Tumor Neovasculature Research Articles

PEG-coated nanoparticles detachable in acidic microenvironments for the tumor-directed delivery of chemo- and gene therapies for head and neck cancer.

Background: Head and neck cancer (HNC) is a major cause of morbidity and mortality and has a poor treatment outcome. Irinotecan, a topoisomerase-I inhibitor, induces cell death by decreasing the religation of double-strand DNA. However, epithelial-mesenchymal transition (EMT), therapy resistance, and systemic toxicity caused by available antineoplastic agents hinder the efficacy and safety of HNC treatment. Chemotherapy combined with gene therapy shows potential application in circumventing therapy resistance and EMT. miR-200 exerts a remarkable suppressing effect on EMT-associated genes. Herein, liposomes and solid lipid nanoparticles (SLNs) modified with a pH-sensitive, self-destructive polyethylene glycol (PEG) shell and different peptides were designed as irinotecan and miR-200 nanovectors to enhance tumor-specific accumulation. These peptides included one ligand targeting the angiogenic tumor neovasculature, one mitochondrion-directed apoptosis-inducing peptide, and one cell-penetrating peptide (CPP) with high potency and selectivity toward cancer cells.Methods: Physicochemical characterization, cytotoxicity analysis, cellular uptake, regulation mechanisms, and in vivo studies on miR-200- and irinotecan-incorporated nanoparticles were performed to identify the potential antitumor efficacy and biosafety issues involved in HNC treatment and to elucidate the underlying signaling pathways.Results: We found that the cleavable PEG layer responded to low extracellular pH, and that the CPP and targeting peptides were exposed to improve the uptake and release of miR-200 and irinotecan into HNC human tongue squamous carcinoma (SAS) cells. The apoptosis of SAS cells treated with the combinatorial therapy was significantly induced by regulating various pathways, such as the Wnt/β-catenin, MDR, and EMT pathways. The therapeutic efficacy and safety of the proposed co-treatment outperformed the commercially available Onivyde and other formulations used in a SAS tumor-bearing mouse model in this study.Conclusion: Chemotherapy and gene therapy co-treatment involving pH-sensitive and targeting peptide-modified nanoparticles may be an innovative strategy for HNC treatment.

Aldoxorubicin-loaded nanofibers are cytotoxic for canine mammary carcinoma and osteosarcoma cell lines in vitro: A short communication

Chemotherapeutic drugs are given parenterally to treat various canine tumors. A limitation of parenteral administration is low drug penetration into the tumor, which reduces tumoricidal activity. Various drug carriers have been used to enhance tumor delivery, including albumin, liposomes and nanoparticles. A novel peptide-based nanofiber precursor (NFP) has been developed that is designed to take advantage of the leaky tumor neovasculature to promote drug delivery after parenteral administration. In this study, we loaded aldoxorubicin, an albumin-bound prodrug version of doxorubicin, onto NFP and tested the in vitro cytotoxicity in canine mammary carcinoma (CMT12, CMT25) and osteosarcoma (HMPOS, D-17, Abrams) cell lines. The half maximal inhibitory concentration (IC50) was determined with a luminescence-based cell viability assay. The IC50 for aldoxorubicin-loaded NFP was lower than free aldoxorubicin or doxorubicin in all cell lines, whereas non-drug loaded NFP had no cytotoxic effects. There were differences in IC50 between the osteosarcoma lines, with lower and higher IC50 for HMPOS and D-17 cells, respectively, with all drugs (aldoxorubicin-loaded NFP, free aldoxorubicin or free doxorubicin). Our results indicate that drug-loaded NFPs are cytotoxic for various canine mammary carcinoma and osteosarcoma cell lines in vitro and hold promise as a mechanism for enhancing delivery of chemotherapeutic agents to canine tumors.

EXTH-32. DEVELOPMENT OF EPHA3 DIRECTED CHIMERIC ANTIGEN RECEPTOR T CELL THERAPY FOR THE TREATMENT OF GLIOBLASTOMA MULTIFORME

Abstract BACKGROUND Efficacy of chimeric antigen receptor T cell (CART) therapy remains limited in solid tumors. Given the heterogeneity of surface receptor expression and immunosuppressive stromal microenvironment, strategies to target tumor neovasculature and tumor stromal cells are needed to help overcome CART inhibition by GBM. Eph receptors are the largest family of receptor tyrosine kinases and are integral to cell adhesion, migration, and axon guidance, during development and homeostasis. EphA3 is a receptor tyrosine kinase which is lowly expressed in adult tissues but is highly expressed in tumor neovasculature and tumor stromal cells in GBM and other solid tumors. EphA3 is over-expressed in up to 40% of GBM samples. We aimed to develop CART cells directed against EphA3 to use in targeting tumor neovasculature and tumor stromal cells in GBM. METHODS we developed a second generation CD28 co-stimulated CAR construct in a third generation lentivirus backbone to generate EphA3 CART cells using the single chain variable fragment of ifabotuzumab, a monoclonal antibody directed against EphA3. Patient derived GBM xenograft cell lines were used in these experiments. RESULTS EphA3 directed CART cells exhibited specific and potent antitumor activity against EphA3+ GBM cell lines with variable transcriptome EphA3 expression indicating its broader applicability in patients with GBM. Killing over 24-hour incubation was significant at low effector: target ratio: 52.5% killing at 1.25:1 against cell lines with 25.05% EphA3 expression and 37.1% killing at 1.25:1and 90% killing at 5:1 ratio against a cell line with 19.28% expression. Conversely, when co-cultured with UTD controls there was significantly lower killing, or growth of tumor cells. CONCLUSION We demonstrate for the first time that targeting EphA3 with CART cells is feasible, efficacious and represents a novel therapeutics strategy to target GBM. Data from in vivo and combinatorial CART experiments will be reported at the meeting.

TMIC-36. ALDH1A2 AS A NOVEL PUTATIVE MARKER OF MACROPHAGE DIFFERENTIATION IN GBM

Abstract Tumor-associated macrophages (TAM) are abundant in glioblastoma (GBM), composing up to 30% of the total tumor mass. However, their genotype/phenotype and exact role in tumor progression and immune suppression are not fully understood. Macrophages are believed to be polarized along a spectrum spanning from M0, M1, and M2 states. M1 TAMs are proinflammatory while M2 TAMs are associated with anti-inflammatory, pro-tumor responses. For example, using gelatin zymography we saw an increase in MMP 2 and 9 activities in co-culture of GBM cells and M2 polarized macrophages. In search for factors determining M2 type TAMs, we found Aldehyde Dehydrogenase 1 family A2 (ALDH1A2) to be highly over-expressed in M2 polarized THP1 cells using a gene expression array. We also performed single cell sequencing on CD45+ cells isolated from GBM tumors using antibody-conjugated magnetic beads. Monocytic/macrophage cells were found in 2 clusters and ALDH1A2 expression was increased in 2.1% of CD68+/CD163+ cells in Cluster 1 and 2.7% of CD68+/CD163+ cells in Cluster 2. Notably, ALDH1A2 was not detected in peripheral blood mononuclear cells. Analysis of the single cell sequencing data has led to identification of several genes whose expression is increased in ALDH1A2+ cells compared to ALDH1A2- cells. These genes are associated with lipid metabolism, regulation of neoplastic transformation, and insulin growth factor signaling. To further validate these results, we co-stained GBM tumor sections for CD163 and ALDH1A2 and observed that ALDH1A2 is co-localized in M2 macrophages. We have noticed a propensity of ALDH1A2+ cells to be associated with tumor neovasculature. Being that ALDH1A2 is the main enzyme in retinoic acid (RA) synthesis, it is plausible that this could represent another possible function of these enzyme-positive TAMs. Taken together these data suggest a role for ALDH1A2 as a novel putative marker of a subset of M2 TAM phenotype and function in GBM.

PH-Responsive PEG-Shedding and Targeting Peptide-Modified Nanoparticles for Dual-Delivery of Irinotecan and microRNA to Enhance Tumor-Specific Therapy.

Irinotecan is one of the main chemotherapeutic agents for colorectal cancer (CRC). MicroRNA-200 (miR-200) has been reported to inhibit metastasis in cancer cells. Herein, pH-sensitive and peptide-modified liposomes and solid lipid nanoparticles (SLN) are designed for encapsulation of irinotecan and miR-200, respectively. These peptides include one cell-penetrating peptide, one ligand targeted to tumor neovasculature undergoing angiogenesis, and one mitochondria-targeting peptide. The peptide-modified nanoparticles are further coated with a pH-sensitive PEG-lipid derivative with an imine bond. These specially-designed nanoparticles exhibit pH-responsive release, internalization, and intracellular distribution in acidic pH of colon cancer HCT116 cells. These nanoparticles display low toxicity to blood and noncancerous intestinal cells. Delivery of miR-200 by SLN further increases the cytotoxicity of irinotecan-loaded liposomes against CRC cells by triggering apoptosis and suppressing RAS/β-catenin/ZEB/multiple drug resistance (MDR) pathways. Using CRC-bearing mice, the in vivo results further indicate that irinotecan and miR-200 in pH-responsive targeting nanoparticles exhibit positive therapeutic outcomes by inhibiting colorectal tumor growth and reducing systemic toxicity. Overall, successful delivery of miR and chemotherapy by multifunctional nanoparticles may modulate β-catenin/MDR/apoptosis/metastasis signaling pathways and induce programmed cancer cell death. Thus, these pH-responsive targeting nanoparticles may provide a potential regimen for effective treatment of colorectal cancer.

Renal Cell Carcinoma: the Oncologist Asks, Can PSMA PET/CT Answer?

To critically review the potential clinical applications of prostate-specific membrane antigen (PSMA) radioactive ligands in renal cell carcinoma (RCC). Radioactive probes targeting PSMA hold promise in several malignancies in addition to prostate cancer, owing to the expression of PSMA by tumor neovasculature. The majority of clear cell RCCs (ccRCC), the most malignant RCC subtype, express PSMA on tumor-associated neovasculature. The endothelium of less aggressive RCC subtypes is PSMA positive in a lower, but still significant percentage of cases. PSMA might therefore represent an interesting theragnostic target in RCC. The preliminary data available suggest a potential role for PSMA-targeting radiopharmaceuticals in complementing conventional imaging for staging ccRCC patients at risk of nodal involvement and oligometastatic disease. Additional applications of PSMA imaging may be the selection and the response assessment of patients receiving anti-angiogenic treatments. The effectiveness of PSMA-targeting radionuclide therapy should also be investigated.

PSMA-Targeting Positron Emission Agents for Imaging Solid Tumors Other Than Non-Prostate Carcinoma: A Systematic Review.

Despite its name, prostate-specific membrane antigen (PSMA) has been shown using immunohistochemistry (IHC) to also be over-expressed in the tumor neovasculature of a wide variety of solid tumors other than prostate carcinoma. Accordingly, positron-emitting radiolabeled small molecules targeting PSMA, initially developed for positron emission tomography in prostate carcinomas, are currently being explored for their staging and restaging potential as an alternative imaging modality in other solid tumor types where 18-F-fluorodeoxyglucose (FDG)-PET imaging has low diagnostic accuracy. In this paper, the currently available literature in this field is reviewed. Preliminary, mainly retrospective studies are encouraging, with evidence of improved diagnostic sensitivity and specificity in clear cell renal carcinoma, glioma, and hepatocellular carcinoma, leading to a change in patient management in several patients. However, the results published thus far warrant confirmation by larger prospective studies additionally assessing the longitudinal impact on patient outcomes.

SEL1L plays a major role in human malignant gliomas.

Suppressor of Lin‐12‐like (C. elegans) (SEL1L) participates in the endoplasmic reticulum‐associated protein degradation pathway, malignant transformation and stem cell biology. We explored the role of SEL1L in 110 adult gliomas, of different molecular subtype and grade, in relation to cell proliferation, stemness, glioma‐associated microglia/macrophages (GAMs), prognostic markers and clinical outcome. SEL1L protein expression was assessed by immunohistochemistry and Western blotting. Genetic and epigenetic alterations were detected by molecular genetics techniques. SEL1L was overexpressed in anaplastic gliomas (World Health Organization [WHO] grade III) and in glioblastoma (GB, WHO grade IV) with the highest labelling index (LI) in the latter. Immunoreactivity was significantly associated with histological grade (p = 0.002) and cell proliferation index Ki‐67/MIB‐1 (p = 0.0001). In GB, SEL1L co‐localised with stemness markers Nestin and Sox2. Endothelial cells and vascular pericytes of proliferative tumour blood vessels expressed SEL1L suggesting a role in tumour neo‐vasculature. GAMs consistently expressed SEL1L. SEL1L overexpression was significantly associated with TERT promoter mutations (p = 0.0001), EGFR gene amplification (p = 0.0013), LOH on 10q (p = 0.0012) but was mutually exclusive with IDH1/2 mutations (p = 0.0001). SEL1L immunoreactivity correlated with tumour progression and cell proliferation, conditioning poor patient survival and response to therapy. This study emphasises SEL1L as a potential biomarker for the most common subgroup of TERT mutant/EGFR amplified/IDH‐WT GBs.

Antitumor effects of chitin specific lectin from Praecitrullus fistulosus by targeting angiogenesis and apoptosis

In the present study, chitin specific lectin was purified from fruit exudates of Praecitrullus fistulosus. The lectin was purified and analyzed using affinity chromatography, RP-HPLC and electrophoretic studies. Furthermore, protein was identified by MALDI-MS/MS and peptide mass fingerprinting. Purified lectin (PfL) effectively agglutinates RBC, lymphocytes and displayed strong cytotoxicity against colon cancer (line HT29) cells among screened cells. PfL induced apoptosis by altering the expression of apoptotic proteins via caspadse-3 dependent pathway. In vivo studies using EAC mice model proves the efficacy of PfL by activating apoptosis and inhibiting the tumor neovasculature by targeting the MVD, VEGF and MMP's secretion. More importantly, the PfL treatment leads to effective inhibition of tumor growth and a ∼2.71 fold increase in the lifespan of EAC mice. Collectively, our study provides comprehensive evidence that the role of dietary lectins with significant cytotoxic potential by targeting tumor angiogenesis and activating apoptosis in cancer study models.

Evidence of Prostate-Specific Membrane Antigen Expression in Hepatocellular Carcinoma Using 68Ga-PSMA PET/CT.

Nineteen HCC patients who underwent F-fluorodeoxyglucose (F-FDG) positron emission tomography (PET) as part of restaging procedure also underwent Ga-PSMA PET. F-FDG PET and Ga-PSMA findings were compared visually as well as quantitatively using maximized standardized uptake values (SUVmax). FDG was positive in 15 patients while 16 patients demonstrated PSMA expression. The only extrahepatic finding was one metastatic lymph node detected by both tracers. Mean SUVmax of liver lesions on FDG PET/CT was 8.3 ± 2.3 and mean tumor to background ratio was 2.3 ± 1.5. Respective values for Ga-PSMA PET/CT were 17.4 ± 9 and 3.3 ± 2.2. On visual and quantitative evaluation uptake was higher with PSMA in nine patients and higher with FDG in four patients. PSMA and FDG activity were similar in three patients. One of the FDG positive patients was PSMA negative whereas two patients were PSMA positive but FDG negative. Heterogeneous uptake pattern was observed in three patients. Comparison of mean SUVmax and T/B values between PET studies revealed no statistically significant difference (P > 0.1). The mean survival was 25 months (range: 18-32 months) and SUVmax of PSMA (P = 0.05) and FDG (P = 0.012) showed medium strength of correlation with overall survival. PSMA expression in advanced HCC can be demonstrated by Ga-PSMA PET but is not superior to FDG PET however it could be useful for identifying patients with limited therapeutic options.

Abstract 2396: Development of a new generation of dimeric, highly cytotoxic fusion proteins containing active GrB and VEGF targeting tumor neovasculature: Incorporating IgG heavy chain Fc fragments improves cytotoxicity, stability and pharmacokinetics

Abstract VEGF121 is a naturally-occurring splice variant that binds to VEGF receptors R-1 and R-2, which are over-expressed on the endothelium of tumor vasculature but not normal vasculature. The serine protease granzyme B (GrB) is capable of inducing intense cellular apoptosis through both caspase-dependent and caspase-independent multiple-cascade mechanisms. We have previously reported the expression of a fusion protein composed of Granzyme B and VEGF121 (GrB/VEGF121) and its subsequent in vitro and in vivo characterization. Here we report on the development and characterization of GrB-Fc-VEGF, which fuses the two domains via an IgG heavy chain Fc-fragment. We hypothesized that this modification, resulting in a relatively high molecular weight (133 kDa vs 80 kDa) would result in improved in vivo circulation and efficacy. VEGF inter-chain dimerization domains were modified such that dimerization occurred only through the Fc domain. GrB-Fc-VEGF121was expressed in HEK-293E cells under serum-free conditions and purified from the media by immobilized metal affinity chromatography (25 mg/L). Western blotting confirmed incorporation of both VEGF121 and GrB moieties into the construct. Comparison of GrB-Fc-VEGF121 with its expected molecular weight suggests significant glycosylation when produced in HEK293E cells. The enzymatic activity of GrB in GrB-Fc-VEGF121 was comparable to that of human GrB. In vitro studies of GrB-Fc-VEGF121 suggests improved cytotoxicity against cell lines expressing high levels of VEGFR-1 or VEGFR-2, unlike GrB/VEGF121, which targeted VEGFR-2+ cells more efficiently. Thus, the GrB-Fc-VEGF121 produced in HEK293E cells is active and has significant potential as a targeted therapeutic. In vitro cytotoxicity against an expanded panel of cells, internalization, and in vivo pharmacokinetics and efficacy against various tumor models are currently ongoing and will be presented. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Ana Alvarez-Cienfuegos, Michael G. Rosenblum. Development of a new generation of dimeric, highly cytotoxic fusion proteins containing active GrB and VEGF targeting tumor neovasculature: Incorporating IgG heavy chain Fc fragments improves cytotoxicity, stability and pharmacokinetics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2396.

Nerve growth factor (NGF) has an anti-tumor effects through perivascular innervation of neovessels in HT1080 fibrosarcoma and HepG2 hepatitis tumor in nude mice

This study investigated whether NGF prevents tumor growth by promoting neuronal regulation of tumor blood flow. HT1080 fibrosarcoma cells or HepG2 hepatitis cells were subcutaneously implanted into nude mice. On Day 21 after the implantation of tumor cells, human NGF (40 or 80 ng/h for 14 days) was administered using a micro-osmotic pump. Growth rates of both tumors were significantly inhibited by the treatment of NGF, and the survival rate was also extended. Significant suppression of HT1080 tumor growth lasted after withdrawing NGF. NGF markedly increased the density of α-smooth muscle actin (α-SMA)-immunoreactive (ir) cells without changing neovessel density in HT1080 tumor tissues. Double immunostaining demonstrated protein gene product (PGP) 9.5-ir nerves around α-SMA-ir cells were found in HT1080 tumor tissue treated with NGF. The blood flow in HepG2 tumors treated with saline was significantly higher than in the non-tumor control area, but the tumor blood flow was markedly reduced by NGF treatment. In in vitro studies, NGF significantly accelerated migration of aortic smooth muscle cells but not endothelial cells, whereas NGF had no cytotoxic action on both cells. NGF inhibits tumor growth via indirect action, probably through innervation and maturation of tumor neovasculature, which regulates blood flow into tumor tissues.

Cowpea mosaic virus nanoparticles for cancer imaging and therapy.

Nanoparticle platforms are particularly attractive for theranostic applications due to their capacity for multifunctionality and multivalency. Some of the most promising nano-scale scaffold systems have been co-opted from nature including plant viruses such as cowpea mosaic virus (CPMV). The use of plant viruses like CPMV as viral nanoparticles is advantageous for many reasons; they are non-infectious and nontoxic to humans and safe for use in intravital imaging and drug delivery. The CPMV capsid icosahedral shape allows for enhanced multifunctional group display and the ability to carry specific cargoes. The native tropism of CPMV for cell-surface displayed vimentin and the enhanced permeability and retention effect allow them to preferentially extravasate from tumor neovasculature and efficiently penetrate tumors. Furthermore, CPMVs can be engineered via several straightforward chemistries to display targeting and imaging moieties on external, addressable residues and they can be loaded internally with therapeutic drug cargoes. These qualities make them highly effective as biocompatible platforms for tumor targeting, intravital imaging and cancer therapy.

Positron Emission Tomography Imaging of Platelet-Derived Growth Factor Receptor β in Colorectal Tumor Xenograft Using Zirconium-89 Labeled Dimeric Affibody Molecule.

Platelet-derived growth factor receptor β (PDGFRβ) is overexpressed in a variety of malignant cancers, plays a critical role in tumor angiogenesis, and has been proven as a valuable target for cancer treatment. In this pilot study, a dimeric affibody molecule, ZPDGFRβ, was prepared and radiolabeled with positron emission radionuclide zirconium-89 for PET imaging of colorectal tumors by targeting PDGFRβ expression in vivo. The PDGFRβ-binding capability of dimeric affibody was evaluated by flow cytometry, immunofluorescent staining, and whole-body optical imaging. Then, ZPDGFRβ was conjugated with DFO-Bn-NCS and radiolabeled with 89Zr. Targeted binding capability of 89Zr-DFO-ZPDGFRβ to PDGFRβ expressing cells was investigated by cellular assay in vitro and microPET/CT imaging in vivo. Dimeric ZPDGFRβ affibody had specifically higher binding capability with PDGFRβ expressing pericytes rather than LS-174T cancer cells, and well colocalized with tumor neovasculature by flow cytometry and immunofluorescent assay. ZPDGFRβ was successfully labeled with 89Zr by DFO chelating with yield of 94.1 ± 3.53%. 89Zr-DFO-ZPDGFRβ indicated preserved specific binding ability with PDGFRβ expressing cells and effective inhibiting capability to PDGF-β ligands ( P < 0.05) in vitro. Biodistribution indicated that tumor uptake of 89Zr-DFO-ZPDGFRβ reached the peak of 6.93 ± 0.64%ID/g, and the tumor-to-blood ratio was 5.5 ± 0.6 at 2 h post-injection. LS-174T xenografts were clearly visualized by microPET/CT imaging through 1 to 4 h post-injection of 89Zr-DFO-ZPDGFRβ affibody conjugate. In conclusion, the 89Zr-DFO-ZPDGFRβ conjugate demonstrated specific and high binding ability with colorectal tumor, which indicated its use as a potential radiopharmaceutical for diagnostic imaging of tumor associate vasculatures with PET/CT.

Physiologic MR imaging of the tumor microenvironment revealed switching of metabolic phenotype upon recurrence of glioblastoma in humans

Treating recurrent glioblastoma (GB) is one of the challenges in modern neurooncology. Hypoxia, neovascularization, and energy metabolism are of crucial importance for therapy failure and recurrence. Twenty-one patients with initially untreated GB who developed recurrence were examined with a novel MRI approach for noninvasive visualization of the tumor microenvironment (TME). Imaging biomarker information about oxygen metabolism (mitochondrial oxygen tension) and neovascularization (microvascular density and type) were fused for classification of five different TME compartments: necrosis, hypoxia with/without neovascularization, oxidative phosphorylation, and glycolysis. Volume percentages of these TME compartments were compared between untreated and recurrent GB. At initial diagnosis, all 21 GB showed either the features of a glycolytic dominant phenotype with a high percentage of functional neovasculature (N = 12) or those of a necrotic/hypoxic dominant phenotype with a high percentage of defective tumor neovasculature (N = 9). At recurrence, all 21 GB revealed switching of the initial metabolic phenotype: either from the glycolytic to the necrotic/hypoxic dominant phenotype or vice-versa. A necrotic/hypoxic phenotype at recurrence was associated with a higher rate of multifocality of the recurrent lesions. Our MRI approach may be helpful for a better understanding of treatment-induced metabolic phenotype switching and for future studies developing targeted therapeutic strategies for recurrent GB.

Abstract A179: Targeting UCP2 pathway in melanoma cells reprograms the tumor microenvironment and initiates antitumor immune cycle

Abstract Immune checkpoint blockade treatment displays promising therapeutic efficiency in different cancer types. However, a significant proportion of patients are refractory to this treatment due to lack of T-cell infiltration in tumors, a phenotype known as “cold tumors.” To improve the therapeutic benefits of checkpoint blockade, it is crucial to address how to escalate T-cell infiltration into tumors. Here, we characterized melanoma patients in The Cancer Genome Atlas (TCGA) into either high or low T-cell antitumor responses. We then identified the mRNA expression level of mitochondrial uncoupling protein 2 (UCP2) positively correlates with T-cell antitumor immune responses and prolonged patients survival. By using murine melanoma model, we revealed that overexpressing UCP2 in melanoma cells elevates CD8+ T-cell infiltration into the tumor microenvironment (TME) in a conventional type 1 dendritic cell (cDC1)-dependent manner and normalize tumor neovasculature. Mechanistically, UCP2 induction in tumor cells alters the cytokine milieu in tumors and stimulates the recruitment of cDC1 through a IRF5-CXCL10 chemokine axis. Hence, enhancing the expression of UCP2 in melanoma cells provides immunostimulatory shift by engaging the cDC1-CD8+ T-cell dependent antitumor immune cycle in the TME. Interestingly, we demonstrated that inducing UCP2 expression in melanoma cells by genetic approach or administration of rosiglitazone, an approved antidiabetic PPAR agonist, can sensitize PD-1 blockade-resistant melanomas to anti-PD-1 antibody treatment. Accordingly, our data indicate that targeting UCP2 in melanoma cells can be a potent strategy to increase antitumor response to checkpoint blockades in non-T-cell-inflamed tumors. Citation Format: Wan-Chen Cheng. Targeting UCP2 pathway in melanoma cells reprograms the tumor microenvironment and initiates antitumor immune cycle [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A179.

Imaging of Prostate-Specific Membrane Antigen with Small-Molecule PET Radiotracers: From the Bench to Advanced Clinical Applications

In recent years, small-molecule inhibitors of prostate-specific membrane antigen (PSMA) labeled with radionuclides that allow for positron emission tomography (PET) imaging have been extensively studied in many clinical contexts in men with prostate cancer (PCa). The high sensitivity and specificity of these agents for identifying sites of PCa has quickly led to their widespread adoption as a de facto clinical standard of care throughout much of the world. PSMA-targeted PET radiotracers have been particularly well-studied in preoperatively staging men with high-risk PCa, evaluating biochemical recurrence following definitive therapy, and guiding metastasis-directed therapy in patients suspected of having oligorecurrent/oligometastatic disease. Furthermore, the expression of PSMA on the tumor neovasculature of many nonprostate malignancies has enabled a burgeoning subfield concentrated on delineating the potential utility of PSMA-targeted PET agents for imaging other cancers. In this review, we highlight the preclinical development of key small molecules that are now being clinically utilized for PCa imaging, discuss the roles of PSMA-targeted agents in guiding patient management, and consider the role these compounds may play in imaging nonprostate cancers.

Antibody-based Delivery of TNF to the Tumor Neovasculature Potentiates the Therapeutic Activity of a Peptide Anticancer Vaccine.

There is a growing interest in the use of tumor antigens for therapeutic vaccination strategies. Unfortunately, in most cases, the use of peptide vaccines in patients does not mediate shrinkage of solid tumor masses.Experimental Design: Here, we studied the opportunity to boost peptide vaccination with F8-TNF, an antibody fusion protein that selectively delivers TNF to the tumor extracellular matrix. AH1, a model antigen to investigate CD8+ T-cell immunity in BALB/c mice, was used as vaccine. Peptide antigens alone exhibited only a modest tumor growth inhibition. However, anticancer activity could be substantially increased by combination with F8-TNF. Analysis of T cells in tumors and in draining lymph nodes revealed a dramatic expansion of AH1-specific CD8+ T cells, which were strongly positive for PD-1, LAG-3, and TIM-3. The synergistic anticancer activity, observed in the combined use of peptide vaccination and F8-TNF, was largely due to the ability of the fusion protein to induce a rapid hemorrhagic necrosis in the tumor mass, thus leaving few residual tumor cells. While the cell surface phenotype of tumor-infiltrating CD8+ T cells did not substantially change upon treatment, the proportion of AH1-specific T cells was strongly increased in the combination therapy group, reaching more than 50% of the CD8+ T cells within the tumor mass. Because both peptide vaccination strategies and tumor-homing TNF fusion proteins are currently being studied in clinical trials, our study provides a rationale for the combination of these 2 regimens for the treatment of patients with cancer.

Microbubble-enhanced ultrasound for the antivascular treatment and monitoring of hepatocellular carcinoma.

Background and Objective: Hepatocellular carcinoma (HCC) is the most common primary liver malignancy, and its current management relies heavily on locoregional therapy for curative therapy, bridge to transplant, and palliative therapy. Locoregional therapies include ablation and hepatic artery therapies such as embolization and radioembolization. In this study we evaluate the feasibility of using novel antivascular ultrasound (AVUS) as a noninvasive locoregional therapy to reduce perfusion in HCC lesions in a rat model and, monitor the effect with contrast-enhanced ultrasound imaging.Methods: HCC was induced in 36 Wistar rats by the ingestion of 0.01% diethylnitrosamine (DEN) for 12 weeks. Two therapy regimens of AVUS were evaluated. A primary regimen (n = 19) utilized 2-W/cm2, 3-MHz ultrasound (US) for 6 minutes insonation with 0.7 ml of microbubbles administered as an intravenous bolus. An alternate dose at half the primary intensity, sonication time, and contrast concentration was evaluated in 11 rats to assess the efficacy of a reduced dose. A control group (n = 6) received a sham therapy. Tumor perfusion was measured before and after AVUS with nonlinear contrast ultrasound (NLC) and power Doppler (PD). The quantitative perfusion measures included perfusion index (PI), peak enhancement (PE), time to peak (TTP), and perfusion area from NLC and PD scans. Total tumor area perfused during the scan was measured by a postprocessing algorithm called delta projection. Tumor histology was evaluated for signs of tissue injury and for vascular changes using CD31 immunohistochemistry.Results: DEN exposure induced autochthonous hepatocellular carcinoma lesions in all rats. Across all groups prior to therapy, there were no significant differences in the nonlinear contrast observations of peak enhancement and perfusion index. In the control group, there were no significant differences in any of the parameters after sham treatment. After the primary AVUS regimen, there were significant changes in all parameters (p ≤ 0.05) indicating substantial decreases in tumor perfusion. Peak enhancement in nonlinear contrast scans showed a 37.9% ± 10.1% decrease in tumor perfusion. Following reduced-dose AVUS, there were no significant changes in perfusion parameters, although there was a trend for the nonlinear contrast observations of peak enhancement and perfusion index to increase.Conclusion: This study translated low-intensity AVUS therapy into a realistic in vivo model of HCC and evaluated its effects on the tumor vasculature. The primary dose of AVUS tested resulted in significant vascular disruption and a corresponding reduction in tumor perfusion. A reduced dose of AVUS, on the other hand, was ineffective at disrupting perfusion but demonstrated the potential for enhancing tumor blood flow. Theranostic ultrasound, where acoustic energy and microbubbles are used to monitor the tumor neovasculature as well as disrupt the vasculature and treat lesions, could serve as a potent tool for delivering noninvasive, locoregional therapy for hepatocellular carcinoma.

Remissions in Relapse/Refractory Acute Myeloid Leukemia Patients Following Treatment with NKG2D CAR-T Therapy without a Prior Preconditioning Chemotherapy

▪Introduction:CYAD-01 is a chimeric antigen receptor T-cell (CAR-T) product based on the receptor NKG2D with specificity for a broad range of ligands (MICA, MICB and ULBP1-6) expressed on most tumors. In vivo preclinical studies showed long-term anti-tumor activity of CYAD-01, whilst not only targeting tumor cells but also cells from the tumor neo-vasculature and immunosuppressive environment in the absence of pre-conditioning therapy.Methods:Exploiting this unique mode of action of CYAD-01, the THINK trial (NCT03018405) is an open-label Phase I study assessing the safety and clinical activity of multiple CYAD-01 administrations without prior preconditioning in 2 parallel cohorts: one in patients (pt) with metastatic solid tumors and the other one in hematological malignancies, including relapsing/refractory (r/r) acute myeloid leukemia (AML), multiple myeloma (MM) and myelodysplastic syndrome (MDS). The dose escalation segment of the study evaluates 3 dose levels (DL; 3x108, 1x109 and 3x109 cells per injection) of one cycle of 3 CYAD-01 administration with 2-weeks intervals. The study has been amended at the stage of DL-2 to authorize a second cycle of 3 CYAD-01 administrations in case of no progressive disease after 2 months.Results:As of July 31, 2018, 12 pts in the hematological cohort (8 AML, 3 MM and 1 MDS) have been enrolled at the 3 DLs (6 pts in DL-1, 3 in DL-2 and 3 in DL-3) without prior preconditioning. Median age was 64 (range 29-83) and median number of prior therapies was 3. DL-3 (3 pts) has been fully accrued as of data cutoff. Over 34 injections, 5 pts experienced grade (G) 3/4 treatment-related AEs: in DL-1, one pt experienced G3 lymphopenia and a second pt experienced G4 lymphopenia and G4 pneumonitis in DL-2, one pt experienced G3 lymphopenia and G3 thrombocytopenia and two other pts experienced G3 cytokine release syndrome (CRS). Treatment related AEs occurring in ≥ 1pt include pyrexia, CRS, hypoxia, lymphopenia, fatigue and nausea. CRS occurred in 5 pts, three G1/2 and two G3 AEs, with rapid resolution to appropriate treatment including tocilizumab. No neurotoxicity AEs have been observed to date.Out of the 8 r/r AML pts enrolled, 7 were response evaluable (2 at DL-1, 3 at DL-2 and 2 at DL-3). The third DL-3 pt has just initiated the first cycle of CYAD-01 treatment. Overall response rate in r/r AML pts was 42% (3/7 patients) with 1 complete remission with partial hematologic recovery (CRh) in DL-1 and 2 CR with incomplete marrow recovery (CRi; 1 in DL-1 and 1 in DL-3). All responding pts achieved response by day 29 (i.e. after 2 CYAD-01 administrations). The AML pt with CRh in DL-1 was bridged to allogeneic hematopoietic stem cell transplantation (allo-HSCT) on day +97 post CYAD-01 and is in durable complete molecular remission (CRMRD-) for more than 1 year (ongoing). The two other responding pts had CRi for 1 month. Two other AML patients at DL-2 had clinical benefit/disease stabilization with hematologic improvement and bone marrow blasts decrease: one pt for 3 months and with a decrease from 24% to 10% and the second pt for at least 4 months (ongoing) and with a decrease from 9.8% to 5.5%. The first patient in CRh had a relative increase in the systemic levels of SDF-1, RANTES and MCP-1 which correlated with the timing of injections. CYAD-01 engraftment kinetics, NKG2D ligand expression (including blasts and soluble ligand expression), and the kinetics of cytokine induction will be correlated with patient's responses. The 3 MM and the MDS pt, all in DL-1, did not present any sign of clinical activity.Conclusions:We have demonstrated the feasibility and safety of multiple injections of CYAD-01 without preconditioning chemotherapy. We evidenced promising anti-leukemic activity with 42% ORR in r/r AML with 5/7 pts having clinical benefit. Rates of G3/4 CRS were low and manageable. Updated safety, activity and correlative science data of the complete dose-escalation segment will be presented. DisclosuresSallman:Celgene: Research Funding, Speakers Bureau. Kerre:Celyad: Consultancy; BMS: Consultancy; Celgene: Consultancy, Research Funding. Davila:Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees. Wang:Jazz: Speakers Bureau; Jazz: Speakers Bureau; Amgen: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Amgen: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Dekker:Celyad: Employment. Snykers:Celyad: Employment. Sotiropoulou:Celyad: Employment. Breman:Celyad: Employment. Braun:Celyad: Employment. Lonez:Celyad: Employment. Verma:Celyad: Employment. Lehmann:GSK: Patents & Royalties; Celyad: Employment, Honoraria, Patents & Royalties.