocarriers not only increases the bioavailability and solubility of hydrophobic drugs but also improves the pharmacokinetic properties of drugs, avoids the degradation of drugs in vivo circulation, and delivers one or more drugs to the lesion site to achieve the controlled release of drugs. Nanotechnology helps to improve the targeting efficiency, has received increasing attention, and has been used in a variety of treatments, such as chemotherapy, radiotherapy and immunotherapy [145] (Fig. 5).

In recent years, stimulus-sensitive or intelligent nanocarriers have attracted much attention because of their advantages in controlling the release of drugs. By replying to external stimuli (e.g., ultrasound and magnetism) or internal stimuli (e.g., temperature, pH, and H₂O₂), medicine can be controllably released from smart nanocarriers at the therapeutic target in a desired manner [146‐148].

Nanocarriers can improve the solubility of drugs, increase the stability of drugs, reduce toxicity and side effects, and specifically deliver medicinal molecules to cancers by heightened targeting effects or retention (EPR) effects and permeability [149, 150]. Moreover, nanocarriers can encapsulate various anticancer drugs with different treatment mechanisms and deliver them at the same time; therefore, they are very beneficial to the combined therapy of cancers [104, 105]. Zhang et al. demonstrated through in vivo experiments that targeted nanoparticles can remove pro-cancer cells and stimulate anti-cancer effector cells by loading immunomodulators (such as lipid nanoparticles coated with cancer-targeting peptides IRGD and PI3K inhibitors) [106]. This not only helps to reduce the toxicity and side effects of antineoplastic medicine, but also helps to exert the congenerous effect of different antineoplastic medicines [151].

In addition to the above effects, the material of the nanoparticles themselves also has a direct impact on the TME. Jiang et al. found that functionalized micelles (FucOMDs) target and have good blood circulation, which can inhibit the adhesion of activated endothelial cells to CTCs in triple-negative breast cancer mice and reverse the abnormal expression of several key marker proteins in the pre-metastatic niche [107]. Chen et al. discovered that a new type of cancer matrix-targeted nanocarrier (FH-SSL-Nav) can specifically remove CAFs from the liver cancer model to promote the penetration of nanodrugs into the cancer and cut off the support of the matrix to cancer cells [108]. In addition, Zhen and others also put forward their own point of view, indicating that the selective killing of CAFs and nanoparticle-based photoimmunotherapy (nano-PIT) can increase the invasiveness of T cells, thus effectively inhibiting cancer, which has also been proven by in vivo experiments [109].

At present, research on nanoparticle systems is mainly focused on accurate drug delivery, precise controlled release, and the efficacy of the material itself. As an ideal adjuvant therapy, there is no denying its advantages and bright prospects in clinical application. However, everything has two sides, and we do not have a deep understanding of it nor are we very clear about its side effects. Therefore,we should evaluate it more comprehensively and examine it before it is used in the clinical setting.

Traditional Chinese medicine (TCM) has a long history and unique advantages. Based on the needs of the comprehensive treatment of clinical cancers, various natural products from TCM have been investigated in the therapy of cancers (Fig. 6).

Curcumin (diferuloylmethane) is an active ingredient in plant turmeric spices [152]. It suppresses tumour progression by affecting many aspects of the microenvironment. Based on in vivo and in vitro studies of ovarian cancer, curcumin interferes with the TME and affects tumour progression by inhibiting transcription factor nuclear factor-NB (NF-NB), signal transduction, activation of transcriptional activator 3 and expression of angiogenic cytokines [110]. For pancreatic cancer, preclinical studies have found that a new curcumin synthesis derivative (CDF) can inhibit VEGF, IL-6 and tumour stem cells, thus affecting the TME of pancreatic cancer [111]. In addition, a randomized phase 1 placebo-controlled trial of the APG-157 botanical drug made from curcumin was conducted. A total of 13 normal subjects and 12 oral cancer subjects participated in the study, of which 12 were treated with placebo and 13 were treated with the active drug APG-157. It was found that the infiltrating expression of immune cells (CD4⁺ and CD8⁺ cells) and the expression of PD-1 and PD-L1 were significantly increased in patients receiving APG-157, implying that APG-157 therapy has the potential to attract immune cells into the TME and provides a strong theoretical basis for using immune checkpoints to block the interaction between T cells and cancer cells (PD-1/PD-L1 axis) [112]. In a randomized, double-blind, placebo-controlled trial, 97 patients were randomly divided into a curcumin group (n = 49) and a placebo group (n = 48). Oral curcumin for 6 months had no significant effect on the overall withdrawal time of prostate cancer patients with intermittent androgen deprivation, and curcumin intake inhibited the increase in prostate-specific antigen (PSA) during curcumin administration. It has been suggested that curcumin has a potential beneficial effect on patients with prostate cancer [113]. Curcumin not only intervenes the TME but also improves the safety and tolerance of patients to a certain extent, suggesting that while traditional Chinese medicine plays an anti-cancer role, it is also helpful for the self-protection of patients.

By interfering with the target or signalling pathway in the microenvironment, TCM can inhibit the microenvironment aiding cancer through immunosuppression, the EMT and cancer stem cells. Through in vitro experiments, it was found that sophoridine can inhibit macrophage-mediated immunosuppression through the TLR4/IRF3 pathway and then up-regulate the killing effect of CD8⁺ T cells on gastric cancer, thus reshaping the immune microenvironment of gastric cancer, which provides a preclinical basis for the clinical application of sophoridine [114]. Research by Li et al. indicated that ginsenoside Rh2 (G-Rh2) can improve the TME by regulating the phenotype of TAMs in lung cancer tissues [115]. Huang et al. demonstrated that berberine can mediate Smad-independent and Smad-dependent TGF-β signalling pathways, thereby inhibiting EMT and promoting apoptosis [116]. Zhao et al. found that wogonin inhibits EMT in the inflammatory microenvironment by interfering with the IL-6/STAT3 signalling pathway in mice with lung cancer [153]. Li et al. found that for the mouse model of metastasis of human colon cancer, Bigelovin inhibits the EMT by interfering with the expression of N-and E-cadherin, inhibits colony formation through the STAT3 pathway, and reduces cell invasion through the cofilin pathway [117]. Jin et al. demonstrated that cordycepin regulates the TME and inhibits cancer growth by targeting CSCs, upregulating cancer cell apoptosis and eliciting cell cycle arrest [118].

The TCM can reduce or block the communication between cells in the TME, thus destroying the microenvironment and inhibiting cancer metastasis. The TCM can affect the communication between cells in the TME, thus inhibiting cancer proliferation. Wei et al. provided evidence that shikonin checks the diffusion of a breast cancer cell line (MCF-7) by reducing the exosome of cancer origin [119]. Through in vivo experiments, Liu et al. showed that the triptolide exosome delivery system (TP-Exos) can reduce the apoptosis and cytotoxicity of TP on SKOV3 cells and enhance the inhibitory effect of TP on cell proliferation [154].

Some studies have also shown the effect of natural products from TCM on improving the TME by promoting vascular normalization. Zhong et al. found that 6-gingerol (6G) reduces cancer growth and metastasis by promoting cancer vascular normalization, reducing microvascular structure entropy (MSE), improving the TME and reducing cancer metastasis in a patient-derived cancer xenotransplantation (PDTX) model [155]. Yang et al. demonstrated that salvianolic acid A can block the secretion of glucose-regulated protein 78 (GRP78) to inhibit cancer-related angiogenesis [156]. Zhuang et al. demonstrated that dihydrodiosgenin (DYDIO) can inhibit platelet activation and reduce endothelial cell-derived factor VIII (FVIII) to inhibit HCC metastasis [157].

There are many types of TCMs that can act on different signalling pathways in the TME, inhibit cancer recurrence and metastasis, have high safety, and can be treated according to the patient’s physique, syndrome differentiation and precise treatment. However, there are few modern pharmacological studies of TCM, and most of them are preclinical studies. Therefore, more in-depth exploration is needed for broad clinical practice.

To better intervene in the TME, combination therapy may be a good strategy, such as the combination of two different chemotherapy drugs or a combination of chemotherapy drugs and natural products from TCM (Fig. 7).