Deciphering hepatoma cell resistance to tyrosine kinase inhibitors: insights from a Liver-on-a-Chip model unveiling tumor endothelial cell mechanisms.

Liver cancer represents a significant global burden in terms of cancer-related mortality, with resistance to anti-angiogenic drugs such as Sorafenib and Lenvatinib presenting a formidable challenge. Tumor angiogenesis, characterized by the formation of new blood vessels within tumors, plays a pivotal role in cancer progression and metastasis. Tumor endothelial cells, specialized endothelial cells lining tumor blood vessels, exhibit unique phenotypic and functional traits that drive aberrant vessel formation and contribute to therapy resistance. CD105, a cell-surface glycoprotein that is highly expressed on endothelial cells during angiogenesis, including tumor endothelial cells, regulates endothelial cell proliferation, migration, and vessel formation by modulating transforming growth factor-beta (TGF-ß) signaling pathways. Elevated CD105 expression on tumor endothelial cells correlates with increased angiogenic activity and poor prognosis in cancer patients. Targeting CD105 with antibodies presents a promising strategy to inhibit tumor angiogenesis and disrupt tumor vasculature, offering potential therapeutic benefits by interfering with the tumor microenvironment and inhibiting its progression. This study investigates tumor angiogenesis through a three-dimensional (3D) microfluidic co-culture system incorporating endothelial cells and hepatocellular carcinoma (HCC) cells. The primary focus is on the role of CD105 expression within the liver tumor microenvironment and its contribution to increased chemoresistance. Additionally, this research examines the influence of CD105 expression on the efficacy of tyrosine kinase inhibitors (TKIs) and its pivotal function in facilitating angiogenesis in liver tumors. The proposed microfluidic chip model investigates liver cancer cell interactions within a microfluidic chip model designed to simulate aspects of liver tumor angiogenesis.

Antagonizing CD105 and androgen receptor to target stromal-epithelial interactions for clinical benefit.

Androgen receptor signaling inhibitors (ARSIs) are standard of care for advanced prostate cancer (PCa) patients. Eventual resistance to ARSIs can include the expression of androgen receptor (AR) splice variant, AR-V7, expression as a recognized means of ligand-independent androgen signaling. We demonstrated that interleukin (IL)-6-mediated AR-V7 expression requires bone morphogenic protein (BMP) and CD105 receptor activity in both PCa and associated fibroblasts. Chromatin immunoprecipitation supported CD105-dependent ID1- and E2F-mediated expression of RBM38. Further, RNA immune precipitation demonstrated RBM38 binds the AR-cryptic exon 3 to enable AR-V7 generation. The forced expression of AR-V7 by primary prostatic fibroblasts diminished PCa sensitivity to ARSI. Conversely, downregulation of AR-V7 expression in cancer epithelia and associated fibroblasts was achieved by a CD105-neutralizing antibody, carotuximab. These compelling pre-clinical findings initiated an interventional study in PCa patients developing ARSI resistance. The combination of carotuximab and ARSI (i.e., enzalutamide or abiraterone) provided disease stabilization in four of nine assessable ARSI-refractory patients. Circulating tumor cell evaluation showed AR-V7 downregulation in the responsive subjects on combination treatment and revealed a three-gene panel that was predictive of response. The systemic antagonism of BMP/CD105 signaling can support ARSI re-sensitization in pre-clinical models and subjects that have otherwise developed resistance due to AR-V7 expression.

Endoglin in the Spotlight to Treat Cancer.

A spotlight has been shone on endoglin in recent years due to that fact of its potential to serve as both a reliable disease biomarker and a therapeutic target. Indeed, endoglin has now been assigned many roles in both physiological and pathological processes. From a molecular point of view, endoglin mainly acts as a co-receptor in the canonical TGFß pathway, but also it may be shed and released from the membrane, giving rise to the soluble form, which also plays important roles in cell signaling. In cancer, in particular, endoglin may contribute to either an oncogenic or a non-oncogenic phenotype depending on the cell context. The fact that endoglin is expressed by neoplastic and non-neoplastic cells within the tumor microenvironment suggests new possibilities for targeted therapies. Here, we aimed to review and discuss the many roles played by endoglin in different tumor types, as well as the strong evidence provided by pre-clinical and clinical studies that supports the therapeutic targeting of endoglin as a novel clinical strategy.

Endoglin Targeting: Lessons Learned and Questions That Remain.

Approximately 30 years ago, endoglin was identified as a transforming growth factor (TGF)-ß coreceptor with a crucial role in developmental biology and tumor angiogenesis. Its selectively high expression on tumor vessels and its correlation with poor survival in cancer patients led to the exploration of endoglin as a therapeutic target for cancer. The endoglin neutralizing antibody TRC105 (Carotuximab®, Tracon Pharmaceuticals (San Diego, CA, USA) was subsequently tested in a wide variety of preclinical cancer models before being tested in phase I-III clinical studies in cancer patients as both a monotherapy and in combination with other chemotherapeutic and anti-angiogenic therapies. The combined data of these studies have revealed new insights into the role of endoglin in angiogenesis and its expression and functional role on other cells in the tumor microenvironment. In this review, we will summarize the preclinical work, clinical trials and biomarker studies of TRC105 and explore what these studies have enabled us to learn and what questions remain unanswered.

Bioenergetic effects of hydrogen sulfide suppress soluble Flt-1 and soluble endoglin in cystathionine gamma-lyase compromised endothelial cells.

Endothelial dysfunction is a hallmark of preeclampsia, a life-threatening complication of pregnancy characterised by hypertension and elevated soluble Fms-Like Tyrosine Kinase-1 (sFlt-1). Dysregulation of hydrogen sulfide (H2S) by inhibition of cystathionine γ-lyase (CSE) increases sFlt-1 and soluble endoglin (sEng) release. We explored whether compromise in CSE/H2S pathway is linked to dysregulation of the mitochondrial bioenergetics and oxidative status. We investigated whether these effects were linked to CSE-induced sFlt-1 and sEng production in endothelial cells. Here, we demonstrate that CSE/H2S pathway sustain endothelial mitochondrial bioenergetics and loss of CSE increases the production of mitochondrial-specific superoxide. As a compensatory effect, low CSE environment enhances the reliance on glycolysis. The mitochondrial-targeted H2S donor, AP39, suppressed the antiangiogenic response and restored the mitochondrial bioenergetics in endothelial cells. AP39 revealed that upregulation of sFlt-1, but not sEng, is independent of the mitochondrial H2S metabolising enzyme, SQR. These data provide new insights into the molecular mechanisms for antiangiogenic upregulation in a mitochondrial-driven environment. Targeting H2S to the mitochondria may be of therapeutic benefit in the prevention of endothelial dysfunction associated with preeclampsia.

Targeting Endoglin-Expressing Regulatory T Cells in the Tumor Microenvironment Enhances the Effect of PD1 Checkpoint Inhibitor Immunotherapy.

PURPOSE: Endoglin is a coreceptor for TGFß ligands that is highly expressed on proliferating endothelial cells and other cells in the tumor microenvironment. Clinical studies have noted increased programmed cell death (PD)-1 expression on cytotoxic T cells in the peripheral blood of patients with cancer treated with TRC105, an endoglin-targeting antibody. In this study, we investigated the combination of endoglin antibodies (TRC105 and M1043) with an anti-PD1 antibody. EXPERIMENTAL DESIGN: The combination anti-endoglin/anti-PD1 antibodies was tested in four preclinical mouse models representing different stages of cancer development. To investigate the underlying mechanism, Fc-receptor-knockout mice were used complemented with depletion of multiple immune subsets in mice. Tumor growth and the composition of immune infiltrate were analyzed by flow cytometry. Finally, human colorectal cancer specimens were analyzed for presence of endoglin-expressing regulatory T cells (Treg). RESULTS: In all models, the combination of endoglin antibody and PD1 inhibition produced durable tumor responses, leading to complete regressions in 30% to 40% of the mice. These effects were dependent on the presence of Fcγ receptors, indicating the involvement of antibody-dependent cytotoxic responses and the presence of CD8+ cytotoxic T cells and CD4+ Th cells. Interestingly, treatment with the endoglin antibody, TRC105, significantly decreased the number of intratumoral Tregs. Endoglin-expressing Tregs were also detected in human colorectal cancer specimens. CONCLUSIONS: Taken together, these data provide a rationale for combining TRC105 and anti-PD1 therapy and provide additional evidence of endoglin's immunomodulatory role.

Inhibition of Endoglin Exerts Antitumor Effects through the Regulation of Non-Smad TGF-ß Signaling in Angiosarcoma.

Angiosarcoma is a rare malignant tumor derived from endothelial cells, and its prognosis is poor because advanced angiosarcoma is often resistant to taxane therapy. Endoglin (CD105) acts as a coreceptor for TGF-ß signaling and is overexpressed in tumor-associated endothelial cells and enhances tumor angiogenesis. Numerous clinical trials are testing the effectiveness of anti-endoglin antibodies in various types of malignancies. Here, we investigated the role of endoglin in the pathogenesis of angiosarcoma and whether endoglin inhibition results in antitumor activity. Endoglin was overexpressed in angiosarcoma, and its inhibition was effective in promoting apoptosis and the suppression of migration, invasion, tube formation, and Warburg effect in angiosarcoma cells. Knockdown of endoglin activated caspase 3/7 that is essential for apoptosis, reduced survivin levels, and decreased paxillin and vascular endothelial cadherin phosphorylation and matrix metalloproteinase 2 and matrix metalloproteinase 9 activities in angiosarcoma cells. Although endoglin is a coreceptor that regulates TGF-ß signaling, the antitumor effect of endoglin in angiosarcoma was not based on Smad signaling regulation but on non-Smad TGF-ß signaling. Taken together, these results indicated that endoglin could be a novel therapeutic target for angiosarcoma.

Decreased sEng plasma levels in hypertensive patients with endothelial dysfunction under chronic treatment with Perindopril.

Purpose: Endoglin is a transmembrane glycoprotein which plays an important role in maintaining cardiovascular homeostasis. One of its forms, soluble endoglin (sEng), a molecule with antiangiogenic properties, has been found overexpressed in patients suffering from hypercholesterolemia, diabetes mellitus and hypertension, and is proposed as a marker of endothelial damage. Accordingly, we aimed to quantify the efficacy of various antihypertensive regimens on sEng levels, in hypertensive patients with endothelial dysfunction. Patients and methods: 323 patients were enrolled, and there were 99 patients with normal blood pressure values, 106 hypertensive patients under chronic treatment with different types of antihypertensive molecules (beta blockers, calcium channel blockers, and diuretics) in monotherapy, and 118 hypertensive patients under chronic treatment with perindopril. sEng plasma levels were quantified and were correlated with classical methods of assessing the endothelial damage. Results: Patients under chronic treatment with perindopril had lower sEng plasma levels compared with the other group of hypertensive patients under different regimens of antihypertensive treatment (sEng: 4.73±1.39 versus 5.63±2.33, p<0.01). Conclusion: Decreased sEng plasma levels were found in patients under chronic treatment with perindopril, when compared with other antihypertensive regimens of treatment (beta blockers, calcium channel blockers, and/or diuretics).

Anti-CD105 Antibody Eliminates Tumor Microenvironment Cells and Enhances Anti-GD2 Antibody Immunotherapy of Neuroblastoma with Activated Natural Killer Cells.

PURPOSE: We determined whether elimination of CD105+ cells in the tumor microenvironment (TME) with anti-CD105 antibodies enhanced anti-disialoganglioside (GD2) antibody dinutuximab therapy of neuroblastoma when combined with activated natural killer (aNK) cells. EXPERIMENTAL DESIGN: The effect of MSCs and monocytes on antibody-dependent cellular cytotoxicity (ADCC) mediated by dinutuximab with aNK cells against neuroblastoma cells was determined in vitro. ADCC with anti-CD105 mAb TRC105 and aNK cells against MSCs, monocytes, and endothelial cells, which express CD105, was evaluated. Anti-neuroblastoma activity in immunodeficient NSG mice of dinutuximab with aNK cells without or with anti-CD105 mAbs was determined using neuroblastoma cell lines and a patient-derived xenograft. RESULTS: ADCC mediated by dinutuximab with aNK cells against neuroblastoma cells in vitro was suppressed by addition of MSCs and monocytes, and dinutuximab with aNK cells was less effective against neuroblastomas formed with coinjected MSCs and monocytes in NSG mice than against those formed by tumor cells alone. Anti-CD105 antibody TRC105 with aNK cells mediated ADCC against MSCs, monocytes, and endothelial cells. Neuroblastomas formed in NSG mice by two neuroblastoma cell lines or a patient-derived xenograft coinjected with MSCs and monocytes were most effectively treated with dinutuximab and aNK cells when anti-human (TRC105) and anti-mouse (M1043) CD105 antibodies were added, which depleted human MSCs and murine endothelial cells and macrophages from the TME. CONCLUSIONS: Immunotherapy of neuroblastoma with anti-GD2 antibody dinutuximab and aNK cells is suppressed by CD105+ cells in the TME, but suppression is overcome by adding anti-CD105 antibodies to eliminate CD105+ cells.

A role for the Tgf-ß/Bmp co-receptor Endoglin in the molecular oscillator that regulates the hair follicle cycle.

The hair follicle is a biological oscillator that alternates growth, regression, and rest phases driven by the sequential activation of the proliferation/differentiation programs of resident stem cell populations. The activation of hair follicle stem cell niches and subsequent entry into the growing phase is mainly regulated by Wnt/ß-catenin signalling, while regression and resting phases are mainly regulated by Tgf-ß/Bmp/Smad activity. A major question still unresolved is the nature of the molecular switch that dictates the coordinated transition between both signalling pathways. Here we have focused on the role of Endoglin (Eng), a key co-receptor for members of the Tgf-ß/Bmp family of growth factors. Using an Eng haploinsufficient mouse model, we report that Eng is required to maintain a correct follicle cycling pattern and for an adequate stimulation of hair follicle stem cell niches. We further report that ß-catenin binds to the Eng promoter depending on Bmp signalling. Moreover, we show that ß-catenin interacts with Smad4 in a Bmp/Eng-dependent context and both proteins act synergistically to activate Eng promoter transcription. These observations point to the existence of a growth/rest switching mechanism in the hair follicle that is based on an Eng-dependent feedback cross-talk between Wnt/ß-catenin and Bmp/Smad signals.

Preclinical Efficacy of Endoglin-Targeting Antibody-Drug Conjugates for the Treatment of Ewing Sarcoma.

PURPOSE: Endoglin (ENG; CD105) is a coreceptor of the TGFß family that is highly expressed in proliferating endothelial cells. Often coopted by cancer cells, ENG can lead to neo-angiogenesis and vasculogenic mimicry in aggressive malignancies. It exists both as a transmembrane cell surface protein, where it primarily interacts with TGFß, and as a soluble matricellular protein (sENG) when cleaved by matrix metalloproteinase 14 (MMP14). High ENG expression has been associated with poor prognosis in Ewing sarcoma, an aggressive bone cancer that primarily occurs in adolescents and young adults. However, the therapeutic value of ENG targeting has not been fully explored in this disease. EXPERIMENTAL DESIGN: We characterized the expression pattern of transmembrane ENG, sENG, and MMP14 in preclinical and clinical samples. Subsequently, the antineoplastic potential of two novel ENG-targeting monoclonal antibody-drug conjugates (ADC), OMTX503 and OMTX703, which differed only by their drug payload (nigrin-b A chain and cytolysin, respectively), was assessed in cell lines and preclinical animal models of Ewing sarcoma. RESULTS: Both ADCs suppressed cell proliferation in proportion to the endogenous levels of ENG observed in vitro. Moreover, the ADCs significantly delayed tumor growth in Ewing sarcoma cell line-derived xenografts and patient-derived xenografts in a dose-dependent manner. CONCLUSIONS: Taken together, these studies demonstrate potent preclinical activity of first-in-class anti-ENG ADCs as a nascent strategy to eradicate Ewing sarcoma.

Endoglin inhibition by sodium acetate and flutamide ameliorates cardiac defective G6PD-dependent antioxidant defense in gestational testosterone-exposed rats.

Gestational androgen excess has been implicated in the development of cardiac dysfunction with poor mechanistic delineation. The role of sodium acetate on cardiac uric acid (UA) production and glucose-6-phosphate dehydrogenase (G6PD)-dependent antioxidant defense in pregnancy is not known. The study therefore sought to test the hypothesis that rats exposed to elevated testosterone in late pregnancy would have increased cardiac UA production and defective G6PD-dependent antioxidant defense. We also hypothesized that sodium acetate (SAc) or androgen receptor blocker, flutamide (Flu) would ameliorate these effects through endoglin inhibition. Twenty-four pregnant Wistar rats were treated (sc) with olive oil, testosterone propionate (0.5 mg/kg) singly or in combination with SAc (200 mg/kg; po) or Flu (7.5 mg/kg; po) in the late gestation between gestational day 14 and 19. The results showed that in the late gestation, testosterone exposure led to increased plasma and cardiac endoglin. In the heart of rats exposed to gestational testosterone there were elevated lactate dehydrogenase, adenosine deaminase, xanthine oxidase, uric acid (UA), cardiac injury markers and decreased G6PD-dependent antioxidant defense. However, either SAc or Flu comparably ameliorated these testosterone-induced effects. The data from the present study revealed that testosterone exposure in the late gestation causes elevated cardiac Eng that is accompanied by increased UA production and defective G6PD-dependent anti-oxidant defenses. Besides, the findings also suggest that the inhibitory effect of SAc or Flu on endoglin attenuates UA production and enhances the G6PD-dependent anti-oxidant barrier, thereby implying that endoglin may be a potentially novel therapeutic intervention for cardiac dysfunction particularly in pregnancy.

Modulation of Circulating Protein Biomarkers in Cancer Patients Receiving Bevacizumab and the Anti-Endoglin Antibody, TRC105.

TRC105 is an anti-endoglin antibody currently being tested in combination with VEGF inhibitors. In the phase Ib trial, 38 patients were treated with both TRC105 and bevacizumab (BEV), and improved clinical outcomes were observed, despite the fact that 30 patients (79%) were refractory to prior anti-VEGF therapy. Plasma samples were tested for angiogenic and inflammatory biomarkers at baseline and on-treatment. To provide broader context of this combination biomarker study, direct cross-study comparisons were made to biomarker studies previously conducted in patients treated with either BEV or TRC105 monotherapy. Upon treatment with BEV and TRC105, pharmacodynamic changes in response to both BEV (PlGF increase) and TRC105 (soluble endoglin increase) were noted. In addition, distinct patterns of change were identified (similar, opposing, neutralizing). Similar patterns were observed when the combination elicited similar effects to those observed with monotherapy treatment (i.e., decreases of Ang-2, increases of IL6 and VCAM-1). Opposing patterns were observed when the combination led to opposing effects compared with monotherapy treatment (i.e., TGFß1, PDGF-AA and PDGF-BB, PAI-1). Lastly, neutralizing patterns were observed when one drug led to increase, whereas the other drug led to decrease, and the combination elicited no overall effect on the marker (i.e., VEGF-A, VEGF-D, and IGFBP-3). Patients achieving partial responses or stable disease from the combination exhibited significantly lower expression of E-Cadherin, HGF, ICAM-1, and TSP-2 at baseline. Taken together, the novel biomarker modulations identified may deepen our understanding of the underlying biology in patients treated with BEV and TRC105 compared with either drug alone. Mol Cancer Ther; 17(10); 2248-56. ©2018 AACR.

Intravital Monitoring of Vasculature After Targeted Gene Therapy Alone or Combined With Tumor Irradiation.

Vascular-targeted therapies exhibit radiosensitizing effects by remodeling tumor vasculature, thus facilitating the increased oxygenation of the remaining tumor tissue. To examine these phenomena, the effects of antiendoglin gene therapy alone and in combination with irradiation were monitored for 5 consecutive days on a murine mammary adenocarcinoma (TS/A) tumor model growing in a dorsal window chamber. The vascularization of the tumors was assessed by the determination of the tumor vascular area and by measurement of tumor perfusion by using laser Doppler flowmetry to provide insight into intratumoral gene electrotransfer effects. The changes in the vascular area after this specific therapy correlated with laser Doppler measurements, indicating that either of the methods can be used to demonstrate the induced changes in the vascularization and perfusion of tumors. Gene electrotransfer with an endothelial-specific promoter resulted in a vascular-targeted effect on tumor vasculature within the first 24 hours and did not restore within 5 days. The combination with the irradiation did not result in a more pronounced vascular effect, and irradiation alone only abrogated the formation of new vessels and prevented an increase in the tumor perfusion over time. The results indicate that tumors grown in a dorsal window chamber facilitate intravital measurements of the vascularization of tumors and blood perfusion, enabling the monitoring of the antiangiogenic or vascular disruptive effects of different therapies.

Antagonizing CD105 enhances radiation sensitivity in prostate cancer.

Radiation therapy is the primary intervention for nearly half of the patients with localized advanced prostate cancer and standard of care for recurrent disease following surgery. The development of radiation-resistant disease is an obstacle for nearly 30-50% of patients undergoing radiotherapy. A better understanding of mechanisms that lead to radiation resistance could aid in the development of sensitizing agents to improve outcome. Here we identified a radiation-resistance pathway mediated by CD105, downstream of BMP and TGF-ß signaling. Antagonizing CD105-dependent BMP signaling with a partially humanized monoclonal antibody, TRC105, resulted in a significant reduction in clonogenicity when combined with irradiation. In trying to better understand the mechanism for the radio-sensitization, we found that radiation-induced CD105/BMP signaling was sufficient and necessary for the upregulation of sirtuin 1 (SIRT1) in contributing to p53 stabilization and PGC-1α activation. Combining TRC105 with irradiation delayed DNA damage repair compared to irradiation alone. However, in the absence of p53 function, combining TRC105 and radiation resulted in no reduction in clonogenicity compared to radiation alone, despite similar reduction of DNA damage repair observed in p53-intact cells. This suggested DNA damage repair was not the sole determinant of CD105 radio-resistance. As cancer cells undergo an energy deficit following irradiation, due to the demands of DNA and organelle repair, we examined SIRT1's role on p53 and PGC-1α with respect to glycolysis and mitochondrial biogenesis, respectively. Consequently, blocking the CD105-SIRT1 axis was found to deplete the ATP stores of irradiated cells and cause G2 cell cycle arrest. Xenograft models supported these findings that combining TRC105 with irradiation significantly reduces tumor size over irradiation alone (p value = 10-9). We identified a novel synthetic lethality strategy of combining radiation and CD105 targeting to address the DNA repair and metabolic addiction induced by irradiation in p53-functional prostate cancers.

Efficient targeted tumor imaging and secreted endostatin gene delivery by anti-CD105 immunoliposomes.

BACKGROUND: Anti-CD105 mAb-conjugated immunoliposomes, loaded with secreted mouse endostatin gene, were developed for targeted tumor imaging and antiangiogenic gene therapy. METHODS: The liposomes were investigated for size, zeta-potential, lipid content, antibody binding ability, and pcDNA loading capacity. The ability of immunoliposomes to target tumor-derived endothelial cells and perform gene transfer in vitro was measured and their basic biocompatibility was evaluated. A nude mouse/breast cancer xenograft model was used to examine the tumor internalization of fluorescent-labeled liposomes and the clinical potential of immnuoliposomes loaded with pcDNA3.1-CSF1-endostatin. RESULTS: Loaded immunoliposomes were homogenously distributed with a well-defined spherical shape and bilayer, diameter of 122 ± 11 nm, and zeta potential + 1.40 mV. No significant differences were observed in body weight, liver index, oxidative stress, or liver and kidney function in mice after liposomes exposure. The addition of CD105 mAb to liposomes conferred the ability to target tumor-derived endothelial cells in vitro and in vivo. Systemic intravenous administration of fluorescent immunoliposomes in the xenograft model resulted in selective and efficient internalization in tumor vasculature. Treatment of mice with pcDNA3.1-CSF1-endostatin-loaded immunoliposomes suppressed tumor growth by 71%. CONCLUSIONS: These data demonstrate the advantages of using anti-CD105 mAb-conjugated immunoliposomes to enhance tumor targeting, imaging, and gene transfer applications.

Endoglin interacts with VEGFR2 to promote angiogenesis.

Endoglin, a TGF-ß coreceptor predominantly expressed in endothelial cells, plays an important role in vascular development and tumor-associated angiogenesis. However, the mechanism by which endoglin regulates angiogenesis, especially during tip cell formation, remains largely unknown. In this study, we report that endoglin promoted VEGF-induced tip cell formation. Mechanistically, endoglin interacted with VEGF receptor (VEGFR)-2 in a VEGF-dependent manner, which sustained VEGFR2 on the cell surface and prevented its degradation. Endoglin mutants deficient in the ability to interact with VEGFR2 failed to sustain VEGFR2 on the cell surface and to promote VEGF-induced tip cell formation. Further, an endoglin-targeting monoclonal antibody (mAb), TRC105, cooperated with a VEGF-A targeting mAb, bevacizumab, to inhibit VEGF signaling and tip cell formation in vitro and to inhibit tumor growth, metastasis, and tumor-associated angiogenesis in a murine tumor model. This study demonstrate a novel mechanism by which endoglin initiates and regulates VEGF-driven angiogenesis while providing a rationale for combining anti-VEGF and anti-endoglin therapy in patients with cancer.-Tian, H., Huang, J. J., Golzio, C., Gao, X., Hector-Greene, M., Katsanis, N., Blobe, G. C. Endoglin interacts with VEGFR2 to promote angiogenesis.

Durable remission for a woman with refractory choriocarcinoma treated with anti-endoglin monoclonal antibody and bevacizumab: A case from the New England Trophoblastic Disease Center, Brigham and Women's Hospital and Dana-Farber Cancer Institute.

A 36-year-old with metastatic and refractory choriocarcinoma following single- and multi-agent chemotherapy and surgical metastectomy experienced a durable remission after receiving therapy with an anti-endoglin monoclonal antibody and bevacizumab. Treatment options and scientific advances in this disease are highlighted.

5-FU resistant EMT-like pancreatic cancer cells are hypersensitive to photochemical internalization of the novel endoglin-targeting immunotoxin CD105-saporin.

BACKGROUND: Development of resistance to 5-fluorouracil (5-FU) is a major problem in treatment of various cancers including pancreatic cancer. In this study, we reveal important resistance mechanisms and photochemical strategies to overcome 5-FU resistance in pancreatic adenocarcinoma. METHODS: 5-FU resistant (5-FUR), epithelial-to-mesenchymal-like sub-clones of the wild type pancreatic cancer cell line Panc03.27 were previously generated in our lab. We investigated the cytotoxic effect of the endosomal/lysosomal-localizing photosensitizer TPCS2a (fimaporfin) combined with light (photochemical treatment, PCT) using MTS viability assay, and used fluorescence microscopy to show localization of TPCS2a and to investigate the effect of photodamage of lysosomes. Flow cytometric analysis was performed to investigate uptake of photosensitizer and to assess intracellular ROS levels. Expression and localization of LAMP1 was assessed using RT-qPCR, western blotting, and structured illumination microscopy. MTS viability assay was used to assess the effect of combinations of 5-FU, chloroquine (CQ), and photochemical treatment. Expression of CD105 was investigated using RT-qPCR, western blotting, flow cytometry, and fluorescence microscopy, and co-localization of TPCS2a and anti-CD105-saporin was assessed using microscopy. Lastly, the MTS assay was used to investigate cytotoxic effects of photochemical internalization (PCI) of the anti-CD105-immunotoxin. RESULTS: The 5-FUR cell lines display hypersensitivity to PCT, which was linked to increased uptake of TPCS2a, altered lysosomal distribution, lysosomal photodamage and increased expression of the lysosomal marker LAMP-1 in the 5-FUR cells. We show that inhibition of autophagy induced by either chloroquine or lysosomal photodamage increases the sensitivity to 5-FU in the resistant cells. The three 5-FUR sub-clones overexpress Endoglin (CD105). Treatment with the immunotoxin anti-CD105-saporin alone significantly reduced the viability of the CD105-expressing 5-FUR cells, whereas little effect was seen in the CD105-negative non-resistant parental cancer cell lines. Strikingly, using the intracellular drug delivery method photochemical internalization (PCI) by combining light-controlled activation of the TPCS2a with nanomolar levels of CD105-saporin resulted in strong cytotoxic effects in the 5-FUR cell population. CONCLUSION: Our findings suggested that autophagy is an important resistance mechanism against the chemotherapeutic drug 5-FU in pancreatic cancer cells, and that inhibition of the autophagy process, either by CQ or lysosomal photodamage, can contribute to increased sensitivity to 5-FU. For the first time, we demonstrate the promise of PCI-based targeting of CD105 in site-specific elimination of 5-FU resistant pancreatic cancer cells in vitro. In conclusion, PCI-based targeting of CD105 may represent a potent anticancer strategy and should be further evaluated in pre-clinical models.

Endoglin inhibition leads to intussusceptive angiogenesis via activation of factors related to COUP-TFII signaling pathway.

Angiogenesis is a highly coordinated, extremely complex process orchestrated by multiple signaling molecules and blood flow conditions. While sprouting mode of angiogenesis is very well investigated, the molecular mechanisms underlying intussusception, the second mode of angiogenesis, remain largely unclear. In the current study two molecules involved in vascular growth and differentiation, namely endoglin (ENG/CD105) and chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) were examined to unravel their specific roles in angiogenesis. Down- respectively up-regulation of both molecules tightly correlates with intussusceptive microvascular growth. Upon ENG inhibition in chicken embryo model, formation of irregular capillary meshwork accompanied by increased expression of COUP-TFII could be observed. This dynamic expression pattern of ENG and COUP-TFII during vascular development and remodeling correlated with formation of pillars and progression of intussusceptive angiogenesis. Similar findings could be observed in mammalian model of acute rat Thy1.1 glomerulonephritis, which was induced by intravenous injection of anti-Thy1 antibody and has shown upregulation of COUP-TFII in initial phase of intussusception, while ENG expression was not disturbed compared to the controls but decreased over the time of pillar formation. In this study, we have shown that ENG inhibition and at the same time up-regulation of COUP-TFII expression promotes intussusceptive angiogenesis.