Crosstalk between glioblastoma and tumor microenvironment drives proneural–mesenchymal transition through ligand-receptor interactions

Glioblastoma (GBM) is the most common intrinsic and aggressive primary brain tumor in adults, with a median survival of approximately 15 months. GBM heterogeneity is considered responsible for the treatment resistance and unfavorable prognosis. Proneural-mesenchymal transition (PMT) represents GBM malignant progression and recurrence, which might be a breakthrough to understand GBM heterogeneity and overcome treatment resistance. PMT is a complicated process influenced by crosstalk between GBM and tumor microenvironment, depending on intricate ligand-receptor interactions. In this review, we summarize the autocrine and paracrine pathways in the GBM microenvironment and related ligand-receptor interactions inducing PMT. We also discuss the current therapies targeting the PMT-related autocrine and paracrine pathways. Together, this review offers a comprehensive understanding of the failure of GBM-targeted therapy and ideas for future tendencies of GBM treatment.


Introduction
Glioblastoma (GBM) is one of the most common, intrinsic, and aggressive primary brain tumors in adults, which is characterized by diffuse infiltration throughout the brain, making it impossible to cure with surgery. 1 The prognosis of recurring tumors has somewhat improved since the tumor treatment field (TTF) device in 2015 for the treatment of recurrent or refractory GBM. 2 Immunotherapy, focused ultrasound, cell delivery methods, and nanomedicine are a few other recent medicinal advancements that have garnered interest. 3,4The quality of life and prognosis of GBM patients remain unfavorable, with an average median survival of approximately 15 months, 5 despite advancements in treatment methods and supportive care.The difficulties that underlie therapeutic failure are primarily due to the heterogeneity of GBM.GBM heterogeneity is fueled by genetic, epigenetic, and microenvironmental factors that influence cellular processes. 6GBM has previously been divided into four subclasses by The Cancer Genome Atlas (TCGA) transcriptomics: proneural (PN), mesenchymal (MES), neural (NL), and classical (CL). 7With the establishment and improvement of single-cell RNA-sequencing (scRNA-seq), the NL subtype was identified as normal neural lineage contamination and dropped from the most recent GBM categorization. 8roneural-mesenchymal transition (PMT), is the term used to describe the gradual change of GBM from the PN to MES subtype.PN is characterized by common mutations in IDH1, PDGFRA, and TP53, as well as a large amplification of chromosome 7 and deletion of chromosome 10 (>50%), which is associated with younger individuals. 8A more recent theory asserts that GBM cells can be divided into separate subregions, with PN subtypes distributed along the tumor margin. 9MES had significant expression of CHI3L1, MET, CD44, the MERTK, TNF-a superfamily, and NF-kB pathways, as well as inactivation of NF1 (37%), TP53 (32%), and PTEN (32%). 8This subtype is known to be associated with high angiogenesis and invasiveness, typically enriched in hypoxic, necrotic, and high glycolysis locations. 9,10STAT3, C/EBP, TAZ, NF-kB, Wnt, PI3K/AKT/mTOR, MAPK, and JNK in GBM cells are demonstrated to be directly associated with PMT.11e16 Additionally, changes in the microenvironment (artificial treatment, metabolism, mRNA splicing) activate these pathways. 6,17BM microenvironment is primarily made up of different types of solid tissue cells, soluble cytokines, and extracellular matrix (ECM). 18One of the primary mechanisms for cell communication in the microenvironment is receptoreligand interaction.Receptors can be classified as membrane, cytoplasmic, or nuclear receptors based on the various binding locations. 19This review focuses on cell membrane receptors as important targets of neoadjuvant therapy, as they constitute a significant proportion.Tumor-associated macrophages (TAMs) are the predominant non-tumor cells in the environment. 18The anti-tumor M1 subtype and the protumor M2 subtype are the two major subtypes in the GBM microenvironment. 20,21M2 TAM infiltration was identified as a significant contributor to PMT in the microenvironment. 21oreover, angiogenesis, hypoxia, and high glucose are the keys to the variables that influence the conversion of TAMs to the M2 phenotype, which can lead to PMT and then harm patients. 22,23In addition, a number of other cells, including endothelial cells, T lymphocytes, oligodendrocytes, and mesenchymal stem cells, participate in the microenvironment's crosstalk through receptoreligand interactions. 24he GBM microenvironment is complex, and understanding the relationships between cells and their interactions is crucial to understanding biological systems.Unfortunately, the current literature on ligand-receptor interactions driving PMT in the GBM microenvironment is fragmented.However, single-cell sequencing and spatial transcriptomics technologies have allowed for a shift in the study of cellecell communication (CCC) towards understanding cell interactions rather than just the existence of cells.With the use of single-cell transcriptomics, new opportunities for exploring cellecell communication have arisen. 25Recent studies have identified several mechanisms of tumor progression, such as chemotactic and cytokine signaling, context receptor signaling, survival and resistance signaling, DNA and membrane repair, avoidance of apoptosis induction, decreased drug efficacy, evasion of immunosurveillance, environmental control of survival, and cooperation of signaling programs. 26To better understand the development of tumors, it is important to consider the various types of tumors and the dynamic changes that occur in their internal and external environments.
Based on the new research demonstrated above, we frame the receptor-ligand interactions-based autocrine and paracrine pathways of the GBM microenvironment, offering a comprehensive understanding of the failure of GBM targeted therapy and ideas for future trends in GBM treatment.

Autocrine pathways
Autocrine is when the same cell expresses the matching receptor protein on its surface as the ligand expressed by the sending cell.This mechanism is critical to the onset and development of various disorders. 25GBM cells secrete prooncogenic molecules that activate downstream signals through autocrine receptors, resulting in the maintenance of multiple malignant biological properties of GBM pathology.
The GBM-mediated autocrine pathways of PMT are complex.They involve autocrine ligands such as neurotransmitters, growth factors, chemokines, hormones, and other substances.Activation of multiple pathways, including NF-kB, JAK/STAT3, and PIK3/Akt, in tumor cells promotes PMT.These pathways control the mesenchymal transition and cause various biological functions, such as increased tumor cell invasion, altered metabolism, apoptosis inhibition, and angiogenesis, etc.
The following is an introduction to some classic examples of recent research (Fig. 1 and Table 1).

Neurotransmitter autocrine pathways
ACh and AChR (M3) Neurotransmitter acetylcholine (ACh) is involved in synaptic transmission.The co-expression of choline transporter, choline acetyltransferase, and vesicular acetylcholine transporter in GBM cells was demonstrated by Thompson et al using information from the Repository for Molecular Brain Neoplasia Data (REMBRANDT) collection. 27The interaction between Ach and AChR (M3) was found to create an autocrine loop that promotes GBM invasion and triggers mesenchymal transition responses.Activation of AChR (M3) also enhances the activity of the matrix metalloproteinase MMP-9. 27 Glu and ionic Glu receptor (NMDAR, AMPAR, TRPM7, P2X7R) In the brain, glutamate (Glu) serves as a significant neurotransmitter. 28Glutamate (Glu) has been found to drive tumor PMT in GBM by activating calcium-permeable ion receptors, including AMPARs, NMDARs, TRPM7, and P2X7R, in an autocrine manner.Kyung-Seok Han et al have reported that Glu release from GBM cells increases intracellular Ca 2þ levels. 29The activation of the Akt pathway is promoted by Ca 2þ release mediated by AMPARs and NMDARs, while the activation of the Notch and STAT3 signaling pathways is promoted by the TRPM7 receptor, and the MEK/ERK pathway is promoted by the P2X7R receptor. 28,29 2D and TRPC6 Transient receptor potential channel 6 (TRPC6) is a member of the TRPC family. 29By promoting a persistent rise in intracellular Ca 2þ levels, which is essential for glioma proliferation and migration, induction of TRPC6 promotes the aggressive phenotype. 30Moreover, in hypoxic conditions, the hypoxia-inducible factor (HIF-1) is controlled by TRPC6 signaling, pointing to its connection with mesenchymal characteristics. 30 and DRD2 or DRD5 (proneural subtype secretion) The neurotransmitter dopamine (DA) is crucial for cerebral function. 31Dopamine receptor 2 (DRD2), one of the DA receptors, is overexpressed on the surface of GBM cells and exhibits a potent autocrine tumorigenic impact. 32Recent research has demonstrated that it is directly related to the development of GBM tumors.Targeted DRD2 therapies are also being developed. 33Additionally, according to data from Seamus et al, DA changed the metabolism of GBM cells to increase glycolysis, and the fact that DA is linked to hypoxia and the NF-kB pathway suggested a clear connection to mesenchymal properties. 34owth factor autocrine pathways VEGF and VEGFR2 (KDR) Angiogenesis is another characteristic of mesenchymal GBM.Vascular endothelial growth factor (VEGF), a wellknown angiogenic growth factor, participates in PMT as a vital ligand. 35VEGFR2 (kinase insert domain receptor, KDR) mediates the autocrine pathway of VEGF in the GBM autocrine pathways. 35Knizetova et al proved that VEGFR2 is predominantly expressed on the cell surface of CD133 þ human GSCs (glioma stem cells), whose viability, selfrenewal, and tumorigenicity depend on signaling through the VEGF-VEGFR2-neuropilin-1 (NRP1) axis. 35As another significant pro-angiogenic factor, NRP1 binds to and stabilizes VEGFR2, enhancing the VEGF-VEGFR2 binding effect and promoting angiogenesis, furthering the pro-mesenchymal angiogenic action of VEGF-VEGFR2. 35Another study also has demonstrated that PMT-related pathways such as c-Raf/MAPK and PI3K/Akt are co-activated after ligand-receptor interaction of VEGF-VEGFR2. 36Fs and FGFR1-4 It has been discovered that fibroblast growth factors (FGFs) control several processes, including growth, differentiation, survival, and migration.37 Four transmembrane receptor (FGFR1-4) tyrosine kinases work together to mediate the signaling caused by FGFs.37 The development, proliferation, and migration of GBM are all tightly correlated with the autocrine pathways of the FGF5/FGFR1-4 axis, which also appears to promote the expression of MAPK, PI3K, and JAK/STAT pathways.37 Additional research has demonstrated that FGF2/FGFR1 signaling is an autocrine positive feedback mechanism that supports angiogenesis, cell proliferation, and self-renewal in GBM.38 A disintegrin known as ADAMDEC1 (a disintegrin and metalloprotease domain-like decysin 1) has been demonstrated to positively regulate this autocrine loop.38 The research also demonstrated a favorable relationship between FGF and ZEB1. Inthe intracellular molecular pathway of PMT, ZEB1 is one of the important actors, 38 which strongly suggests that FGF can encourage PMT.

PDGF and PDGFR
Another growth factor, platelet-derived growth factor (PDGF), mediating autocrine signaling through PDGF receptor (PDGFR) tyrosine kinases, regulates mitogenic pathways in GBM cells. 39Moreover, this autocrine loop has a high correlation with hypoxia, and VEGF receptors, which may indicate a connection with the production of mesenchymal-like characteristics. 40Furthermore, according to research by Stommel et al, PDGFR and EGFR (epidermal growth factor receptor) can co-express in GBM cells and activate RTK and PI3K signaling pathways, which promote the development and proliferation of tumors. 41emokine autocrine pathways CXCL8 and CXCR1/2 Interleukin-8 (IL-8), also known as CXCL8, is an important inflammatory mediator and chemokine that is functional through engaging with its receptors, CXCR1 and CXCR2. 42XCL8 up-regulation has been seen in GBM, and it has been shown that Bcl-xl-induced CXCL8 up-regulation in GBM cells is mediated through the NF-kB-dependent mechanism, consistent with other tumor research.43 This NF-kBdependent mechanism occurs as a result of the JAK/STAT1/ HIF-1/Snail signaling pathways in tumor cells.44 Additionally, CXCL8 is related to several MES-related markers, including CD44 and Twist.45 Moreover, some studies have shown that necrotic cells in the tissue cause GBM cells to secrete CXCL8, induced by HIF-1-mediating hypoxia, 46 which also indicates that CXCL8 is connected to mesenchymal characteristics.

CXCL12 and CXCR4
Chemokine ligand 12 (CXCL12) is one kind of chemokine.It was shown by Gatti et al that GBM released CXCL12, which binds to the CXCR4 auto-receptor.Then, several biochemical pathways connected to PMT are activated and altered as a result of a change in the three-dimensional conformation of CXCR4.When the ligand was bound to the CXCR4 receptor, the receptor separated into the ai and bg subunits. 47The downstream PMT master regulators including PYK2, NF-kB, JAK/STAT, and PIK3/Akt pathways were activated by the ai subunit.Similarly, bg subunit modifies the cell cycle via activating the Ras pathway. 47oreover, it has been demonstrated that the CXCL12/ CXCR4 axis supports MMPs activity in GBM, suggesting a connection between these pathways and mesenchymal transition. 48he expression of CXCR4 and CXCR7 are the hallmark of mesenchymal GBM cells, which are typically found in the hypoxic necrotic regions of the tumor. 47The expression of CXCL12 and CXCR4 was also found to be boosted by HIF-1 to aid this autocrine loop. 47Moreover, numerous molecules, including VEGF, are expressed in tandem with the CXCL12/ CXCR4 axis, which is regulated by TNF-a, IL-4, and IL-6. 49N and integrin aVb3 or variant forms of CD44 Osteopontin (OPN) (also known as secreted phosphorylated protein-1, SPP1) is a chemotactic factor and a glycophosphoprotein.50 OPN is a ligand widely expressed by a variety of cell types, such as osteoblasts, macrophages, epithelial cells, smooth muscle cells, and cancer cells.51 The receptors of OPN include several CD44 isoforms as well as the integrins aVb3, aVb5, avb1, and aVb1.52e54 The degree of angiogenesis and GBM is correlated with OPN expression levels.55 The link between OPN and mesenchymal transition is revealed via OPN-mediated autocrine and paracrine pathways.The continued stemness of tumor cells, which is sustained by the OPN/CD44 autocrine loop, triggers the Akt pathways. 56Additionally, there is proof that OPN also triggers the NF-kB pathway and that integrin receptors, including aVb3, are responsible for this effect.54 Hormone autocrine pathways EPO and EPOR Erythropoietin (EPO) is a hormone that increases red blood cell production.57 In GBM, EPO functions as PMT in an autocrine manner, which activates the JAK2, STAT5, and Akt pathways.57 Mohyeldin et al demonstrated that the hypoxic areas and invasive margins of glioma specimens acquired through biopsy showed expression of both EPO and EPOR, and the expression of EPOR was correlated with the tumor's stage.58 EPO supports angiogenesis, ensures the survival of cancer cells, and encourages their growth.59 EPO was distinguished by a high level of expression in the necrotic regions of GBM, facilitated tumor invasion via MMP-2, and regulated by HIF-a.60

AM and CLR/RAMP2, CLR/RAMP3
The adrenal glands produce the stress hormone adrenomedullin (AM). 61AM is linked to a subgroup of GBM that is expressed in regions of hypoxic necrosis. 62Additionally, studies have shown that AM promoted angiogenesis by interacting with its receptors, CLR/RAMP2 and CLR/ RAMP3. 62Moreover, AM expression is positively associated with VEGF expression. 62These AM characteristics indicate its role in the mesenchymal transition.

STC1 and CaSR
Stanniocalcin-1(STC1) is a secreted glycoprotein hormone that, through autocrine and paracrine activities, mediates the PMT of GBM. 63The role of STC1 as a new promoter of GBM metastasis was found to be regulated by four micro-RNAs, including miR-29B, miR-34a, miR-101, and miR-137, as described by Sakata et al. 63 Other research has demonstrated that STC1 activates cyclin 1 and cyclin-dependent kinase 2 (CDK2) to support proliferation in the autocrine loop. 64STC1 participated in the spread of GBM tumors by activating the PI3K/Akt and JNK signaling pathways. 65More significantly, HIF-1 promotes STC1 production in hypoxic environments. 64her molecular autocrine pathways TNC and integrin or CD44 Tenascin C (TNC), an extracellular matrix that rarely expresses in adults and typically expresses during embryonic development. 66TNC is highly expressed in tumor cells and acts in a variety of ways by binding to integrin receptors. 66n the MES-GBM subtype with strong NF-kB signaling activity, TNC is found to be up-regulated, according to research by Angel et al. 67 Furthermore, TNC was shown to be positively correlated with the expression of MES markers such as STAT3, TGF-b, and CD44, while negatively correlated with PN phenotypic markers like OLIG2, DLL3, and ASCL1. 67NC was demonstrated to have a strong relationship with HIF-1, harming endothelial cells and encouraging GBM angiogenesis, invasion, and proliferation. 68TNC silencing attenuated GSCs' proliferation, migration, and renewal ability. 67he interplay of CD44 receptors in GBM and TNC in ECM further supports the link between PMT and TNC. 67Gupta et al showed that CD44 and TNC are co-expressed and linked to YBX1, a potential regulator of tumor invasion, in GBM. 69hese demonstrate that GBM can be affected by TNC in the ECM to encourage mesenchymal transformation.

Ephrin-B2 and EPHB4
The TNC stated above can stimulate the expression of Ephrin-B2. 67Angel et al also discovered that TNC played a novel autocrine function in the plasticity of glioma cells and the production of TDECs (tumor-derived endothelial cells) by activating the NOTCH signaling pathway and up-regulating and secreting the pro-angiogenic EphrinB2 signaling axis. 67Other studies also verified that Ephrin-B2 is the main element of TNC-induced angiogenesis. 68Thus, TNC and Ephrin-B2 are significant GBM mesenchymal transition factors.

Chemerin and CMKLR1
Chemerin, also known as retinoic acid receptor responder protein 2 (RARRES2), is a released protein that interacts with CMKLR1 and influences the development of different tumors. 70The mesenchymal phenotype of GBM cells is reinforced by an autocrine and paracrine pathway that is mediated by chemerin, according to research by Wu et al. 71 Chemerin, which is primarily expressed in TAMs and partially expressed in GBM cells, promoted the mesenchymal characteristics of GBM by inhibiting the ubiquitinproteasomal degradation of CMKLR1, increasing NF-kB pathway activation in the process. 71Studies have demonstrated a strong positive correlation between the expression of mesenchymal markers such as NeCa, CD44, and VIM and the expression of chemerin produced by GBM tumor cells. 71Additionally, the effect of chemerin on the mesenchymal transition can be increased by TNF-a production. 71M and PIP5K1A A type of non-glycosylated protein called b2-microglobulin (b2M) plays a crucial role in the major histocompatibility complex-1 (MHC-1) for the presentation of antigens.72 Li et al showed that b2M was also a crucial regulator of GSC proliferation, maintenance, and self-renewal, through both autocrine and paracrine effects interacting with PIP5K1A and activating the PI3K/AKT/mTOR pathways.73 Additionally, b2M also promoted the synthesis and secretion of TGF-b1, demonstrating that it is a regulator essential for the mesenchymal transition.73

WISP1 and integrin a6b1
Wnt/b-catenin signaling is extremely active in GSCs in GBM, promoting tumor growth and malignant progression. 74Tao et al found that the Wnt-induced signaling protein 1 (WISP1) released by GSCs in GBM stimulated the remodeling of the tumor microenvironment and preserved the stem cell characteristics of GBM through autocrine and paracrine processes. 74WISP1 binds to the integrin a6b1 receptor in the autocrine pathways and stimulates the PIK3-Akt pathways to mediate self-renewal and proliferation, related to mesenchymal features. 74Another study discovered that inhibiting WISP1 induced apoptosis and cell cycle arrest in glioblastoma cells and inhibited their ability to proliferate, migrate, and invade. 75TAMs M2 polarization and persistence have also been shown to be supported by WISP1/a6b1 pathway (described below).
HMGB1 and TLR2/TLR4/RAGE/CXCR4/CXCR12 High mobility group box-1 protein 1 (HMGB1) is a DNA chaperone, which is often secreted by immune cells. 76MGB1 has been linked to a number of biological activities, including DNA repair, transcription, cell proliferation, and migration. 76According to Cheng et al, HMGB1 is an independent prognostic biomarker for GBM patients. 16In the tumor microenvironment, HMGB1 released from GBM cells interacts with several receptors, including TLR2/TLR4 and RAGE. 16As a result of HMGB1-RAGE binding, the AKT and ERK signaling cascades were activated, which promoted GBM cell invasion. 16Another research demonstrated that the interaction of HMGB1 and TLR2/4 promoted NF-kB activation. 77Moreover, hypoxia also up-regulated the expression of HMGB1. 78

EREG and EGFR
The oncogene Rab27b, a member of the Rab family, facilitates the growth and invasion of some kinds of tumors. 79ab27b mediated the radioresistance of GBM cells, as demonstrated by Nishioka et al. 80 Following radiation, Rab27b causes the release of epithelial regulatory protein (EREG), which interacts with EGFR to encourage the development of neighboring cells.80 The study also showed that PMT signaling pathways, such as NF-kB, STAT3, and MAPK, are downstream of EREG.80 Additionally, they demonstrated that the release of substances via the EREG/ EGFR pathway following radiation modified the subtypes and/or transdifferentiation of cancer cells in the GBM tumor microenvironment, 80 suggesting a connection to PMT.Other research has also demonstrated that the EREG/ EGFR pathway, which is mediated by Rab27b, has significant impacts on cancer cell proliferation, survival, invasion, and microenvironment modification.81

Paracrine pathways
Paracrine occurs when the transmitting ligand is expressed in a different cell than the matching recipient.GBM cells modify the microenvironment by secreting various molecules, including cytokines, chemokines, colony-stimulating factors, and growth factors.Meanwhile, normal cells in the microenvironment introduce pro-oncogenic molecules to paracrine receptors in GBM cells.This crosstalk through paracrine pathways constructs the foundation of GBM pathology by inducing angiogenesis, proliferation, invasion, and metastasis. 82This study summarizes the current paracrine pathways related to PMT in GBM and aims to provide direction for future research (Fig. 2 and Table 2).

Paracrine pathways of GBM and tumor-associated macrophages
Tumor-associated macrophages (TAMs) are main non-tumor cells in the tumor microenvironment (TME), which have a complex paracrine pathway with GBM cells. 83In addition, it is confirmed that the mesenchymal phenotype and the M2 phenotype of TAMs are related significantly. 21Not only do GBMs through paracrine pathways recruit and polarize M2 TAMs, but M2 TAMs also through paracrine pathways facilitate the malignant mesenchymal progression in GBM. 22,23In conclusion, investigating the interactions between GBM and TAM might help to clarify PMT and provide fresh guidance for the treatment of GBM.
Paracrine pathways of GBM recruiting and polarizing TAMs sICAM and LFA-1 of TAMs Soluble intercellular adhesion molecule-1 (sICAM-1) is a transmembrane glycoprotein that functions as an intercellular adhesion ligand. 84Yoo et al demonstrated that GBM cells generated sICAM-1 following radiation therapy, which worked in conjunction with the LFA-1 receptor on TAMs to encourage macrophage infiltration and enrich the tumor microenvironment with inflammatory macrophages. 84Additionally, their research demonstrated that the binding of sICAM and LFA-1 initiated the signaling pathway of WNT3A, a member of the family of airfoil-free MMTV integration sites, resulting in GBM mesenchymal transformation. 84X and integrin a1b1 of TAMs Lysyl oxidases (LOX) secreted by GBM cells function as a potent macrophage chemoattractant via activation of the integrin a1b1-PYK2 pathway in macrophages.85 These infiltrating macrophages secrete OPN, which sustains glioma cell survival and stimulates angiogenesis (described below).In different research, Chen et al used functional Crosstalk between glioblastoma and tumor microenvironment studies and analysis of glioma cells in a GBM pleomorphism model to discover that PTEN deficiency activates YAP1, directly promotes the LOX/integrin a1b1 pathway, and concurrently increases the expression of GBM mesenchymal molecules CD44 and VIM.85

CSF-1 and CSF-1R of TAMs
The colony-stimulating factor-1 (CSF-1) released by GBM influences immunological aggregation and infiltration features by binding with the CSF-1R receptor of TAMs. 86esearch has demonstrated that the combination of CSF-1 and CSF-1R can promote various pro-tumor anti-inflammatory responses, including maintaining the M2 phenotype of TAMs and accelerating the formation of GBM. 86These evidences point to a connection with GBM's mesenchymal characteristics.
WISP1 and integrin a6b1 of TAMs PIK3-Akt is activated by the autocrine pathways of WISP1 to encourage GBM self-renewal and proliferation, as previously mentioned. 74Additionally, WISP1, which is secreted by GBM cells, interacts with the integrin a6b1 receptor on TAMs to support polarization and maintenance of M2-type TAMs. 74This offers more proof that WISP1 and PMT are related.
b2M and PIP5K1A, LILRB1 of TAMs As demonstrated by Li et al, GBM cells produced b2M, interacting with PIP5K1A receptors on TAMs and activating the SMAD and PI3K/Akt signaling pathways. 73Crucially, this paracrine b2M/PIP5K1A pathway polarized TAMs to the M2 phenotype, mediating immune infiltration with mesenchymal characteristics. 73urthermore, leukocyte immunoglobulin-like receptor B1 (LILRB1) on TAMs and b2M have been found to interact to send immunological escape signals. 73emerin and CMKLR1 of TAMs Chemerin mentioned above also participates in the PMTrelated paracrine pathway.Chemerin has been demonstrated to be involved in microenvironmental TAM recruitment by Wu et al. 71 Moreover, it mediates the immunosuppressive milieu and encourages the conversion of the TAM phenotype to the M2 phenotype primarily through the CMKLR1/NF-kB axis.71 Furthermore, when TAMs are recruited and polarized by GBM overexpressing chemerin, inflammatory factors such as IL-1b and TNF-a, as well as immunosuppressive factors such as PD-L1 and TGF-b, are up-regulated.71,87 In conclusion, the chemerin/CMKLR1 axis plays a role in both the associated intracellular PMT pathways and the activation of the external immunosuppressive milieu.

OPN and integrin aVb3 of TAMs or MDSCs
Through the paracrine pathway, GBM-released OPNs attach to integrin aVb3 of TAMs to encourage the recruitment of macrophages and the differentiation of the mesenchymal phenotype through angiogenesis. 53,88As a result, TAMs produce more OPN and draw more TAMs into the microenvironment. 53Some studies claim that OPN can cause M2 polarization and sustain TAMs, but its primary purpose is to uphold and consolidate the M2 phenotype. 53Another discovery showed that by activating STAT3, the master factor of mesenchymal transition, OPN encourages the growth of myeloid-derived suppressor cells (MDSCs) and suppresses anti-tumor immunity. 89

MARCO of TAMs
According to a recent study, recombinant macrophage receptor with collagenous structure (MARCO) in TAMs plays a role in PMT.Highly expressed MARCO TAMs cause GSCs to phenotypically change to a mesenchymal form, boosting invasion and proliferation activities as well as radiation treatment resistance. 90,91Also, it greatly sped up in vivo tumor implantation and development. 90Moreover, when PTEN is lost, the PI3K pathway is more commonly abnormal in TAM cancers with high levels of MARCO. 90O and EPOR of microglia and oligodendrocytes EPO also mediates paracrine pathways.59 Additionally, the mesenchymal characteristics of GBM are mediated by the EPO secreted by GBM, which can act on the EPOR of microglia and oligodendrocytes in the microenvironment, promote tumor survival, proliferation, and angiogenesis.57,92 Paracrine pathways of TAMs promoting GBM OPN of TAMs and integrin aVb3 of GBM As previously mentioned, LOX mediates OPN secretion.85 OPN released by M2 TAMs is a ligand of integrin aVb3 receptors in GBM cells. 88OPN/integrin aVb3 paracrine pathway attenuated tumor cell death.53,88 Wei et al discovered that the paracrine mode of interaction between a subset of OPN þ TAMs and cancer cells promoted GBM mesenchymal transition and that this effect is related to hypoxia.93 TGF-b1 of TAM and TGF-bR2 of CD133 D GBM Transforming growth factor-b1 (TGF-b1), an immunosuppressor, is primarily secreted by microglia in TAMs.94 M2type TAMs have been shown to have a significant impact on TGF-b1 secretion, and microglia and monocyte-macrophages are found to be the major contributors to early and late secretion.94 In the paracrine pathway, TGF-bR2 on CD133 þ GSC can bind with TGF-b1 released by TAMs, and mediate a number of responses. 94 Fily, it promoted GBMs' MMP-9 expression and invasion.94,95 Secondly, TGF-bR2 promoted GBM self-renewal by transactivating TGF-bR1, which then activates ERK, PI3K-Akt, and p38 intracellular signals.94 These facts imply that TGF-b1 secreted by TAMs, is crucial for sustaining and developing mesenchymal features.

PTN of TAM and PTPRZ1 of GBM
A liver-binding glycoprotein called polytrophin (PTN) is expressed by the CD11b þ CD163 þ M2 TAMs, stimulating the growth and proliferation of cancer cells by binding to the protein PTPRZ1 (protein tyrosine phosphatase, receptor type Z1) on GBM cells. 96Studies have shown that PTN-PTPRZ1 signaling controls the phosphorylation of Fyn, activating the AKT pathway for the maintenance of GSCs. 97he PTN-PTPRZ1 paracrine pathway induced by M2 TAMs seems to be another manner in which M2 TAMs facilitate GBM mesenchymal transition. 97M of TAMs and OSMR, LIFR, IL-6R of GBM cell or PDGFRA D oligodendrocytes Oncostatin M (OSM) is a member of the IL-6 family.During routine bodily functions, neutrophils and macrophages release OSM.98 OSM is mostly secreted in inflammatory settings, and studies have shown that it is related to the growth of malignancies and has a significant function in PMT.99 OSM possesses the most comprehensive downstream signaling pathways of the IL-6 family, including the Jak-STAT3, ERK1/ ERK2, PI3K/Akt, NF-B, and Jak-STAT3 pathways.99,100 The most frequently mentioned OSM downstream signaling pathway among them is Jak-STAT3.101 In the GBM milieu, microglia in TAMs are primarily responsible for secreting OSM.In line with this, its receptors OSMR and LIFR are expressed in PDGFRA þ oligodendrocytes and GBM tumor cells.101 OSM-OSMR is a critical pathway for promoting tumor cell malignancy.Hara et al demonstrated that OSM activates STAT3 by binding to OSMR, LIFR, and IL-6R, and subsequently triggers various intracellular pathways.102 The normal function of STAT3 is essential for this process. Stdies showed that several chemokines, including CCL3/ 3L1/4/4L2/5/7/8, CSF1, and CXCL2/3/8, co-express with OSM, 101 indicating that these chemokines might play a role in PMT.103 In conclusion, the OSM-OSMR pathway promotes PMT depending on the crosstalk between GBM and TAMs.

Paracrine pathways between GBM and endothelial cells
Angiogenesis, particularly in the MES subgroup of GBM, results from communication between GBM and nearby vascular endothelial cells.These cells, along with TAMs, have a paracrine network induced by ligand/receptor interactions with GBM in the TME. 104,105As a result, a method for understanding PMT may also be found in the paracrine pathway of endothelial cells and GBM.

Paracrine pathways of GBM promoting angiogenesis
CXCL-8 and CXCR2 of endothelial cells Shen et al found that CXCL-8 plays a critical regulatory function in angiogenic tissue repair by inducing vascular endothelial cells to secrete cytokines like VEGF and SOD when skin injury happens. 106Through binding to CXCR2 on endothelial cells, CXCL-8 secreted by GBM cells promotes GBM angiogenesis and mesenchymal characteristics. 107oreover, up-regulation of Bcl-xL protein in GBM triggers the CXCL-8/CXCR2 axis, which leads to the stimulation of biological processes like cell proliferation, endothelial cell migration, and the formation of vital vascular structures in the central lumen. 43GF and VEGFR of endothelial cells Vascular endothelial growth factor (VEGF) possesses a proangiogenic impact that has been well documented.108 Along with an autocrine cycle, VEGF also mediates paracrine pathways.VEGF released by GBM interacts with VEGFR of vascular endothelial cells to increase angiogenesis and vascular permeability, which facilitates the transformation of the tumor into a mesenchymal phenotype.36 CXCL-12 and CXCR4/7 of endothelial cells As previously stated, CXCL12 and its receptor, CXCR4, were directly related to hypoxia. 47  addition to driving angiogenesis in GBM through autocrine activation of PMT-related pathways, CXCL12 secreted by GBM also interacts with CXCR4 in vascular endothelial cells through paracrine actions.109 After this, vascular endothelial cells raise the transcript of CXCR4, which facilitates the binding of CXCL-12 and CXCR4.110 Additionally, the migration of GBM tumors induced by CXCL12 also relies on endothelial cells that express CXCR7 in hypoxic environments.110 FGF5 and FGFR1(IIIc)of endothelial cells Fibroblast growth factor-5 (FGF5) is another ligand that plays a role in mesenchymal transition through paracrine signaling.FGF5 released by GBM is a vital ligand of FGFR1 (IIIc) in vascular endothelial cells, and the activation of the FGF5/FGFR1 (IIIc) pathway enhances the angiogenesis in GBM.37 STC1 and CaSR of endothelial cells STC1, which was already stated, also interacted with CaSR in endothelial cells.63 Sakata et al showed that STC1 activated the VEGF signaling pathways of endothelial cells and increased the levels of eNOs, VEGF, and VEGFR2 in both mRNA and protein.63 Paracrine STC1 can act on endothelial cells to promote angiogenesis.63 Ephrin-B2 and EPHB2 of endothelial cells As previously mentioned, ephrin-B2 also mediates the paracrine pathway.Paracrine ephrin-B2 in GBM cells is regulated by TNC and binds to EPHB2 receptors in vascular endothelial cells, inducing angiogenesis and regulating the mesenchymal transition of GBM.68 Paracrine pathways of endothelial cells on GBM progression DLL4/jaggd-1/eNOs, IL-6 of endothelial cells and Notch1/2, CXCR1/2 of GBM Sharma et al linked four ligands, including DLL4, jaggd-1, eNOs, and IL-6, to endothelial cell-mediated angiogenic effects in their research.111 The first three improved the GBM stem cell phenotype by influencing Notch signaling by acting on Notch1/2 receptors, promoting neurosphere formation, and inducing tumorigenicity in vivo.111 Additionally, CXCR1/2 receptors in GBM cells bound to IL-6 secreted by endothelial cells mediate the formation of additional perivascular niches.111 These four binding functions promote the development of perivascular niches, which may pave the way for future research on the mesenchymal transition.
Paracrine pathways between GBM and others DA and transferrin (TF) of PN-GBM and TFR1 of MES-GBM Numerous researches have shown that metabolic rewiring of specific tumor cells leads to intra-tumoral metabolic symbiosis, which is also known as "mutualism" and "commensalism". 112This biochemical co-existence promotes tumor heterogeneity by allowing metabolites and signaling molecules to circulate between different kinds of tumor cells. 112Vo et al have demonstrated that PN GBM cells secrete DA to foster self-development. 113Additionally, through a paracrine mechanism, DA also activates TFR1 receptors in tumor cells with the MES phenotype in a different tumor subtype region.TF then mediates iron uptake by MES subtype cells and lowers the risk of ferroptosis by acting on TFR1. 113u and NMDAR/AMPAR of neurons GBM-produced Glu stimulates Ca 2þ -permeable NMDAR and AMPAR not only in GBM cells but also in nearby cells, including neurons.114 It was demonstrated that this paracrine pathway of Glu acted on nearby neurons' NMDAR/ AMPAR receptors, promoted excitotoxic neuronal death, made room for movement, and significantly boosted tumor growth, migration, and tumorigenic potential, eventually promoting mesenchymal phenotype.114 Kinins of MSCs and kinins-B1/B2 of GBM The kallikrein-kinin system is an endogenous metabolic pathway that results in the release of kinins, regulating a number of physiological functions.115 Pillat et al demonstrated the communication between mesenchymal stem cells (MSCs) and GBM cells is facilitated by kinin ligands secreted by MSCs and the GBM kinin B1 and B2 receptors.116 These signals play a role in the migration of GBM cells and angiogenesis.116 B1 receptors in particular have been explicitly proven to be crucial for GBM mesenchymal phenotype.116 C5a of MSCs and C5aR1 of GBM Complement C5a, which MSCs secrete, is both a chemokine for GBM and a molecule with mesenchymal characteristics.117 According to the study of Lim et al, C5a binding to GBM C5aR1 induces the elevation of ZEB1, which is a crucial regulator of the mesenchymal process. 117The p38 MAPK pathways are then activated by ZEB1 to promote invasiveness and shift to mesenchymal phenotype.117 IL-6/IL-11 of CAFs and IL-6R/IL-11R of GBM CAFs (cancer-associated fibroblasts) are responsible for GBM mesenchymal characteristics.118 Studies have demonstrated that CAFs cause the release of interleukins like IL-6 and IL-11 interact with IL-6R and IL-11 on GBM cells, activating the intracellular JAK/STAT3 pathway or the CXCL12/CXCR4 signaling pathway and increasing the activity of cancer cells in terms of invasion, migration, and metastasis.118,119 CXCL-12 of CAFs and CXCR4 of GBM CAFs in the tumor microenvironment secrete more CXCL12 as a result of increased hypoxia, which causes GBM to express more CXCR4 (as described previously).Additionally, the CXCL12/CXCR4 axis can recruit arterial endothelial cells directly, aiding in angiogenesis and mediating the mesenchymal transition.47 HA of ECM and CD44, RHAMM of GBM Hyaluronic acid (HA), which is prevalent in invasive cancer cells, makes up a significant portion of the brain ECM.29 According to So et al, HA interacted with the GBM CD44mediated hyaluronic acid mediates movement receptor (RHAMM) to promote the growth, migration, and invasion of GBM. 29 Moreover, HA plays a role in MMP secretion, indicating a connection to mesenchymal transition.29,120 Therapies targeting autocrine and paracrine pathways inducing proneuralemesenchymal transition The increasingly interaction complex mechanism between GBM and the microenvironment has led to the development of receptor and ligand-related drugs that target the microenvironment. 121These therapies inhibit PMT-related targets, such as EGFR, TGF-b, VEGF, PDGFR, and FGFR (Table 3). 121owever, the bloodebrain barrier significantly hinders drug delivery and effectiveness for GBM in the brain. 121Clinical research on GBM has focused on studying cases with recurrence, refractoriness, and drug resistance, which may have acquired a mesenchymal phenotype.Here are some recent clinical study findings on targeted therapeutic medicines from autocrine and paracrine perspectives.

Therapies targeting autocrine pathways EGFR
A popular molecule for targeted treatment is the epidermal growth factor receptor (EGFR), which is connected to PMT. 122 The EGFRvIII mutant indicates a poor prognosis for GBM. 123Research has focused on immunotherapy and targeted medications, such as Afatinib, which demonstrated limited single-agent activity in unselected individuals with recurrent GBM. 124Depatuxizumab mafodotin (depatux-m) did not provide survival benefits over placebo in phase III clinical study. 125A 2021 trial using depatux-m to treat EFGR-amplified recurrent GBM had no success. 126Pulse high-dose lapatinib is a safe regimen for newly-diagnosed GBM, but it may cause lymphopenia. 127Immunotherapy, such as EGFRvIII-targeted peptide vaccines or anti-EGFR immunoliposomes, has shown progress in some patients but not all. 128,129Rindopepimut, an anti-EGFRvIII vaccine, did not extend survival time in patients with freshly diagnosed glioblastoma. 130Targeted treatment for EGFR requires more study, particularly in relapsed or refractory patients.

Therapies targeting paracrine pathways TGF-b
The paracrine-related molecule TGF-b, which is closely linked to mesenchymal transformation, was previously noted. 94Studies targeting TGF-b are also being done, but they are still tiny and ineffective. 131,132One explanation is that there is a greater interplay between this molecule's release and acceptance, and as a result, the therapeutic therapy impact is not what was anticipated.

Therapies targeting autocrine and paracrine pathways
VEGF VEGF is a key molecule in the angiogenesis of MES-GBM, and previous clinical studies have targeted its paracrine effect with the monoclonal antibody bevacizumab. 121Recent research focuses on combining bevacizumab with other treatments, such as irinotecan and temozolomide. 133owever, other VEGF-targeting combinations like ponatinib, vorinostat, and trebananib appear ineffective, and trebananib may even have harmful interactions with bevacizumab.134e136 Another research that attempted to establish whether the addition of bevacizumab would result in patients with initial progression of malignant gliomas having longer overall survival than lomustine alone came up empty-handed. 137Patients with recurrent glioblastoma can use aflibercept (VEGF trap), a recombinant fusion protein that eliminates VEGF and placental growth factors. 138owever, a study found that aflibercept monotherapy has moderate toxicity and little indication of single-agent activity in unselected patients with recurrent malignant glioma. 138In conclusion, a more thorough study is still needed on VEGF-targeted therapy for GBM.Phase II in newly-diagnosed GBM: tolerable and safe regimen, but higher rates of lymphopenia should be noted (ClinicalTrial.govNCT01591577) 129

TGF-b
Galunisertib Phase II of with Galunisertib with temozolomide-based radiochemotherapy (TMZ/RTX) in newly diagnosed malignant GBM: no differences in efficacy, safety or pharmacokinetic variables were observed between the two treatment arms (ClinicalTrials.govNCT01220271) 133

Aflibercept
Phase II in recurrent malignant GBM: moderate toxicity and minimal evidence of single-agent activity (ClinicalTrials.govNCT00369590) 140

FGFR
The available targeted pharmacological therapy for FGFR is currently limited, even though FGFR is also a common effector receptor of autocrine and paracrine. 37Nintedanib showed no effect on relapsed high-grade gliomas, regardless of prior bevacizumab therapy, according to Norden et al's investigation of these tumors. 139Dovitinib was found to not affect PFS in patients with relapsed GBM despite early anti-angiogenic therapy (including bevacizumab), according to another trial of relapsed gliomas at the same target. 140In actuality, neither of these two medications particularly targets FGFR.Poor clinical benefit could have several causes, including low specificity.

PDGFR
The PDGFR autocrine pathway in GBM is the subject of some clinical research as well.Targeting VEGFR1-3, FGFR1-3, and PDGFR-a/b, nintedanib (BIBF 1120) is a small, orally accessible, triple angiokinase inhibitor that is in phase III development. 139However, in patients with recurrent GBM who had not responded to 1/2 lines of prior therapy, single-agent nintedanib (200 mg bid) showed limited but clinically insignificant antitumor activity. 139dditionally, Muhic et al attested to nintedanib's ineffectiveness. 141Another medication that targets PDGFR is dasatinib.However, despite efforts to broaden the community and raise the dose, Dasatinib was unable to show efficacy as a monotherapy for recurrent GBM, according to research by Lassman et al. 142 All in all, most clinical studies indicate that the prospects for the use of targeted drugs in the therapy of GBM are dim.The mechanism of immunotherapy resistance may involve the fluctuating and uncontrollable spliceosome and an immunosuppressive microenvironment.Additionally, the multithreading of receptor ligands may also contribute to drug resistance. 6If there is a promising future for molecularly targeted drugs with other PMT-related receptor ligands in the microenvironment, more clinical practice and study are required.

Conclusion
GBM treatment resistance remains a challenge, and new strategies are needed to confront its heterogeneity and replace traditional cytotoxic chemotherapy.Technologies such as scRNA-seq and spatial transcriptome sequencing have increased our understanding of tumor heterogeneity.This review shows that GBM achieves PMT through complicated autocrine and paracrine systems, with multiple signals and various types of cells that mutually complement each other.Inhibiting a single target or pathway might not be sufficient.
GBM medication resistance induced by PMT is influenced by various extrinsic and intrinsic factors, including tumor heterogeneity, hypermutation, altered metabolomics, and oncologically activated alternative splicing pathways.In addition, immunotherapy often fails in GBM due to hypoxia and an immune-suppressive tumor microenvironment.Efforts are currently focused not only on reducing immunotolerance but also on preventing tumor cell escape mechanisms from treatment, which are caused by interand intra-tumoral heterogeneity.
To overcome the treatment resistance resulting from intricate autocrine and paracrine systems, two potential directions are worth studying: one is to explore novel effective therapeutic regimens, and the other is to combine multiple therapies.As we summarized, TAMs play a crucial role in PMT through paracrine manner, immunotherapy especially for TAMs including engineered immune cells such as chimeric antigen receptor-macrophage (CAR-M) might be a promising approach. 143Preclinical studies of combination therapies were also performed to confront the PMT.What's more, combination strategies are attracting accumulating attention.For example, dual targeting of polyunsaturated fatty acid synthesis and EGFR signaling has shown a combinatorial cytotoxic effect on GSCs. 144Our former study also demonstrated that the combination of targeting spliceosome and NF-kB therapy significantly inhibited PMT. 145Although the security of these combination therapies still needed to be confirmed, a combination strategy might be prospective to overcome the mechanisms of mutual complement in the autocrine and paracrine systems.
The proposed receptoreligand interaction pathways in this review highlight the complexity of the GBM microenvironment, involving various molecules such as neurotransmitters, chemokines, hormones, growth factors, and secreted glycoproteins.We classified these ligands and described their associations with PMT.In addition to receptors and ligands, cell-to-cell connections also include direct contact, gap junctions, intercellular nanotube tunnels, and exosomal vesicles, which require further in-depth study.
In conclusion, this study reviews the molecular pathways of PMT mediated by receptor and ligand binding in the tumor microenvironment, and it is hoped to provide help and support for future work in related fields.ogy Projects in Guangzhou, Guangdong

Figure 1
Figure 1 Autocrine mechanism controls PMT.Different ligands bind to their respective receptors to activate corresponding pathways and up-regulate the expression of related molecules to promote tumor progression in a mesenchymal manner.

Figure 2
Figure 2 Paracrine mechanism controls PMT.This diagram demonstrates how cellecell ligand-receptor binding affects PMT modulation in the GBM microenvironment.TAMs, endothelial cells, mesenchymal stem cells, and fibroblasts are the main paracrine mechanisms that engage with tumor cells in the GBM microenvironment.

Table 1
Molecular pathways of GBM autocrine promoting ProneuraleMesenchymal Transition.

Table 2
Molecular pathways of GBM paracrine promoting ProneuraleMesenchymal Transition.

Table 3
Therapies targeting autocrine and paracrine pathways inducing proneuralemesenchymal transition.