Transmembrane‐4 L‐six family member‐1 (TM4SF1) is an atypical tetraspanin that is highly and selectively expressed in proliferating endothelial cells and plays an essential role in blood vessel development. TM4SF1 forms clusters on the cell surface called TMED (TM4SF1‐enriched microdomains) and recruits other proteins that internalize along with TM4SF1 via microtubules to intracellular locations including the nucleus. We report here that tumor growth and wound healing are inhibited in Tm4sf1‐heterozygous mice. Investigating the mechanisms of TM4SF1 activity, we show that 12 out of 18 signaling molecules examined are recruited to TMED on the surface of cultured human umbilical vein endothelial cells (HUVEC) and internalize along with TMED; notable among them are PLCγ and HDAC6. When TM4SF1 is knocked down in HUVEC, microtubules are heavily acetylated despite normal levels of HDAC6 protein, and, despite normal levels of VEGFR2, are unable to proliferate. Together, our studies indicate that pathological angiogenesis is inhibited when levels of TM4SF1 are reduced as in Tm4sf1‐heterozygous mice; a likely mechanism is that TM4SF1 regulates the intracellular distribution of signaling molecules necessary for endothelial cell proliferation and migration.

Transmembrane-4 L-six family member-1 (TM4SF1) is a small cell surface glycoprotein that is highly and selectively expressed on endothelial cell and mesenchymal stem cell surfaces. TM4SF1 regulates cellular functions by forming protein complexes called TMED (TM4SF1-enriched microdomains) that either recruit growth-factor activated proteins and internalize them via microtubules to distribute the recruited molecules intracellularly or support the formation of nanopodia for intercellular interactions extracellularly. Through a genetically manipulated mouse model for global Tm4sf1 gene knockout, we demonstrate here that TM4SF1 is essential for blood vessel development. Tm4sf1-null embryos fail to develop blood vessels and experience lethality at E9.5. Tm4SF1-heterozygous embryos are smaller in body size during early embryonic development, and almost half die in utero due to intracranial hemorrhage in the intraventricular and subarachnoid space, which becomes apparent by E17.5. Surviving Tm4SF1-heterozygotes do not display overt phenotypic differences relative to wild type littermates postnatally. Together, these studies demonstrate that TM4SF1, through its molecular facilitator and nanopodia formation roles in TMED, intimately regulates blood vessel formation during embryonic development.

Transmembrane-4 L-six family member-1 (TM4SF1) is a small cell surface glycoprotein that is highly and selectively expressed on endothelial cells and mesenchymal stem cells. TM4SF1 regulates cellular functions by forming protein complexes called TMED (TM4SF1-enriched microdomains) that internalize via microtubules from the cell surface and transport recruited proteins to intracellular locations including the nucleus. Through a genetically manipulated mouse model, we demonstrate here that Tm4sf1 is essential for blood vessel development. Tm4sf1 null embryos fail to develop blood vessels and experience lethality at E9.5. Tm4SF1 heterozygous embryos are smaller in body size during early embryonic development, and almost half die in utero due to intracranial hemorrhage in the intraventricular and subarachnoid space which becomes apparent by E17.5. Surviving Tm4SF1 heterozygotes do not display overt phenotypic differences relative to wild type littermates postnatally. These findings confirm that Tm4sf1 is essential for embryonic blood vessel development.

Endothelial cell proliferation, migration, and intercellular interactions for blood vessel formation require coordinated signaling by a myriad of molecules. Following endothelial cell activation by growth factors and cytokines, a variety of signaling molecules are activated on the surface and transported intracellularly by TM4SF1-enriched microdomains (TMEDs), 100–300 nm diameter protein–lipid complexes recruited by the transmembrane protein TM4SF1. TMEDs internalize via microtubules from the cell surface toward the microtubule-organizing center (MTOC) and then enter the nucleus via nuclear pores (see Graphic Illustration). This internalization pathway permits delivery of activated proteins and other signaling molecules from the cell surface to the nucleus, which directly translates extracellular stimuli to modulation of gene expression. Molecules transported by this route include phospholipase C, gamma 1 (PLCγ1), histone deacetylase 6 (HDAC6), and importins. In the absence of TMEDs, endothelial cells lose the ability to divide into cultures in vitro and to support blood vessel formation in mouse embryos in vivo. We liken TMEDs to cable cars, which take in passengers at the cell surface, travel along microtubule cables, and deliver their passengers to various locations, including the “city center”, the nucleus. This commentary aims to elucidate the functions of TMEDs in endothelial cells, to show that cells, like busy cities, need efficient transport systems to deliver molecules to the destinations where they perform their cellular functions. TMEDs offer a novel and curated transport system providing selected molecules with access to the nucleus.

Antibody–drug conjugates (ADCs) are a promising therapeutic modality for treating cancer. TM4SF1 is an integral membrane protein that internalizes from the cell surface along microtubules to the nucleus and is highly expressed on the surface of both tumor endothelium and tumor cells. We previously reported that in human tumor xenografts in mice, an ADC directed to mouse TM4SF1 (2A7A-LP2) effectively regressed tumors through an anti-vascular mechanism, and an ADC directed to human TM4SF1 (v1.10-LP2) effectively regressed tumors through an anti-tumor cell mechanism. In this study, we investigated the actions of the mouse TM4SF1-directed ADC on VEGF-A-provoked angiogenic vessels. We employed an adenovirus expressing mouse VEGF-A¹⁶⁴ (Ad-VEGF-A) to induce surrogate tumor blood vessels in the ears of nude mice. We showed that an immune effector function-ablated ADC, 3m2A7A-LP2, was better tolerated than its parent 2A7A-LP2. Homing of 3m2A7A to Ad-VEGF-A-induced new blood vessels became evident within six hours after intraperitoneal injection. A single dose of 3m2A7A-LP2 at 3 mg/kg disrupted evolving Ad-VEGF-A-provoked blood vessels within forty-eight hours, and three doses of 3m2A7A-LP2 at 48 h intervals caused striking local ear necrosis; in each case, there was no apparent harm to vessels in the corresponding control virus-injected ears and the surrounding tissues of the same mice. Our studies demonstrate that an ADC-directed against mouse TM4SF1 specifically targeted the newly formed blood vessels induced by Ad-VEGF-A at multiple stages of their development. Thus, TM4SF1-directed ADCs, through their ability to target angiogenic vessels, represent an alternative anti-angiogenic approach for treating solid tumors.

Proliferating cells with volumes of 2,000–4,000 μm3 contain about ten billion protein molecules, making it challenging, in a crowded cellular environment filled with vesicles and organelles, for proteins to find their interaction partners and coordinate signaling. In endothelial cells, we discovered a key molecule transport pathway mediated by TM4SF1-enriched microdomains (TMED). TMED are 100-300 nm diameter proteinlipid complexes formed by clusters of TM4SF1, a small plasma membrane glycoprotein with tetraspanin topology. TMED internalize via microtubules from the cell surface toward the microtubule organizing complex and then enter the nucleus via nuclear pores (see Graphic Illustration). This internalization pathway permits direct communication of extracellular stimuli to intracellular gene expression regulation. Key molecules involved in cell proliferation and cell migration such as phospholipase C, gamma 1 (PLCγ1) and histone deacetylase 6 (HDAC6) are among the signaling proteins transported. In the absence of TMED, endothelial cells lose the ability to divide in vitro and to support blood vessel formation in vivo. We propose that TMED act like cable cars, take in passengers at the cell surface, ride along microtubule cables, and deliver their passengers to various locations including the “city center”, the nucleus. This commentary aims to illustrate this analogy through TMED in endothelial cells, to show that cells, like busy cities, need efficient transport systems to deliver passenger molecules to their proper destinations to conduct signaling.