Oncogenes in the RAS family (K-, H-m, and N-RAS) are the most mutated and play a key regulatory role in important signaling pathways that promote tumor growth (2, 81) ( Figure 3 ). Under the pressure of nutritional deficiency, cells expressing oncogenic K-RAS can directly activate Ras protein to internalize and degrade extracellular proteins through a macropinocytic uptake mechanism (82). In recent years, increasing number of cancer cell types that express oncogenic RAS genes have been confirmed to obtain extracellular proteins through macropinocytosis in a hungry state. In pancreatic cancer cells, macropinocytosis has been shown to involve in active Ras protein signaling (17, 83). Pancreatic ductal adenocarcinoma (PDA) is observably hypervascular, and macropinocytosis is deemed to facilitate amino acid supply and survival of the tumor cells. In prostatic cancer cells, Mishra et al. found that epigenetic activation of RAS genes induced macropinocytosis involved in albumin uptake and amino acid release (primarily glutamine) to promote metabolism and differentiation of cancer cells (84). In non-small cell lung cancers (NSCLC), Hodakoski et al. confirmed a subpopulation of cell lines that can internalize and degrade extracellular proteins by macropinocytosis to maintain their survival in glucose deficient environment (85). Extracellular proteins are degraded into amino acids (86) that feed these cancer cells to meet the high nutritional requirements of cell proliferation (82, 87). For example, internalized proteins can be degraded to produce alanine, which participates in the tricarboxylic acid (TCA) cycle and the production of glycolytic intermediates. In this process, alanine transaminase 2 (ALT2) converts alanine to pyruvate, which is essential for the survival of cancer cells during glucose deficiency. Macropinocytosis in these cells is adjusted by Rac-Pak signaling activated by PI3K. Cell proliferation was inhibited when macropinocytosis was inhibited.

In recent years, research on the molecular mechanism of macropinocytosis induced by the oncogenic RAS genes has gradually intensified. Ramirez et al. proved that V-ATPase was involved in regulating RAS genes induced macropinocytosis (22). Tumorigenic RAS genes drive the V-ATPase transfer between the endomembrane and plasma membranes, which needs excitation of protein kinase A from solvable adenylate cyclase relying on bicarbonate. The collection of V-ATPase in the cell membranes facilitates the binding of Rac1 relying on cholesterol to the cell membrane, which is the primary condition for stimulating membrane ruffles and macropinocytosis. A recent study showed that macropinocytosis in PDA cells lack of glutamine needed to be driven by K-RAS gene, the activation of K-RAS gene could increase position of the transmembrane proteoglycan receptor protein, SDC1 on the plasma membrane. SDC1 could further assembly form a signaling complex that could activate Rac1 GTPase, which could stimulate PAK1 and thereby activate macropinocytosis (23). It delivered the proteins to lysosomes and degraded them into amino acids that were then used by cells.

In the microenvironment of solid tumors, vascular system, fibrotic tissue, and immune cell infiltration are specific. When nutrients and growth regulators in the tumor microenvironment change, tumor cells can make adaptive responses to maintain survival. Colin et al. studied whether the expression of the key factors associated with macropinocytosis was modified in human glioma datasets (88). Thirty-eight genes associated with macropinocytosis were identified from the mRNA levels of GBM. As markers of GBM, EGFR and platelet-derived growth factor receptor (PDGFR) may trigger macropinocytosis ( Figure 3 ). The stimulation of EGFR and PDGFR induces the activation of K-RAS and H-RAS gene. Lee et al. found that EGFR could regulate the interaction between macropinocytosis and nutrients in PDA (25). In the absence of glutamine, activation of EGFR signaling pathway can activate RAS genes following Rac1 and serine-threonine kinase PAK1 activation, thereby causing changes in the membrane skeleton, promoting macropinocytosis, and increasing the acquisition of glutamine. Glutamine can produce the TCA circulating intermediate, alpha-ketoglutarate, which is then demineralized to form glutamic acid. Supplementation with alpha-ketoglutarate or glutamic acid can eliminate macropinocytosis induced by glutamine deficiency. It can therefore be inferred that the decrease in glutamine metabolism, rather than the decrease in glutamine levels, is responsible for macropinocytosis. In addition, in pancreatic ductal adenocarcinoma (PDAC) cells, the lack of glutamine can lead to autophagy. This autophagy process is a macropinocytosis-associated autophagy pathway. Its activation can provide glutamine for the TCA cycle, which is beneficial to the maintenance of intracellular glutamine levels in PDAC cells (89).

There are several theories regarding the sensor that links glutamine level to the EGF signal. The first is the GCN2-eIF2α-ATF4 mechanism (90). At first, glutamine starvation results in the appearance of uncharged tRNAs that activate GCN2. Next, the level of ATF4 transcription factor increased, promoting the expression of genes related to metabolic amino acids and transduction of cytokine signals. The second is AMP-activated protein kinase (AMPK) (90). Intracellular energy levels change when TCA circulatory function is impaired and nutrient uptake is increased but utilization is reduced. In prostate cancer cells, reduced energy levels activate AMPK through direct and indirect mechanisms that induce macropinocytosis and the internalization of necrotic fragments for lipids and proteins synthesis. In addition, Kim et al. found that loss of PTEN, the most frequently deleted tumor suppressor gene in prostate cancer, can activate AMPK, which supports RAC1 and PAK1 activation and macropinosomes formation of extracellular proteins in nutritional pressure (21).

As a nutrient-sensitive signaling protein, mTORC1 can react to environmental changes to regulate cell survival (91) ( Figure 3 ). Under conditions of sufficient amino acids, the activation of mTORC1 can promote cancer cell growth. However, in a recent study, the authors found that when cells rely on exogenous proteins(through macropinocytosis) instead of exogenous amino acids as the source of amino acids, the activation of mTORC1 inhibits cell proliferation (19). Therefore, in the case of insufficient amino acids, inhibiting the activity of mTORC1 will increase the macropinocytosis of extracellular proteins to meet the needs of cell survival. Kasitinon et al. found that TRPML1, a lysosomal cation channel, whose activation under nutritional deficiency is beneficial to the occurrence of macropinocytosis and cell proliferation and survival of melanoma cells (92). The main mechanism is that TRPML1 reversely adjusts and weakens mitogen-activated protein kinase (MAPK) pathways and mTORC1 signal of melanoma cells, maintains the occurrence of macropinocytosis, and reduces or blocks protein toxic stress.

In short, oncogenic RAS genes, GFRs, and mTORC1 are of great significance for tumor cells in a nutrient-deficient environment to rely on macropinocytosis for survival. Based on this, further design of related anticancer drugs will have certain research value.

Oncogenic mutations in RAS genes are found in about 30% of human cancers, so RAS genes are considered to be of great significance for the development of cancers. Chi et al. reported that in GBM and gastric cancer cells, abnormal activation of H-RAS oncoprotein (G12/V) was found to induce a large number of vacuoles and caspase-independent cell death (5, 15, 16). The appearance of cell vacuoles is similar to macropinocytosis. This type of cell death is called methuosis (35). In follow-up studies, methuosis has been found in other tumor cells such as osteosarcoma cells and HEK293 cells (121). This new type of cell death, which is different from apoptosis, can achieve anti-tumor effect by inhibiting the K-RAS gene mutation in normal human epithelial cells. Therefore, the over-activation of K-RAS gene has considerable promise for the treatment of cancers that can express K-RAS gene (122).

In the normal process of macropinocytosis, the plasma membrane ruffles produce macropinocytic cups, which close to form macropinosomes. After the macropinosome enters the cell, some macropinosomes circulate back to the plasma membrane surface under the action of macropinosome cyclic receptors (123, 124), while other macropinosomes fuse with lysosomes (74, 125), and are lysed (126), thereby releasing extracellular materials and nutrients (4). In GBM cells with over-activated RAS genes, the process of macropinocytosis is partly different from the normal macropinocytosis pathway. Overmeyer et al. labeled newly formed macropinosomes with fluorescent dextran and found that they did not loop back to the plasma membrane or merge with lysosomes (16). As more and more macropinosomes appeared, they began to fuse with each other to form larger macropinosomes. Following fusion, the macropinosomes exhibit the characteristics of late endosomes, which are positive for LAMP1 and Rab7. However, due to the differences between the properties of macropinosomes and advanced endosomes, some researchers believe that macropinosomes can be seen as nonfunctional advanced endosomes (16, 127). Excessive accumulation of macropinosomes results in extreme vacuolization of cells, followed by cell death.

After the onset of macropinocytosis, Ras GTPases are activated and act as mediators to promote macropinocytosis-related actin assembly (3). The key players in this process are Arf and Rab GTPases (94, 126). Over time, continuous high expressing of RAS (G12V) with subsequent slow stimulating of Rac1 causes extreme vacuolation of tumor cells, eventually leading to methuosis (128). One step that takes effect in methuosis is that the activity of macropinosomes recycled to the plasma membrane surface is reduced during the over-activated macropinocytosis process. This causes more and more macropinosomes to aggregate inside the cell, and they fuse into larger macropinosomes, eventually filling the entire space of the cell, forming extreme vacuolation and endangering cell viability. In over-activated macropinocytosis, chronic Rac1 excitement affects the other one GTPase, Arf6, which mainly causes of defects in macropinosome recycling. Arf6 plays an important role during the circulation mechanism of cytidine-uncovered endosomes (6). The authors found the active condition of endogenous Rac1 and Arf6 to be opposite. As Rac1 activation level increased, Arf6 activation was inhibited. Another important step in the process of methuosis is that the huge vacuoles formed by the fusion of macropinosomes do not fuse with lysosomes and are not degraded. Therefore, due to the lack of effective lysosomal clearance and the inability of macropinosomes to recycle to the plasma membrane, macropinosomes continue to accumulate, leading to extreme vacuolation in the cells and cell death ( Figure 5 ).

Tyrosine kinase receptor A (TrkA) is a receptor for neurotrophin (125), which mainly exists in endosomes induced by clathrin-dependent endocytosis and eventually degrades in the lysosome to promote the survival and differentiation of normal neuronal cells (129). In contrast, the effects of its induction on tumor cells macropinocytosis is reflected in the promotion of cell death (130) ( Figure 5 ). Li et al. discovered a new mode of cell death associated with ectopically expressed TrkA activation-induced macropinocytosis in myeloma tumor cells (131). When TrkA expression was inhibited by casein kinase 1 (CK1) inhibitor, macropinocytosis and cell death were inhibited. The above-mentioned cell death is similar to methuosis induced by RAS genes overactivation. It is characterized by excessive vacuolation of macropinosomes, leading to extreme vacuolization of cells and forcing the cell to die. The most different aspect is that the macropinosomes induced by RAS genes overactivation cannot fuse with lysosomes after maturation, whereas macropinosomes induced by ectopic expression of TrkA can fuse with some lysosomes partly (35). To prove the relationship between the above mode of cell death and autophagy, Li et al. downregulated beclin-1, Atg5, LC3, and other autophagy proteins in medulloblastoma cells and found that it did not inhibit the extreme vacuolation and cell death (131). In contrast, some macropinosomes bind to LC3 protein markers and then fuse with lysosomes. It has been reported that this paradox may be due to the ability of macropinosomes to collect aberrantly expressed LC3 protein markers to regulate the downstream mechanism of the fusion process for endosomes and lysosomes (132).

Abnormal expression and activation of genes and proteins involved in the process of normal macropinocytosis may contribute to the occurrence of methuosis. 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) is a key point in the glycolysis pathway. In the sarcomatoid and epithelioid cells, gene suppression of PFKFB3 can lead to methuosis (133). The inhibition of PIKFYVE (a class III phosphoinositide (PI) kinase) can also induce methuosis (134). In addition, the up-regulation of Rac1 mRNA and Rac1 protein expression can cause methuosis in human nasopharyngeal carcinoma cells (135). More detailed mechanisms and more gene and protein targets related to methuosis need further research.

In 2011, researchers discovered a class of small-sized indoles, the typical representatives of which are the synthetic indole-based chalcones (MIPP, MOMIPP) (136). They can rapidly cause vacuolization of cells similar to methuosis induced by overactivation of RAS genes at very low concentrations in U251 GBM cells and a wide range of other cells (137). Using time-lapse microscopy and extracellular fluorescent yellow, the authors found that MIPP and MOMIPP rapidly induced plasma membrane shrinkage, leading to the formation of macropinosomes. The newly formed macropinosomes fuse rapidly and neither participate in membrane circulation nor undergo lysosomal fusion. MOMIPP induces inchoate disruption of amino acid assimilation and glycolytic metabolism in GBM and other cell lines (26). At the same time of these metabolic changes, the JNK1/2 stress kinase pathway is activated by MOMIPP, thus causing phosphorylation of some sites, such as Bcl-2, C-Jun and Bcl-XL. PIKFYVE (134), a class III phosphoinositide kinase, is responsible for methuosis-inducing activity. The phosphorylation of surviving members of the Bcl-2 family is mediated by JNK, and this process and interference with glucose uptake play a key role in methuosis. It has been reported that MIPP and MOMIPP have a high degree of structural specificity in inducing macropinocytosis (138), and their specific structures may be involved in the textural constitution of early or late endocytosis pathway in macropinocytosis (139). Some researchers have proposed that MIPP drugs disturb the normal macropinocytosis process by disrupting the regulatory cycle of Rab5 GTPase, thereby causing extreme vacuolation in cells and leading to methuosis (140). After treating cells with MIPP, they quantitatively measured the amount of active Rab5-GTP and Rab7-GTP on the macropinosome membrane, and found that the former was significantly downregulated, while the latter increased significantly. Based on these results, it is speculated that MIPP drugs command the components of the Rab5 complex in a direct or indirect manner.

MIPP and MOMIPP-induced methuosis have similarities and differences with Ras-related methuosis. One difference is that MIPP drugs induce macropinocytosis about 10 times faster than Ras. The macropinocytosis process induced by MIPP drugs not only disrupts the membrane recovery and fusion phase with lysosomes, but also destroys the nascent process of macropinosomes in the later stages. MIPP and MOMIPP also have a characteristic manifestation in that the macropinocytosis induced in a short time (4 h) is reversible (35). Another difference is that the target points of action of MIPP-induced macropinocytosis are the downstream endocytosis factors of Rac1 and Arf6 rather than Rac1 and Arf6 that are acted upon by Ras protein. To study the mechanism of MIPP-induced macropinocytosis, researchers treated cells with bafilomycin A1 (Baf-A) combined with MIPP, and found that Baf-A not only increased the lysosomal pH value, but also interfered with the conversion in early endosomes and late endosomes (141). The potential molecular mechanism of methuosis needs further understanding, and the application of MIPP compounds in clinical anti-tumor therapy has promising prospects (142).

The main role of well-known methamphetamine (METH) is to alter the integrality of dopamine teleneurons and disrupt nerve conduction. METH has been reported to cause vacuolation of lysosomes (143). In studying the neurotoxicity of METH, Nara et al. found a specific cell death manner like methuosis (144). They cultured neuroblastoma cells in METH for 24 h and found that macropinocytosis appeared, followed by cell death. The author traced the liquid in the macropinosomes induced by METH, and observed that the macropinosomes did not arise from the endoplasmic reticulum or autophagosome, and the source was like that of RAS genes-induced and MIPP-induced macropinosomes. At the same time, in the process of macropinocytosis induced by METH, there was also a disorder of fusion of macropinosomes and lysosomes. In 2012, Nara et al. demonstrated that METH-induced macropinocytosis involved the activation of Rac and Rac1 (145). To study the correlation between Ras and Rac1 activation and METH-induced macropinocytosis, the authors also treated the cells with inhibitors of Ras and Rac and found that the production of macropinosomes was inhibited. During the formation of the macropinosomes, the lysosomal function was defective, and the authors speculated that the defective lysosomal function may be the cause of cell death. Nara et al. found that in METH-induced macropinocytosis, some FITC-dextran-labeled vacuoles co-localized with LAMP1, indicating fusion of macropinosomes and lysosomes (145). Meanwhile, it has been reported that LAMP1 can also be detected on the membranes of advanced endosomes and mature macropinosomes (125). It is speculated that the cause of cell death is an impediment to the transport of mature macropinosomes to the lysosome.

The behavior of METH-induced macropinocytosis is different from that induced by RAS genes and MIPP. For instance, there is no caspase activation-induced cell death in the process of METH-induced macropinocytosis. In cells where RAS genes are over-activated and treated with MIPP, caspase activation-induced cell death coexists with methuosis; however, methuosis dominates between these two types of cell deaths (35). Autophagosomes also exist in the process of METH-induced methuosis. However, macropinosomes induced by RAS genes overactivation are not associated with autophagosomes. In their study, Nara et al. used an autophagy inhibitor to inhibit the methuosis induced by excessive activation of RAS and observed that it did not prevent cell death (144).

Vacquinol-1 (146), a quinolone derivative, shows promising therapeutic potential by inducing rapid cell death in GBM. Vacquinol-1-induced cell death is similar to methuosis (147). Vacquinol-1 has well drug metabolism capability in both vascular tissues and brain tissues. In animal models of GBM multiform, treatment with Vacquinol-1 has been shown to prolong patient survival. In a major finding, as low as 1 μM of exogenous ATP concentration could regulate Vacquinol-1-induced cell death (148). A study found that Z−VAD−FMK, a specific caspase inhibitor, can inhibit the function of hepatocellular carcinoma cell death caused by Vacquinol-1, which suggests that cell death caused by Vacquinol-1 is related to apoptosis (89). Therefore, the specific types of vacuolation and cell death induced by Vacquinol-1 are controversial, and more evidence is needed.

AS1411 is an anticancer drug that specifically disturbs growth and mediates death in cancer cells. Its essence is a guanine-rich oligodeoxynucleotide, which can be used as an aptamer to bind to nucleosides (149–151). Reyes-Reyes et al. found that in cancer cells treated with AS1411 (152), the drug binds to nucleoproteins and changes the subcellular localization of nucleoproteins (153), causing cell swelling and cytoplasmic vacuoles, leading to excessive macropinocytosis similar to methuosis (154).

F14512, a cancer cell-targeting drug, is essentially a polyamine-modified topoisomerase II inhibitor (155). The anticancer mechanism of F14512 involves killing of cancer cells by blocking the liberation of intermediate DNA breakage during the action of the enzyme (156). F14512 has been reported to have anticancer properties resulting from the accumulation of numerous multilamellar vesicles and vacuoles like methuosis (157). However, unlike classic methuosis, in the cytotoxic process induced by F14512, the cells enter a doddery status as revealed by β-galactosidase dye. Therefore, the relationship between F14512-induced cytoplasmic vacuolation in the cancer cells and methuosis needs further studies.

As a silmitasertib precursor, CX-4945 can inhibit the activity of a protein kinase—CK2. Lertsuwan et al. demonstrated that CX-4945 can inhibit cell survival and induce cell death like methuosis through CK2-independent passages in cholangiocarcinoma and other cancer cell lines (158). In colorectal cancer cells, Silmitasertib inhibits the binding of CK2 to its target, Akt, thereby inhibiting the PI3K/Akt/mTORC1 signaling pathway, and tumor growth (159). During Silmitasertib treatment, tumor cells produce numerous catastrophic cytoplasmic vacuoles like methuosis, which was revealed by molecular markers. But the specific mechanism needs further research.

CD99 is one of the target molecules of Ewing sarcoma (EWS) (160). CD99 triggered by mAb can cause IGF-1R/Ras/Rac1 complex and produce Rab5-positive endocytic vesicles. In subsequent pathways, the IGF-1R/Ras/Rac1 complex dissociates and the IGF-1R circulates back to the membranes. Ras/Rac1 and CD99 continue to exist on the isolated immature LAMP-1-positive vacuoles, which would continue to accumulate and cause methuosis. Therefore, CD99 targeted therapy has great potential in the treatment of EWS patients with resistance to apoptotic drugs.

Sun et al. reported a small molecule derived from ursolic acid that may mediate cell death by macropinocytosis overactivation (161). The death was prevented after amiloride treatment, a specific inhibitor of macropinocytosis, suggesting that the cell death pattern caused by this small molecule is consistent with methuosis.

In conclusion, as a new form of cell death, methuosis needs further studies. Although the importance of RAS genes and TrkA is known, the molecular mechanisms associated with methuosis remain unclear. In addition, there are more stimulants of methuosis waiting to be discovered for clinical applications.

As mentioned earlier, some drugs have the ability to induce macropinocytosis, but they are only a minority. Therefore, researchers have begun to shift their focus to the combination of substances that can activate cell surface receptors of macropinocytosis and non-macropinocytosis-dependent anticancer drugs. Through the formation of this kind of drug conjugates, the macropinocytosis pathway is used as a way of anticancer drug presentation to promote cancer treatment (162). Here, the drug conjugates that can cause macropinocytosis are introduced as follows according to the types of some raw materials ( Figure 6 ).

Based on the role of EVs (including exosomes) in cell information communication, the potential of EVs as drug carriers has been discovered (108). Takenaka et al. reported that EVs as carriers of gefitinib can improve the therapeutic effect of EGFR-mutant NSCLC tumor patients through macropinocytosis (104). Nakase et al. designed EVs loaded with ribosome-inactivating protein saporin with EGF that can produce stronger tumor suppression (111). Subsequent studies found that EVs modified with arginine-rich cell penetrating peptide can induce macropinocytosis and promote the delivery of saporin (188). Particularly, engineered exosomes (iExosomes), also known as mimic exosomes, are artificially designed exosomes carrying functional molecules or drugs that are used for cancer treatment. Lou et al. confirmed that iExosomes loaded with miR-199a derived from mesenchymal stem cells can enhance the sensitivity of liver cancer to chemotherapy drugs by targeting the mTOR pathway (189). Kamerkar et al. reported that artificially designed iExosomes from normal fibroblast-like mesenchymal cells carrying short interfering RNA or short hairpin RNA specific to oncogenic KrasG12D can be internalized by macropinocytosis and inhibit the development of pancreatic cancer in mice (104). As carriers, iExosomes can also be used in the treatment of neurological diseases such as schwannoma, glioma and Parkinson’s disease (190). Targeted cells can absorb EVs through a variety of endocytosis pathways, including macropinocytosis, caveolin-mediated uptake, phagocytosis, and lipid raft-mediated endocytosis, etc. (191, 192).

In conclusion, macropinocytosis for delivering antitumor drugs holds immense potential for research and clinical implications. Macropinocytosis can present substances including monomer drugs, drug NPs and other drug conjugates, and EVs. Through macropinocytosis, antitumor drugs can quickly and efficiently enter tumor cells to exert antitumor effects.

The first point is to inhibit tumor metabolism by inhibiting macropinocytosis in tumor cells (193) ( Figure 7 ). When cells are in an amino acid-deficient environment, tumor cells upregulate macropinocytosis. Macropinocytosis, in turn, promotes the endocytosis of extracellular proteins by affecting mTORC1 in cancer cells, thereby maintaining tumor cell survival (194). It has been reported that hydroxychloroquine (HCQ) can indirectly inhibit macropinocytosis in tumor cells by inhibiting lysosomal acidification (195). Following inhibition of macropinocytosis, the ability of tumor cells to metabolize extracellular proteins and eATP is weakened, and the lack of metabolic substrates limits the viability of tumor cells (196). The mTOR pathway is often disrupted in cancers, and the intersection between mTOR and TPCs suggests intriguing therapeutic possibilities (197). In cancers associated with skin, breast, lung and cervix, and rhabdomyosarcoma (RMS), Srivastava et al. found that the use of dual mTORC1/mTORC2 inhibitors, OSI-027 and PP242, resulted in over-activation of macropinocytosis, inhibiting tumor survival (24). In further experiments, xenograft RMS in mice treated with OSI-027 showed reduced tumor growth, reduced proliferation, and excessive macropinocytosis in cancer cells. These results suggested that dual inhibitors may be useful in the treatment of refractory or recurrent cancers.

Identifying the adaptive metabolic pathways in cancers may provide novel targets for cancer therapy. RAS genes-induced macropinocytosis of extrinsic proteins in glucose-deficient tumor cells, an adaptive metabolic pathway for cancers, may be a potential target for anticancer strategy (85). Liu et al. demonstrated significantly greater uptake of NPs by cancer cells that have activating mutations of K-RAS gene (86). Such an NP-based therapy that targets K-RAS gene-induced macropinocytosis is a handy way toward improved transmitting into K-RAS gene-induced tumors. Research on the inhibition of macropinocytosis in this strategy may contribute to the improvement of the curative effect of cancers (198). Similarly, Ramirez et al. found that V-ATPase, as one of the regulators of RAS genes mediated macropinocytosis, is closely related to nutritional supply (22). Thus, V-ATPase inhibitors may inhibit the metabolic adaptation of RAS genes mutant cancer cells.

The second point is to make macropinocytosis abnormal through the interference of the physiological process of macropinocytosis, thereby incapacitating it to help cancer cells absorb beneficial substances. ( Figure 7 ). Currently, most of the research in this area is centered around interfering with macropinocytosis by interfering with the phosphoinositide biochemical pathway. Thapa et al. reported that when Iγi2, a specific PI4P5K, was knocked down using RNA interference (RNAi) in breast cancer cells, the viability of the breast cancer cells was suppressed (199). Similarly, Araki and Teranishi et al. found that in pancreatic cancer cells, the process of macropinocytosis was disrupted following treatment with the fungal metabolite, wortmannin, which blocks PI3K (200, 201). Consequently, the viability and metastasis of the pancreatic cancer cells were inhibited. Salloum et al. found that macropinocytosis induced by tyrosine kinase receptors excitement was strongly relying on a PI3K subtype, PI3Kβ (202). In the early stage of macropinocytosis, PI3Kβ is involved in regulating the formation of circular folds. In the late stage of macropinocytosis, PI3Kβ participates in the activation of Rac1-activated downstream molecules. Similarly, PI3Kβ is involved in regulating macropinocytosis in tumor cells with defective PTEN tumor suppressor genes. Therefore, pharmacological inhibitors of PI3Kβ may have great potential in anticancer treatment. In addition to the phosphoinositide pathway, methods to interfere with other pathways of macropinocytosis remain to be studied, and their significance in tumor treatment will become increasingly clear in the future.

In GBM cells, the presence of macropinocytosis is conducive to drug presentation, and persistent macropinocytosis may eventually cause methuosis. Therefore, Colin et al. hypothesized that the combined use of macropinocytosis inducers might achieve the dual effects of drug presentation and methuosis promotion (88). SN21 was combined with membrane cleavage peptides by Arafiles et al. to realize the internalization through macropinocytosis and release of endosomes of extracellular materials (128). This strategy has been shown to be effective in promoting cellular uptake of anticancer drugs. Cao et al. found that eATP uptake by macropinocytosis is involved in early metastasis steps such as EMT of tumor cells (94). Tumor survival was inhibited in nude mice with macropinocytosis-associated SNX5 gene knockout. These results demonstrate that the importance of eATP in tumor metastasis, suggesting that intracellular energy balance biochemical equation requires new consideration, and indicating that eATP internalization through macropinocytosis may be an effective anticancer drug therapy target.

In short, whether the inhibition of macropinocytosis is achieved through inhibition of the macropinocytosis process itself, or through the associated inducible and regulatory factors, as long as the macropinocytosis activity in cancer cells is inhibited, it may interfere with the survival of the cancer cells and inhibit cancer. Therefore, elucidating the molecular mechanism of macropinocytosis in cancer cells is of great significance for cancer therapy.