Inhibition Of Tumor Angiogenesis Research Articles

Muscone abrogates breast cancer progression through tumor angiogenic suppression via VEGF/PI3K/Akt/MAPK signaling pathways

BackgroundAngiogenesis strongly reflects poor breast cancer outcome and an important contributor to breast cancer (BC) metastasis; therefore, anti-angiogenic intervention is a potential tool for cancer treatment. However, currently used antibodies against vascular endothelial growth factor A (VEGFA) or inhibitors that target the VEGFA receptor are not effective due to weak penetration and low efficiency. Herein, we assessed the anti-BC angiogenic role of muscone, a natural bioactive musk constituent, and explored possible anti-cancer mechanisms of this compound.MethodsCCK-8, EdU, scratch and Transwell assessments were employed to detect the muscone-mediated regulation of breast cancer (BC) and human umbilical vein endothelial cells (HUVECs) proliferation and migration. Tube formation, matrigel plug assay and zebrafish assay were employed for assessment of regulation of tumor angiogenesis by muscone. In vivo xenograft mouse model was constructed to compare microvessel density (MVD), vascular leakage, vascular maturation and function in muscone-treated or untreated mice. RNA sequencing was performed for gene screening, and Western blot verified the effect of the VEGFA-VEGFR2 pathway on BC angiogenic inhibition by muscone.ResultsBased on our findings, muscone suppressed BC progression via tumor angiogenic inhibition in cellular and animal models. Functionally, muscone inhibited BC cell proliferation and migration as well as tumor cell-conditioned medium-based endothelial cell proliferation and migration. Muscone exhibited a strong suppressive influence on tumor vasculature in cellular and animal models. It abrogated tumor cell growth in a xenograft BC mouse model and minimized tumor microvessel density and hypoxia, and increased vascular wall cell coverage and perfusion. Regarding the mechanism of action, we found that muscone suppressed phosphorylation of members of the VEGF/PI3K/Akt/MAPK axis, and it worked synergistically with a VEGFR2 inhibitor, an Akt inhibitor, and a MAPK inhibitor to further inhibit tube formation.ConclusionOverall, our results demonstrate that muscone may proficiently suppress tumor angiogenesis via modulation of the VEGF/PI3K/Akt/MAPK axis, facilitating its candidacy as a natural small molecule drug for BC treatment.

Marine algae-derived characterized bioactive compounds as therapy for cancer: A review on their classification, mechanism of action, and future perspectives.

In 2022, there were around 20 million new cases and over 9.7 million cancer-related deaths worldwide. An increasing number of metabolites with anticancer activity in algae had been isolated and identified, which were promising candidates for cancer therapy. Red algae are well-known for the production of brominated metabolites, including terpenoids and phenols, which have the capacity to induce cell toxicity. Some non-toxic biological macromolecules, including polysaccharides, are distinct secondary metabolites found in many algae, particularly green algae. They possess anticancer activities by inhibiting tumor angiogenesis, stimulating the immune response, and inducing apoptosis. However, the structure-activity relationship between these components and antitumor activity, as well as certain taxa within the algae, remains relatively unstudied. This work is based on the reports published from 2003 to 2024 in PubMed and ISI Web of Science databases. A comprehensive review of the characterized algal anticancer active compounds, together with their structure and mechanism of action was performed. Also, their structure-activity relationship was preliminarily summarized to better assess their potential properties as a natural, safe bioactive product to be used as an alternative for the treatment of cancers, leading to new opportunities for drug discovery.

Eugenol suppresses VEGF-dependent angiogenesis by JAK2/STAT3 pathway in non-small cell lung cancer

Background: Non-small cell lung cancer (NSCLC) is a highly vascularized solid tumor, and tumor angiogenesis is closely associated with the metastasis and progression of NSCLC. Antiangiogenic drugs can target the tumor microenvironment to degrade existing tumor blood vessels while inhibiting tumor angiogenesis and have become one of the indispensable treatments for patients with advanced NSCLC. Although various new drugs have been tested in different settings of NSCLC, none of them have shown the desired therapeutic effects so far. Therefore, the search for new and effective therapeutic modalities has become a new goal for treating NSCLC. Objective: Eugenol is a phenolic aromatic compound derived from Eugenia caryophyllata, Cinnamomum cassia, etc., which has historically been used for various medical purposes. Studies have shown that eugenol exhibits significant anticancer effects against several types of cancer; however, its therapeutic effect on angiogenesis remains a mystery. In this study, the in vitro and in vivo antiangiogenic effects of eugenol in NSCLC and the underlying molecular mechanism were explored, which could provide a promising strategy for the treatment of NSCLC. Methods: The effects of eugenol on the proliferative capacity of human umbilical vein endothelial cells (HUVECs) and A549 cells were examined by methyl thiazolyl tetrazolium assay. The migration and invasion of eugenol-treated HUVECs were evaluated by wounding-healing and transwell assay, and the angiogenesis was measured by tube formation assay. The expression of angiogenesis-related genes and proteins, as well as the JAK2/STAT3 pathway, was evaluated by real-time quantitative PCR and western blot. Flow cytometry was performed to detect the effect of eugenol on the apoptotic profile of A549 cells. Finally, the A549 tumor-bearing nude mice were constructed to evaluate the in vivo anti-NSCLC activity of eugenol. Results: Eugenol inhibited the migration, invasion, and tube formation of HUVECs. Meanwhile, eugenol blocked the phosphorylation of vascular endothelial growth factor reporter-2 and inhibited the expression of other angiogenesis-related proteins. In addition, eugenol suppressed the expression of p-JAK2 and p-STAT3 in HUVECs and A549 cells. Eugenol also suppressed the proliferation of A549 cells by promoting apoptosis and inhibited tumor growth and microvessel formation in A549 cell xenograft-bearing nude mice. Conclusions: Eugenol could be a potential lead compound for the treatment of NSCLC by blocking the vascular endothelial growth factor/vascular endothelial growth factor reporter-2 and JAK2/STAT3 signaling pathways.

Updated results from ALTER-S002: A single-arm multicenter trial of the combination of anlotinib with chemotherapy in patients with stage IIB classic osteosarcoma of the extremity.

11529 Background: The overexpression of multiple tyrosine kinase receptors in osteosarcoma indicated a promising therapy targeting these receptors. Anlotinib is a multi-targeted tyrosine kinase inhibitor that potentially inhibits tumor angiogenesis. The combination of anti-angiogenic drugs with chemotherapeutic agents is proposed to act synergistically to achieve favorable tumor control, especially as the neoadjuvant therapy. We had reported the neoadjuvant therapy data of anlotinib in combination with chemotherapy for treatment-naive stage IIB classical osteosarcoma of the extremity in the 2023 ESMO congress. Here we report an update on the effectiveness and safety of the follow-up. Methods: In this open-label, single-arm, multicenter phase II clinical trial. Eligible patients(pts) were aged 12-40 years with histologically confirmed primary localized extremity classic osteosarcoma, stage IIB, operable. Pts received anlotinib (10mg, po, d1-14, q3w), doxorubicin (A) (20-25mg/m2 iv, d1-3, q3w) and cisplatin (P) (70-90mg/m2, iv, d1, q3w) for the first nine weeks. Pts underwent radical surgery after the exclusion of contraindications at week 10. After surgery, pts received A+P at weeks 12-14, and anlotinib plus A+P at weeks 15-20 with the drug dose unchanged. Since week 21, anlotinib (12mg, po, d1-14, q3w) monotherapy was maintained until week 104 or the occurrence of an EFS event. The primary endpoint was 24-month EFS rate, secondary endpoints were 36-month EFS rate, local recurrence rate, lung metastasis rate, 3-year OS rate and safety. Results: From May 2020 to Apr 2022, 52 eligible pts were enrolled, while 51 pts underwent surgery after 3 cycles of neoadjuvant therapy. At the data cut-off date (Oct. 2023), the median EFS was not reached, 12 and 24-month EFS rates were 84.18% (95% CI :70.82, 91.77) and 73.43% (95% CI :57.40, 84.21), respectively. The stratified analysis of EFS showed that the length and diameter of the target lesion are independent prognostic factors. The median OS was not reached, the 24-month OS rate was 95.92% (95% CI :84.65, 98.96). Among the 51 pts who underwent surgical treatment, 12(23.53%, 95% CI: 12.79, 37.49) pts experienced recurrence or metastasis. Most treatment-related adverse events (TRAEs) were grade 1-2. Grade 3/4 TRAEs (≥20%) included neutropenia (51.92%), leukopenia (38.46%), thrombocytopenia (36.54%) and anemia (32.69%). Conclusions: The updated results suggested that anlotinib combined with doxorubicin and cisplatin in the perioperative period showed favorable efficiency and manageable adverse events in Stage IIB classic osteosarcoma of the extremity. Clinical trial information: ChiCTR 2000033298.

Apigenin inhibits tumor angiogenesis by hindering microvesicle biogenesis via ARHGEF1

Extracellular vesicles are essential for intercellular communication and are involved in tumor progression. Inhibiting the direct release of extracellular vesicles seems to be an effective strategy in inhibiting tumor progression, but lacks of investigation. Here, we report a natural flavonoid compound, apigenin, could significantly inhibit the growth of hepatocellular carcinoma by preventing microvesicle secretion. Mechanistically, apigenin primarily targets the guanine nucleotide exchange factor ARHGEF1, inhibiting the activity of small G protein Cdc42, which is essential in regulating the release of microvesicles from tumor cells. In turn, this inhibits tumor angiogenesis related to VEGF90K transported on microvesicles, ultimately impeding tumor progression. Collectively, these findings highlight the therapeutic potential of apigenin and shed light on its anticancer mechanisms through inhibiting microvesicle biogenesis, providing a solid foundation for the refinement and practical application of apigenin.

Targeting ONECUT2 inhibits tumor angiogenesis via down-regulating ZKSCAN3/VEGFA

OC-2 plays a vital role in tumor growth, metastasis and angiogenesis, but molecular mechanism how OC-2 regulates angiogenic factors is unclear. We found that OC-2 was highly expressed in HepG2, COLO, MCF-7, SKOV3 cells and rectum carcinoma tissues, and angiogenic factors levels were positively related to OC-2. Then OC-2 KD inhibited the tumor growth, metastasis and angiogenesis process in vitro and vivo. ChIP-Seq showed that 228 target genes of OC-2 were identified and they were associated with tumor growth, metastasis, angiogenesis and signal transduction; OC-2 bound to ZKSCAN3 at promoter region. Luciferase assays showed that ZKSCAN3 was identified as target gene of OC-2 and VEGFA was identified as target gene of ZKSCAN3; OC-2 promoted VEGFA expression via activating ZKSCAN3 transcriptional program. Importantly, OC-2 KD down-regulated VEGFA secretion to suppress tumor angiogenesis of HUVECs. Besides VEGFA, OC-2 was positively correlated with other angiogenic factors HIF-1α, FGF2, EGFL6 and HGF. Meanwhile, ERK1/2 and Smad1 signaling pathways might be related to function of OC-2 driving tumor aggressiveness. We revealed that OC-2 might regulate tumor growth, metastasis, angiogenesis via ERK1/2, Smad1 signaling pathways and regulate VEGFA expression for tumor angiogenesis via activating ZKSCAN3 transcriptional program, indicating that OC-2 was a convincing target to develop novel anti-tumor drugs based on angiogenesis.

Hybrid cell membranes camouflaged copper-loaded nano-prodrug for tumor angiogenesis inhibition and cell cuproptosis

The establishment of hybrid cell membrane-camouflaged nanotherapeutics is crucial to preserve and impart their biological functionality inherited from the source cells. In this study, one side relies on the intrinsic affinity between the intracellular domain of Band 3 in the red blood cells membrane (Rm) and P4.2 peptide-modified nanoparticles (Rm-Que/Cu), promoting the self-assembly coupling-driven encapsulation of Rm on the surface of nanodrugs. On the other side, it involves integrating the bioengineered cell membrane with high Del-1 expression (Dm) with Rm-Que/Cu to form RDm-Que/Cu, targeting the role of integrin αvβ3 on tumor neovascularization and endothelial cells (ECs). This approach simultaneously integrates the multi-functions, including self-assembly encapsulation, targeted drug delivery, and on-demand drug release. Both in vitro and in vivo experimental results confirmed that RDm-Que/Cu were able to disrupt the cellular copper homeostasis, inhibit tumor angiogenesis, and induce cuproptosis. This discovery provides a feasible platform for the biomedical application of hybrid cell membrane-camouflaged biomimetic nanotechnology.

Steering Piezocatalytic Therapy for Optimized Tumoricidal Effect

AbstractPiezocatalysts, because of their mechano‐electrical conversion properties, are exploited for various medical applications, such as sterilization, tissue engineering, biosensing, and disease theranostics. In particular, based on the unique advantage of the piezoelectric effect, piezocatalytic therapy (PCT) has been developed as a novel and promising candidate for tumor therapy. To optimize the utilization of piezocatalysts in tumor therapy, a comprehensive understanding of the antitumor mechanism associated with these materials is imperative. Here, the piezocatalytic action principle is elucidated by investigating piezocatalysts, reactants, energy inputs, and products. Subsequently, the antitumor mechanisms of PCT have been extensively discussed and are recapitulative as follows: restraining cell proliferation, inducing cell programmed death, hindering tumor metastasis, inhibiting tumor angiogenesis, and enhancing antitumor immunity. Additionally, the optimized therapeutic outcomes of PCT‐centric synergistic cancer therapy are systematically described. Finally, the main challenges and future research directions of piezocatalysis applied in cancer therapy are envisioned. It is believed that PCT will serve as a new‐generation ingenious tool for cancer treatment.

PH-responsive drug-loaded peptides enhance drug accumulation and promote apoptosis in tumor cells

The efficacy of chemotherapeutic drugs in tumor treatment is limited by their toxicity and side effects due to their inability to selectively accumulate in tumor tissue. In addition, chemotherapeutic agents are easily pumped out of tumor cells, resulting in their inadequate accumulation. To overcome these challenges, a drug delivery system utilizing the amphiphilic peptide Pep1 was designed. Pep1 can self-assemble into spherical nanoparticles (PL/Pep1) and encapsulate paclitaxel (PTX) and lapatinib (LAP). PL/Pep1 transformed into nanofibers in an acidic environment, resulting in longer drug retention and higher drug concentrations within tumor cells. Ultimately, PL/Pep1 inhibited tumor angiogenesis and enhanced tumor cell apoptosis. The use of shape-changing peptides as drug carriers to enhance cancer cell apoptosis is promising.

Screening and construction of nanobodies against human CD93 using phage libraries and study of their antiangiogenic effects.

Cluster of Differentiation 93 (CD93) plays an important role in angiogenesis and is considered an important target for inhibiting tumor angiogenesis, but there are currently no therapeutic antibodies against CD93 in the clinic. Thus, we describe the screening of novel nanobodies (Nbs) targeting human CD93 from a phage library of shark-derived Nbs. Screening and enrichment of phage libraries by enzyme-linked immunosorbent assay (ELISA). Anti-CD93 Nbs were purified by expression in E. coli. The binding affinity of anti-CD93 Nbs NC81/NC89 for CD93 was examined by flow cytometry (FC) and ELISA. The thermal stability of NC81/NC89 was examined by ELISA and CD spectroscopy. Afterward, the anti-angiogenic ability of NC81/NC89 was examined by MTT, wound healing assay, and tube formation assay. The expression level of VE-cadherin (VE-Ca) and CD93 was detected by Western Blot (WB). The binding sites and binding forms of NC81/NC89 to CD93 were analyzed by molecular docking. The anti-CD93 Nbs were screened in a phage library, expressed in E. coli, and purified to >95% purity. The results of FC and ELISA showed that NC81/NC89 have binding ability to human umbilical vein endothelial cells (HUVECs). The results of ELISA and CD spectroscopy showed that NC81/NC89 retained the ability to bind CD93 at 80°C and that the secondary structure remained stable. In vitro, the results showed that NC81 and NC89 significantly inhibited the proliferation and migration of human umbilical vein endothelial cells (HUVECs) as well as tube formation on Matrigel. Western Blot showed that NC81 and NC89 also inhibited the expression of VE-Ca thereby increasing vascular permeability. It was found during molecular docking that the CDR regions of NC81 and NC89 could be attached to CD93 by strong hydrogen bonds and salt bridges, and the binding sites were different. We have successfully isolated NC81 and NC89, which bind CD93, and both Nbs significantly inhibit angiogenesis and increase vascular permeability. These results suggest that NC81 and NC89 have potential clinical applications in angiogenesis-related therapies.

Metronomic Chemotherapy for Pediatric Refractory Solid Tumors: A Retrospective Single-center Study.

Metronomic chemotherapy (MC) is based on chronic administration of chemotherapeutic agents at minimally toxic doses without prolonged drug-free breaks, that inhibits tumor angiogenesis and induces tumor dormancy. This study aimed to determine the efficacy of MC for pediatric refractory solid tumors. We retrospectively analyzed the data of pediatric patients with relapsed/refractory solid tumors who received treatment, including low-dose continuous administration of anticancer drugs, at our institute. Of the 18 patients, the disease statuses at the initiation of MC were complete remission (n=2), partial remission/stable disease (n=5), and progressive disease (n=11). The overall survival rate was 61% at 12 months and 34% at 24 months, and the progression-free survival rate was 21% at 12 and 24 months. Although only 5 of the 18 patients showed certain tumor regression or maintained remission, tumors that stabilized, maintained remission/stable disease, and showed certain advantages in terms of overall survival rate, even if limited to progressive disease. Approximately half of the patients demonstrated temporal tumor stabilization and improved survival time. Overall, previous reports and the present study support the conclusion that MC has the potential to play an important role in pediatric cancer treatment during the advanced stage.

Pre-coating cRGD-modified bovine serum albumin enhanced the anti-tumor angiogenesis of siVEGF-loaded chitosan-based nanoparticles by manipulating the protein corona composition

Chitosan-based nanoparticles inevitably adsorb numerous proteins in the bloodstream, forming a protein corona that significantly influences their functionality. This study employed a pre-coated protein corona using cyclic Arg-Gly-Asp peptide (cRGD)-modified bovine serum albumin (BcR) to confer tumor-targeting capabilities on siVEGF-loaded chitosan-based nanoparticles (CsR/siVEGF NPs) and actively manipulated the serum protein corona composition to enhance their anti-tumor angiogenesis. Consequently, BcR effectively binds to the nanoparticles' surface, generating nanocarriers of appropriate size and stability that enhance the inhibition of endothelial cell proliferation, migration, invasion, and tube formation, as well as suppress tumor proliferation and angiogenesis in tumor-bearing nude mice. Proteomic analysis indicated a significant enrichment of serotransferrin, albumin, and proteasome subunit alpha type-1 in the protein corona of BcR-precoated NPs formed in the serum of tumor-bearing nude mice. Additionally, there was a decrease in proteins associated with complement activation, immunoglobulins, blood coagulation, and acute-phase responses. This modification resulted in an enhanced impact on anti-tumor angiogenesis, along with a reduction in opsonization and inflammatory responses. Therefore, pre-coating of nanoparticles with a functionalized albumin corona to manipulate the composition of serum protein corona emerges as an innovative approach to improve the delivery effectiveness of chitosan-based carriers for siVEGF, targeting the inhibition of tumor angiogenesis.

MAPK4 facilitates angiogenesis by inhibiting the ERK pathway in non-small cell lung cancer.

Angiogenesis plays an important role in the occurrence and development of non-small cell lung cancer (NSCLC). The atypical mitogen-activated protein kinase 4 (MAPK4) has been shown to be involved in the pathogenesis of various diseases. However, the potential role of MAPK4 in the tumor angiogenesis of NSCLC remains unclear. Adult male C57BL/6 wild-type mice were randomly divided into the control group and p-siMAPK4 intervention group, respectively. The cell proliferation was analyzed withflow cytometry and immunofluorescence staining. The vascular density in tumor mass was analyzed by immunofluorescence staining. The expressions of MAPK4 and related signaling molecules were detected by western blot analysis and immunofluorescence staining, and so on. We found that the expression of MAPK4, which was dominantly expressed in local endothelial cells (ECs), was correlated with tumor angiogenesis of NSCLC. Furthermore, MAPK4 silencing inhibited the proliferation and migration abilities of human umbilical vein ECs (HUVECs). Global gene analysis showed that MAPK4 silencing altered the expression of multiple genes related to cell cycle and angiogenesis pathways, and that MAPK4 silencing increased transduction of the extracellular regulated protein kinases 1/2 (ERK1/2) pathwaybut not Akt and c-Jun n-terminal kinase pathways. Further analysis showed that MAPK4 silencing inhibited the proliferation and migration abilities of HUVECs cultured in tumor cell supernatant, which was accompanied with increased transduction of the ERK1/2 pathway. Clinical data analysis suggested that the higher expression of MAPK4 and CD34 were associated with poor prognosis ofpatients with NSCLC. Targeted silencing of MAPK4 in ECs using small interfering RNA driven by the CD34 promoter effectively inhibited tumor angiogenesis and growth of NSCLC in vivo. Our results reveal that MAPK4 plays an important role in the angiogenesis and development of NSCLC. MAPK4 may thus represent a new target for NSCLC.

Abstract LB447: Drug delivery and efficacy testing using vascularized tumor-on-a-chip

Abstract The inhibition of tumor angiogenesis is considered a promising strategy to impede cancer progression, as the vasculature associated with tumors plays a crucial role in supplying blood and facilitating metastasis. Therefore, the in vitro replication of vascularized tumors is essential for comprehending cancer pathology and discerning the mechanisms governing tumor cell proliferation, metastasis, and response to pharmaceutical interventions. In this study, we fabricated vascularized tumor-on-a-chip to recapitulate the pathological features of solid tumors. We first developed hybrid spheroids consisting of tumor and endothelial cells, resulting in self-assembled intratumoral vessels. This approach enhanced the uniformity of the spheroids and peritumoral angiogenic capacity compared to spheroids composed of only cancer cells. RNA sequencing analysis revealed that the tumor-EC hybrid spheroids exhibited expression profiles associated with aggressive behaviors, such as cancer progression, invasion and metastasis. The blood vessels sprouting around the hybrid spheroids on the chip displayed the distinctive characteristics of leaky tumor vessels. We further validated the suppressive effects of axitinib on tumor growth and angiogenesis, depending on exposure dose and time. Notably, we successfully induced both lymphangiogenesis and angiogenesis around the tumor hybrid spheroids by promoting interstitial flow. In summary, our findings demonstrated that vascularized tumor-on-a-chip reproduces various characteristics of vascularized tumors. Consequently, our model stands as a valuable platform for investigating interactions between within tumor microenvironments and exploring therapeutic strategies in cancer. Citation Format: Da-Hyun Kim. Drug delivery and efficacy testing using vascularized tumor-on-a-chip [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB447.

In situ assembly FeS2-tetrathiomolybdate nanosheets for imaging-guided tumor vascular collapse and amplified catalytic therapy

Catalytic therapy has recently gained considerable attention as a novel tumor microenvironment (TME)-responsive treatment. While conventional nano-catalysts have demonstrated the ability to induce the death of tumor cells through oxidative damage via the generation of reactive oxygen species (ROS), their therapeutic efficacy is impeded by several factors, including limited tissue penetration depth and suboptimal catalytic efficiency. Herein, the FeS2-tetrathiomolybdate (FeS2-TTM) nanosheet was synthesized via in-situ self-assembled in the specific redox TME using metal precursors of divalent iron and tetrathiomolybdate, and these metal precursors could easily enter tumor cells through vascular penetration to avoid the blood-brain barrier. This nanosheet has been proved to possess peroxidase (POD)-like catalytic activity, which overcomes the limitations of traditional nanoparticals by the bimetallic electron transfer effect in the TME to amplify the catalytic treatment effect. Importantly, it retains the chelating copper characteristics of TTM, and inhibits tumor angiogenesis by depriving the necessary copper in cells and angiogenesis, resulting in vascular embolization to cause tumor blood vessels to collapse. Additionally, it can also act as a nanoprobe that supports both real-time fluorescence imaging monitoring and T1/T2 dual-modality magnetic resonance imaging (MRI). As a promising nanomaterial, this new type of in-situ self-assembled nanosheet provides more varieties for the construction of an integrative platform of tumor diagnosis and treatment.

Developing a Copper(II) Isopropyl 2-Pyridyl Ketone Thiosemicarbazone Compound Based on the IB Subdomain of Human Serum Albumin-Indomethacin Complex: Inhibiting Tumor Growth by Remodeling the Tumor Microenvironment.

To develop a next-generation metal agent and dual-agent multitargeted combination therapy, we developed a copper (Cu) compound based on the properties of the human serum albumin (HSA)-indomethacin (IND) complex to remodel the tumor microenvironment (TME). We optimized a series of Cu(II) isopropyl 2-pyridyl ketone thiosemicarbazone compounds to obtain a Cu(II) compound (C4) with significant cytotoxicity and then constructed an HSA-IND-C4 complex (HSA-IND-C4) delivery system. IND and C4 bind to the hydrophobic cavities of the IB and IIA domains of HSA, respectively. In vivo, the HSA-IND-C4 not only showed enhanced antitumor efficacy relative to C4 and C4 + IND but also improved their targeting ability and decreased their side effects. The antitumor mechanism of C4 + IND involved acting on the different components of the TME. IND inhibited tumor-related inflammation, while C4 not only induced apoptosis and autophagy of cancer cells but also inhibited tumor angiogenesis.

Abstract 6817: Control of breast cancer via pro-resolving mediators

Abstract Tumor recurrence is the principal cause of mortality in breast cancer, yet the underlying mechanisms are not well understood. Anti-estrogen therapy, including tamoxifen, reduces tumor burden by inducing apoptosis and necrosis of cancerous tissue. The resulting cellular debris may stimulate tumor growth by disrupting inflammation resolution and creating pro-inflammatory and pro-tumorigenic microenvironments. Compared to estrogen receptor (ER)-positive breast cancer, ER-negative behaves aggressively, and patients have a poor prognosis due to the lack of treatment options. Here, we demonstrate that breast tumor cells killed by cytotoxic anti-estrogen therapy or chemotherapy (“tumor cell debris”) stimulate primary tumor growth when co-injected with a subthreshold (nontumorigenic) inoculum of tumor cells by triggering a macrophage-derived pro-inflammatory and pro-angiogenic “cytokine storm.” Thus, tumor cell debris is a critical risk factor for aggressive breast cancer growth. To assess whether stimulating the clearance of debris would impact breast cancer progression in vivo, we utilized maresin 1 (MaR1), maresin conjugates in tissue regeneration (MCTR1, MCTR2) and protectin conjugates in tissue regeneration (PCTRs), which are specialized pro-resolving lipid autacoid mediators (SPMs). We treated established models of ER-positive (EO771) and negative (4T1) breast cancer. Each maresin sharply reduced tumor growth in debris-stimulated and spontaneous (e.g. MMTV-PyMT) breast cancer models at nanogram concentrations without toxicity. Notably, PCTR1 inhibited orthotopic triple-negative (4T1) growth and proliferation compared to chemotherapy (paclitaxel) or immunotherapy (anti-cytotoxic T-lymphocyte-associated protein 4, CTLA4). Similarly, PCTR1 alone or in combination with chemotherapy (paclitaxel) reduced gene expression and protein levels of pro-angiogenic factor CXCL12/SDF1 in the ER-negative (4T1) tumor microenvironment. In ER-positive (EO771), a triple combination of PCTR1, chemotherapy (paclitaxel), and immunotherapy (anti-CTLA4) treatment potentiated the immune checkpoint blockade. Maresins also stimulated macrophage phagocytosis of therapy-generated breast tumor cell debris, inhibited tumor angiogenesis, and dampened a therapy-induced cytokine storm, including TNF-α, MIP-2/CXCL2, CCL2/MCP-1, IL- 1ra/IL-1F3, and G-CSF. Expression of SPM receptors (e.g. resolvin D1 (RvD1) receptor ALX/FPR2, resolvin D2 (RvD2) receptor GPR18, maresin 1 (MaR1) receptor LGR6, and resolvin E1 (RvE1) receptor ChemR23) were specifically identified in breast cancer tissue. Taken together, the maresin and protectin pathway mediators may represent a new therapeutic approach to stimulate the resolution of inflammation in breast cancer. Citation Format: Sarina A. Virani, Michael Gillespie, Haixia Yang, Diane R. Bielenberg, Mark W. Kieran, Sui Huang, Charles N. Serhan, Dipak Panigrahy. Control of breast cancer via pro-resolving mediators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6817.

Abstract 1211: Biomarker analysis of pembrolizumab and ramucirumab neoadjuvant therapy for PD-L1-positive stage IB-IIIA lung cancer: EAST ENERGY trial

Abstract Background: Neoadjuvant treatments with novel combination immunotherapies for resectable non-small cell lung cancer (NSCLC) are being developed. Combination therapy with antiangiogenic agents and anti-PD-1/PD-L1 antibody has shown enhanced antitumor effects. EAST ENERGY is a single-arm multicenter phase 2 trial of neoadjuvant pembrolizumab and ramucirumab in patients with PD-L1-positive stage IB-IIIA NSCLC (NCT04040361). In this study, we aimed to identify biomarkers that predicts the efficacy of the neoadjuvant therapy. Furthermore, we aimed to elucidate the effect of the neoadjuvant therapy on the tumor microenvironment (TME). Methods: Patients enrolled in the EAST ENERGY trial (n=24) for whom pre- and post-treatment specimens were available (n=20) were included in this study. RNA-sequencing and multiplex immunohistochemistry (mIHC) were used to examine the association between the immunological phenotype of the TME and major pathological response (MPR), and changes in the TME before and after treatment. Results: MPR was achieved in 50.0% (12/24) of patients. Biomarker analyses with RNA-sequencing revealed that high expression of cancer-associated genes, and upregulation of interferon-gamma and interferon-alpha response in pre-treatment tumor were associated with MPR (q value<0.001). mIHC in pre-treatment samples revealed that abundance of CD8+ T cells in the pre-treatment TME was associated with MPR (p=0.039). On the other hand, infiltration of immunosuppressive cells such as Treg or M2-macrophages were not associated with resistance for the treatment (p=0.836, 0.639, respectively). Next, to clarify the effect of the neoadjuvant therapy on the TME, comparative analyses of pre- and post-treatment samples was performed. In gene set enrichment analysis, the neoadjuvant therapy enhanced gene set associated with interferon-gamma response (q value=0.051). Furthermore, angiogenesis-related gene set were downregulated after the treatment (q value=0.090). mIHC analyses revealed that the number of CD8+ T cells in the TME was significantly increased after the neoadjuvant therapy (p<0.001). Notably, Tregs/CD8+ T cells ratio and the number of M2-macrophage in the TME were significantly decreased after the neoadjuvant therapy (p=0.032, 0.003, respectively). Conclusion: In the neoadjuvant therapy with pembrolizumab and ramucirumab, immune-activated status before treatment serves as a potential biomarker for predicting treatment response. Moreover, combining ramucirumab with pembrolizumab reduced immunosuppressive cells in the TME and inhibited tumor angiogenesis. These results suggest that this combination therapy modifies the TME and augments antitumor immunity. Larger clinical trial is warranted to assess the efficacy of neoadjuvant therapy with ramucirumab and pembrolizumab in patients with PD-L1-positive NSCLC. Citation Format: Kotaro Nomura, Shogo Kumagai, Shohei Koyama, Keiju Aokage, Yoshihisa Shimada, Kiyotaka Yoh, Masashi Wakabayashi, Miki Fukutani, Hideki Furuya, Tomohiro Miyoshi, Kenta Tane, Joji Samejima, Tetsuro Taki, Takuo Hayashi, Jun Matsubayashi, Genichiro Ishii, Norihiko Ikeda, Hiroyoshi Nishikawa, Masahiro Tsuboi. Biomarker analysis of pembrolizumab and ramucirumab neoadjuvant therapy for PD-L1-positive stage IB-IIIA lung cancer: EAST ENERGY trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1211.

Abstract 2732: NB203: A Novel therapeutic bispecific antibody targeting CEACAM1/VEGF for tumor immunity and angiogenesis modulation in the tumor microenvironment

Abstract The carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is detected on leukocytes, epithelia and endothelia, and involved in cell adhesion through homophilic or heterophilic binding. In various gastrointestinal cancers, NSCLC (non-small cell lung cancer), and melanoma, CEACAM1 is highly expressed on tumor cells and tumor stroma, with its levels increasing as the disease progresses. Notably, CEACAM1 has been found to dampen T and NK cell functions while promoting tumor angiogenesis and metastasis. In this study, we present NB203, a novel CEACAM1/VEGF bispecific antibody developed using Neologics CEACAM-plus, a bifunctional molecule engineering platform designed to target and modulate the tumor microenvironment (TME). NB203 effectively accumulated in the TME and hindered the homophilic or heterophilic interactions of CEACAM1. It demonstrated a significant enhancement in T cell activities and NK cell-mediated killing of CEACAM1-expressing tumor cells, and the inhibition of tumor cell migration and polyploidy through macrophages. More importantly, NB203 was specifically designed to impede tumor angiogenesis. It exhibited an increased binding capacity to both CEACAM1 and VEGF in pro-angiogenic TME, and undermined intratumoral vessel maturation by blocking VEGF and negatively regulating myeloid cell-dependent tumor angiogenesis. In summary, our preclinical data indicate that NB203 exhibits effective anti-tumor activity by mediating NK and cytotoxic T cell activities, improving the immune-suppressive TME, and suppressing tumor angiogenesis. The use of NB203 holds promise in overcoming resistance to anti-VEGF therapy and substantially improving the safety of therapeutic targeting of angiogenesis in cancer treatment. Citation Format: Jinyu Dong, Binbin Wang, Dong Wang, Jie Ni, Tingting Bu, Haojie Wang, Bishnu Nayak, Baiyang Wang, Xin Dong, Yu Zhang. NB203: A Novel therapeutic bispecific antibody targeting CEACAM1/VEGF for tumor immunity and angiogenesis modulation in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2732.

Abstract 3045: PPAR-α antagonist enhances immunotherapy and anti-angiogenic therapy to inhibit murine renal cancer

Abstract Peroxisome proliferator activated receptor alpha (PPARα) is a transcription factor and master regulator of fatty acid oxidation. PPARα ligands modulate the switch between co-activator and co-repressor transcription complexes at PPARα-controlled target genes. In addition, PPARα has been shown to transrepress NF-κB signaling and inhibit angiogenesis. Renal cell carcinoma (RCC) expresses high levels of PPAR-α and is a highly angiogenic cancer. Current frontline treatments for RCC include chemotherapy, anti-angiogenics, and immunotherapy but are limited by the immune suppressive state of the tumor microenvironment. TPST-1120 is a novel PPARα antagonist for the treatment of cancer patients that binds in the ligand binding domain, positioning the AF-2 activation helix in an inactive conformation. The inactive conformation has a lower affinity for co-activator motifs and reduces activation of PPARα regulated genes. In a CHO-based reporter cell line, TPST-1120 potently competed against the PPARα-specific agonist GW7647 (IC50 = 36 nM at 20 nM GW7647) and an endogenous PPARα agonist oleoylethanolamide (OEA; IC50 = 15 nM at 30 µM OEA). Furthermore, in an enzyme fragment complementation assay, we demonstrated that TPST-1120 inhibited agonist-induced binding of PPARα to MED1 by destabilizing the co-activator complex thereby stabilizing the inactive conformation of PPAR-α. In a murine model of renal cell adenocarcinoma (RENCA), TPST-1120 treatment for 12 days (30 mg/kg qd) reduced tumor growth by 52% as a monotherapy (p<0.0001). Consistent with these findings, TPST-1120 inhibited tumor angiogenesis and proliferation as quantified by Ki67 levels. Combination treatment with current frontline therapeutics resulted in synergistic tumor inhibition of 74% with anti-PD1 (200 ug Q3D for 12 days) and 81% with cabozantinib (15 mg/kg QD for 15 days) (p<.0001). Flow cytometry-based analysis of tumors demonstrated increased CD8+ T cell infiltration in the TME as well as decreases in M2 and elevations in M1 macrophages. Importantly, we demonstrate that TPST-1120 can reverse an immunosuppressive class of immune cells to promote anti-tumor immunity and anti-angiogenesis in kidney cancer in the absence of overt toxicity. These data support advancement of TPST-1120 into RCC. Citation Format: Michael Gillespie, Valerie Chen, Isabella Howard, Keira Smith, Dave Freund, Nathan Standifer, Dara Burdette, Thomas W. Dubensky, Sam Whiting, Dipak Panigrahy. PPAR-α antagonist enhances immunotherapy and anti-angiogenic therapy to inhibit murine renal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3045.