Tumor Vascular Perfusion Research Articles

Tumor perfusion enhancement by focus ultrasound-induced blood-brain barrier opening to potentiate anti-PD-1 immunotherapy of glioma

ObjectiveTo demonstrate the feasibility of using focused ultrasound to enhance delivery of PD-1 inhibitors in glioma rats and determine if such an approach increases treatment efficacy. MethodsC6 glioma in situ rat model was used in this study. Transcranial irradiation with FUS combined with microbubbles was administered to open the blood-brain barrier (BBB). The efficacy of BBB opening was evaluated in normal rats. The rats with glioma were grouped to evaluate the role of PD-1 inhibitors combined with FUS-induced immune responses in suppressing glioma when the BBB opens. Flow cytometry was used to examine the changes of immune cell populations of lymphocytes in peripheral blood, tumor tissue and spleen tissue of the rats. A section of rat brain tissue was also used for histological and immunohistochemical analysis. The survival of the rats was then monitored; the tumor progression and changes in blood perfusion of tumor were dynamically observed in vivo using multimodal MRI. ResultsFUS combined with microbubbles could enhance the blood perfusion of tumors by increasing the permeability of BBB (p < 0.0001), thus promoting the infiltration of CD4+ T lymphocytes (p < 0.01). Compared with the control group, the combination treatment group had increased in the infiltration number of CD4+(p < 0.05) and CD8+ T (p < 0.05); the tumor volume of the combined treatment group was smaller than that of the control group (p < 0.01) and the survival rate of the rats was prolonged (p < 0.05). ConclusionsIn this study, we demonstrated that the transient opening of the BBB induced by FUS enhanced tumor vascular perfusion and facilitated the delivery of PD-1 inhibitors, ultimately improving the therapeutic efficacy for glioblastoma.

Imaging-Guided Metabolic Radiosensitization of Pediatric Rhabdoid Tumors.

Tumor hypoxia leads to increased resistance to radiation therapy (RT), resulting in markedly worse clinical outcomes in the treatment and management of pediatric malignant rhabdoid tumors (MRT). To alleviate hypoxia in MRT, we repurposed an FDA approved, mitochondrial oxidative phosphorylation (OXPHOS) inhibitor, Atovaquone (AVO), to inhibit oxygen consumption and thereby enhance the sensitivity of tumor cells to low dose RT in MRT by hypoxia alleviation. Additionally, to better understand the tumor response induced by AVO and optimize the combination with RT, we employed an emerging, noninvasive imaging modality, known as multispectral optoacoustic tomography (MSOT), to monitor and evaluate real-time dynamic changes in tumor hypoxia and vascular perfusion. Oxygen-Enhanced (OE)-MSOT could measure the change of tumor oxygenation in the MRT xenograft models after AVO and RT treatments, indicating its potential as a response biomarker. OE-MSOT showed that treating MRT mouse models with AVO resulted in a transient increase in oxygen saturation (ΔsO 2 ) in tumors when the mice were subjected to oxygen challenge, while RT or saline treated groups produced no change. In AVO+RT combination groups, the tumors showed an increase in ΔsO 2 after AVO administration followed by a significant decrease after RT, that correlated with a strong anti-tumor response, demarcated by complete regression of tumors, with no relapse on long-term monitoring. These observations were histologically validated. In MRT models of acquired AVO resistance, combination therapy failed to alleviate tumoral hypoxia and elicit any therapeutic benefit. Together, our data highlights the utility of repurposing anti-malarial AVO as an anticancer adjuvant for enabling low dose RT for pediatric patients.

Abstract 700: Impact of heterogeneity for mismatch repair activity on colon tumor development and therapeutic response

Abstract Background: Colorectal cancers (CRC) that are deficient for DNA mismatch repair (dMMR) proteins have high levels of genetic instability and consequently high mutational burden, creating numerous neoantigens that can elicit an immune response. Therefore, dMMR CRC are the best candidates for immune checkpoint inhibition (ICI) compared to proficient DNA mismatch repair (pMMR) CRC. Early clinical trials revealed that only 30-55% metastatic dMMR CRC responded to ICI. Recent studies utilizing allograft models have demonstrated that tumor immune landscape can be primed with radiopharmaceutical therapy (RPT). Although early results have been quite promising, allograft models often fail to accurately predict efficacy for many reasons, but most often are due to a lack of heterogeneity. Methods: To better understand how heterogenous MMR expression impacts tumor development and response to ICI alone or in combination with RPT, we developed a new mouse model that develops three tumor types: (1) homotypic dMMR tumors that express green fluorescent protein, (2) homotypic pMMR tumors that express red fluorescent protein, and (3) heterotypic tumors with a mixture of dMMR and pMMR cells. Surveillance bright field and fluorescent colonoscopies allow us to assess treatment response by measuring tumor size and proportion of dMMR and pMMR tumor cells in real-time. These outcomes are verified through ex vivo imaging and histological analysis. Results: Tumor response to anti-PD-L1 ICI was dependent on MMR status. Homotypic dMMR tumors exhibited the strongest response: significantly smaller in treated mice than controls, exhibited areas of vascular congestion and increased CD8+ cytotoxic T-cells infiltration. Response of heterotypic MMR tumors varied depending on percentage of dMMR cells; tumors with a high percentage of dMMR cells remained small, whereas tumors with a low percentage of dMMR had an outgrowth of pMMR cells. Treated and untreated homotypic pMMR tumors were indistinguishable. To enhance the response of all three tumor types, mice were treated with dual ICI treatment, anti-PD-L1 and anti-CTLA-4, or RPT. Animals administered the tri-therapy (dual ICI + RPT) survived significantly longer with fewer tumors than animals treated with either dual ICI or RPT alone. Conclusions: Our innovative mouse model allows for the highly detailed characterization of tumor response to therapies. Simplistic allograft models may not adequately account for the dynamic effects that neighboring cell death may have on radio-resistant cells, on tumor vascular perfusion and oxygenation, or on tumor immune infiltration and adaptive immune recognition of remaining cells. We begin to fill these critical gaps in knowledge and therefore the results from our studies will help guide the translation and integrated use of ICIs and RPTs in clinic. Citation Format: Santina M. Snow, Dawn Albrecht, Paul A. Clark, Caroline P. Kerr, Joseph J. Grudzinski, Justin J. Jeffery, Hansel Comas Rojas, Reinier Hernandez, Kristina A. Matkowskyj, Jamey P. Weichert, Zachary S. Morris, Richard B. Halberg. Impact of heterogeneity for mismatch repair activity on colon tumor development and therapeutic response [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 700.

Hypoxia-induced miR-181a-5p up-regulation reduces epirubicin sensitivity in breast cancer cells through inhibiting EPDR1/TRPC1 to activate PI3K/AKT signaling pathway

Breast carcinoma (BC) ranks as a predominant malignancy and constitutes the second principal cause of mortality among women globally. Epirubicin stands as the drug of choice for BC therapeutics. Nevertheless, the emergence of chemoresistance has significantly curtailed its therapeutic efficacy. The resistance mechanisms to Epirubicin remain not entirely elucidated, yet they are conjectured to stem from diminished tumor vascular perfusion and resultant hypoxia consequent to Epirubicin administration. In our investigation, we meticulously scrutinized the Gene Expression Omnibus database for EPDR1, a gene implicated in hypoxia and Epirubicin resistance in BC. Subsequently, we delineated the impact of EPDR1 on cellular proliferation, motility, invasive capabilities, and interstitial-related proteins in BC cells, employing methodologies such as the CCK-8 assay, Transwell assay, and western blot analysis. Our research further unveiled that hypoxia-induced miR-181a-5p orchestrates the regulation of BC cell duplication, migration, invasion, and interstitial-related protein expression via modulation of EPDR1. In addition, we identified TRPC1, a gene associated with EPDR1 expression in BC, and substantiated that EPDR1 influences BC cellular dynamics through TRPC1-mediated modulation of the PI3K/AKT signaling cascade. Our findings underscore the pivotal role of EPDR1 in the development of BC. EPDR1 was found to be expressed at subdued levels in BC tissues, Epirubicin-resistant BC cells, and hypoxic BC cells. The overexpression of EPDR1 curtailed BC cell proliferation, motility, invasiveness, and the expression of interstitial-related proteins. At a mechanistic level, the overexpression of hypoxia-induced miR-181a-5p was observed to inhibit the EPDR1/TRPC1 axis, thereby activating the PI3K/AKT signaling pathway and diminishing the sensitivity to Epirubicin in BC cells. In summation, our study demonstrates that the augmentation of hypoxia-induced miR-181a-5p diminishes Epirubicin sensitivity in BC cells by attenuating EPDR1/TRPC1 expression, thereby invigorating the PI3K/AKT signaling pathway. This exposition offers a theoretical foundation for the application of Epirubicin in BC therapy, marking a significant contribution to the existing body of oncological literature.

ROS impairs tumor vasculature normalization through an endocytosis effect of caveolae on extracellular SPARC

BackgroundThe accumulation of reactive oxygen species (ROS) in tumor microenvironment (TME) is an important player for tumorigenesis and progression. We aimed to explore the outcomes of ROS on tumor vessels and the potential regulated mechanisms.MethodsExogenous H2O2 was adopted to simulate the ROS setting. Immunofluorescence staining and ultrasonography were used to assess the vascular endothelial coverage and perfusions in the tumors inoculated with Lewis lung cancer (LLC) and melanoma (B16F10) cells of C57BL/6 mice, respectively. ELISA and western-blot were used to detect the expression of secreted acidic and cysteine-rich protein (SPARC) and Caveale-1 in human umbilical vein endothelial cells (HUVEC) extra- and intracellularly. Intracellular translocation of SPARC was observed using electron microscopy and immunofluorescence approaches.ResultUnder the context of oxidative stress, the pericyte recruitment of neovascularization in mouse lung cancer and melanoma tissues would be aberrated, which subsequently led to the disruption of the tumor vascular architecture and perfusion dysfunction. In vitro, HUVEC extracellularly SPARC was down-regulated, whereas intracellularly it was up-regulated. By electron microscopy and immunofluorescence staining, we observed that SPARC might undergo transmembrane transport via caveale-1-mediated endocytosis. Finally, the binding of SPARC to phosphorylated-caveale-1 was also detected in B16F10 tissues.ConclusionIn the oxidative stress environment, neovascularization within the tumor occurs structural deterioration and decreased perfusion capacity. One of the main regulatory mechanisms is the migration of extracellular SPARC from the endothelium to intracellular compartments via Caveolin-1 carriers.

Evaluation of performance trade-offs when using mechanically swept 1D linear arrays for 3D DCE-US.

Dynamic contrast enhanced ultrasound imaging (DCE-US) may be used to characterise tumour vascular perfusion using metrics derived from time amplitude curves (TAC). Three-dimensional (3D) DCE-US enables generation of 3D parametric maps of TAC metrics that may inform on how perfusion varies across the entire tumour. The aim of this work was to understand the effect of low temporal sampling (i.e., < 1 Hz) typical of 3D imaging using a swept 1D array transducer on the evaluation of TAC metrics and the effect of transducer motion in combination with flow on 3D parametric maps generated using both plane wave imaging (7 angles) and focused imaging. Correlation maps were introduced to evaluate the spatial blurring of TAC metrics. A research ultrasound scanner and a pulse-inversion algorithm were used to obtain DCE-US. 2D (frame rate 10 Hz) and 3D (volume rate 0.4 Hz) images were acquired of a simple wall-less vessel phantom (flow phantom) and a cartridge phantom. Volumetric imaging provided similar TACs to that of the higher 2D sampling rate. Varying sweep speed and acceleration/deceleration had little influence on the 3D TAC compared to 2D for both focused and plane wave imaging. Sweeping motion and limited temporal sampling (0.4 Hz) did not change the spatial correlation of TAC metrics measured using focused imaging whereas a small increase in correlation across the cartridge phantom was observed for plane wave imaging. This was attributed to grating lobe artefacts, broad beam spatial blurring and incoherent compounding caused by motion. Increased correlation will reduce the spatial resolution with which inhomogeneity of vascular perfusion can be mapped supporting the choice of focused imaging for DCE-US.

CT perfusion combined with energy spectrum imaging to evaluate the short-term efficacy of bronchial arterial chemoembolization for lung cancer

Objective: To evaluate the clinical value of dynamic volumetric CT perfusion combined with energy spectrum imaging in bronchial arterial chemoembolization (BACE) in patients with lung cancer. Methods: The data of 31 patients with lung cancer confirmed by pathology and treated with BACE in Lishui Central Hospital from January 2018 to February 2022 were retrospectively collected, including 23 men and 8 women, aged 31-84 (67) years. All patients received perfusion scans of lesion sites within 1 week before surgery and 1 month after surgery. We collected and compared the changes in preoperative and postoperative perfusion parameters such as blood flow (BF), blood volume (BV), mean through time (MTT), permeability surface (PS) and energy spectrum parameters including arterial phase CT value (CTA), venous phase CT value (CTV), arterial phase iodine concentration (ICA), venous phase of iodine concentration (ICV), arterial standardization iodine concentration (NICA), and intravenous standardized iodine concentration (NICV) to confirm the significance of these parameters in evaluating the short-term efficacy of BACE in the treatment of advanced lung cancer. Data normality was tested using the Kolmogorov-Smirnov test and normally distributed measurement data are expressed here as mean ± standard deviation; the independent-samples t-test was used for comparisons between two groups. The measurement data that were not normally distributed are expressed as median (interquartile interval) [M (Q1, Q3)], and the comparison between the two groups used the Kruskal-Wallis test. Count data are expressed as cases (%), and comparisons between groups used the χ2 test. Results: The objective response rate (ORR) and disease control rate (DCR) at 1 month after BACE were 54.8% (17/31) and 96.8% (30/31), respectively. CT perfusion parameters and energy spectrum parameters of patients before and after BACE treatment were compared. The results showed that BF, BV, MTT, ICA, ICV and NICV were significantly decreased after BACE treatment compared with before treatment, and the differences were statistically significant[58.06 (40.47,87.22) vs.23.57(10.92, 36.24) ml·min-1·100g-1,3.33(2.86,6.09) vs.2.12(1.96,3.61)ml/100g,2.70(2.19,3.88) vs.1.53 (1.12,2.25)s, 3.51 (3.11,4.14)vs.1.74 (1.26,2.50)mg/ml,2.00 (1.30,2.45) vs.1.32(0.92,1.76)mg/ml,0.51(0.42,0.57) vs.0.33(0.23,0.39)](all P<0.05). At the same time, compared with the non-remission group, the study results showed that the difference of parameters in remission group before and after BACE was more obvious, including ΔBF, ΔBV, ΔMTT, ΔPS, ΔCTA, ΔCTV, ΔICA, ΔICV, ΔNICA, ΔNICV were significantly increased, and the difference was statistically significant [36.82(32.38, 45.34) vs.9.50(-1.43, 12.34) ml·min-1·100g-1,4.46(2.52, 5.79) vs.0.22(-0.76, 4.09) ml/100g,4.22(2.25, 6.77) vs.0.43(-2.53, 1.88) s,10.07 (2.89, 13.13) vs.-2.01(-6.77, 4.28) ml·min-1·100g-1,14.22(11.88, 20.57) vs.4.18(-5.25, 6.37) HU, 34.6(14.88, 43.15) vs.11.60(0.26, 25.05) HU,0.95(0.54, 1.47) vs.0.11(0.20, 0.59) mg/ml,1.57(1.10, 2.38) vs. 0.26(-0.21, 0.63) mg/ml,0.05(0.03, 0.08) vs.-0.02(-0.04, 0.01),0.18(0.13, 0.21)vs. 0.11(-0.06, 0.16)](all P<0.05). Conclusions: CT perfusion combined with spectral imaging could effectively evaluate the changes in tumor vascular perfusion in patients with advanced lung cancer before and after BACE treatment, which has important value in judging the short-term efficacy after treatment.

Spatiotemporal image reconstruction for photoacoustic computed tomography of tumor vascular perfusion in murine models

Monitoring critical physiological processes in murine models like tumor vascular perfusion and its response to prospective anti-cancer treatments is a significant potential use case of dynamic photoacoustic computed tomography (PACT). Previously reported studies of dynamic PACT are based on a frame-by-frame image reconstruction (FBFIR) procedure in which full-view measurement data are assumed to be rapidly acquired. However, many commercial three-dimensional PACT imagers acquire measurements at each tomographic view rotating the object in discrete steps. The time to collect the full-view data is limited by the rotation speed of the object holder and the laser repetition rate. Therefore, FBFIR techniques are not applicable, and there is a critical need for accurate and efficient spatiotemporal image reconstruction (STIR) techniques that can account for spatiotemporal redundancies in the object’s features. To address this, we propose a low-rank matrix estimation-based STIR technique in which the sought-after dynamic image is approximated using a semiseparable approximation in space and time. To validate the proposed method, we also develop a virtual imaging framework for PACT employing dynamic numerical mouse phantoms with a physiologically realistic respiratory motion and perfusion model. The studies demonstrated that the proposed method accurately recovers the tumor vascular perfusion and object motion.

Gastric Cancer Angiogenesis Assessment by Dynamic Contrast Harmonic Imaging Endoscopic Ultrasound (CHI-EUS) and Immunohistochemical Analysis-A Feasibility Study.

Tumor vascular perfusion pattern in gastric cancer (GC) may be an important prognostic factor with therapeutic implications. Non-invasive methods such as dynamic contrast harmonic imaging endoscopic ultrasound (CHI-EUS) may provide details about tumor perfusion and could also lay out another perspective for angiogenesis assessment. Methods: We included 34 patients with GC, adenocarcinoma, with CHI-EUS examinations that were performed before any treatment decision. We analyzed eighty video sequences with a dedicated software for quantitative analysis of the vascular patterns of specific regions of interest (ROI). As a result, time-intensity curve (TIC) along with other derived parameters were automatically generated: peak enhancement (PE), rise time (RT), time to peak (TTP), wash-in perfusion index (WiPI), ROI area, and others. We performed CD105 and CD31 immunostaining to calculate the vascular diameter (vd) and the microvascular density (MVD), and the results were compared with CHI-EUS parameters. Results: High statistical correlations (p < 0.05) were observed between TIC analysis parameters MVD and vd CD31. Strong correlations were also found between tumor grade and 7 CHI-EUS parameters, p < 0.005. Conclusions: GC angiogenesis assessment by CHI-EUS is feasible and may be considered for future studies based on TIC analysis.

Candesartan induces tumor vascular normalization to improve the efficacy of radiotherapy in the therapeutic window.

BackgroundThis study sought to examine whether candesartan induces tumor vascular normalization in a narrow therapeutic window to increase perfusion and oxygen content, and thus enhance the effect of radiotherapy (RT).MethodsCandesartan was administered to tumor-bearing mice to induce the normalization of tumor vessels, and the time window for normalization was determined. Then, tumor vascular density, perfusion, pericyte coverage, hypoxia of tumor-bearing mice was detected at 3, 6, 9, 12 days, respectively. In the treatment window, RT was administered to enhance the anti-tumor effect, which was evaluated based on relevant anti-tumor indicators. The tumor volume and tumor weight were recorded. Next, tumor growth inhibition rate was calculated according to the tumor weight. The Tunnel and Ki-67 expression of the tumor tissues of every group was measured to evaluate the effects on tumor proliferation and apoptosis.ResultsBased on tumor vascular density, perfusion, pericyte coverage, hypoxia, and other indicators, the tumor vascular normalization window occurred 9 days after the administration of candesartan. The anti-tumor results showed that administering RT during the therapeutic window enhanced anti-tumor efficacy, with a tumor growth inhibition rate of 66.67%. The results of Tunnel and Ki-67 expression showed that window-period RT significantly promoted tumor cell apoptosis and limited proliferation compared to the control group (P<0.05 in Tunnel expression and P<0.01 in Ki-67 expression).ConclusionsCandesartan regulates the normalization of tumor vessels and increases the sensitivity of RT in the therapeutic window.

Endostar Rebuilding Vascular Homeostasis and Enhancing Chemotherapy Efficacy in Cervical Cancer Treatment.

BackgroundThe incidence rate of cervical cancer is the highest in the reproductive tract and is not sensitive to chemotherapy. An appropriate amount of anti-angiogenic agents can reconstruct tumor blood vessels in a short period of time and form vascular homeostasis, increase the function of blood vessel perfusion and reverse the multidrug resistance of chemotherapy, which is also called “vascular normalization.” Endostar (a recombinant human endostatin) was developed by China and as a multi-target anti-angiogenesis agent. Many reports about endostar involved the treatment of non-small cell lung cancer, fewer reports are on cervical cancer.PurposeTo determine whether endostar can rebuild tumor vascular homeostasis and enhance chemotherapy effects for patients with cervical cancer.MethodsIn this study, the patients with cervical cancer within stage IIB2 were selected, endostar combined with cisplatin+paclitaxel neoadjuvant chemotherapy (NACT) before radical surgical operation was adopted, patients outcome and adverse reaction were followed up. The changes of tumor vascular structure and perfusion function before and after endostar given were evaluated by histopathology and dynamic contrast-enhanced magnetic resonance imaging (DEC-MRI). VEGF-Notch signal pathway was detected for the regulating mechanism of vascular proliferation in different groups. GraphPad Prism 6 software was used for statistical analysis of the study results.ResultsEndostar enhanced the short-term (2 year) overall survival (OS), progression-free survival (PFS) rates for cervical cancer patients. All the same, endostar increased long-term (5 year) OS for cervical cancer patients. Endostar therapy exhibited with mild adverse reaction. MRI showed endostar+NACT further reduce tumor volume than NACT alone. The parameters of Ktrans, Ve for DEC-MRI in endostar group exhibited obviously increase than NACT group. Tumor vascular maturation index α-SMA/CD31 in endostar group increased obviously than NACT group, correspondingly Ki67 staining for tumor proliferative rates, lymphovascular space invasion in endostar group further declined than NACT group. The genes and proteins expression of VEGFR2, Notch1, Notch4, Dll4, Jag1 were obviously downregulated in endostar group comparing to NACT group.ConclusionEndostar restored vascular homeostasis in cervical cancer temporarily, enhanced chemotherapeutic agents effects in cervical cancer, increased patient OS ratio. Endostar+NACT treatment may provide a new target therapy for cervical cancer.

Abstract 97: The effect of hyaluronan degradation on the uptake and cytotoxic activity of liposome-encapsulated doxorubicin in 3D tumor spheroid models

Abstract The accumulation of hyaluronan (HA) in the solid tumor microenvironment (TME) can be associated with poor prognosis in several human cancers. Accumulation of HA in the TME can increase interstitial pressure that compresses local blood vessels and impairs the access of anti-cancer therapy. In preclinical models, enzymatic degradation of HA by pegvorhyaluronidase alfa (PEGPH20; PVHA), a PEGylated form of recombinant human hyaluronidase PH20 (rHuPH20), is associated with decreased tumor interstitial pressure, improved tumor vascular perfusion, and increased access and anti-tumor efficacy of cytotoxic and immunotherapies. The functional effect of HA degradation on the TME has been mostly characterized in animal tumor models. Here, we describe the utilization of 3D tumor spheroid models to evaluate the effects of HA degradation on the cytotoxic effect of anti-cancer therapies. A number of commonly used human colorectal cancer (CRC) cell lines were assessed for HA accumulation and tumor spheroid morphology: HCT 116, HT-29 parental, and HT-29/HAS3 (HT-29 cells engineered to over-express hyaluronan synthase 3). CRC 3D tumor spheroid models were developed to evaluate the uptake and cytotoxic activity of liposome-encapsulated doxorubicin (DOX) using the IncuCyte® S3 Live-Cell Analysis System. The effect of HA degradation on DOX uptake and cytotoxic activity was initially tested with rHuPH20 treatment in all three models, and further evaluated with PVHA treatment in HT-29 parental and HT-29/HAS3 models. A dose-dependent uptake and cytotoxic activity of DOX was observed in HCT 116 tumor spheroids. HA degradation after rHuPH20 treatment enhanced DOX uptake by an average of 6.1% (up to 7.7%) and cytotoxicity by an average of 37.9% (up to 58.2%) over the 5-day duration of the experiment compared with DOX alone. Dose-dependent uptake and cytotoxic activity of DOX in HT-29/HAS3 tumor spheroids were enhanced with rHuPH20 treatment to a greater extent than in HT-29 parental tumor spheroids. Specifically, rHuPH20 treatment led to an average increase in DOX uptake of 9.2% (up to 14.6%) with HT-29/HAS3 spheroids and 2.6% (up to 5.1%) with HT-29 parental spheroids. The addition of rHuPH20 resulted in an average increase in DOX cytotoxicity of 101% (up to 227.6%) with HT-29/HAS3 spheroids and 24.9% (up to 63.9%) with HT-29 parental spheroids. Enhanced uptake and cytotoxic activity of DOX was also observed with PVHA treatment in HT-29 and HT-29/HAS3 models. These data corroborate our previous findings with in vivo tumor models, in which the combination of PVHA-mediated HA degradation and other anti-cancer therapies display enhanced anti-tumor activity compared with either agent alone. Further, 3D tumor spheroid models may be used to evaluate the effect of extracellular matrix remodeling such as HA degradation and serve as a bridge between 2D cell-based assays and in vivo tumor models. Citation Format: Feng Gao, Susan Zimmerman, Kelly Chen, Daniel C. Maneval, Chunmei Zhao. The effect of hyaluronan degradation on the uptake and cytotoxic activity of liposome-encapsulated doxorubicin in 3D tumor spheroid models [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 97.

Overcoming Biological Barriers in Neuroblastoma Therapy: The Vascular Targeting Approach with Liposomal Drug Nanocarriers.

Neuroblastoma is a rare pediatric cancer characterized by a wide clinical behavior and adverse outcome despite aggressive therapies. New approaches based on targeted drug delivery may improve efficacy and decrease toxicity of cancer therapy. Furthermore, nanotechnology offers additional potential developments for cancer imaging, diagnosis, and treatment. Following these lines, in the past years, innovative therapies based on the use of liposomes loaded with anticancer agents and functionalized with peptides capable of recognizing neuroblastoma cells and/or tumor-associated endothelial cells have been developed. Studies performed in experimental orthotopic models of human neuroblastoma have shown that targeted nanocarriers can be exploited for not only decreasing the systemic toxicity of the encapsulated anticancer drugs, but also increasing their tumor homing properties, enhancing tumor vascular permeability and perfusion (and, consequently, drug penetration), inducing tumor apoptosis, inhibiting angiogenesis, and reducing tumor glucose consumption. Furthermore, peptide-tagged liposomal formulations are proved to be more efficacious in inhibiting tumor growth and metastatic spreading of neuroblastoma than nontargeted liposomes. These findings, herein reviewed, pave the way for the design of novel targeted liposomal nanocarriers useful for multitargeting treatment of neuroblastoma.

PO-263 Assessment of the accumulation of hyaluronan in the tumour microenvironment (TME) of solid tumours

IntroductionHyaluronan (HA), a primary component of the extracellular matrix, can accumulate in a variety of solid malignancies, where histological analyses of patient tumour tissues demonstrated that HA levels may correlate with patient outcomes. HA accumulation in the TME may be associated with poor prognosis. Preclinical studies demonstrated elevated TME HA levels increased tumour interstitial pressure, reduced tumour blood flow, and resulted in poor penetration of anticancer agents and immune cells. A PEGylated recombinant human hyaluronidase, pegvorhyaluronidase alfa (PEGPH20), depolymerizes HA, increases tumour vascular perfusion, and may increase access and anti-tumour efficacy of cytotoxic and immunotherapies.Material and methodsPegvorhyaluronidase alfa is being evaluated in clinical studies for pancreatic, breast, lung, cholangiocarcinoma, and gastric cancers. To broaden the spectrum of possible tumour types that may benefit from pegvyorhyaluronidase alfa, we assessed the prevalence and accumulation of HA in 3633 human histological samples in 22 solid tumour types. HA levels were measured using tissue microarrays (TMAs) stained with a proprietary HA probe, HTI–601, a recombinant immunoadhesin containing a modified HA–specific link domain from TNF-Stimulated Gene 6 protein (TSG–6) and the Fc portion of human IgG1. The resulting HA probe was biotinylated for use in an affinity-based histochemistry method. HA staining was evaluated with research-grade histological image analysis software. HA levels associated with distinct tumour compartments (stroma or tumour cells) were assessed. HA accumulation associated with immune infiltrates present in the TMA cores was also evaluated.Results and discussionsDistribution of HA accumulation across compartments fell into 4 main arrangements:- For desmoplastic tumour indications (pancreatic ductal adenocarcinoma, cholangiocarcinoma), the stroma was associated with high percentage of surface area stained with HTI-601 relative to the tumour cells;- Comparable distribution of HA staining in both tumour cells and stroma (gall bladder and glioblastoma);- Sub-clusters of HA staining within a specific tumour type (NSCLC non-squamous);- Distribution bias towards increased or decreased HA intensity staining (head and neck excluding nasopharyngeal cavities and renal cell carcinoma, respectively).ConclusionUnderstanding HA prevalence and accumulation in solid tumours may potentially identify malignancies that may benefit from pegvorhyaluronidase alfa therapy targeting HA.

Chemotherapy and Radiofrequency-Induced Mild Hyperthermia Combined Treatment of Orthotopic Pancreatic Ductal Adenocarcinoma Xenografts

Patients with pancreatic ductal adenocarcinomas (PDAC) have one of the poorest survival rates of all cancers. The main reason for this is related to the unique tumor stroma and poor vascularization of PDAC. As a consequence, chemotherapeutic drugs, such as nab-paclitaxel and gemcitabine, cannot efficiently penetrate into the tumor tissue. Non-invasive radiofrequency (RF) mild hyperthermia treatment was proposed as a synergistic therapy to enhance drug uptake into the tumor by increasing tumor vascular inflow and perfusion, thus, increasing the effect of chemotherapy. RF-induced hyperthermia is a safer and non-invasive technique of tumor heating compared to conventional contact heating procedures. In this study, we investigated the short- and long-term effects (~20 days and 65 days, respectively) of combination chemotherapy and RF hyperthermia in an orthotopic PDAC model in mice. The benefit of nab-paclitaxel and gemcitabine treatment was confirmed in mice; however, the effect of treatment was statistically insignificant in comparison to saline treated mice during long-term observation. The benefit of RF was minimal in the short-term and completely insignificant during long-term observation.

Accelerated tumour metastasis due to interferon-γ receptor-mediated dissociation of perivascular cells from blood vessels.

Angiostasis mediated by interferon (IFN)-γ is a key mechanism of anti-tumour immunity; however, the effect of IFN-γ on host vascular endothelial growth factor A (VEGFA)-expressing cells during tumour progression is still elusive. Here, we developed transgenic mice with IFN-γ receptor (IFNγR) expression under control of the Vegfa promoter (V-γR). In these mice, the IFN-γ responsiveness of VEGFA-expressing cells led to dramatic growth suppression of transplanted lung carcinoma cells. Surprisingly, increased mortality and tumour metastasis were observed in the tumour-bearing V-γR mice, in comparison with the control wild-type and IFNγR-deficient mice. Further study showed that perivascular cells were VEGFA-expressing cells and potential IFN-γ targets. In vivo, tumour vascular perfusion and pericyte association with blood vessels were massively disrupted in V-γR mice. In vitro, IFN-γ inhibited transforming growth factor-β signalling by upregulating SMAD7, and therefore downregulated N-cadherin expression in pericytes. Importantly, IFN-γ neutralization in vivo with a monoclonal antibody reduced tumour metastasis. Together, the results suggest that IFNγR-mediated dissociation of perivascular cells from blood vessels contributes to the acceleration of tumour metastasis. Thus, the inhibition of tumour growth via IFN-γ-induced angiostasis might also accelerate tumour metastasis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

18F-Fluoromisonidazole Kinetic Modeling for Characterization of Tumor Perfusion and Hypoxia in Response to Antiangiogenic Therapy.

Multiparametric imaging of tumor perfusion and hypoxia with dynamic 18F-fluoromisonidazole (18F-FMISO) PET may allow for an improved response assessment to antiangiogenic therapies. Cediranib (AZD2171) is a potent inhibitor of tyrosine kinase activity associated with vascular endothelial growth factor receptors 1, 2, and 3, currently in phase II/III clinical trials. Serial dynamic 18F-FMISO PET was performed to investigate changes in tumor biomarkers of perfusion and hypoxia after cediranib treatment. Methods: Twenty-one rats bearing HT29 colorectal xenograft tumors were randomized into a vehicle-treated control group (0.5% methylcellulose daily for 2 d [5 rats] or 7 d [4 rats]) and a cediranib-treated test group (3 mg/kg daily for 2 or 7 d; 6 rats in both groups). All rats were imaged before and after treatment, using a 90-min dynamic PET acquisition after administration of 42.1 ± 3.9 MBq of 18F-FMISO by tail vein injection. Tumor volumes were delineated manually, and the input function was image-derived (abdominal aorta). Kinetic modeling was performed using an irreversible 1-plasma 2-tissue compartmental model to estimate the kinetic rate constants K1, K1/k2, and k3-surrogates for perfusion, 18F-FMISO distribution volume, and hypoxia-mediated entrapment, respectively. Tumor-to-blood ratios (TBRs) were calculated on the last dynamic frame (80-90 min). Tumors were assessed ex vivo by digital autoradiography and immunofluorescence for microscopic visualization of perfusion (pimonidazole) and hypoxia (Hoechst 33342). Results: Cediranib treatment resulted in significant reduction of mean voxelwise 18F-FMISO TBR, K1, and K1/k2 in both the 2-d and the 7-d groups (P < 0.05). The k3 parameter was increased in both groups but reached significance only in the 2-d group. In the vehicle-treated groups, no significant change in TBR, K1, K1/k2, or k3 was observed (P > 0.2). Ex vivo tumor analysis confirmed the presence of hypoxic tumor regions that nevertheless exhibited relatively lower 18F-FMISO uptake. Conclusion:18F-FMISO kinetic modeling reveals a more detailed response to antiangiogenic treatment than a single static image is able to reveal. The reduced mean K1 reflects a reduction in tumor vascular perfusion, whereas the increased k3 reflects a rise in hypoxia-mediated entrapment of the radiotracer. However, if only late static images are analyzed, the observed reduction in 18F-FMISO uptake after treatment with cediranib may be mistakenly interpreted as a global decrease, rather than an increase, in tumor hypoxia. These findings support the use of 18F-FMISO kinetic modeling to more accurately characterize the response to treatments that have a direct effect on tumor vascularization and perfusion.

Tumor physiological changes during hypofractionated stereotactic body radiation therapy assessed using multi-parametric magnetic resonance imaging.

Radiation therapy is a primary treatment for non-resectable lung cancer and hypoxia is thought to influence tumor response. Hypoxia is expected to be particularly relevant to the evolving new radiation treatment scheme of hypofractionated stereotactic body radiation therapy (SBRT). As such, we sought to develop non-invasive tools to assess tumor pathophysiology and response to irradiation. We applied blood oxygen level dependent (BOLD) and tissue oxygen level dependent (TOLD) MRI, together with dynamic contrast enhanced (DCE) MRI to explore the longitudinal effects of SBRT on tumor oxygenation and vascular perfusion using A549 human lung cancer xenografts in a subcutaneous rat model. Intra-tumor heterogeneity was seen on multi-parametric maps, especially in BOLD, T2* and DCE. At baseline, most tumors showed a positive BOLD signal response (%ΔSI) and increased T2* in response to oxygen breathing challenge, indicating increased vascular oxygenation. Control tumors showed similar response 24 hours and 1 week later. Twenty-four hours after a single dose of 12 Gy, the irradiated tumors showed a significantly decreased T2* (-2.9±4.2 ms) and further decrease was observed (-4.0±6.0 ms) after 1 week, suggesting impaired vascular oxygenation. DCE revealed tumor heterogeneity, but showed minimal changes following irradiation. Rats were cured of the primary tumors by 3×12 Gy, providing long term survival, though with ultimate metastatic recurrence.

Abstract P4-02-02: Increased tumor perfusion following treatment with trastuzumab as measured by contrast-enhanced ultrasound

Abstract Introduction: The primary purpose of this study is to determine if the order of dosing in standard-of-care (SOC) combination therapy for HER2+ breast cancer has an effect on the perfusion characteristics within the tumor. Improving the intratumoral delivery of cytotoxic systemic therapy is a significant challenge in advancing cancer treatment. Knowledge of the vascular changes following SOC treatments could enable optimizing their order and timing, potentially leading to significantly improved response. Currently, one SOC regimen for HER2+ breast cancer treatment is doxorubicin administered for 3-4 cycles prior to trastuzumab. Our goal is to quantitatively map the changes in perfusion in response to different combinations of trastuzumab plus doxorubicin treatment through imaging in a murine model of HER2+ breast cancer. Experimental Design: BT474 breast cancer cells (1×107) were subcutaneously implanted into mice (n = 12) and randomly assigned into three treatment groups: two doses of trastuzumab (10 mg/kg) followed by doxorubicin (1.5 mg/kg), doxorubicin prior to trastuzumab (same total drug dosage as group 1), and saline. After tumors reached ~225 mm3, animals were imaged with contrast-enhanced ultrasound (CEUS) (VisualSonics Vevo 770, Definity microbubbles) before treatment (day 0), and on days 1, 3, 4, and 7. Treatment occurred on days 0, 3 and 4. Percent change (from baseline, day 0 scans) of the CEUS signal intensity quantified from the functional vasculature (surrogate for vessel perfusion) following contrast injection were measured for each animal for each day. Tumors were extracted on day 7, and sectioned, paraffin-embedded, and stained with CD31, alpha-SMA and H&amp;E. Results: Tumors treated with trastuzumab initially exhibited a significant increase in CEUS signal intensity (from the functional vasculature) on day 1 compared to tumors initially treated with doxorubicin (p &amp;lt; 0.01). Additionally, compared to the control tumors, tumors treated with trastuzumab prior to doxorubicin revealed a significant increase in perfusion (change in signal intensity of functional vasculature) of contrast agent on days 3 (p = 0.01), 4 (p = 0.001) and day 7 (p &amp;lt; 0.01). There were no significant differences in the doxorubicin treated first group and the controls on any of the days (p &amp;gt; 0.25). Qualitative differences were noted between control and treated groups for alpha-SMA, no apparent differences were noted in microvessel density. Conclusion: Trastuzumab significantly improves a tumor's vascular perfusion in this HER2+ breast cancer model. Doxorubicin dosing prior to treatment with trastuzumab may potentially be hindering the intratumoral delivery of the subsequently delivered, targeted therapy. Improving the tumor's functional vasculature by altering the order of dosing of these combination therapies by giving trastuzumab prior to cytotoxic therapy has potential to enhance both the delivery and the effectiveness of these combination therapies. These data indicate a potential pathway to optimize therapeutic efficacy for individual HER2+ breast cancer patients. Citation Format: Sorace AG, Barnes SL, Quarles CC, McIntyre JO, Yankeelov TE. Increased tumor perfusion following treatment with trastuzumab as measured by contrast-enhanced ultrasound [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-02-02.

Multiphoton imaging reveals that nanosecond pulsed electric fields collapse tumor and normal vascular perfusion in human glioblastoma xenografts

Despite the biomedical advances of the last century, many cancers including glioblastoma are still resistant to existing therapies leaving patients with poor prognoses. Nanosecond pulsed electric fields (nsPEF) are a promising technology for the treatment of cancer that have thus far been evaluated in vitro and in superficial malignancies. In this paper, we develop a tumor organoid model of glioblastoma and apply intravital multiphoton microscopy to assess their response to nsPEFs. We demonstrate for the first time that a single 10 ns, high voltage electric pulse (35–45 kV/cm), collapses the perfusion of neovasculature, and also alters the diameter of capillaries and larger vessels in normal tissue. These results contribute to the fundamental understanding of nsPEF effects in complex tissue environments, and confirm the potential of nsPEFs to disrupt the microenvironment of solid tumors such as glioblastoma.